Toxicological assessment of enzyme-treated Zizania latifolia extract: Oral toxicology and genotoxicity in rats.

Zizania latifolia Turcz. has long been used as a food source in Southeast Asia. The grains, stems, and leaves of Z. latifolia and its major component, tricin, have also been studied to determine their biological activities. Previously, we hydrolyzed the aerial part of Z. latifolia using an enzyme mixture to maximize the tricin content of the Z. latifolia extract. However, the safety of enzyme-treated Z. latifolia extract (ETZL; DermaNiA™) has not yet been determined. In this study, we performed an in vivo 90-day repeated-dose evaluation and genotoxicity study to assess the toxicological potential of ETZL. EZTL did not exhibit genotoxicity in the bacterial reverse mutation test, in vitro chromosomal aberration assay, or in vivo micronucleus test. Moreover, no changes in body weight or hematological and serum biological parameters were observed in male or female rats under high-dose EZTL treatment (5000 mg/kg body weight (bw)/day) for 90 days with a 4-week recovery period. Significant changes were noted in the forestomach, kidneys, and adrenal glands in the test groups, but these changes, or tendency for recovery, were not observed in the recovery group. Based on these data, the no adverse effect level was determined to be 1250 mg/kg bw/day in rats.

Anti-inflammatory activity of palmitoylethanolamide ameliorates osteoarthritis induced by monosodium iodoacetate in Sprague-Dawley rats.

Novel treatment strategies are urgently required for osteoarthritis (OA). Palmitoylethanolamide (PEA) is a naturally occurring fatty acid amide with analgesic and anti-inflammatory effects. We aimed to examine its effect on OA and elucidate the molecular mechanism of actions in monosodium iodoacetate (MIA)-induced OA Sprague-Dawley rats. The experimental animals were divided into normal control group (injected with saline + treated with phosphate-buffered saline (PBS), NOR), control group (injected with MIA + treated with PBS, CON), 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day, PEA50 or PEA100), and positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day, DiC). The changes in blood parameters, body parameters, gene expression of inflammatory mediators and cytokines, knee thickness, and joint tissue were observed. Oral administration of PEA had no adverse effects on the BW, liver, or kidneys. PEA reduced knee joint swelling and cartilage degradation in MIA-induced OA rats. The serum levels of leukotriene B4, nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and prostaglandin E2 considerably reduced in the PEA100 group compared with those in the CON group. In the synovia of knee joints, the mRNA expression of iNOS, 5-Lox, Cox-2, Il-1ß, Tnf-α, and Mmp-2, -3, -9, and -13 apparently increased with MIA administration. Meanwhile, Timp-1 mRNA expression apparently decreased in the CON group but increased to the normal level with PEA treatment. Thus, PEA can be an effective therapeutic agent for OA.

Protection against UVB-Induced Wrinkle Formation in SKH-1 Hairless Mice: Efficacy of Tricin Isolated from Enzyme-Treated Zizania latifolia Extract.

Tricin, a flavone found mainly in rice bran and sugarcane, has various beneficial effects. It has proven to be a clinically safe and selective potent inhibitor of different cancer cell lines. In this study, we evaluated the efficacy of enzyme-treated Zizania latifolia (ETZL) and its major active compound tricin on skin photoaging in SKH-1 hairless mice. Tricin (0.3 mg/kg) and ETZL (50, 150, and 300 mg/kg) were orally administrated to mice for 14 weeks; no cytotoxicity was observed during the entire experimental period. After UVB exposure, we observed significant increases in keratinization, coarse wrinkles, loss of moisture, thickened epidermis, and collagen fiber degradation in the dorsal skin. These features of photoaging were significantly suppressed after oral administration of tricin or ETZL. In addition, the protein expression of collagen effectively increased in ETZL (150 and 300 mg/kg)-treated mice, while the increased metalloproteinase (MMP)-1 and MMP-3 expressions were reduced after exposure to tricin or ETZL, although the effects were not dose-dependent. These data indicate that ETZL may be effective for attenuation of UVB-induced skin damage and photoaging in hairless mice, possibly by inhibiting MMPs expression.

In vivo evaluation of hot water extract of Acorus gramineus root against benign prostatic hyperplasia.

BACKGROUND: Acorus gramineus has been reported to exhibit various pharmacological effects including inhibition of cholesterol synthesis, enhancement of lipid metabolism, prevention of dementia and inhibition of mast cell growth. According to the Chinese compendium of materia media, it has been reported that Acorus spp. is effective for sedation, dementia prevention as well as diuretic effect. In addition, it showed more than equivalent activity compared to furosoemide, a drug known to be effective in diuretic action in animal model study. However, their effectiveness against benign prostatic hyperplasia (BPH) of Acorus gramineus has not been reported. This study was designed to evaluate the effect of Acorus gramineus root hot water extract (AG) against BPH in vivo. METHODS: Male rats, 10 weeks of age and weighing 405 g ± 10 g, were used for this study. Biomarkers were evaluated including prostate weight, prostate weight ratio, hormonal changes, 5-α reductase type II androgen receptor (AR) of the prostate gland and anti-oxidant activation factors related to BPH. These biomarkers were measured in vivo test. RESULTS: AG showed significant effect at the 250 and 500 mg/kg/day in rats. Groups treated with AG displayed significantly lower levels of prostate gland weight (0.79 g) compared to the BPH induced group (1.19 g). Also, dihydrotestosterone (DHT) level was decreased from 61.8 to 100% and androgen receptor expression level was decreased from 111 to 658%. Any hematological toxicity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level wasn't observed. CONCLUSION: This study indicated that AG was effective for reducing BPH symptoms. TRIAL REGISTRATION: Not applicable.

Pharmacokinetics of Ginsenoside Rb1, Rg3, Rk1, Rg5, F2, and Compound K from Red Ginseng Extract in Healthy Korean Volunteers.

Individual differences in ginsenoside pharmacokinetics following ginseng administration in humans are still unclear. We aimed to investigate the pharmacokinetic properties of various ginsenosides, including Rb1, Rg3, Rg5, Rk1, F2, and compound K (CK), after a single oral administration of red ginseng (RG) and bioconverted red ginseng extract (BRG). This was a randomized, open-label, single-dose, single-sequence crossover study with washout every 1 week, and 14 healthy Korean men were enrolled. All subjects were equally assigned to two groups and given RG or BRG capsules. The pharmacokinetic parameters of ginsenosides were measured from the plasma drug concentration-time curve of individual subjects. Ginsenosides Rg3, Rk1 + Rg5, F2, and CK in the BRG group showed a higher C max, AUC(0-t), and AUC(0-∞) and shorter T max (for CK) than those in the RG group. These results suggest that BRG may lead to a higher absorption rate of bioactive ginsenosides. This study provides valuable information on the pharmacokinetics of various bioactive ginsenosides, which is needed to enhance the therapeutic efficacy and pharmacological activity of ginseng.

Gynostemma pentaphyllum extract and Gypenoside L enhance skeletal muscle differentiation and mitochondrial metabolism by activating the PGC-1α pathway in C2C12 myotubes.

BACKGROUND/OBJECTIVES: Peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α) has a central role in regulating muscle differentiation and mitochondrial metabolism. PGC-1α stimulates muscle growth and muscle fiber remodeling, concomitantly regulating lactate and lipid metabolism and promoting oxidative metabolism. Gynostemma pentaphyllum (Thumb.) has been widely employed as a traditional herbal medicine and possesses antioxidant, anti-obesity, anti-inflammatory, hypolipemic, hypoglycemic, and anticancer properties. We investigated whether G. pentaphyllum extract (GPE) and its active compound, gypenoside L (GL), affect muscle differentiation and mitochondrial metabolism via activation of the PGC-1α pathway in murine C2C12 myoblast cells. MATERIALS/METHODS: C2C12 cells were treated with GPE and GL, and quantitative reverse transcription polymerase chain reaction and western blot were used to analyze the mRNA and protein expression levels. Myh1 was determined using immunocytochemistry. Mitochondrial reactive oxygen species generation was measured using the 2'7'-dichlorofluorescein diacetate assay. RESULTS: GPE and GL promoted the differentiation of myoblasts into myotubes and elevated mRNA and protein expression levels of Myh1 (type IIx). GPE and GL also significantly increased the mRNA expression levels of the PGC-1α gene (Ppargc1a), lactate metabolism-regulatory genes (Esrra and Mct1), adipocyte-browning gene fibronectin type III domain-containing 5 gene (Fndc5), glycogen synthase gene (Gys), and lipid metabolism gene carnitine palmitoyltransferase 1b gene (Cpt1b). Moreover, GPE and GL induced the phosphorylation of AMP-activated protein kinase, p38, sirtuin1, and deacetylated PGC-1α. We also observed that treatment with GPE and GL significantly stimulated the expression of genes associated with the anti-oxidative stress response, such as Ucp2, Ucp3, Nrf2, and Sod2. CONCLUSIONS: The results indicated that GPE and GL enhance exercise performance by promoting myotube differentiation and mitochondrial metabolism through the upregulation of PGC-1α in C2C12 skeletal muscle.

Tannase-Converted Green Tea Extract with High (-)-Epicatechin Inhibits Skeletal Muscle Mass in Aged Mice.

The objective of this study was to investigate the effects of tannase-converted green tea extract on body composition, muscle oxidative stress-related factors, and differentiation-related factors. The mean bone-related parameters and body composition were determined by the live dual-energy X-ray absorptiometry (DEXA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to determine mRNA expression and protein levels, respectively. The results of total mass testing in the epicatechin control (EC) and middle concentration tannase-converted green tea extract (T 1) intake groups were not significantly different compared with those in the control group; however, the high-concentration tannase-converted green tea extract (T 2) group showed a significantly higher effect to the lean than that of all other groups (p < 0.05). The results of the assay of muscle differentiation-related genes indicated that the expression levels in the EC and T 1 groups (p < 0.05) and the expression levels in the T 2 group (p < 0.01) were significantly different in the bicep femoris compared with that in the control group. The results of the SOD gene assay indicate that the expression levels in the EC and T 1 groups (p < 0.05) and the expression level in the T 2 group (p < 0.01) were significantly different in the bicep femoris compared with that in the control group. Additionally, SOD gene expression in the T 2 group was significantly increased (p < 0.05) in the soleus compared with that in the control, EC and T 1 groups. Our results suggest that tannase-converted green tea extract prevents muscle loss and regulates the quantity and quality of muscle by the levels of antioxidant stress-related enzymes and muscle differentiation factors to a greater extent than the administration of epicatechin and middle dose green tea extract.

Enzyme-Treated Zizania latifolia Ethanol Extract Protects from UVA Irradiation-Induced Wrinkle Formation via Inhibition of Lysosome Exocytosis and Reactive Oxygen Species Generation.

Ultraviolet A (UVA) is a risk factor for photoaging and wrinkle formation. Zizania latifolia is an herbaceous perennial plant. It contains many bioactive compounds such as tricin that show antioxidative and anti-inflammatory effects. The aim of this study was to investigate the antiwrinkle effect of a mixture of hydrolytic enzyme (cellulase, hemicellulase and pectinase)-treated Z. latifolia extract (ZLE) and tricin on UVA-irradiated human dermal fibroblasts (HDFs) and SKH-1 hairless mice. Treatment of UVA-irradiated HDF cells with ZLE and tricin significantly decreased UVA induced-plasma membrane rupture, generation of ROS, expression levels of total and secreted lysosomal associated membrane protein (LAMP-1), cathepsin B and metalloproteinases (MMPs) and inhibited NF-κB activation. In the animal study, UVA-damaged epidermal and dermal tissues were repaired by the ZLE and tricin treatments. Administration of ZLE or tricin to UVA-irradiated animals recovered skin surface moisture and collagen fiber in dermal tissue. Treatment of ZLE or tricin decreased wrinkle formation, secretion of MMPs and expression levels of vascular endothelial growth factor (VEGF) and cathepsin B, and increased the expression level of collagen-1 in UVA-irradiated animals. Overall, the ZLE and tricin treatments decreased the skin damage induced by UVA irradiation via inhibition of lysosomal exocytosis and ROS generation. Therefore, ZLE and tricin are promising as antiwrinkle and antiphotoaging agents.

Effects of tannase-converted green tea extract on skeletal muscle development.

BACKGROUND: The aim of this study was to investigate the effect of tannase-converted green tea extract with a high (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and gallic acid (GA) content on myotube density and fusion in normal and oxidative stress-induced C2C12 skeletal muscle cells. Although the use of green tea extract is considered beneficial, cellular and molecular mechanisms of action of tannase-converted green tea extracts that are used as potential muscle growth materials have not been thoroughly studied. METHODS: This study used histological analysis and molecular biology techniques, and compared the results with those for AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) and green tea extracts. RESULTS: The myotube density of normal and oxidative stress-induced C2C12 cells was significantly higher in the tannase-converted green tea extract-treated group than that observed in the other groups (normal cells: P < 0.01; oxidative stress-induced cells: P < 0.05). In addition, tannase-converted green tea extract and green tea extract treatments significantly upregulated the genetic expression of myogenin, Myf5, and MyoD (P < 0.05). The levels of AMP-activated protein kinase-α (AMPKα) and muscle RING-finger protein-1 (MuRF-1) in the tannase-converted green tea extract group were higher than those in the AICAR and green tea extract groups (P < 0.05). CONCLUSIONS: Taken together, our findings describe that the high levels of EC, EGC, and GA in the tannase-converted green tea extract are attributable to the morphological changes in C2C12 cells and intercellular signaling pathways. Therefore, tannase-converted green tea extract can be used in the treatment of sarcopenia.

Effect of Enzymatic Treatment of Chrysanthemum indicum Linné Extracts on Lipid Accumulation and Adipogenesis in High-Fat-Diet-Induced Obese Male Mice.

Enzyme treatment of the foods and herbs has been used to improve the absorption rate the efficiency of plant extracts by converting the glycosides of the plant into aglycones. In this study, we examined the obesity-inhibitory effect of Chrysanthemum indicum Linné (CI) treated with enzymes such as viscozyme and tannase, which are highly efficient in converting glycosides to aglycones and then compared with untreated CI extract. The enzyme-treated CI ethanol extract (CIVT) was administered orally at various doses for 7 weeks in the high fat diet (HFD)-fed male mice. CIVT administration reduced the body weights, the food efficiency and the serum levels of lipid metabolism-related biomarkers, such as triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and leptin in the dose-dependent manner but not those high-density lipoprotein cholesterol (HDL-c) and adiponectin. CIVT also reduced considerably the total lipid amount in the liver and the size of adipocytes in the epididymal white adipose tissue (eWAT). CIVT effectively downregulated the adipogenesis-related transcription factors such as peroxisome proliferation activated receptor (PPAR)-γ and CCAAT/enhancer binding protein-α (C/EBP-α) but up-regulated PPAR-α, in the liver and eWAT. In addition, when compared to the enzyme-untreated CI 50% ethanol extract (CIEE), CIVT enhanced the reduction of body weight and lipid accumulation. Moreover, the viscozyme and tannase treatment of CI increased the flavonoid contents of the aglycone form. Therefore, our results support that the enzymatic treatment induced the production of aglycones for potentially suppressing the adipogenesis and lipid accumulation in HFD-fed mice. It suggests that CIVT might be an effective candidate for attenuating the over-weight and its related diseases.

Gynostemma Pentaphyllum Extract Ameliorates High-Fat Diet-Induced Obesity in C57BL/6N Mice by Upregulating SIRT1.

Gynostemma pentaphyllum is widely used in Asia as a herbal medicine to treat type 2 diabetes, dyslipidemia, and inflammation. Here, we investigated the anti-obesity effect and underlying mechanism of G. pentaphyllum extract (GPE) enriched in gypenoside L, gypenoside LI, and ginsenoside Rg3 and obtained using a novel extraction method. Five-week-old male C57BL/6N mice were fed a control diet (CD), high-fat diet (HFD), HFD + 100 mg/kg body weight (BW)/day GPE (GPE 100), HFD + 300 mg/kg BW/day GPE (GPE 300), or HFD + 30 mg/kg BW/day Orlistat (Orlistat 30) for 8 weeks. The HFD-fed mice showed significant increases in body weight, fat mass, white adipose tissue, and adipocyte hypertrophy compared to the CD group; but GPE inhibited those increases. GPE reduced serum levels of triglyceride, total cholesterol, and LDL-cholesterol, without affecting HDL-cholesterol. GPE significantly increased AMPK activation and suppressed adipogenesis by decreasing the mRNA expression of CCAAT/enhancer binding protein-α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein-1c (SREBP1c), PPARγ coactivator-1α, fatty acid synthase (FAS), adipocyte protein 2 (AP2), and sirtuin 1 (SIRT1) and by increasing that of carnitine palmitoyltransferase (CPT1) and hormone- sensitive lipase (HSL). This study demonstrated the ameliorative effect of GPE on obesity and elucidated the underlying molecular mechanism.