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* Laboratory of Metabolic Disorders and Alternative Medicine, Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources;
Retrovirology Research Laboratory,
** Department of Tropical Medicine and Medical Microbiology and
Department of Medical Biotechnology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii; and
Hospital for Sick Children, University of Toronto, Toronto, Canada
3To whom correspondence should be addressed. E-mail: pratibha{at}hawaii.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bitter melon • hyperlipidemia • apolipoprotein B • transcription factors • triglycerides
The incidence and prevalence of type 2 diabetes is rapidly increasing globally. Current pharmacologic agents used for treating type 2 diabetes improve glycemic control but have varying effects on dyslipidemia, which is commonly associated with this disease (1). Therefore, therapeutic approaches that would ameliorate diabetic dyslipidemia would form an important cornerstone in treating these patients. Type 2 diabetic patients are at high risk for developing cardiovascular diseases (CVD)4 due to associated hyperlipidemia and increased levels of plasma apolipoprotein B (apoB)-containing VLDL and LDL (2). Among conventional treatment strategies, a combination of 2 or more drugs is usually necessary to achieve the target glucose and lipid levels; this likely affects compliance and the quality of life due to possible drug-drug interactions (3). Therefore, regardless of enormous advances in medical care, alternative therapies have become increasingly popular over the past several years, including medicinal herbs and functional foods (4).
Momordica charantia, also known as bitter melon, balsam pear, or karela, is widely cultivated in Asia, Africa, and South America and extensively used in folk medicines as a remedy for diabetes, specifically in India, China, and Central America [(5) and references therein]. Freeze-dried bitter melon capsules are widely available and marketed in health food stores across North America and Western European countries. All parts of the plant (fruit, seed, and leaves) were shown to possess hypoglycemic properties in normal and diabetic animal models (6–11). To date, only a few, nonrandomized, clinical studies conducted using bitter melon juice (BMJ) or fried fruit have demonstrated a significant decrease in blood glucose levels among type 2 diabetic patients (12–15).
Although empirically used to lower blood glucose, studies in animal models now indicate that bitter melon may also lower hepatic and serum lipids (7,9,10,16,17). Feeding of freeze-dried BMJ to rats fed a high-cholesterol or a normal diet, significantly decreased hepatic triglyceride (TG) and cholesterol and increased serum HDL cholesterol (7,10). Rats fed BMJ along with a high-fat diet demonstrated not only reduced adiposity, but also lower serum insulin and leptin levels and normalized glucose tolerance (9). In streptozotocin-induced diabetic rats, BMJ decreased both hepatic and serum TG and cholesterol (16). In alloxan-induced diabetic rats, as well as untreated control rats, aqueous extracts of bitter melon fruit and seeds significantly reduced VLDL levels, suggesting an effect on lipoprotein metabolism (17). Overall, the effects of bitter melon on various lipid variables in animals are novel findings, but the underlying mechanisms are unknown. To date, only one study demonstrated an upregulation of peroxisome proliferator-activated receptors (PPAR
and 
) and acyl CoA oxidase in rat hepatoma cells, H4IIEC3, which could be involved in the lipid-lowering effects of BMJ (8). However, no clinical studies have yet investigated the effects of bitter melon on serum lipids in humans.
Among the various lipoproteins, apoB is the major protein component of VLDL, intermediate density lipoproteins, and LDL. These particles are linked in a delipidation cascade in which TG-rich VLDL, released from the liver, is converted to cholesterol-rich LDL (18). Abnormalities in the metabolism of apoB-containing lipoproteins are responsible for the generation of hyperlipidemia and the associated increased risk of developing coronary heart disease (19). Because bitter melon lowers plasma lipids and VLDL levels in diabetic rats (17), the objective of this study was to investigate the effects of bitter melon on apoB secretion in vitro, using the human hepatoma cell line, HepG2. Our results indicate that BMJ significantly inhibits the synthesis and secretion of cellular TG as well as apoB secretion in HepG2 cells. Understanding the mechanisms of traditional functional foods such as bitter melon may help to identify new molecular targets in the treatment of type 2 diabetes and hyperlipidemia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preparation of BMJ. The Chinese variety of young bitter melons (raw and green) was obtained from a local farmer’s market, washed, and deseeded. BMJ was extracted according to the published protocol (20) using a household juicer and centrifuged at 560 x g at 4°C for 30 min. The supernatant BMJ was stored in aliquots at –80°C until further analysis.
Cell culture conditions for HepG2 cells. HepG2 cells were used up to passage 10. Typically, cells were seeded in 6-well culture plates, grown to 80–90% confluence, and then incubated in serum free medium containing 1% BSA for 24 h. After 24 h, cells were further incubated with either the control medium (Eagle’s MEM with 1% BSA) or experimental media (Eagle’s MEM with 1% BSA and varying concentrations of BMJ for 24 h, with or without 0.8 mmol/L oleate and/or 40 mg/L ALLN, a proteasome inhibitor). At the end of the experiment, the media were harvested to measure cellular cytotoxicity and apoB levels, and the cells were used for the analyses of microsomal triglyceride transfer protein (MTP) gene expression and sterol regulatory element-binding protein 1c (SREBP-1c).
Cytotoxicity and apoB ELISA. HepG2 cells were treated with concentrations of BMJ varying from 0.5 to 5% (v:v) for 24 h. The release of lactate dehydrogenase (LDH) into the culture media was used as a measure of cell death due to damaged membrane and was measured fluorimetrically using the CytoTox-ONETM Assay kit (Promega). ApoB levels in the culture media were quantitated using the ApoB Microwell ELISA Assay Kit (AlerCHEK) (21). Albumin in the media was measured with the Human Serum Albumin ELISA Kit (Bethyl Laboratories) (22).
TG synthesis and secretion. Rates of cellular TG synthesis and secretion were measured according to a published protocol (23). In brief, cells were labeled with 10 mCi/L [2-3H]glycerol (200 Ci/mol; Perkin Elmer Life Science Research Products) for the last 6 h of a 24-h treatment. Lipids from the media and cells were extracted 3 times with hexane:isopropanol (3:2, v:v) and separated by TLC. Cells were solubilized with 0.5 mol/L NaOH, and the lysates were used to measure total cellular proteins. Radioactivity associated with TG was expressed as dpm/mg protein.
Semiquantitation of human MTP gene expression. MTP gene expression was determined by RT-PCR. Total RNA was extracted using RNAzolTM B (TEL-TEST). RNA (2 µg) was reverse transcribed into complementary DNA (cDNA) and human MTP gene expression levels were quantified by RT-PCR using published primers: MTP; forward: 5'-GGACTTTTTGGATTTCAAAAGTGAC-3' and reverse: 5'-GGAGAAACGGTCATAATTGTG-3', and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); forward: 5'-ACAGCCGCATCTTCTTGTGCAGTG-3' and reverse: 5'-GGCCTTGACTGTGCCGTTGAATTT-3' (24). Various cyclingconditions were initially used to standardize amplification of the MTP gene in the log phase of the RT-PCR reaction. The final cycling condition was based on half of the maximum saturation of the amplicons. A two-step PCR was performed with the following conditions: 94°C for 2.5 min followed by 29 cycles of 94°C for 40 s, 53°C for 1 min, 68°C for 2 min, 1 cycle of 68°C for 8 min and 4°C hold. The PCR amplicon was size-fractionated on a 2% agarose gel and visualized with ethidium bromide staining. MTP gene expression was semiquantitated with Kodak 1D image analysis software. The intensity of the amplicon was then expressed as a ratio of the gene of interest against a housekeeping gene, GAPDH.
Immunoblot analysis of SREBP. Nuclear extracts were prepared as previously described and stored in aliquots at –80°C until further analysis (25). Protein concentrations were measured using the Bradford method according to manufacturer’s instructions (Bio-Rad Laboratories). Nuclear proteins (20 µg) were separated on 8% SDS-PAGE and transferred to nitrocellulose membrane, blocked in 5% milk, and incubated in SREBP-1 primary antibody (Santa Cruz Biotechnology) at room temperature. After washing, blots were probed with rabbit IgG secondary antibody (Santa Cruz Biotechnology). Proteins were detected by commercially available electrochemiluminescence kit, Pierce SuperSignalTM West Femto Maximum Sensitivity Substrate (Amersham Biosciences).
Statistical analysis.
All data are presented as means ± SD. Three different sets of experiments were performed in triplicate and group means were compared using ANOVA. ApoB modulators, oleate, and/or ALLN were incorporated into multifactor ANOVA models. Normality and homogeneity of variance were checked using multivariate log-normal plots. When appropriate, the data were transformed using a logarithm or square root function. Post hoc comparisons were done via the 2-stage Ryan-Einot-Gabriel-Welsch Multiple Range Test. P-values 
0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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63 ± 8% cell death, whereas 0.5, 1, and 2% caused 5, 8, and 20% cell death, respectively, compared with untreated controls. No dose dependent cytotoxicity was observed at 48 h of BMJ treatment. We therefore used 0.5 and 1.0% BMJ in all our studies. Overall, BMJ decreased apoB secretion in HepG2 cells treated with oleate and/or ALLN for 24 h, compared with untreated control cells, as analyzed by a three-way ANOVA model (P < 0.05, Fig. 1). Among BSA-treated cells, BMJ inhibited apoB secretion by 17 ± 4% at 0.5% and by 32 ± 4% at 1% BMJ. However, only 1% BMJ concentration was significantly different from control (Fig. 1A). ALLN increased apoB secretion in control cells (148 ± 13%, P < 0.05) indicating an inhibition of apoB degradation (Fig. 1B). However, in the presence of BMJ, ALLN did not significantly increase apoB secretion (95 ± 9 and 89 ± 10 at 0.5 and 1% BMJ, respectively) compared with BMJ-treated with only 1% BSA (Fig. 1A,B). These results suggest that BMJ has no effect on the proteasomal degradation pathway in HepG2 cells, and other factors such as lipid bioavailability could be responsible for the BMJ-associated decrease in apoB secretion.
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BMJ inhibits synthesis and secretion of cellular TG. Because lipid bioavailability was one of the factors affecting the BMJ-associated decrease in apoB (Fig. 1), the rate of incorporation of [3H] glycerol into cellular TG was measured in HepG2 cells treated with BMJ for 24 h. BMJ tended to decrease (P = 0.109) cellular TG synthesis by 17–21%, but both 0.5 and 1% BMJ inhibited TG secretion (38 ± 5 at 0.5% 44 ± 6 at 1% BMJ) compared with untreated control cells (P < 0.05, Fig. 2). However, no dose-dependent effects of BMJ were observed. The data suggest that a reduction in TG secretion could be one of the factors in the apoB-reducing effects of BMJ.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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MTP plays a pivotal role in the assembly and secretion of apoB-containing lipoproteins in the presence of lipids. Because BMJ decreases apoB secretion in the presence of lipids, we tested the effect of BMJ on MTP gene expression. HepG2 cells treated with BMJ for 24 h in the presence or absence of oleate, had a dose-dependent decrease in MTP mRNA expression (Fig. 3, P < 0.05). MTP expression and lipoprotein secretion are regulated by nuclear transcription factors such as SREBP-1, which are also involved in lipid homeostasis (27). Our findings indicate that BMJ increases the nuclear translocation of mature SREBP-1 (68-kDa protein) as analyzed by Western immunoblot (Fig. 4, P < 0.05). It is further interesting to note that although SREBP-1 is involved in fatty acid and TG synthesis, our preliminary data indicate that BMJ decreases cellular TG synthesis and secretion, suggesting differential effects and alternative mechanisms, such as upregulation of LDL receptor or inhibition of diacylglycerol acyltransferase, an enzyme involved in TG synthesis. Recently, Chao and Huang (8) demonstrated that H4IIEC3 rat liver cells treated with 100 and 150 mg/L of BMJ extract for 72 h activated transcriptional factors such as PPAR and PPAR, probably contributing to its hypolipidemic and hypoglycemic effects. However, they did not measure the effects of BMJ on cellular lipid levels (8). In our study, HepG2 cells were treated with crude undiluted BMJ at 0.5 and 1% for 24 h (corresponding to 20 and 40 mg/L BMJ protein), which demonstrated a decrease in cellular TG synthesis and secretion independent of SREBP increase. Although a direct comparison between our study and that of Chao and Hung (8) is difficult, due to differences in bitter melon preparations and exposure time, it is highly possible that BMJ exerts its hypoglycemic and hypolipidemic effects through differential regulation of nuclear transcriptional factors, because not only PPAR but also SREBP-1 was shown to regulate insulin effects (28). BMJ concentrations of 0.5 and 1% used in our study were within the range of freeze dried BMJ used in animal studies (7,9). However, our results may reflect acute effects due to differences in absorption and metabolism of BMJ in HepG2 cells compared with in vivo animal models and humans.
Most of the studies conducted to date have used whole fruit juices or crude preparations of bitter melon, and the chemical profile of bitter melon is not well characterized. Therefore, the exact nature of the active ingredients that are responsible for various health-promoting effects is not known. Nevertheless, most preparations from various laboratories were able to demonstrate the beneficial effects of bitter melon in numerous animal studies. In contrast, only a few clinical studies have investigated the effects of bitter melon in humans. Welihinda and co-workers (12) demonstrated an improved glucose tolerance in 73% of type 2 diabetic patients consuming 57 g BMJ/d. In another study, consumption of 15 g/d of the aqueous extract of bitter melon led to a 54% decrease in postprandial glucose levels and a 17% reduction in glycosylated hemoglobin among type 2 diabetes patients (29). Only one study demonstrated that feeding fried bitter melon (rather than raw juice) as a dietary supplement significantly improved glucose tolerance in humans (13). However, these clinical studies were small and they were not randomized. Therefore, adequately powered, randomized, placebo-controlled clinical trials are essential before bitter melon can be recommended as an effective complementary therapy. However, due to its hypoglycemic properties, use of bitter melon with other hypoglycemic agents must occur with medical supervision and monitoring. Some negative side effects such as diarrhea and hepatotoxicity in humans were noted (30); these could be due to excessive consumption. Further studies are warranted to characterize and identify the active ingredients of bitter melon and understand the pharmacokinetics in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by U.S. Public Health Service grants (G12 RR003061 and P20 RR011091) from the Research Centers in Minority Institutions Program, National Center for Research Resources.
4 Abbreviations used: ALLN, oleic acid and N-acetyl-leucyl-leucyl-norleucinal; apoB, apolipoprotein B; BMJ, bitter melon juice; BSA, bovine serum albumin; Con, control; CVD, cardiovascular diseases; ER, endoplasmic reticulum; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; MTP, microsomal triglyceride transfer protein; PPAR, peroxisome proliferator-activated receptor; SREBP-1c, sterol regulatory element-binding protein 1c; TG, triglyceride.
Manuscript received 27 September 2004. Initial review completed 22 October 2004. Revision accepted 2 January 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. DeFronzo, R. A. (1999) Pharmacologic therapy for type 2 diabetes mellitus. Ann. Intern. Med. 131:281-303.
2. Krentz, A. J. (2003) Lipoprotein abnormalities and their consequences for patients with type 2 diabetes. Diabetes Obes. Metab. 5(suppl. 1):S19-S27.
3. Snow, V., Aronson, M. D., Hornbake, E. R., Mottur-Pilson, C. & Weiss, K. B., the Clinical Efficacy Assessment Subcommittee of the American College of Physicians (2004) Lipid control in the management of type 2 diabetes mellitus: a clinical practice guideline from the American College of Physicians. Ann. Intern. Med. 140:644-649.
4. Eisenberg, D. M., Kessler, R. C., Van Rompay, M. I., Kaptchuk, T. J., Wilkey, S. A., Appel, S. & Davis, R. B. (2001) Perceptions about complementary therapies relative to conventional therapies among adults who use both: results from a national survey. Ann. Intern. Med. 135:344-351.
5. Grover, J. K., Yadav, S. & Vats, V. (2002) Medicinal plants of India with anti-diabetic potential. J. Ethnopharmacol. 81:81-100.[Medline]
6. Srivastava, Y., Venkatakrishna-Bhatt, H. & Verma, Y. (1988) Effects of Momordica charantia Linn. pomous aqueous extract on cataractogenesis in murrin alloxan diabetics. Pharmacol. Res. Commun. 20:201-209.[Medline]
7. Jayasooriya, A. P., Sakono, M., Yukizaki, C., Kawano, M., Yamamoto, K. & Fukuda, N. (2000) Effects of Momordica charantia powder on serum glucose levels and various lipid parameters in rats fed with cholesterol-free and cholesterol-enriched diets. J. Ethnopharmacol. 72:331-336.[Medline]
8. Chao, C. Y. & Huang, C. J. (2003) Bitter gourd (Momordica charantia) extract activates peroxisome proliferator-activated receptors and up regulates the expression of the acyl CoA oxidase gene in H4IIEC3 hepatoma cells. J. Biomed. Sci. 10:782-791.[Medline]
9. Chen, Q., Chan, L.L.Y. & Li, E.T.S. (2003) Bitter melon (Momordica charantia) reduces adiposity, lowers serum insulin and normalizes glucose tolerance in rats fed a high fat diet. J. Nutr. 133:1088-1093.
10. Senanayake, G. V., Maruyama, M., Shibuya, K., Sakono, M., Fukuda, N., Morishita, T., Yukizaki, C., Kawano, M. & Ohta, H. (2004) The effects of bitter melon (Momordica charantia) on serum and liver triglyceride levels in rats. J. Ethnopharmacol. 91:257-262.[Medline]
11. Miura, T., Itoh, Y., Iwamoto, N., Kato, M. & Ishida, T. (2004) Suppressive activity of the fruit of Momordica charantia with exercise on blood glucose in type 2 diabetic mice. Biol. Pharm. Bull. 27:248-250.[Medline]
12. Welihinda, J., Karunanayake, E. H., Sheriff, M. H. & Jayasinghe, K. S. (1986) Effect of Momordica charantia on the glucose tolerance in maturity onset diabetes. J. Ethnopharmacol. 17:277-282.[Medline]
13. Leatherdale, B. A., Panesar, R. K., Singh, G., Atkins, T. W., Bailey, C. J. & Bignell, A. H. (1981) Improvement in glucose tolerance due to Momordica charantia (karela). Br. Med. J. 282:1823-1824.
14. Ahmed, N., Hassan, M. R., Halder, H. & Bennoor, K. S. (1999) Effect of Momordica charantia (Karolla) extracts on fasting and postprandial serum glucose levels in NIDDM patients. Bangladesh Med. Res. Counc. Bull. 25:11-13.[Medline]
15. Akhtar, M. S. (1982) Trial of Momordica charantia Linn (Karela) powder in patients with maturity-onset diabetes. J. Pak. Med. Assoc. 32:106-107.[Medline]
16. Ahmed, I., Lakhani, M. S., Gillett, M., John, A. & Raza, H. (2001) Hypotriglyceridemic and hypocholesterolemic effects of anti-diabetic Momordica charantia (karela) fruit extract in streptozotocin-induced diabetic rats. Diabetes Res. Clin. Pract. 51:155-161.[Medline]
17. Virdi, J., Sivakami, S., Shahani, S., Suthar, A. C., Banavalikar, M. M. & Biyani, M. K. (2003) Antihyperglycemic effects of three extracts from Momordica charantia. J. Ethnopharmacol. 88:107-111.[Medline]
18. Wang, A. B., Liu, D. P. & Liang, C. C. (2003) Regulation of human apolipoprotein B gene expression at multiple levels. Exp. Cell Res. 290:1-12.[Medline]
19. Walldius, G. & Jungner, I. (2004) Apolipoprotein B and apolipoprotein A-I: risk indicators of coronary heart disease and targets for lipid-modifying therapy. J. Intern. Med. 255:188-205.[Medline]
20. Raza, H., Ahmed, I., Lakhani, M. S., Sharma, A. K., Pallot, D. & Montague, W. (1996) Effect of bitter melon (Momordica charantia) fruit juice on the hepatic cytochrome P450-dependent monooxygenases and glutathione S-transferases in streptozotocin-induced diabetic rats. Biochem. Pharmacol. 52:1639-1642.[Medline]
21. Scharnagl, H., Schinker, R., Gierens, H., Nauck, M., Wieland, H. & Marz, W. (2001) Effect of atorvastatin, simvastatin, and lovastatin on the metabolism of cholesterol and triacylglycerides in HepG2 cells. Biochem. Pharmacol. 62:1545-1555.[Medline]
22. Casaschi, A., Maiyoh, G. K., Rubio, B. K., Li, R. W., Adeli, K. & Theriault, A. G. (2004) The chalcone xanthohumol inhibits triglyceride and apolipoprotein B secretion in HepG2 cells. J. Nutr. 134:1340-1346.
23. Theriault, A., Wang, Q., Van Iderstine, S. C., Chen, B., Franke, A. A. & Adeli, K. (2000) Modulation of hepatic lipoprotein synthesis and secretion by taxifolin, a plant flavonoid. J. Lipid Res. 41:1969-1979.
24. Sellers, J. A. & Shelness, G. S. (2001) Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein. J. Lipid Res. 2001:1897-1904.
25. Dowell, P., Flexner, C., Kwiterovich, P. O. & Lane, M. D. (2000) Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors. J. Biol. Chem. 275:41325-41332.
26. Dixon, J. L., Furukawa, S. & Ginsberg, H. N. (1991) Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting early intracellular degradation of apolipoprotein B. J. Biol. Chem. 266:5080-5086.
27. Sato, R., Miyamoto, W., Inoue, J., Terada, T., Imanaka, T. & Maeda, M. (1999) Sterol regulatory element-binding protein negatively regulates microsomal triglyceride transfer protein gene transcription. J. Biol. Chem. 274:24714-24720.
28. Kim, S. Y., Kim, H. I., Kim, T. H., Im, S. S., Park, S. K., Lee, I. K., Kim, K. S. & Ahn, Y. H. (2004) SREBP-1c mediates the insulin-dependent hepatic glucokinase expression. J. Biol. Chem. 279:30823-30829.
29. Srivastava, Y., Venkatakrishna-Bhatt, H., Verma, Y., Venkaiah, K. & Raval, B. H. (1993) Antidiabetic and adaptogenic properties of Momordica charantia extract. An experimental and clinical evaluation. Phytother. Res. 7:285-289.
30. Basch, E., Gabardi, S. & Ulbricht, C. (2003) Bitter melon (Momordica charantia): a review of efficacy and safety. Am. J. Health Syst. Pharm 60:356-359.
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