Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel.

Introduction: Mammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the study: The present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methods: Sperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. Results: A significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20-100 µg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 µM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. Conclusions: PJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

Chemical constituents of the Ajuga multiflora bunge and their protective effects on dexamethasone-induced muscle atrophy in C2C12 myotubes.

Ajuga multiflora Bunge is a perennial ornamental herb and has been used for the treatment of fever in Korean folk medicine. In the course of searching for protective agents associated with the potential of A. multiflora against dexamethsone (DEX)-induced muscle atrophy, a new phytoecdysteroid, 29-hydroxyprecyasterone (1), together with four known compounds (2-5), were isolated from A. multiflora. The structures of the compounds were determined by spectroscopic analyses, including 1D-, 2D-NMR and HR-MS interpretation. To elucidate the effects of obtained compounds on DEX-induced muscle atrophy, the myotubes diameter, myosin heavy chain (MyHC) positive area, and fusion index were evaluated by immunofluorescence staining. Overall, each compound treatment effectively prevented the atrophic myotubes through an increase of MyHC-positive myotubes and the number of nuclei. Particularly, the measurement of myotube diameter showed that compounds 1 and 5 treatment significantly alleviated the myotube thickness.

In vitro assessment of Prunus japonica seed extract on human spermatozoa hypermotility and intracellular alkalization.

Prunus japonica var. nakaii is used in traditional Korean medicine to treat various conditions; however, it has not been investigated for treating male infertility. In this study, we investigated the in vitro effects of the ethanolic extract of P. japonica seeds on human sperm motility and identified its mechanism of action. Eleven male volunteers were selected, and the effects of the extract on human spermatozoa were assessed through a computer-assisted semen analysis. The P. japonica seed extract increased the percentage of total and progressive motility of spermatozoa. To understand the mechanism of action, we monitored intracellular alkalization using flow cytometry and obtained electrophysiological recordings of human voltage-gated proton channels hHv1 that were overexpressed in HEK-293 cells. The extract shifted the activation curves in a concentration-dependent manner. Two major constituents of the extract, linoleic acid and oleic acid, exhibited proton channel activity. Our in vitro experiments suggested that P. japonica seed extract could be potentially used to rescue sperm motility in idiopathic infertility patients via pharmacological modulation of the proton channels during capacitation. Therefore, our results indicate the therapeutic potential of P. japonica seed extract for treating male infertility.

The Effect of Curcuma phaeocaulis Valeton (Zingiberaceae) Extract on Prion Propagation in Cell-Based and Animal Models.

Prion diseases are neurodegenerative disorders in humans and animals for which no therapies are currently available. Here, we report that Curcuma phaeocaulis Valeton (Zingiberaceae) (CpV) extract was partly effective in decreasing prion aggregation and propagation in both in vitro and in vivo models. CpV extract inhibited self-aggregation of recombinant prion protein (PrP) in a test tube assay and decreased the accumulation of scrapie PrP (PrPSc) in ScN2a cells, a cultured neuroblastoma cell line with chronic prion infection, in a concentration-dependent manner. CpV extract also modified the course of the disease in mice inoculated with mouse-adapted scrapie prions, completely preventing the onset of prion disease in three of eight mice. Biochemical and neuropathological analyses revealed a statistically significant reduction in PrPSc accumulation, spongiosis, astrogliosis, and microglia activation in the brains of mice that avoided disease onset. Furthermore, PrPSc accumulation in the spleen of mice was also reduced. CpV extract precluded prion infection in cultured cells as demonstrated by the modified standard scrapie cell assay. This study suggests that CpV extract could contribute to investigating the modulation of prion propagation.

Application of Linear Gradient Solvent System in Centrifugal Partition Chromatography Facilitating Bioassay-Guided Fractionation of Yongdamsagan-Tang, Traditional Oriental Decoction.

As important pharmaceutical resources, traditional herbal medicines retain continuous attention. To do that, isolation and identification of bioactive molecules from traditional herbal decoction are important. However, conventional fractionation through octadecyl silica column faces irreversible sample adsorption that causes a bias in bioactivity assessment. However, liquid-liquid chromatographic system suffers tedious K value calculation as well as insufficient capacity in separation power when crude extract composed of widely ranging polarities. Here, we developed a comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) to aid bioassay-guided isolation. The lower aqueous phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary, whereas its upper organic phase followed by the upper phase of ethyl acetate-acetonitrile-water and water-saturated n-butanol-acetonitrile-water in the same ratio were eluted in a linear gradient mode, thereby increasing polarity in the mobile phase. The HPLC profiling of CPC fraction showed that proposed gradient CPC was suitable to separate metabolites from Yongdamsagan-Tang, a traditional medicinal decoction made of ten herbal plants. Exhibiting a high recovery yield of 98.3%, antioxidant response element (ARE) luciferase-inducing assay in HepG2 cells indicated that the fractions composed of baicalein and wogonin, the marker natural products of Scutellaria baicalensis, were to be the most effective molecules from Yongdamsagan-Tang. The presented results demonstrated that bioassay-guided separation that assisted with a linear gradient CPC is an incomparable alternative to HPLC and biphasic CPC in terms of higher yield rate and redundant K value calculation, respectively, which led to an unbiased/time-saving separation and identification of bioactive molecules from the complex crude extract of natural products.

Bioassay-Guided Isolation of Two Eudesmane Sesquiterpenes from Lindera strychnifolia Using Centrifugal Partition Chromatography.

In this study, a centrifugal partition chromatography (CPC) separation was applied to identify antioxidant-responsive element (ARE) induction molecules from the crude extract of Lindera strychnifolia roots. CPC was operated with a two-phase solvent system composed of n-hexane-methanol-water (10:8.5:1.5, v/v/v) in dual mode (descending to ascending), which provided a high recovery rate (>95.5%) with high resolution. Then, ARE induction activity of obtained CPC fractions was examined in ARE-transfected HepG2 cells according to the weight ratios of the obtained fractions. The fraction exhibiting ARE-inducing activity was further purified by preparative HPLC that led to isolation of two eudesmane type sesquiterpenes as active compounds. The chemical structures were elucidated as linderolide U (1) and a new sesquiterpene named as linderolide V (2) by spectroscopic data. Further bioactivity test demonstrated that compounds 1 and 2 enhanced ARE activity by 22.4-fold and 7.6-fold, respectively, at 100 µM concentration while 5 µM of sulforaphane induced ARE activity 24.8-fold compared to the control.

Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves.

Dendropanax morbifera leaves (DML) have long been used as traditional medicine to treat diverse symptoms in Korea. Ethyl acetate-soluble extracts of DML (DMLE) rescued HT22 mouse hippocampal neuronal cells from glutamate (Glu)-induced oxidative cell death; however, the protective compounds and mechanisms remain unknown. Here, we aimed to identify the neuroprotective ingredients and mechanisms of DMLE in the Glu-HT22 cell model. Five antioxidant compounds were isolated from DMLE and characterized as chlorogenic acid, hyperoside, isoquercitrin, quercetin, and rutin by spectroscopic methods. Isoquercitrin and quercetin significantly inhibited Glu-induced oxidative cell death by restoring intracellular reactive oxygen species (ROS) levels and mitochondrial superoxide generation, Ca2+ dysregulation, mitochondrial dysfunction, and nuclear translocation of apoptosis-inducing factor. These two compounds significantly increased the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the presence or absence of Glu treatment. Combinatorial treatment of the five compounds based on the equivalent concentrations in DMLE showed that significant protection was found only in the cells cotreated with isoquercitrin and quercetin, both of whom showed prominent synergism, as assessed by drug-drug interaction analysis. These findings suggest that isoquercitrin and quercetin are the active principles representing the protective effects of DMLE, and these effects were mediated by the Nrf2/HO-1 pathway.

Dibenzocyclooctadiene Lignans in Plant Parts and Fermented Beverages of Schisandra chinensis.

The fruit of Schisandra chinensis, Omija, is a well-known traditional medicine used as an anti-tussive and anti-diarrhea agent, with various biological activities derived from the dibenzocyclooctadiene-type lignans. A high-pressure liquid chromatography-diode array detector (HPLC-DAD) method was used to determine seven lignans (schisandrol A and B, tigloylgomisin H, angeloylgomisin H, schisandrin A, B, and C) in the different plant parts and beverages of the fruit of S. chinensis grown in Korea. The contents of these lignans in the plant parts descended in the following order: seeds, flowers, leaves, pulp, and stems. The total lignan content in Omija beverages fermented with white sugar for 12 months increased by 2.6-fold. Omija was fermented for 12 months with white sugar, brown sugar, and oligosaccharide/white sugar (1:1, w/w). The total lignan content in Omija fermented with oligosaccharide/white sugar was approximately 1.2- and 1.7-fold higher than those fermented with white sugar and brown sugar, respectively. A drink prepared by immersion of the fruit in alcohol had a higher total lignan content than these fermented beverages. This is the first report documenting the quantitative changes in dibenzocyclooctadiene-type lignans over a fermentation period and the effects of the fermentable sugars on this eco-friendly fermentation process.

MOTILIPERM Ameliorates Immobilization Stress-Induced Testicular Dysfunction via Inhibition of Oxidative Stress and Modulation of the Nrf2/HO-1 Pathway in SD Rats.

It is well established that physiological stress has an adverse effect on the male reproductive system. Experimental studies have demonstrated the promising effects of MOTILIPERM in male infertility. MOTILIPERM extract is composed of three crude medicinal herbs: Morinda officinalis How (Rubiaceae) roots, Allium cepa L. (Liliaceae) outer scales, and Cuscuta chinensis Lamark (convolvulaceae) seeds. The present study aimed to investigate the possible mechanisms responsible for the effects of MOTILIPERM on testicular dysfunction induced by immobilization stress. Fifty male Sprague Dawley rats were divided into five groups (10 rats each): a normal control group (CTR), a control group administered MOTILIPERM 200 mg/kg (M 200), an immobilization-induced stress control group (S), an immobilization-induced stress group administered MOTILIPERM 100 mg/kg (S + M 100), and MOTILIPERM 200 mg/kg (S + M 200). Stressed rats (n = 30) were subjected to stress by immobilization for 6 h by placing them in a Perspex restraint cage, while controls (n = 20) were maintained without disturbance. Rats were administrated 100 or 200 mg/kg MOTILIPERM once daily for 30 days 1 h prior to immobilization. At the end of the treatment period, we measured body and reproductive organ weight; sperm parameters; histopathological damage; reproductive hormone levels; steroidogenic acute regulatory protein (StAR); biomarkers of oxidative stress; and apoptosis markers. MOTILIPERM treatment improved testicular dysfunction by up-regulating (p < 0.05) sperm count, sperm motility, serum testosterone level, StAR protein level, Johnsen score, and spermatogenic cell density in stressed rats. MOTILIPERM decreased oxidative stress by increasing (p < 0.05) testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione peroxidase-4 (GPx 4), catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) levels and decreasing (p < 0.05) malondialdehyde (MDA) and reactive oxygen species/reactive nitrogen species (ROS/RNS) levels. Furthermore, MOTILIPERM down-regulated (p < 0.05) cleaved caspase 3 and BCL2 associated X protein (Bax) levels; increased pro caspase-3 and B-cell lymphoma 2 (Bcl-2) levels; and upregulated testicular germ cell proliferation in stressed rats. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels also significantly (p < 0.05) decreased after pretreatment with MOTILIPERM in stressed rats. Collectively, our results suggest that, in immobilization-mediated stress-induced testicular dysfunction, MOTILIPERM sustains normal spermatogenesis via antioxidant and anti-apoptotic activities by activating the NRF/HO-1 signaling pathway.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

The ameliorative effect of monotropein, astragalin, and spiraeoside on oxidative stress, endoplasmic reticulum stress, and mitochondrial signaling pathway in varicocelized rats.

BACKGROUND: Monotropein, astragalin, and spiraeoside (MAS) are active compounds extracted from medicinal herbs; monotropein from Morinda officinalis How (Rubiaceae), astragalin (kaempferol 3-O-glucoside) from Cuscuta chinensis Lamark (Convolvulaceae) and spiraeoside from the outer scales of Allium cepa L. (Liliceae) in a ratio of 6.69:0.41:3.61. Monotropein, astragalin, and spiraeoside are well-known antioxidants, anti-inflammatory, and antinociceptive agents. The current investigation aims to study the molecular mechanism of varicocele-induced male infertility and the underlying pharmacological mechanisms of MAS. METHODS: Four groups were included: control (CTR), MAS 200 group (MAS 200 mg/kg), varicocele group (VC), and VC + MAS 200 group (MAS 200 mg/kg). Sprague-Dawley (SD) rats were treated with 200 mg/kg MAS or vehicle once daily for 28 days. The possible signaling mechanism and effects of MAS were measured via histological staining, immunohistochemistry, western blot, and biochemical assays. RESULTS: Parameters such as sperm motility and count, Johnsen's scores, spermatogenic cell density, serum testosterone, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and expression of the steroidogenic acute regulatory protein (StAR) improved significantly in the VC + MAS 200 group compared with the VC group. MAS treatment of varicocele-induced group significantly decreased the levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as testicular interleukin-6 (IL6), tumor necrosis factor-α (TNF-α), ROS/RNS, and malondialdehyde (MDA). It also decreased the apoptotic index and reduced the expression of endoplasmic reticulum (ER) protein levels (Grp78, p-IRE1α, and p-JNK) and apoptotic markers such as cleaved caspase-3 and Bax/Bcl2 ratio. CONCLUSION: This study suggests that the crosstalk between oxidative stress, ER stress, and mitochondrial pathway mediates varicocele-induced testicular germ cell apoptosis. MAS promotes spermatogenesis in varicocele-induced SD rat, probably by decreasing cytokines (IL-6, TNF-α) levels, regulating abnormal sex hormones, and decreasing oxidative stress, ER stress, and apoptosis.

Cross-talk between ER stress and mitochondrial pathway mediated adriamycin-induced testicular toxicity and DA-9401 modulate adriamycin-induced apoptosis in Sprague-Dawley rats.

BACKGROUND: DA-9401 was prepared as a mixture of Chinese medicinal herb extracts from roots of Morinda officinalis How (Rubiaceae), outer scales of Allium cepa L. (Liliceae) and seeds of Cuscuta chinensis Lamark (Convolvulaceae). The present study was designed to investigate the possible protective role of DA-9401 in adriamycin (ADR)-induced testicular toxicity associated with oxidative stress, endoplasmic reticulum (ER) stress, and apoptosis. METHODS: Fifty healthy 8-week-old male Sprague-Dawley rats were equally divided into five groups. The first CTR group was treated with normal saline 2 ml/day by gavage. The second was treated with DA-100 (DA-9401 100 mg/kg/day). The third (ADR) group received ADR (2 mg/kg/once a week) intraperitoneally, while the combination of ADR and DA-9401 was given to the fourth ADR + DA-100 (100 mg/kg/day p.o) group and fifth ADR + DA-200 (200 mg/kg/day p.o) group. At the end of the 8-week treatment period, body weight, reproductive organ weights, fertility rate, pups per female were recorded, and serum were assayed for hormone concentrations. Tissues were subjected to semen analysis, histopathological changes, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), oxidative stress markers and expression levels of endoplasmic reticulum (ER) stress markers, apoptosis markers, tight junction protein markers, steroidogenic acute regulatory protein (StAR), cation channel of sperm (CatSper) and glycogen synthase kinase-3 (GSK-3) by western blot. RESULTS: DA-9401 administration to ADR-treated rats significantly decreased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, interleukin-6, TNF-α, MDA level, ROS/RNS level, ER stress response protein levels, tunnel positive cells, cleaved caspase-3, and Bax/Bcl2 ratio. Moreover, pretreatment with DA-9401 significantly increased body weight, reproductive organ weights, fertility rate, pups per female, Johnsen's score, spermatogenic cell density, sperm count and sperm motility, serum testosterone concentration, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), tight junction protein markers, star protein level, CatSper, and GSK-3 level. CONCLUSIONS: ADR treatment can markedly impair testicular function and induce testicular cell death presumably by causing significant changes in oxidative stress, ER stress, and mitochondrial pathway. DA-9401 exerts beneficial effects against oxidative stress, ER stress, and mitochondria-mediated cell death pathway in testis tissue by up-regulating expression levels of tight junction protein markers, steroidogenic acute regulatory protein, GSK-3 alpha, and cation channels of sperm.

Application of Centrifugal Partition Chromatography for Bioactivity-Guided Purification of Antioxidant-Response-Element-Inducing Constituents from Atractylodis Rhizoma Alba.

Activity-guided separation of antioxidant response element (ARE)-inducing constituents from the rhizomes of Atractylodis Rhizoma Alba was performed by the combination of centrifugal partition chromatography (CPC) and an ARE luciferase reporter assay. From 3 g of the active n-hexane fraction, one polyacetylene, (6E,12E)-tetradeca-6,12-dien-8,10-diyne-1,3-diyl diacetate (47.3 mg), and two sesquiterpenes, atractylenolide I (40.9 mg), and selina-4(14),7(11)-dien-8-one (6.0 mg) were successfully isolated by CPC with n-hexane⁻ethyl acetate⁻methanol⁻water (8:2:8:2, v/v). The chemical structures of the isolated compounds were determined by ¹H- and 13C-NMR and ESI-MS. Among the isolated compounds, (6E,12E)-tetradeca-6,12-diene-8,10-diyne-1,3-diol diacetate and selina-4(14),7(11)-dien-8-one increased ARE activity 32.9-fold and 16.6-fold, respectively, without significant cytotoxicity, when 5 µM sulforaphane enhanced ARE activity 27.1-fold. However, atractylenolide I did not increase ARE activity at 100 µM, and showed cytotoxicity at concentrations over 10 µM.

Protective effect of MOTILIPERM in varicocele-induced oxidative injury in rat testis by activating phosphorylated inositol requiring kinase 1α (p-IRE1α) and phosphorylated c-Jun N-terminal kinase (p-JNK) pathways.

CONTEXT: MOTILIPERM was prepared as a mixture of extracts of three medicinal herbs [roots of Morinda officinalis How (Rubiaceae), outer scales of Allium cepa L. (Liliaceae) and seeds of Cuscuta chinensis Lamark (Convolvulaceae)]. OBJECTIVE: To investigate the role of reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress in a rat model of varicocele and the therapeutic efficacy of MOTILIPERM in this model. MATERIALS AND METHODS: Sixty male rats were divided into five experimental groups: a normal control group (CTR + vehicle), a control group administered MOTILIPERM 200 mg/kg (CTR + M 200), a varicocele-induced control group (VC + vehicle) and two varicocele-induced groups administered MOTILIPERM 100 (VC + M 100) or 200 (VC + M 200) mg/kg for 4 weeks. Testis weights were recorded and serums were assayed for hormone concentrations. Tissues were subjected to semen analysis, histopathology, analyses of ER response protein expression levels and oxidative stress were assessed by measuring ROS, reactive nitrogen species (RNS), malondialdehyde (MDA) level and ratios of total glutathione (GSH)/oxidized GSH (GSSG). RESULTS: MOTILIPERM treatment of varicocele-induced groups significantly increased left testis weight, testosterone level, sperm motility, count and spermatogenic cell density. ER-response protein expression levels were dose-dependently decreased in VC + M 200 group compared with VC + vehicle group. MOTILIPERM treatment also decreased MDA and ROS/RNS level but increased GSH/GSSG ratio. DISCUSSION AND CONCLUSIONS: This study suggests that ROS-related ER stress may play a major role in varicocele-induced infertility and MOTILIPERM, a novel compound targeting ROS-based ER stress, may be therapeutically useful in treatment of varicocele, or as a supplement for the treatment of infertility.

Protective effect of DA-9401 in finasteride-induced apoptosis in rat testis: inositol requiring kinase 1 and c-Jun N-terminal kinase pathway.

Finasteride is used to treat male pattern baldness and benign prostatic hyperplasia. This study investigated the toxicity of finasteride and recovery by DA-9401 using Sprague Dawley (SD) rats. Forty adult male SD rats were assigned to four groups: control (CTR), finasteride 1 mg/kg/day (F), finasteride 1 mg/kg + DA-9401 100 mg/kg/day (F + DA 100) and finasteride 1 mg/kg + DA-9401 200 mg/kg/day (F + DA 200). Treatments were by oral delivery once daily for 90 consecutive days. The gross anatomical parameters assessed included: genital organ weight; vas deferens sperm count and sperm motility; testosterone, dihydrotestosterone (DHT) and malondialdehyde levels; and histological and terminal deoxynucleotidyl transferase enzyme mediated dUTP nick-end labeling (TUNEL) staining of testis for spermatogenic cell density, Johnsen's score and apoptosis. Testicular tissue was also used for evaluating endoplasmic reticulum (ER) stress and apoptotic proteins. Epididymis weight, seminal vesicle weight, prostate weight, penile weight and vas deferens sperm motility showed significant differences between the F group and the CTR, F + DA 100 and F + DA 200 groups. There was no significant change in the testosterone level. DHT level decreased significantly in the F group compared with the CTR group. Testis tissue revealed significant changes in spermatogenic cell density, Johnsen's score and apoptotic index. Western blot showed significant changes in the ER stress and apoptotic markers. Finasteride resulted in reduced fertility and increased ER stress and apoptotic markers, which were recovered by administration of DA-9401 in the SD rats.

A new diacetylated flavonol triglycoside from the aerial parts of Actinidia polygama.

Phytochemical investigation of a methanolic extract of aerial parts Actinidia polygama Miq. led to the isolation of one new diacetylated flavonol triglycoside, kaempferol 3-O-[α-L-rhamnopyranosyl(1→3)-(4-O-acetyl)-O-α-L-rhamnopyranosyl(1→6)-(2-Oacetyl)-O-ß-D-galactopyranoside] (1) along with 12 known compounds (2-13). The chemical structures were determined using their spectroscopic data including 1D and 2D NMR. To the best of our knowledge, this is the first time that compounds 2, 4, 6, 7, 8, 9, 12 and 13 are isolated from this plant. All purified compounds were tested for free radical scavenging effect using DPPH and ABTS assays. Our results showed that compounds 4, 6, 7 and 13 have potential antioxidative effect for scavenging both DPPH· and ABTS·+ radicals that are comparable with those of ascorbic acid used as positive control, whereas compounds 1 and 2, which are di- and mono- acetylated flavonol triglycoside respectively, were not found to be potent scavengers of free radicals.

Angelica dahurica Extracts Improve Glucose Tolerance through the Activation of GPR119.

G protein-coupled receptor (GPR) 119 is expressed in pancreatic ß-cells and intestinal L cells, and is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, respectively. Therefore, the development of GPR119 agonists is a potential treatment for type 2 diabetes. We screened 1500 natural plant extracts for GPR119 agonistic actions and investigated the most promising extract, that from Angelica dahurica (AD), for hypoglycemic actions in vitro and in vivo. Human GPR119 activation was measured in GeneBLAzer T-Rex GPR119-CRE-bla CHO-K1 cells; intracellular cAMP levels and insulin secretion were measured in INS-1 cells; and GLP-1 release was measured in GLUTag cells. Glucose tolerance tests and serum plasma insulin levels were measured in normal C57BL6 mice and diabetic db/db mice. AD extract-treated cells showed significant increases in GPR119 activation, intracellular cAMP levels, GLP-1 levels and glucose-stimulated insulin secretion as compared with controls. In normal mice, a single treatment with AD extract improved glucose tolerance and increased insulin secretion. Treatment with multiple doses of AD extract or n-hexane fraction improved glucose tolerance in diabetic db/db mice. Imperatorin, phellopterin and isoimperatorin were identified in the active fraction of AD extract. Among these, phellopterin activated GPR119 and increased active GLP-1 and insulin secretion in vitro and enhanced glucose tolerance in normal and db/db mice. We suggest that phellopterin might have a therapeutic potential for the treatment of type 2 diabetes.

Potent Anti-inflammatory and Analgesic Actions of the Chloroform Extract of Dendropanax morbifera Mediated by the Nrf2/HO-1 Pathway.

Dendropanax morbifera LEVEILLE (DP) has been used in traditional Korean medicines to treat a variety of inflammatory diseases. Although the in vitro anti-inflammatory potential of this plant is understood, its in vivo efficacy and underlying molecular mechanism of anti-inflammatory effects are largely unknown. We elucidated the anti-inflammatory and analgesic activities and the underlying molecular mechanisms of DP using in vitro and in vivo models. Lipopolysaccharide (LPS)-stimulated murine macrophages were used to analyze the in vitro anti-inflammatory potential of DP extract and to elucidate the underlying mechanisms. In vivo animal models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and acetic acid-induced writhing response tests were used to analyze the in vivo anti-inflammatory effects and anti-nociceptive effects of DP extract, respectively. Methanolic extract of DP (DPME) significantly inhibited the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-activated macrophages. Among the five sub-fractions, the chloroform fraction (DP-C) showed the most potent suppressive effects against pro-inflammatory mediators and cytokines in LPS-stimulated macrophages. These effects were attributed to inhibition of nuclear factor-κB (NF-κB) nuclear translocation and c-Jun N terminal kinase (JNK) 1/2 phosphorylation and to activation of NF-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling. DP-C exhibited strong protective in vivo effects in TPA-induced ear edema mouse model and acetic acid-induced writhing response test. Our data suggest that DP-C has potent anti-inflammatory and analgesic activities and may be a promising treatment against a variety of inflammatory diseases.

Preparative separation of sesamin and sesamolin from defatted sesame meal via centrifugal partition chromatography with consecutive sample injection.

A preparative separation method using consecutive sample injection centrifugal partition chromatography (CPC) was developed to obtain sesamin and sesamolin from defatted sesame meal extracts. A two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (8:2:8:2, v/v) was applied in reversed-phase mode (descending mode). Preliminary experiments with an SCPC-100 (column volume: 100mL) were performed to select the appropriate two-phase solvent system and sample injection times; these parameters were then used with an SCPC-1000 (column volume: 1000mL) in a 10-fold scale-up preparative run. A sample containing 3g of crude extract was consecutively injected four times onto the SCPC-1000, which yielded 328mg of sesamin and 168mg of sesamolin. These compounds were analyzed by high-performance liquid chromatography and determined to have purities of 95.6% and 93.9%, respectively. Sesamin and sesamolin (30µM) increased antioxidant response element (ARE) luciferase activity 2.6-fold and 1.9-fold, respectively.

Heme oxygenase 1-mediated novel anti-inflammatory activities of Salvia plebeia and its active components.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia plebeia R. Br. (SP) has been widely used as a traditional folk medicine for the treatment of infectious diseases and pain. An anti-inflammatory potential of SP has remains largely unknown. AIM OF THE STUDY: We tried to elucidate the principle mechanism and the active ingredients underlying the anti-inflammatory activities of SP. MATERIALS AND METHODS: We investigated the protective activities of SP methanolic extract (SPME) and seven representative ingredients against inflammation. Quantitative analysis using HPLC-DAD-ESI/MS was conducted to determine the relative amounts of these seven active ingredients in SPME. Both in vitro murine macrophages and in vivo mouse models were employed to elucidate SP- and active ingredient-mediated anti-inflammatory effects. RESULTS: SPME significantly reduced inflammatory processes both in vivo in a TPA-induced ear edema model and in vitro in lipopolysaccharide (LPS)-activated macrophages. SPME decreased the release of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS). Seven active components (luteoloside (C1), nepitrin (C2), homoplantagenin (C3), luteolin (C4), nepetin (C5), hispidulin (C6), and eupatorin (C7)) of SPME were analyzed and their relative concentrations were determined, demonstrating that C2, C3, C5 and C6 were present in higher amounts than were C1, C4, and C7. These major compounds inhibited NO and PGE2 production, and iNOS and COX-II protein expression through heme oxygenase-1 (HO-1) induction via activation of nuclear factor erythroid 2-related factor2 (Nrf2). CONCLUSION: Our data demonstrate that SPME possesses potent in vitro and in vivo anti-inflammatory activities. Nepetin and hispidulin, and their glycosides are the major active compounds in SPME, and their effects are mediated by Nrf2/HO-1 signaling. Taken together, we propose that SPME and its active ingredients may serve as novel therapeutic candidates for diseases associated with excessive inflammation.