Comparison of the Extraction Effects of Two Methods on the Total Volatile Oil from Citrus aurantium and Cyperus rotundus in Qizhi Weitong Granule / 中国药房

OBJECTIVE： To compare the extraction effects of steam distillation method （SD） and extraction-azeotropic distillation coupling technology （WER） on the volatile oil from Citrus aurantium and Cyperus rotundus， so as to determine the suitable extraction method and improve the extraction technology of volatile components in Qizhi weitong granule. METHODS： SD and WER were used to extract the total volatile oil from C. aurantium and C. rotundus. t-test was conducted for the yield of volatile oil extracted by the two methods. GC-MS method was used to analyze the volatile oil， and the main components were determined. The relative content of main components was determined compared by area normalization method. GC-MS conditions included that Agilent HP-5 capillary column （30 m×0.25 mm ， 0.25 μm）， inlet temperature of 250 ℃， nitrogen as carrier gas， flow rate of 1 mL/min， split ratio of 20 ∶ 1， sample size of 2 μL， temperature programmed， electron bombardment， electron bombardment energy of 70 eV， scanning range of m/z 50-500. RESULTS： The appearance of volatile oil extracted by WER was more clear， with better product phase than that by SD. The average yield of volatile oil extracted by WER method were significantly higher than SD method （1.78％ vs. 1.48％， P＜0.01）. The volatile oil extracted by WER method and SD method contained 39 and 38 components， involving 38 common components. Among them， D-limonene， acetophenone， ketoenone and α-ketone were the main components of the total volatile oil from C. aurantium and C. rotundus， and the sum of the 4 main components was about 73.40％ （WER method） and 68.46％ （SD method） of the total components.  CONCLUSIONS： Compared with SD method， WER method for extracting volatile oil from C.aurantium and C. rotundus is better in product， phase higher in yield and higher in content of active volatiles， and is more suitable for the extraction of volatile components in Qizhi weitong granule.

Induction of Translocation of Glucose Transporter 4 in Rat Skeletal Muscle Cells by a Water-Soluble Extract from Culture Medium of <i>Ganoderma lucidum</i> Mycelia / 日本補完代替医療学会誌

<b>Objective:</b> The water-soluble extract of <i>Ganoderma lucidum</i> mycelia (WER), which is used as a health food, reduced hyperglycemia and enhanced glucose transporter 4 (GLUT4) translocation to the plasma membrane in skeletal muscle and adipose tissue in KK-<i>A</i>^y<i> </i>mice, a type 2 diabetic animal model with obesity.In order to elucidate the reduction of hyperglycemia by WER, we investigated the translocation of glucose transporter 4, glucose uptake and associating signal transduction in rat skeletal muscle (L6) cells. <b>Method:</b> The glucose uptake was analyzed with radioactive 2-deoxy-D-glucose.The localization of GLUT4 in L6 cells treated with various concentrations of WER was analyzed with immunohistochemical staining and Western blot technique.As a positive control, insulin or troglitazone was used in these experiments.Furthermore, the activation of intracellular signaling pathways by Western blot analysis and the influence of glucose uptake using four kinds of inhibitors (LY294002 as potent PI3K inhibitor, rapamycin as mTOR inhibitor, Gö6983 as broad PKC inhibitor, compound C as AMPK inhibitor) was evaluated. <b></b><b>Results: </b>GLUT4 protein content in the plasma membrane was induced in a dose-dependent manner of WER without increasing the gene expression and amount of total protein in the L6 cells and the glucose uptake was augmented with increasing the amount of GLUT4 translocated on the plasma membrane.The phosphorylation of phosphatidylinositol-3 kinase (PI3K), Akt and acetyl CoA carboxylase (ACC) were induced in a concentration dependent manner and inhibited by the above inhibitors except rapamycin. <b>Conclusion: </b>These results indicate that the hypoglycemic effect of some material(s) in WER may be due to the enhancement of glucose uptake through GLUT4 translocation on the plasma membrane by activating the PI3K/Akt pathway through improving insulin resistance.

Influence of a Water-Soluble Extract from Culture Medium of<i> Ganoderma lucidum</i> Mycelia (WER) on Carbohydrate Metabolism in the Liver of Type 2 Diabetic Mice / 日本補完代替医療学会誌

<b>Objective:</b> Recently, we reported that long-term intake of a water-soluble extract from culture medium of <i>Ganoderma lucidum</i> mycelia (WER) reduced hyperglycemia and enhanced glucose transporter-4 (GLUT4) translocation to the plasma membrane in skeletal muscles and adipose tissue in KK-<i>A^y</i> mice, a type 2 diabetic animal model with obesity. In the present study, we investigated the effect of WER on hepatic carbohydrate metabolism.<br> <b>Methods: </b>Female KK-<i>A^y</i> mice were given free access to water and high-fat food containing 0.5% WER for 8 weeks, and blood glucose levels were assessed every week. At the end of the experimental period, the expression and activities of sugar metabolic enzymes in the liver were determined by Real Time RT-PCR and each activity measurement method. Also, the amount of glycogen was measured by anthrone-sulfuric acid method. Furthermore, the expression level of GLUT2 and activation of AMP kinase (AMPK) and glycogen synthase kinase 3β (GSk3β) was also determined by western blot analysis.<br> <b>Results: </b>The mice with the high-fat ingestion showed a gradual increase in the levels of blood glucose and body weight. In the WER-treated mice, the blood glucose level was suppressed after 2 weeks of intake. The gene expression and enzyme activities of both glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were suppressed, whereas those of glucokinase were increased in the mice with WER intake and pioglitazone administration. The accumulation of glycogen was increased. Moreover the expression of GLUT2 and phosphorylation levels of AMPK and GSk3β were also increased in the mice with WER intake.<br> <b>Conclusion: </b>These results indicate that WER affects hepatic carbohydrate metabolism, which may derive from the suppression of gluconeogenesis through the modulation of related enzymes and enhancement of glucose uptake, glycolysis and glycogen synthesis.<br>

Inhibitory Effects of a Water-Soluble Extract from Culture Medium of Ganoderma lucidum (Rei-shi) Mycelia on Postprandial Blood Glucose Elevation in Type 2 Diabetic Mice and Additional Effect with .ALPHA.-Glucosidase Inhibitors / 日本補完代替医療学会誌

<b>Objective:</b> The water-soluble extract of <i>Ganoderma lucidum</i> mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown with<i> Ganoderma lucidum</i> mycelia. Recently, we reported that WER shows a blood glucose-lowering effect in maltose-loaded non-diabetic mice. Here, we investigated the efficacy of WER in type 2 diabetic state using KK-A^y mice. Moreover, the food-drug interactions of WER with α-glucosidase inhibitors, voglibose or acarbose were examined using both <i>in vitro</i> and <i>in vivo </i>experiments.<br> <b>Methods:</b> The glucose-lowering effects of oral administration <i>in vivo</i> of WER alone, or concomitant administration of WER with voglibose/acarbose on the elevation of blood glucose levels by sugar-tolerance tests were examined in KK-A^y mice. The inhibitory effects on α-glucosidase <i>in vitro</i> were also evaluated.<br> <b>Results:</b> Oral administration of WER (1 g/kg), which did not affect fasting blood glucose, significantly suppressed the hyperglycemia after loading of maltose (18% of decrease in AUC) compared to the water-administrated control mice. <i>In vitro</i> study showed that WER inhibited maltase in concentration-dependent manner. The inhibitory effects of lower concentrations of voglibose or acarbose on α-glucosidase activity were additively enhanced by the presence of WER, but those of higher concentrations were not affected. The glucose-lowering effect of voglibose (0.1 mg/kg) disappeared in maltose-loaded KK-A^y mice when the drug was concomitantly administrated with WER (1 g/kg), whereas acarbose (16 mg/kg) with WER showed no significant change in its effect.<br> <b>Conclusion:</b> These results demonstrated that WER shows the glucose-lowering effect in maltose-loaded KK-A^y, which may be based on inhibition of the α-glucosidase activity. The present study suggests that concomitant intake of WER with voglibose may override the therapeutic effect of voglibose on postprandial hyperglycemia by food-drug interaction in diabetic state.<br>

Hypoglycemic Effects of a Water-Soluble Extract from Culture Medium of Ganoderma lucidum (Rei-shi) Mycelia in Type 2 Diabetic Mice / 日本補完代替医療学会誌

<i>Objective</i>: The water-soluble extract of <i>Ganoderma lucidum</i> mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown with <i>Ganoderma lucidum</i> mycelia. Recently, we have reported that WER had glucose-lowering effect in streptozotosin-induced diabetic mice, an animal model of type 1 diabetes. Here, we investigated whether long-term treatment with WER affects hyperglycemia and insulin resistance in KK-A^y mice, a type 2 diabetic animal model with obesity.<br> <i>Methods</i>: Female KK-A^y mice were given free access to water and high-fat food containing 0.5% WER for 8 weeks, with blood glucose and plasma insulin levels assessed every week. At the end of the experimental period, insulin tolerance test (ITT) was performed, and plasma levels of triglyceride, total cholesterol, HDL-cholesterol, AST, ALT and adiponectin were measured. Furthermore, expression of GLUT4 in skeletal muscle cell membrane and adipocytes was also determined by immunostaining and Western blot analysis.<br> <i>Results</i>: The mice with high-fat ingestion showed a gradual increase in levels of blood glucose and body weight. In the WER-treated mice, the blood glucose level was significantly suppressed after 2 weeks of treatment. WER also reduced plasma levels of ALT and insulin, but did not affect the other parameters. Additionally, ITT revealed that WER improved insulin sensitivity. Moreover, expression of GLUT4 in the plasma membrane of skeletal muscle cells and adipocytes of the WER-treated mice was increased.<br> <i>Conclusion</i>: These results indicate that WER has a glucose-lowering effect in type 2 diabetic mice. WER also improved hyperinsulinemia and insulin sensitivity, which may derive from enhancement of glucose uptake through GLUT4 of skeletal muscle cells and adipocytes.<br>

Antioxidant Properties of a Water-soluble Extract from Culture Medium of Ganoderma lucidum (Rei-shi) Mycelia and Antidiabetic Effects in Streptozotocin-treated Mice / 日本補完代替医療学会誌

<i>Objective</i>: <i>Ganoderma lucidum</i> (Rei-shi) is widely used as an alternative medicine agent to promote health and longevity. The water-soluble extract from culture medium of <i>Ganoderma lucidum</i> mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown for 3–4 months with <i>Ganoderma lucidum</i> mycelia.<br> <i>Design</i>: In this study, we evaluate antioxidant activity of WER <i>in vitro</i>, and examined the effects of oral treatment of WER on oxidative stress in streptozotocin (STZ)-induced diabetic mice.<br> <i>Method</i>: Male ICR mice were treated with STZ (150 mg/kg, i.p.) and were housed for a week for induction of experimental diabetic state. WER (1 g/kg daily) was orally administered for an additional 9 weeks, and the levels of blood glucose, oxidative stress in plasma, liver and kidney of the mice were assessed.<br> <i>Result</i>: WER inhibited generation of superoxide anion and lipid peroxidation in concentration dependent manner<i> in vitro</i>. Oral administration of WER to diabetic mice significantly reduced the levels of blood glucose, hydroperoxides, triglyceride, ALT, and AST. Moreover, WER prevented the increase in lipid peroxide levels and the decrease in activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) in liver and kidney of diabetic mice. In addition, histochemical studies revealed that treatment of WER precluded the sinusoidal dilation in liver and expansion of mesangial matrix in kidney of diabetic mice. Normal mice treated with WER showed no change in any parameters studied.<br> <i>Conclusion</i>: These data suggested that WER can act as an antioxidant <i>in vivo</i>, and show the antidiabetic effects by relieving diabetic hyperglycemia-induced oxidative stress.<br>

Protective Effects of a Water-Soluble Extract from Culture Medium of Ganoderma lucidum Mycelia against Neuronal Damage after Hypoxia-Ischemia in Mice / 日本補完代替医療学会誌

<i>Objective</i>: In this study, the neuroprotective effects of a water-soluble extract from culture medium of <i>Ganoderma lucidum</i> mycelia (WER) on oxidative stress-induced injury were examined using H₂O₂-treated PC12 cells. Additionally, we investigated both the acute and chronic effects of WER on brain necrosis and apoptosis induced by hypoxia/ischemia (H/I) followed by reoxygenation in mice.<br> <i>Methods</i>: Viability and apoptosis index of H₂O₂-treated PC12 cells were determined by 3,4,5-dimethylthiazol-2-yl (MTT) assay and TUNEL staining, respectively. H/I in mice was induced by unilateral ligation of carotid artery and exposure of 8%O₂ for 30 min. Twenty-four hours after H/I, neurological deficits, cerebral infarction volume, and apoptosis level were evaluated.<br> <i>Results</i>: WER–pretreated PC12 cells showed an increased viability evaluated by MTT assay compared to untreated cells. TUNEL staining indicated that WER induced a concentration-dependent decrease of the number of apoptotic cells. In the mouse model of H/I, acute (pre-H/I) treatment of WER (1 g/kg, p.o.) did not affect neurological deficits, total plasma oxidative stress, cerebral lipid peroxidation, and infarction volume assessed 24-h after reoxygenation. However, chronic treatment of WER (1 g/kg, p.o., for 7 days) significantly improved these parameters compared with distilled water-treated mice. Moreover, chronic treatment of WER decreased the levels of apoptosis in two brain areas, the sensori-motor cortex and the CA1 of the hippocampus, analyzed by TUNEL and cleaved caspase-3 immunostaining.<br> <i>Conclusion</i>: These results show that daily intake of WER relieves the cerebral ischemic injury, which may be attributed to decrease of oxidative stress.<br>