Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: Activities: Bulbs of Lilium longiflorum were defatted with pentanes and extracted with 70% ethanol and the extract was washed with ethyl acetate and extracted with butanol to yield the "BuOH extract". In experiment 1, 10 female KK CgAy/J mice were used for each group and both groups were given high fat diets. Group I: control with free access to drinking water; Group II: free access to drinking water with 0.075% of BuOH. Body weight, food and water uptake of the mice were taken on a regular basis. After 7 weeks, all the experimental mice were sacrificed. The weight of parametrial fat, retro-peritoneal fat and brown fat was recorded. Liver, spleen and kidney were removed and weighed as well. Blood glucose, serum insulin, leptin levels, and cytokines including interlukin-6 (IL-6), interlukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in the liver were measured. In experiment 2, 16 Female KK CgAy/J diabetic mice were used except for the Group I, in which female KK mice were used as normal control. Mice in all groups were fed with high fat diets and given free access to drinking water. Group I: Normal control, 20 KK mice; Group II: Diabetic control; Group III: drinking water with 1% of Bulb extract, Group IV: drinking water with 0.1% BuOH; Group V: drinking water with 0.2% of BuOH; Group VI: drinking water with 300 mg/kg metformin (Met). Food and water uptake of the mice were recorded daily. At weeks 3, 16, and 24, 5 mice from each diabetic group and 6 mice from normal control group were sacrificed. Glucose tolerance test and activity of a liver enzyme alanine aminotransferase (ALT) was determined in the serum by commercial kits. Liver was removed, weighed and kept in 80% formalin for future study. Glucose tolerance test. All mice were fasted for 15 h the previous night but allowed free access to water and blood was collected from the tail vein. Immediately after blood collection, diabetic mice received an intraperitoneal injection of glucose (0.1 g/ml/100 g body weight). Blood samples were successively collected time intervals (30 and120 min), and blood glucose levels were determined as mentioned above. Liver Enzyme Study and H&E Staining. Blood sample was collected and centrifuged at 12,500 rpm for an hour. ALT activity was determined by the colorimetric method using Randox Diagnostic No. 146 kit. Livers were observed for any gross appearance and color change and tissues were preserved for histopathological studies. Liver samples were prepared for sectioning and staining using standard methods and observed by light microscopy. Statistical Analysis. All experiments and analyses were performed at least in triplicate. Results are expressed as means plus or minus SE. Statistical analyses were performed using the Student's T-test. PARTICIPANTS: Training for graduate student Wenping Tang TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In experiment 1, the BuOH extract was provided in the drinking water ad libitum for 7 weeks. Mice became obese on the high fat diet and body weight did not differ between the control and treatment group; however, the control group became hyperglycemic with a blood glucose of 233 mg/dL compared to the treatment group with a blood glucose of 166 mg/dL. At the same time, serum insulin level was significantly higher in the BuOH extract treatment group (17 uU/mL) compared to 1 uU/ml in the diabetic group, indicating a potent effect of the BuOH extract on insulin secretion. Moreover, plasma leptin levels in the BuOH treatment group was almost double that in the diabetic control group. Leptin is a hormone that plays a key role in regulating glucose metabolism. Leptin deficiency causes obesity, insulin resistance and diabetes in mice and humans. Despite the high body weight of mice in the lily BuOH extract treatment group, leptin levels were high, stimulating insulin secretion and lowering blood glucose, but lipid parameters were not different between treatments in this study. To determine if lily bulb extract could have an effect on lipid parameters, a longer time course study was done in experiment 2 with both the BuOH extract and a simple aqueous extract of the freeze-dried bulb powder from which the BuOH was derived. In this 24 week experiment, there were no differences in body weight among treatments but there were significant differences in glucose tolerance tests. Both the BuOH extracts and the bulb powder extracts had lower 30 min and 120 minute blood glucose levels following an intraperitoneal injection of glucose. The blood lipid profile for triglycerides (TG), total cholesterol, HDL and LDL were different among treatments for TG. The diabetic control group had TG of 219 mg/dL, the bulb extract 138, and the BuOH extracts at 0.1% and 0.2% were 131 and 146 mg/dL respectively. The diabetic control group had elevated liver enzymes (alanine amino transferase) and the livers were enlarged compared with normal control. All treatments significantly suppressed the increase in liver enzymes and liver weight. Liver tissues were sectioned and stained with H & E. The liver from diabetic mice was swollen and irregularly shaped. Liver histology under 20 X magnification revealed many accumulated lipid droplets in the cytoplasm of hepatocytes in diabetic mice, but very few in the normal control hepatocytes. There was also loss of usual concentric arrangement of liver hepatocytes. Lily extract treatments reduced the damage, with the livers from the BuOH (0.2%) extract resembling normal liver. In conclusion, this study shows that Easter Lily (Lilium longiflorum) bulb extracts reduce symptoms of type 2 diabetes and related liver dysfunctions.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Lily bulb extracts were provided to KKAy female mice for 10 weeks in drinking water ad libitum to determine if the extracts would reduce the symptoms of type 2 diabetes in this spontaneously diabetic mouse model. Lily bulbs contain steroidal glycosides, phenolic compounds and inulin, which may reduce insulin resistance and delay the onset of type 2 diabetes. The bulb extract treatment refers to an aqueous extract of freeze-dried lily bulb powder, and the BuOH extracts were prepared by defatting the bulb powder with pentanes, extracting the residue with 70% ethanol, and removing the ethanol in vacuo. The residue was taken up in water and freeze-dried. The freeze-dried residue was redissolved in water and partitioned with ethyl acetate. The aqueous phase was partitioned with butanol, and the butanol phase evaporated in vacuo, taken up in water and freeze dried. Metformin, a commonly prescribed drug for type 2 diabetes, was included as a positive control. The diabetic control mice and other treatments became obese with about a 20% increase in body weight. Fasting blood glucose (FBG) for this group was surprisingly low given that this mouse model is characteristically hyperglycemic. In fact, the normal control mice had higher FBG than the KKAy mice after 10 weeks. Oral glucose tolerance test data clearly indicated that lily bulb extracts resulted in a significant reduction in blood glucose after 2 hr compared to all treatments. Triglycerides were also significantly reduced by lily extracts and metformin. Liver weight was elevated in the diabetic control, and more than double the weight of the liver from the normal mice. Lily extracts and metformin significantly reduced liver weights, but they remained about 25% greater than for the normal mice. PARTICIPANTS: Graduate student training opportunity TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
More than 20% of the American population harbor the risk factors defined for metabolic syndrome, a condition that is highly correlated with the development of type 2 diabetes and coronary heart disease. Various botanical extracts have been used in traditional medicines to treat the symptoms of diabetes, and many of our common drugs, such as metformin, are of plant origin. The results of this research could lead to the development of L. longiflorum bulbs, or other plant parts, as a functional food or nutritional supplement that may be consumed regularly to prevent the development of metabolic syndrome and the onset of diabetes.
Publications
- Munafo Jr, JP Gianfagna TJ (2011) Antifungal Activity and Fungal Metabolism of Steroidal Glycosides of Easter Lily (Lilium longiflorum) by the Plant Pathogenic Fungus, Botrytis cinerea J. Agric Food Chem 59(11):5945-54
- Munafo, Jr, JP, Gianfagna, TJ (2011) Quantitative Analysis of Steroidal Glycosides in Different Organs of Easter Lily (Lilium longiflorum Thunb.) by LC-MS/MS. J. Agric. Food Chem 59: 995-1004
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Many plants have been used in traditional medicine for treating diseases similar to type 2 diabetes. Feeding extracts of some of these plants to laboratory animals often produces a beneficial hypoglycemic effect. Chemical analysis of active extracts usually reveals a wide range of compounds, but some of the most convincing evidence suggests that saponins are almost always involved in this response. Lily bulbs from a variety of species including Lilium longiflorum, L. brownii, L. henryi and L. candidum contain steroidal saponins and glycoalkaloids. A new steroidal glycoalkaloid and two new furostanol saponins, along with two known steroidal glycosides, were isolated from the bulbs of L. longiflorum. The new steroidal glycoalkaloid was identified as (22R, 25R)-spirosol-5-en-3-beta-yl O-alpha-L-rhamnopyranosyl-(1,2)-O-[6-O-acetyl-beta-D-glucopyranosyl-( 1,4)]-beta-D-glucopyranoside. The new furostanol saponins were identified as (25R)-26-[beta-D-glucopyranosyl]oxy]-22-alpha-hydroxyfurost-5-en-3-be ta-yl O-alpha-L-rhamnopyranosyl-(1,2)-O-[alpha-L-arabinopyranosyl-(1,3)]-be ta-D-glucopyranoside and (25R)-26-[beta-D-glucopyranosyl]oxy]-22alpha-hydroxyfurost-5-en-3-bet a-yl O-alpha-L-rhamnopyranosyl-(1,2)-O-[alpha-L-xylopyranosyl-(1,3)]-beta- D-glucopyranoside. The previously known steroidal glycosides, (22R, 25R)-spirosol-5-en-3-beta-yl O-alpha-L-rhamnopyranosyl-(1,2)-O-[beta-D-glucopyranosyl-(1,4)]-beta- D-glucopyranoside and (25R)-26-[beta-D-glucopyranosyl]oxy]-22alpha-hydroxyfurost-5-en-3-bet a-yl O-alpha-L-rhamnopyranosyl-(1,2)-O-[beta-D-glucopyranosyl-(1,4)]-beta- D-glucopyranoside were identified in L. longiflorum for the first time (Munafo et al., 2010). Partially purified fractions containing steroidal glycosides and other compounds from lily bulbs were obtained and some of these fractions were administered to diabetic mice through their water for 6 weeks after which blood glucose, triglycerides, cholesterol, body weight and abdominal fat were determined. Freeze-dried lily bulbs (fraction 1) were first defatted with pentanes. The residue was extracted with 70% aqueous ethanol (fraction 2). The ethanol extract was dried in vacuo, taken up in water and partitioned with ethyl acetate and butanol (fraction 3). The butanol fraction was enriched for steroidal glycosides by precipitation with chloroform, redissolving in water and freeze-drying (fraction 3A). PARTICIPANTS: The project provided John P. Munafo, a graduate student, with the opportunity to develop skills in natural product pruification and identification TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
From 100 g freeze-dried bulb powder, the yields of fraction 2 was 13.7 g, fraction 3 was 2.4 grams and fraction 3A 1.9g. Each of these fractions when fed to diabetic mice at a dose of 0.4% in water significantly reduced blood glucose. There was also a trend toward lower body weight, abdominal fat and triglycerides but the results were not statistically significant.
Publications
- Munafo Jr., JP, Ramanathan, A, Jimenez, L, Gianfagna TJ (2010) Isolation and Structural Determination of Steroidal Glycosides from the Bulbs of Easter Lily (Lilium longiflorum Thunb.). J. Agric Food Chem 58:8806-8813
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