Abstract: In present study, twelve 6 week age chickens were decapitated; liver extracted, sliced and cultured as primary culture. Effects of hydro extract of mulberry leaf on TG secretion and hepatic TG were determined. Data indicated that hydro extract of mulberry leaf extract decreased triglyceride secretion in a dose dependent manner (as much as 82, 76 and 67% in response to 0.075, 0.05 and 0.015% of hydro extract after 48 h incubation, respectively). Moreover, at 0.075 concentration it decreased TG content as much as 43% after 12 h incubation. Mulberry leaves contain some inhibitory components for accumulation and secretion of TG in chicken hepatocytes.
INTRODUCTION
Excessive accumulation of lipids in the adipose tissue of modern lines of broilers is a major concern for producers, because most fat depots are lost during evisceration of the carcass or processing of the meat. TG storage in avian adipose tissue depends on the availability of plasma lipid substrate, mainly very low density lipoprotein (VLDL), originating from either the diet or lipogenesis in the liver (Hermier, 1997). In young broiler chickens approaching market weight, about 80-85% of the fatty acids that accumulate in the adipose tissue are derived from plasma lipids (Griffin, 1992). Because commercial avian breeds are usually fed lipid-poor diets the liver plays a key role in providing lipids destined to be used by all tissues. Therefore any attempt to modify these metabolic processes must take into consideration the specific features of lipid metabolism in birds (Hermier, 1997).
In the recent years, several plant flavonoids were identified as hypolipidemic agents in tissue culture, animals and humans (Jahromi et al., 1993; Arai et al., 2000; Wilcox et al., 2001; Cassachi et al., 2004; Li et al., 2006). They inhibit the activity of a number of lipogenic enzymes, including hydroxymethylglutaryl (HMG) CoA reductase, acyl CoA:cholesterol acyltranferase (ACAT), microsomal triglyceride transfer protein (MTP) and CoA:diacylglycerol acyltransferase (DGAT) (Wilcox et al., 2001; Theriault et al., 2000; Casaschi et al., 2004).
Mulberry (M. alba L.) leaves containing many nutritional components are the best feed for silkworms and they have been used in traditional Chinese medicine as an antihyperglycemic. Major flavonoids have been extracted from the mulberry leaves are quercetin and its glycosylated forms specially rutin (Jia et al., 2001; Katsube et al., 2006). Andallu (2001) have showed oral administration of mulberry leaves powder cause decrease in blood and urine glucose, TG, LDL-cholesterol and VLDL-cholesterol and Fatty acids in type-2 diabetic patients.
The aim of the present study was to examine the effects of mulberry leaves extract on TG secretion and content in chicken hepatocytes primary culture.
MATERIALS AND METHODS
Mulberry leaves extract preparation: Mulberry leaves were collected from campus of school of Veterinary Medicine, University of Tehran on 12 June, 2004. The leaves were washed three times with tap water. Shade dried leaves were ground to powder. Powder (1 g) was extracted in a Soxhlet extractor with 100 mL distilled water 1 h and filtered. The extract was evaporated to dryness. After weighing, the dry extract was dissolved in distilled water to make 40% (w/w) stock solution.
Cell culture: Isolation and Culture of chicken hepatocytes were performed as previously described by Tarlow and Watkins (1977) by some modification. Briefly, the liver of twelve 6 week-old roosters was extracted after 12 h fast (water ad libitum). The livers were minced finely. Thirty milliliter of 1.5 IU mL-1 collagenase (type IV) solution and 2.5 mL of 400 mM glucose were added to the minced tissue. The tissue suspension was incubated for 20 min at 37°C in a gyratory shaker set at 130 oscillations min-1. The obtained suspension containing dissociated cells was filtered through 50 and 100-mesh nylon screen. The cell pellet was subjected to a procedure for lysing erythrocytes by washing with 20 mL of RBC lysing buffer (8.26 g of NH4Cl, 1.0 g of KHCO3, 0.037 g of Na2-EDTA in 1 L of distilled water). The cell pellet was washed with serum free DMEM medium to remove the RBC derbies. Hepatocytes resuspended in DMEM medium supplemented with FBS (5%), penicillin (100 IU mL-1), streptomycin (100 μg mL-1), insulin (10 μg mL-1). Cell viability, estimated by the trypan blue exclusion test or the well-preserved refringent shape, was always greater than 80%. For experiments, cells were plated out at a density of 1.5x106 cells cm-2 (500 μL well-1) in 17 mm dishes (24-well plates) and preincubated for 3 h at 37°C in 5% CO2. Cells were then changed to 1% BSA in serum-free medium with the indicated treatments and incubated for the different times at 37°C.
Lipid extraction and TG quantification: At the end of the incubation period, the cells were placed on ice and the medium was collected. The cells were washed three times with 1 mL ice-cold PBS and the cell lipids were extracted with 2 mL heptane/isopropanol (3:2, v/v). After 4 h, the extracts were removed and the cells were washed once with an additional 1 mL heptane/isopropanol (3:2, v/v) and added to the previous extract (Cianflone et al., 1992). The soluble cell protein was dissolved in 1 mL 0.1 N NaOH and total cell protein was measured by Bradford method, using BSA as a standard (Bradford, 1976). Media lipids were extracted by adding 1 mL of chloroform/methanol (80/20, v/v) methanol (80/20, v/v), decanting for 10 min and the organic phase was separated for TG assay. TG was measured by the method of Neri and Frings (Neri and Frings, 1973), both in cell extract and media.
Statistical analysis: All values are presented as mean±SD. Statistical significance was tested by one-way ANOVA, SigmaStat 2.03.
RESULTS
Cell viability was determined using the trypan blue exclusion test and cell protein measurement. There was no difference in cell viability between control cells and cells treated with different doses of mulberry leaves extract in different incubation times (data not shown).
Effect of mulberry extract on TG secretion: Different doses of mulberry leaves extract, 0.015, 0.05 and 0.075% decreased TG secretion from chicken hepatocytes compare to control group after 12, 24 and 48 h incubation in a dose dependent manner (p<0.001). 10-9 M glucagon also decreased TG secretion in different incubation times. However 0.075% MLE caused more decreasing in TG secretion compare to 10-9 M glucagon (p<0.005) (Table 1).
Effect of mulberry leaves extract on cell TG content: 0.075% MLE and 10-9 M glucagon decreased chicken hepatocytes TG content after 12, 24 and 48 h incubation times (p<0.001). No significant differences observed between effect of 0.075% MLE and 10-9 M glucagon cell TG content (p>0.05). However, 0.015 and 0.05% extracts showed any effect on cells TG content (p>0.05) (Table 2).
| Table 1: | Effect of MLE and 10-9 M glucagon on TG secretion in chicken hepatocytes primary culture. Values were expressed as μmol L-1 |
| Values were presented as mean±SD. *The mean value differs significantly from the control in one way-ANOVA (p<0.001, n = 12) | |
| Table 2: | Effect of MLE and 10-9 M glucagon on TG content in chicken hepatocytes primary culture. Values were expressed as μmol mg-1 cell protein |
| Values were presented as mean±SD. *The mean value differs significantly from the control in one way-ANOVA test (p<0.001, n = 12) | |
DISCUSSION
Several flavonoids dramatically reduce apoB secretion in cell culture. A number of lipogenic enzymes are involved in the mechanism of action, including HMG CoA reductase, ACAT, MTP and DGAT (Casaschi et al., 2002; Theriault et al., 2000; Wilcox et al., 2001). However, the enzymes predominantly responsible for the assembly of apoB-Lp in the presence of flavonoids have not been thoroughly investigated. Borradaile et al. (2002) made progress in this area by ruling out ACAT activity and cholesterol ester (CE) availability in the regulation of apoB-Lp by nanringenin, a citrus flavonoid, in epG2 cells. Casaschi et al. (2004) showed that Chalcone Xanthohumol a plant polyphenlol, decrease apoB secretion in Hep G2 cells via the decreasing of microsomal triglyceride transfer protein (MTP) and CoA:diacylglycerol acyltransferase (DGAT) activity.
The present study provides evidence that mulberry leaves extract have ability for influencing TG metabolism in the chicken hepatocytes. We have shown a dose dependent inhibitory effect for MLE on TG secretion and cell content (p<0.001). Mulberry leaves contain five flavonol glycosides: rutin, isoquercitrin, quercetin 3-(6-acetylglucoside), astragalin and kaempferol 3-(6-acetylglucoside). From these compounds rutin and Quercetin 3-(6-malonylglucoside) are the most ones in the mulberry leaves (Katsube et al., 2006). In literature review, we found any in vitro study on the effects of whole mulberry leave extract on the synthesis and secretion of TG in hepatocytes. However Andallu et al. (2001) showed oral administration of mulberry leaves powder has a dual effect (decreasing of TG synthesis and increasing of VLDL removing) on serum TG levels in diabetics. In this regard, Casaschi et al. (2002) showed that quercetin inhibited TG synthesis in intestinal cell-line, CaCO2, in part, via DGAT activity. In this respect, Theriault et al. (2000) showed that taxifolin, a plant flavonoid, decreased TG synthesis and secretion in HepG2 cells through inhibition of DGAT and MTP activity.
In present study, although all doses of MLE decreased TG secretion from chicken hepatocytes, TG content only was affected by 0.075% of MLE. Nakamura et al. (2000) reported decrease in serum TG followed by oral administration of rutin in rats without alteration in hepatic TG content. Moreover, we have shown that 0.075% of MLE's effect on the TG secretion and content was greater than 10-9 glucagon (p<0.001). Tarlow and Watkins (1977) showed glucagon decrease TG secretion and synthesis via suppression of de novo lipogenesis in chicken hepatocytes.
Future studies will need to examine the mechanism of mulberry leaves extract induced changes in TG secretion and content of chicken hepatocytes deduce if the effect is due to decreased de novo fatty acid synthesis or increased β-oxidation of fatty acids before administration to rooster in vivo. However these data suggest that mulberry leaves extract administration may be an alternative method to reduce fat mass and providing increased meat yield.
ACKNOWLEDGMENT
We would like to thank Dr. A. Shahriari, Mr. A. Naderloo and Miss. F. Jadidizadeh for their help offered us.