Sdarticle

Accepted Manuscript
Supplementation of whole persimmon leaf improves lipid profiles and sup -

presses body weight gain in rats fed high-fat diet
J.S. Lee, M.K. Lee, T.Y. Ha, S.H. Bok, H.M. Park, K.S. Jeong, M.N. Woo, M.S.
Choi
PII: S0278-6915(06)00156-6
DOI: 10.1016/j.fct.2006.06.014
Reference: FCT 3590
To appear in: Food and Chemical Toxicology
Received Date: 8 February 2006

Revised Date: 27 May 2006
Accepted Date: 7 June 2006
Cite this article as: Lee, J.S., Lee, M.K., Ha, T.Y., Bok, S.H., Park, H.M., Jeong, K.S., Woo, M.N., Choi, M.S.,
Supplementation of whole persimmon leaf improves lipid profiles and suppresses body weight gain in rats fed high-
fat diet, Food and Chemical Toxicology (2006), doi: 10.1016/j.fct.2006.06.014
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ACCEPTED MANUSCRIPT
1 Supplementation of whole persimmon leaf improves lipid profiles and
2 suppresses body weight gain in rats fed high-fat diet
4 J. S. Lee1, M. K. Lee2, T. Y. Ha3, S. H. Bok4, H. M. Park5, K. S. Jeong6, M. N. Woo7, M. S.
5 Choi*7
6
1
7 Ottogi Research Center, 166-4, Pyeongchon-dong, Dong An-ku, Anyang, Kyeonggi-do 430-
8 070, 2Division of Food Sciences, Sunchon National University, Jeonnam, 540-742, South
9 Korea, 3Korea Food Research Institute, Seongnam-si, Gyeonggi-do, 463-746, 4Bionutrigen,
10 Yusong 305-333, Daejon, , 5 Department of Oriental Medicine, Sangji University, Wonju, 220-
11 702, 6Department of Veterinary Medicine, 7Department of Food Science and Nutrition,
12 Kyungpook National University, Daegu, 702-701, South Korea
13
14 *Corresponding author
15 Myung-Sook Choi, Ph.D
16 Department of Food Science and Nutrition,
17 Kyungpook National University, 1370 Sank-Yuk Dong Puk-Ku, 702-701, Daegu, Korea
18 E-mail: mschoi@knu.ac.kr, Tel: +82-53-950-6232, Fax: +82-53-950-6229
19
20 Running title : Persimmon leaf supplement & lipid metabolism
21 Key Words : persimmon leaf, hypolipidemic effect, high-fat diet, lowering body weight,
22 adipose tissue
1
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1 Abstract
2 The objective of this study was to investigate the hypolipidemic effects of powdered
3 whole persimmon leaf supplement in rats fed high-fat diet. Three groups of male Sprague-
4 Dawley rats during 6 weeks were fed different diet: normal control (NC), high-fat (HF), and
5 high-fat supplemented with powdered whole persimmon leaf (PL; 5%, wt/wt) groups. Body
6 weight and relative weight of interscapular brown adipose tissue were significantly lower in
7 the PL group than in the HF group, while plasma leptin concentration was higher. The
8 supplementation of persimmon leaf significantly lowered the plasma total cholesterol and
9 triglyceride concentrations, whereas elevated the ratio of HDL-C/total-C and improved the
10 atherogenic index. Persimmon leaf supplementation led the hepatic cholesterol and
11 triglyceride values to similar levels to the NC group. Accumulation of hepatic lipid droplets
12 and the epididymal white adipocyte size of PL group were diminished comparing to the HF
13 group. Hepatic HMG-CoA and ACAT activities were significantly higher in the PL group
14 than in other groups. Contents of fecal triglyceride, cholesterol and acidic sterol were
15 significantly higher in the PL group than in the HF group. Accordingly, we suggest that
16 supplementation of the powdered whole persimmon leaf improves plasma and hepatic lipid
17 levels profile partly via the increased fecal lipids in high-fat fed rats. These beneficial
18 effects may be due to the properties of its phenolic compounds (1.15 g/100 g) and high fiber
2
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1 (63.48 g/100 g) content in the powdered persimmon leaf.
3 Introduction
4 Dietary fat is one of the most important environmental factors associated with the
5 cardiovascular diseases incidence (McNamara, 2000). Plant polyphenols exert
6 cardiovascular benefits by altering concentrations of blood lipid components and a high intake
7 of polyphenols (flavonoids) can significantly reduce the risk of mortality from cardiovascular
8 diseases (Yugarani et al., 1992; Hartog et al., 1993).
9 In the leaf of persimmon diospyros kaki, flavonoid oligomers, tannins, phenols,
10 organic acids, chlorophyll, vitamin C, and caffeine are present and the leaf is commonly used
11 as a tea in Asia (Jo et al., 2003; Matsuok and Ito, 1978). Since the persimmon leaves have
12 beneficial effects on haemostasis, constipation, hypertension, apoplexy and atherosclerosis,
13 they have been broadly applied in food and medicinal area (Funayama and Hikino, 1979;
14 Kameda et al, 1987; Kotani et al., 2000; Matsumoto et al., 2002; Tanaka et al., 2003). It has
15 been found that condensed tannins and flavonoids isolated from persimmon leaves are mainly
16 responsible for the hypotensive and antioxidative actions (Funayama and Hikino, 1979;
17 Kameda et al, 1987; Uchida et al., 1987). Especially flavonoid aglycones in persimmon
18 leaves, such as catechin, kaempferol, and quercetin reportedly possess strong antioxidative
3
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1 activities acting as oxygen radicals scavenger, metal chelator and lipid peroxidation inhibitor
2 (Morel et al., 1993). Tannin or tannin with gallate group have various physiological
3 functions such as antibacterial, anti-allergic, scavenging free radicals, lowering blood pressure
4 and serum and hepatic cholesterol concentrations and increasing fecal sterol excretion in rats
5 with hypercholesterolemia (Choi, 2000; Kotani et al., 2000; Park et al., 2002).
6 Epidemiological data, as well as in vitro studies, strongly suggest that foods containing
7 phytochemicals with antioxidant potential have strong protective effects against major disease
8 risks including cancer, diabetes, cardiovascular diseases and Alzheimer’s disease (Knekt et al.,
9 1997; Wilett, 2002). Consumption of fruits, vegetables, and teas has been strongly linked to
10 reduced risk of those diseases (Kanekt et al, 1996; Le-Marchand et al, 2000; Xing et al., 2001).
11 A high fat intake itself can contribute to the development of obesity and hyperlipidemia in
12 human and rodents by altering cholesterol and triglyceride levels in plasma and tissues.
13 Accordingly, objective of this study was to investigate the effects of persimmon leaf as whole
14 food on lipid lowering metabolisms in rats fed a high-fat diet.
15
16 Materials and methods
17 Preparation of powdered persimmon leaf and analysis of general composition
18 Persimmon leaf was harvested in Sangju (Korea) and dried in the shade for a week.
4
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1 Then the leaf was powdered and passed through 60 mesh sieves. The crude protein and the
2 crude fat contents were determined by the Kjedahl method and Soxhelt extraction method,
3 respectively. And the carbohydrate and the total fiber contents were analysed using by
4 AOAC (1995) method and the Prosky-AOAC (1988) method, respectively.
6 Determination of total phenolic contents in powdered whole persimmon leaf
7 Total phenolic content was analyzed spectrophotometrically by a modified Folin-
8 Ciocaleu colorimetric method (Singleton et al., 1999; Wolfe et al., 2003). Briefly, 50 g of
9 powdered persimmon leaf was suspended and extracted with 10 volumes of methanol with
10 shaking at room temperature for 15 h. The extracts were filtered through a filter paper and
11 the supernatants were pooled. The residue was re-extracted under the same condition.
12 Pooled extracts were condensed (and methanol removed) to 9.2 g with a rotary evaporator at
13 50°C. Volume of 0.5 mL of deionized water and 0.125 mL of a known dilution of the extract
14 were added to a test tube, followed by addition of 0.125 mL of Folin-Diocalteu reagent.
15 They were mixed well and then allowed to stand 6 min before 1.25 mL of a 7% sodium
16 carbonate solution was added. The mixture was diluted to 3 mL with deionized water. The
17 color was developed for 90 min at room temperature and the absorbance was measured at 760
18 nm using a spectrophotometer (UVIDEC-50, JAS-CO Corporation, Tokyo, Japan). The
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1 measurement was compared to a standard curve of prepared gallic acid solutions and
2 expressed as means (±S.E.) mg of gallic acid equivalents per gram for the triplicate extracts
3 and total phenolic content was expressed as mg phenolics per gram of powdered whole
4 persimmon leaf.
6 Animals and diets
7 Thirty male Sprague–Dawley rats aged 3 weeks (40-50 g) were purchased from Bio
8 Genomics, Inc. (Seoul, Korea). The animals were housed individually in stainless steel
9 cages in a room with a 12:12-h light-dark cycle and an ambient temperature of 24°C. All the
10 rats were fed a pellitized commercial chow diet for 1 week after arrival. They were then
11 randomly divided into 3 groups and fed with a normal control diet (NC, n=10) and two high-
12 fat diets (HF, N=10, PL, n=10) for 6 weeks, respectively. High-fat group was consisted of
13 two groups, without (HF) or with 5% (wt/wt) powdered persimmon leaf (PL). The
14 composition of the experimental diet (Table 1) was based on the AIN-76 semisynthetic diet
15 (American Institute of Nutrition, 1977, 1980). The energies of the high-fat diets in the HF
16 and the PL groups were 497.5 and 482.7 kcal/100 g whereas that of NC group was 385.0
17 kcal/100 g. The animals were given food and distilled water ad libitum during the
18 experimental period. Food consumption and weight gain were measured daily and weekly,
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1 respectively.
2 <Insert table 1 here>
3 Feces were collected during the final 3 days using metabolic cages, and dried feces were
4 used for determination of fecal lipid and sterol levels. At the end of the experimental period,
5 the rats were sacrificed following a 14-h fast by removing the food from the cages at 7 p. m. a
6 day before and collecting the blood samples at 9 a. m. of the next day. Animals were
7 anesthetized with ketamine and blood samples were taken from the inferior vena cava for
8 determination of the plasma lipid profiles and leptin. The livers were removed under an
9 anesthetized condition and rinsed with physiological saline. The adipose tissues (epididymal
10 white adipose tissue, perirenal white adipose tissue, interscapular white adipose tissue, and
11 interscapular brown adipose tissue) were immediately weighed. The livers were removed
12 and rinsed with physiological saline. All samples were stored at –70°C until analyzed. The
13 current study protocol was approved by the Ethics Committee at Kyungpook National
14 University for animal studies.
15
16 Plasma, hepatic, and fecal lipids
17 Plasma total cholesterol (Total-C) and high-density lipoprotein (HDL)-cholesterol
18 concentrations were determined using a commercial kit (Sigma) based on a modification of
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1 the cholesterol oxidase method of Allain et al. (1974). The HDL-fractions were separated
2 using a kit (Sigma) based on the heparin–manganese precipitation procedure (Warnick and
3 Albers, 1978). The plasma triglyceride concentrations were measured enzymatically using a
4 kit (Sigma), a modification of the lipase–glycerol phosphate oxidase method (McGowan et al.,
5 1983). The hepatic and fecal cholesterol and triglycerides were extracted using the
6 procedure developed by Folch et al. (1957) Triton X-100 and a sodium cholate solution
7 were added to 200 µL of the dissolved lipid solution in 1 mL of ethanol to produce final
8 concentrations of 5 g/L and 3 mmol/L, respectively. The cholesterol and triglyceride
9 concentrations of liver and feces were analyzed with the same enzymatic kit as used in the
10 plasma analysis. The fecal bile acid was extracted with methanol and quantified
11 enzymatically with 3-α-hydroxysteroid dehydrogenase (Crowell and Macdonald, 1980).
12
13 Hepatic and fatty morphology
14 Livers and epididymal white adipose tissue were removed from the rats and fixed in a
15 buffer solution of 10% formalin. Fixed tissues were processed routinely for paraffin
16 embedding, and 4-µm sections were prepared and dyed with hematoxylin–eosin; stained areas
17 were viewed using an optical microscope with a magnifying power of ×200.
18
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1 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and acyl-CoA:cholesterol
2 acyltransferase (ACAT) activities
3 The microsomes were prepared according to the method developed by Hulcher and
4 Oleson (1973) with a slight modification. One gram of liver tissue was homogenized in 4
5 mL of an ice-cold buffer (pH 7.0) containing 0.1 mol/L of triethanolamine, 0.02 mol/L of
6 EDTA, and 2 mmol/L of dithiothreitol. The homogenates were centrifuged twice at 10,000 g
7 for 15 min at 4°C. Next, the supernatants were ultracentrifuged twice at 100,000 g for 60
8 min at 4°C. The resulting microsomal pellets were then redissolved in 1 mL of a
9 homogenation buffer for protein determination (Bradford, 1976) and finally analyzed for their
10 HMG-CoA reductase and ACAT activities.
11 The HMG-CoA reductase activities were determined as described by Shapiro et al.
12 (1974) with a slight modification using freshly prepared hepatic microsomes. The
13 incubation mixture (60 µL) containing the microsomes (100~150 µg of protein) and 500 nmol
14 of NADPH (dissolved in a reaction buffer containing 0.1 mol/L of triethanolamine and 10
15 mmol/L of EDTA) was preincubated at 37°C for 5 min. Next, 10 µL of 50 nmol of
16 [14C]HMG-CoA (specific activity, 2.1083 GBq/mmol; NEN™ Life Science Products, Boston,
17 MA) was added, and the incubation was continued for 15 min at 37°C. The reaction was
18 terminated by the addition of 15 µL of 10 mol/L HCl, and the resultant reaction mixture was
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1 incubated at 37°C for an additional 15 min to convert the mevalonate into mevalonolactone.
2 The incubation mixture was centrifuged at 10,000 g for 5 min, and the supernatant was
3 spotted on a Silica Gel 60 F254 thin-layer chromatography plate using mevalonolactone as the
4 standard. The plate was developed in benzene–acetone (1:1, vol/vol) and air-dried. Finally,
5 the ratio of fronts (Rf) 0.3–0.6 region was removed by scraping with a clean razor blade, and
14
6 its C radioactivity was determined using a liquid scintillation counter (Tricarb 1600TR,
7 Packard Instrument, Meriden, CT). The results were expressed as picomoles of mevalonate
8 synthesized per min per mg of protein.
9 The ACAT activities were determined in freshly prepared hepatic microsomes, by the
10 method of Erickson et al. (1980) as modified by Gillies et al. (1986) To prepare the
11 cholesterol substrate, 6 mg of cholesterol and 600 mg of Tyloxapol (Triton WR-1339, Sigma)
12 were each dissolved in 6 mL of acetone, mixed well, and completely dried in N2 gas. The
13 dried substrate was then redissolved in 20 mL of distilled water to a final concentration of 300
14 µg of cholesterol/mL. Next, reaction mixtures containing 20 µL of the cholesterol solution
15 (6 µg of cholesterol), 20 µL of a 1 mol/L of potassium phosphate buffer (pH 7.4), 10 µL of 0.6
16 mmol/L bovine serum albumin, 10 µg of the microsomal fraction, and distilled water (up to
17 180 µL) were preincubated at 37°C for 30 min. The reaction was then initiated by adding 20
18 µL of 5.62 nmol of [14C]oleoyl-CoA (specific activity, 1.9795 GBq/mmol; NEN Life Science
10
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1 Products) to a final volume of 200 µL; the reaction time was 30 min at 37°C. The reaction
2 was stopped by the addition of 500 µL of isopropanol–heptane (4:1, vol/vol), 300 µL of
3 heptane, and 200 µL of 0.1 mol/L potassium phosphate (pH 7.4), and the reaction mixture was
4 allowed to stand at room temperature for 2 min. Finally, an aliquot (200 µL) of the
5 supernatant was subjected to scintillation counting. The ACAT activities were expressed as
6 picomoles of cholesteryl oleate synthesized per min per mg of protein.
8 Plasma leptin concentration
9 Plasma leptin concentration was determined using a Murine Leptin kit (Diagnostic
10 Systems Laboratories Inc., USA).
11
12 Statistical analysis
13 All data were presented as the mean ± standard error of the mean. The data were
14 evaluated by a one-way analysis of variance using a SPSS program, and the differences
15 between the means assessed using Duncan’s multiple-range test. Statistical significance was
16 defined as p< 0.05.
17
18 Results
11
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1 The general composition and the total phenolic content of powered persimmon leaf
2 The general composition and the total phenolic content of powdered persimmon leaf
3 are shown in Table 2. In 100 g of powdered persimmon leaf, following components : 0.91 ±
4 0.01 g carbohydrate, 11.56 ± 0.10 g crude protein, 6.10 ± 0.12 g crude fat and 63.48 ± 1.01 g
5 fiber. Its energy content was 104.78 kcal per 100 g diet. Total phenolic content recovered
6 from 50 g of the powdered whole persimmon leaf was 575 ± 0.5 mg.
9 Food intakes, weight gains and food efficiency ratio
10 Energy density of two high fat diets used was higher than that of normal control diet
11 (498.7 kcal/100 g and 483.7 kcal/100 g vs. 386.2 kcal/100 g) as shown in Table 1. High fat
12 diet fed groups exhibited significantly lower food intake compared to normal diet (Table 3).
13 Interestingly, when compared two high fat diet groups (HF & PL), food intake was
14 significantly lower in the PL group than in the HF.
15 <Insert Table 3 here>
16
17 Initial body weights of three groups were not significantly different (88 ~ 91 g),
18 however, after 6 weeks, body weights were significantly lower in the NC and PL groups than
12
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1 in the HF group (Fig. 1). Average weight gain was significantly higher in the HF group than
2 in the NC or PL group (HF>PF>NC). Food efficiency ratio (FER) was significantly higher
3 in the HF and PL group than in the NC group, and the HF and the PL groups exhibited a same
4 FER value.
5 <Insert Fig. 1 here>
7 Organ weights
8 Organ weights were expressed as its relative weight per body weight. The relative
9 weights of liver and the heart were significantly lower in the PL group than in the NC and HF
10 groups. However, the kidney weight was not significantly different between the groups
11 (Table 4). Epididymal WAT, interscapular WAT, and total adipose tissue weights were the
12 highest in the HF, PL and NC group in order. There was no difference in perirenal WAT
13 weight among the groups. The supplementation of PL significantly lowered the weight of
14 brown adipose tissue compared to the HF group (Table 4).
16
17 Plasma and hepatic lipids
18 Concentrations of plasma and hepatic lipids are shown in Table 5. The
13
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1 supplementation of persimmon leaf significantly lowered plasma total cholesterol
2 concentration by 50.1% and 44.8%, and triglyceride concentration by 32.2% and 38.6%
3 compared to the HF and NC groups, respectively (p<0.05). Although HDL-cholesterol
4 concentration was significantly lower in the PL group than in the NC or HF group, the ratio of
5 HDL-C/Total-C exhibited the highest value in the PL group and the lowest value in the HF
6 group. For this reason, atherogenic index was significantly higher in the HF group than in
7 the NC and PL group. Hepatic cholesterol and triglyceride concentrations also were
8 significantly lower in the PL group than in the HF group by 38.8% and 41.3%, respectively
9 (p< 0.05).
13
14 Morphological comparisons in epididymal adipocytes and hepatocytes
15 Fig. 2 shows the histological appearance and size of epididymal adipose tissue. The
16 sizes of adipocytes in the NC and PL groups were smaller than those of the HF group when
17 compared under the light microscophy (Table 6). Accumulation of hepatic lipid droplets
18 (indicated by the arrow) appeared the highest in HF group, but they were relatively lower in
14
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1 the NC and PL groups (Fig. 3).
4 Hepatic HMG-CoA reductase and ACAT activities
5 Both hepatic HMG-CoA reductase and ACAT activities were significantly higher in
6 the PL group than in the HF or NC group (Table 7). There was no significant difference
7 between the HF and NC groups in these enzyme activities.
10 Fecal lipids and acidic sterol levels
11 Dried fecal weight was significantly higher in the PL group than in the HF group.
12 Persimmon leaf supplementation significantly elevated excretion of fecal cholesterol and
13 triglyceride compared to the HF or NC group (Table 8). Fecal acidic sterol was
14 significantly different between the groups; the PL group is 3.6 fold higher than the HF group
15 and NC group value was between the HF and PL group.
18
15
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1 Plasma leptin concentrations
2 Plasma leptin concentration was significantly higher in the PL group than in the HF
3 group (9.11±0.95 ng/ml vs. 6.13±0.73 ng/ml, Fig. 4). The NC group was not different from
4 PL or HF group in its plasma leptin concentration.
6 Discussion
7 Two high fat diets, HF and PL, are almost isonitrogenous and isocaloric although fiber
8 content was higher in the PL diet than in the HF diet. The lower food intake shown in the PL
9 group than in the HF diet seemed to be due to high fiber content in persimmon leaf. In the
10 present study, the persimmon leaf was effective in lowering body weight gain as well as
11 reducing food intake when compared to the HC group. As shown in Fig. 1, the high-fat
12 diet (approximately 50% energy as fat) feeding to rats for 6 weeks significantly increased
13 body weight by 114% based on the NC group (479.24±7.12 g in the HF group vs.
14 420.34±7.12 g in the NC group, p<0.05). The PL supplementation led the final body weight
15 similar to the rats fed normal diet, thus indicating the persimmon leaf supplementation
16 suppressed the excess body weight gain that could be induced by high-fat feeding.
17 Warwick and Weingarten (1995) reported high-fat diet (60% energy as fat) has been shown
18 to enhance food intake and weight gain relative to a high-carbohydrate diet (76% energy as
16
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1 carbohydrate) when both energy density and palatability are equated. In our study, normal
2 diet was a high-carbohydrate diet (67.5% energy as carbohydrate) and was not isocaloric to
3 high-fat diets (HF or PL diet). Nevertheless high-fat diet itself resulted to reduce the food
4 intake when compared to the normal control diet, body weight gain of HF group was
5 significantly higher. Accordingly, FER was significantly lower in the NC group than in the
6 HF and PL groups. Interestingly, although FER value of PL group is equal to the HF group,
7 persimmon leaf supplementation significantly lowered the weight gain in rats fed high-fat by
8 seemingly suppressing food intake and/or lowering fat-pad weights. Additional dietary fiber
9 in PL diet from whole persimmon leaf rather than energy density difference in PL diet
10 partially can be responsible for lower food intake as well as lower weight gain observed in
11 the PL group compared to the HF group.
12 This current study, plasma leptin concentration was significantly higher in the PL group
13 than in the HF group. Leptin acts primarily on hypothalamic centers to regulate food intake
14 and energy expenditure, and has activities to reduce food intake and body weight in animal
15 models (Pelleymounter et al., 1995; Levin et al., 1996). Leptin is also involved in the
16 regulation of carbohydrate and lipid metabolism, cardiovascular function, reproduction and
17 immune response (Margetic et al., 2002). Accordingly, high plasma leptin concentration
18 induced by persimmon leaf supplementation seemed to play one of important roles on
17
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1 reducing food intake and weight gain although this mode of action needs to be elucidated in
2 detail with respective functional components present in persimmon leaf.
3 There was no abnormality in the growth performance, however, the relative weights of
4 liver and heart were significantly lower in the PL group compared to the HF group.
5 Persimmon leaf supplementation significantly lowered the weight of interscapular brown
6 adipose tissue compared with the HF group (1.38±0.04 mg/g B.W. vs. 1.62±0.10 mg/g B.W.)
7 that seemed to suggest the lower body weight observed in the PL group didn’t seem to be
8 due to the elevated heat production of brown adipose tissue. Although there were no
9 significant differences, persimmon leaf tended to lower the weight of epididymal, perirenal
10 and interscapular white adipose tissue compared to the HF group. In particular, persimmon
11 leaf supplementation seemed to diminish the epididymal adipocyte sizes, that is normally
12 increased in obese condition, similar size to the NC group.
13 An elevated plasma total cholesterol concentration or LDL-cholesterol increases the risk
14 of coronary heart disease (CHD) (Lipid Research Clinics Program, 1984). The clinical
15 complications of atherosclerosis could be diminished when plasma lipids are lowered by
16 hypocholesterolemic agents (Downs et al., 1998). The effect of flavonoids on serum and
17 hepatic lipids and fecal steroid excretions are very relevant to cardiovascular diseases and
18 some cancer (Stangl et al., 2005). In this study, persimmon leaf improved lipid profiles by
18
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1 lowering plasma total cholesterol concentration and triglyceride concentration compared
2 with the HF and NC groups. The plasma HDL-cholesterol concentration was lower in the
3 PL group than in the HF group, however, the ratio of HLD-C/Total-C was significantly
4 increased by 68.9% compared with the HF group. Gorinstein et al. (2000) reported that two
5 diet, fortified with 7% whole dry persimmon and 7% phenolic compound-free dry
6 persimmon, given to cholesterol-fed rats improved plasma lipid levels which suggest
7 persimmon includes not only functional phenolic compounds but other potent components(s)
8 including fiber that are responsible for lipids lowering action. Total fiber content in PL diet
9 was approximately 8.2% compared 5% in HF and NC diet. Our result indicates that the
10 supplementation of persimmon leaf improved high-fat diet induced atherogenic index.
11 Generally, high-fat diet significantly increases the total cholesterol levels in serum and liver
12 compared normal control diet in rats (Ghasi et al., 2000). In present study, persimmon leaf
13 significantly lowered hepatic cholesterol and triglyceride levels by 38.8% and 41.3%
14 compared to the HF group and diminished the accumulation of hepatic droplet.
15 In addition, high-fat diet supplemented with persimmon leaf significantly increased
16 both the HMG-CoA reductase and ACAT activities compared to the other groups. Plasma
17 cholesterol lowering mediated by persimmon leaf supplementation possibly resulted to
18 enhance HMG-CoA reductase and ACAT activities to maintain cholesterol homeostasis.
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1 Similar results were observed in our previous study with rabbit supplemented with a citrus
2 flavonoid that lowered plasma and hepatic cholesterol level (Jeon et al., 2004)
3 Daily fecal weight was significantly higher in the PL group than in the HF group that
4 seemed to be due to additional fiber content in PL diet because persimmon leaf contain 63.48
5 g fiber per 100 g. The increase in fecal weight might vary widely with the type and quantity
6 of dietary fiber being consumed (Shankardass et al., 1990). Chau et al. (2004) reported that
7 fecal dry weight was significantly higher when hamsters were supplemented with the water-
8 insoluble fiber-rich fraction isolated from the feel of Citru sinensis L. cv. Liucheng and
9 cellulose diets, 55~56% increase relative to the fiber-free diet, and suggested that the
10 consumption of insoluble fiber could significantly increase the fecal weight, as well as fecal
11 bulk. Fecal acidic sterol was significantly higher in the NC group by 2.3 fold and the PL
12 group by 4.7 fold than in the HF group. Chisaka et al. (1988) and Matsumoto et al. (1998)
13 suggested that the hypocholesterolemic effect of polyphenols was mainly due to increased
14 fecal excretion of cholesterol and bile acids. Accordingly, combined effect of high fiber
15 and high phenolic content in persimmon leaf itself could enhance the fecal excretion of
16 neutral and acidic sterol. In this study, not only fecal cholesterol but fecal triglyceride
17 concentration was significantly higher in the PL group (9.87±0.56 and 1.20±0.11 mmol/g)
18 than in the HF group (8.45±0.69 and 0.58±0.08 mmol/g). In general, many effects of
20
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1 dietary fibers, such as decreased transit time, higher bile acid adsorption, increased
2 cholesterol biosynthesis, and decreased cholesterol absorption, could lead to a greater
3 excretion of fecal sterol, and subsequently decrease the serum cholesterol level (Lairon,
4 2001; Marlett, 2001). Functional components in mmol/g persimmon leaf that result to
5 regulate these cholesterol and fat metabolism could include various phenolic compounds,
6 fiber or other possible bioactive compounds.
7 Conclusion
8 The supplementation of powdered whole persimmon leaf that is rich in fiber and
9 phenolic compounds seemingly suppressed the body weight gain, and it remarkably lowered
10 plasma and hepatic lipid concentrations as well as fecal lipids in rats fed a high-fat diet.
11 Efficacy test of lipid lowering action of persimmon leaf, suggest that this whole PL food
12 would be beneficial for regulation of lipid metabolism or prevention of hyperlipidemia in
13 experimental animal model.
14
15 Acknowledgements
16 This study was supported by the Korea Research Foundation Grant (KRF-2003-005-
17 C00005) and the Project of Bio-Food Research from the Korea Science and Engineering
18 Foundation (KOSEF) under the Ministry of Science and Technology in Korea.
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1 Table Legends
2 Table 1. Compositions of the experimental diets (%)
4 Table 2. The general composition of powdered persimmon leaf and the phenolic content of its
5 extract
7 Table 3. Effects of powdered persimmon leaf supplementation on food intake, body weight
8 gain and food efficiency ratio in rats fed high-fat diet
10 Table 4. Effects of powdered persimmon leaf supplementation on organs and adipose tissues
11 weights in rats fed high-fat diet
12
13 Table 5. Effects of powdered persimmon leaf supplementation on plasma and hepatic lipid
14 concentration in rats fed high-fat diet
15
16 Table 6. Effects of powdered persimmon leaf supplementation on sizes of epididymal
17 adipocyte in rats fed high-fat diet
18
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1 Table 7. Effects of powdered persimmon leaf supplementation on activities of hepatic ACAT
2 and HMG-CoA reductase in rats fed high-fat diet
4 Table 8. Effects of powdered persimmon leaf supplementation on fecal lipid contents in rats
5 fed high-fat diet
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1 Figure Legends
2 Fig. 1.
3 Effect of powdered persimmon leaf supplementation on body weight changes in high-fat diet
4 supplemented rats. Mean±S.E.M (n=10) The means not sharing a common letter are
5 significantly different (p<0.05) between groups. NC; normal control group, HF; high-fat fed
6 group, PL; high-fat with powdered persimmon leaf fed group
8 Fig. 2.
9 Light micrography of epididymal adipocytes in obesity rats induced by high-fat diet (×200).
10 Representative pictures of hematoxylin and eosin-stained sections of epididymal adipocytes
11 from rats fed a normal control diet (NC) or high-fat diet supplemented with persimmon leaf
12 (PL) show smaller sizes of adipocyte than in rats fed a high-fat diet (HF).
13
14 Fig. 3.
15 Effects of powdered persimmon leaf supplementation on hepatic tissue morphology in rats fed
16 high-fat diet (×200). Fat accumulation, indicated by the arrowheads, in the form of large fat
17 droplet is present in liver of rats fed a high-fat diet (HF). Representative pictures of
18 hematoxylin and eosin-stained sections of liver tissue from rats fed a normal control diet (NC)
24
ACCEPTED MANUSCRIPT
1 or high-fat diet supplemented with persimmon leaf (PL) show few fat droplets.
3 Fig. 4.
4 Effects of powdered persimmon leaf supplementation on plasma leptin level in rats fed high-
5 fat diet. Mean±S.E.M (n=10) The means not sharing a common letter are significantly
6 different (p<0.05) between groups. NC; normal control group, HF; high-fat fed group, PL;
7 high-fat with powdered persimmon leaf fed group
25
ACCEPTED MANUSCRIPT
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11
12
13
14
15
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17
18
33
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1 Table 1. Compositions of the experimental diets (%)
Component NC HF PL
Casein 20 20 20
D, L-methionine 0.3 0.3 0.3
Corn starch 15 15 15
Sucrose 50 27.5 22.5
Cellulose powder 5 5 5
Mineral Mix 1) 3.5 3.5 3.5
Vitamin Mix 2) 1 1 1
Choline bitartate 0.2 0.2 0.2
Corn oil 5 9.9 9.9
Lard - 17.6 17.6
PL 3) - - 5
Total (%) 100 100 100
Kcal/100g diet 386.2 498.7 483.7
Calorie from fat (%) 11.7 49.6 51.9
Calorie from carbohydrate (%) 67.3 34.1 31.8
Calorie from proten (%) 21.0 16.3 17.3

1)
2 AIN-76 mineral mixture contained (in g/kg of mixture): calcium phosphate, dibasic
3 500.0; sodium chloride, 74.0; potassium citrate, monohydrate, 220.0; potassium sulfate, 52.0;
4 magesium oxide, 24.0; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6;
5 cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium
6 sulfate, 0.55; sucrose, finely powdered, 118.03.

2)
7 AIN-76A vitamin mixture contained (in g/kg of mixture): thiamine HCl, 0.6;
34
ACCEPTED MANUSCRIPT
1 riboflavin, 0.6; pyridoxine HCl, 0.7: niacin, 3.0; D-calcium pantothenate, 1.6; folic acid, 0.2:
2 D-biotin, 0.02; cyanocobalamin (vitamin B12), 1.0; dry vitamin A palmitate (500,000 U/g),
3 0.8; dry vitamin E acetate (500 U/g), 10.0; vitamin D3 trituration (400,000 U/g), 0.25;
4 menadione sodium bisulfite complex, 0.15; sucrose, fine powder, 981.08.

3)
5 Powdered persimmon leaf; Five gram persimmon leaf in 100 g diet provides 5.2
6 kcal energy, 46 mg carbohydrate, 578 mg protein, 305 mg fat and 3.2 g fiber based on table
7 2.
35
ACCEPTED MANUSCRIPT
1 Table 2. The general composition of powdered persimmon leaf and the total phenolic content
2 of its extract*
Component Powdered whole persimmon leaf
Energy (kcal/100 g) 104.78±2.23
Carbohydrate (g/100 g) 0.91±0.01
Crude protein (g/100 g) 11.56±0.10
Crude fat (g/100 g) 6.10±0.12
Fiber (g/100 g) 63.48±1.01
Total phenolics (mg/g) 11.49±0.5
*
3 Mean ± S.E.M (n=3).
36
ACCEPTED MANUSCRIPT
1 Table 3. Effects of powdered persimmon leaf supplementation on food intake, body weight
2 gain and food efficiency ratio in rats fed high-fat diet*
. NC1) HF2) PL3)
Food Intake (g/day) 22.34± 1.45 a 20.38± 0.38 b 18.39± 0.47 c
Energy Intake (kcal/day) 86.27± 2.45 a 101.64± 3.16 b 88.85± 2.16 a
Body Weight Gain (g/day) 7.72 ± 0.16 a 9.01 ± 0.16 b 8.13 ± 0.11 a
FER4) 0.34 ± 0.01 a 0.44 ± 0.01 b 0.44 ± 0.01 b

*
3 Means ± S.E.M (n=10)
1)
4 Normal control group
2)
5 High-fat fed group
3)
6 High-fat with powdered persimmon leaf fed group
4)
7 Food efficiency ratio = body weight gain / food intake
a,b
8 Means in the same row not sharing a common superscript are significantly different
9 (p<0.05) between groups.
37
ACCEPTED MANUSCRIPT
1 Table 4. Effects of powdered persimmon leaf supplementation on organs and adipose tissues
2 weights in rats fed high-fat diet*
NC1) HF2) PL3)
Organs (mg/g B.W.)
Liver 34.03 ± 0.78 a 33.73 ± 0.43 a 31.40 ± 0.46 b
Heart 3.15 ± 0.06 a 3.18 ± 0.04 a 2.98 ± 0.04 b
Kidney 6.72 ± 0.10 6.91 ± 0.15 6.57 ± 0.07
Adipose tissue (mg/g B.W.)
Epididymal WAT4) 17.78 ± 0.80 a 21.75 ± 0.99 b 19.94 ± 0.58 ab
Perirenal WAT 26.27 ± 1.43 31.33 ± 1.43 27.61 ± 1.30
Interscapular WAT 5.18 ± 0.49 a 7.00 ± 0.53 b 6.55 ± 0.40 ab
Interscapular BAT5) 1.25 ± 0.06 a 1.58 ± 0.10 b 1.38 ± 0.04 a
Total 50.48 ± 2.96 a 61.66 ± 3.36 b 55.47 ± 2.54 ab

*
1)
2)
3)
4)
7 WAT; white adipose tissue
5)
8 BAT; brown adipose tissue.
a,b
38
ACCEPTED MANUSCRIPT
1 Table 5. Effects of powdered persimmon leaf supplementation on plasma and hepatic lipid
2 concentration in rats fed high-fat diet*
NC1) HF2) PL3)
Plasma
Triglyceride (mmol/L) 1.58 ± 0.13 a 1.41± 0.08 a 0.97± 0.09 b
Total cholesterol (mmol/L) 3.88 ± 0.32 a 4.29 ± 0.23 a 2.14 ± 0.21 b
HDL-cholesterol (mmol/L) 0.83 ± 0.05 a 0.71 ± 0.03 b 0.60 ± 0.03 c
HDL-cholesterol/Total- 21.36± 1.07 b 16.51 ± 1.44 a 27.89 ± 2.23 c
cholesterol (%)
AI4) 3.68 ± 0.23 a 5.06 ± 0.44 b 2.58 ± 0.29 a
Liver
Triglyceride (mmol/g) 2.10 ± 0.28 a 3.27 ± 0.17 b 1.92 ± 0.12 a
Cholesterol (mmol/g) 2.70 ± 0.23 a 3.92 ± 0.14 b 2.40 ± 0.18 a

*
1)
2)
3)
4)
7 Atherogenic index : (Total cholesterol - HDL-cholesterol) / HDL-cholesterol
a, b, c
39
ACCEPTED MANUSCRIPT
1 Table 6. Effects of powdered persimmon leaf supplementation on sizes of epididymal
2 adipocyte in rats fed high-fat diet*
NC1) HF2) PL3)
Epididymal Adipocyte 551.74 ± 21.97 a 689.94 ± 26.31 b 580.79 ± 17.14 a
(um)
*
1)
2)
3)
a, b
40
ACCEPTED MANUSCRIPT
1 Table 7. Effects of powdered persimmon leaf supplementation on activities of hepatic ACAT
2 and HMG-CoA reductase in rats fed high-fat diet*
NC1) HF2) PL3)
ACAT
1.76 ± 0.04 a 1.94 ± 0.10 a 2.21 ± 0.06 b
(pmol/min/mg protein)
HMG-CoA reductase
128.67 ± 7.67 a 129.74 ± 4.16 a 167.74 ± 12.04 b
(pmol/min/mg protein)
*
1)
2)
3)
a,b
41
ACCEPTED MANUSCRIPT
1 Table 8. Effects of powdered persimmon leaf supplementation on fecal lipid contents in rats
2 fed high-fat diet*
NC1) HF2) PL3)
Daily fecal weight (g) 2.24 ± 0.11 ab 2.08 ± 0.13 a 2.73 ± 0.10 b
Triglyceride (mmol/g) 0.54 ± 0.06 a 0.58 ± 0.08 a 1.20 ± 0.11 b
Cholesterol (mmol/g) 8.10 ± 0.71 a 8.45 ± 0.69 a 9.87 ± 0.56 b
Acidic sterol (mg/g) 5.98 ± 0.18 b 2.85 ± 0.10 a 10.22 ± 0.79 c

*
1)
2)
3)
a, b, c
42
ACCEPTED MANUSCRIPT
480 b
a
b a
b
380 b
a
Body weight (g)
a
NC
b a
280 HF
a PL
a
b
a
180 a
80
0 1 2 3 4 5 6
Weeks on diets
1
2 Fig. 1. Effect of powdered persimmon leaf supplementation on body weight changes in high-
3 fat diet supplemented rats. Mean±S.E.M (n=10) The means not sharing a common letter
4 are significantly different (p<0.05) between groups. NC; normal control group, HF; high-fat
5 fed group, PL; high-fat with powdered persimmon leaf fed group.
6
43
ACCEPTED MANUSCRIPT
NC HF PL
1
2 Fig. 2. Light micrography of epididymal adipocytes in obesity rats induced by high-fat diet
3 (×200). Representative pictures of hematoxylin and eosin-stained sections of epididymal
4 adipocytes from rats fed a normal control diet (NC) or high-fat diet supplemented with
5 persimmon leaf (PL) show smaller sizes of adipocyte than in rats fed a high-fat diet (HF).
44
ACCEPTED MANUSCRIPT
NC HF PL
1
2 Fig. 3. Effects of powdered persimmon leaf supplementation on hepatic tissue morphology in
3 rats fed high-fat diet (×200). Fat accumulation, indicated by the arrowheads, in the form of
4 large fat droplet is present in liver of rats fed a high-fat diet (HF). Representative pictures of
5 hematoxylin and eosin-stained sections of liver tissue from rats fed a normal control diet (NC)
6 or high-fat diet supplemented with persimmon leaf (PL) show few fat droplets.
45
ACCEPTED MANUSCRIPT
12
b
Plasma leptin (ng/mL) 10
8
ab a
6
4
2
0
NC HF PL
2
3 Fig. 4. Effects of powdered persimmon leaf supplementation on plasma leptin level in rats fed
4 high-fat diet. Mean±S.E.M (n=10) The means not sharing a common letter are significantly
5 different (p<0.05) between groups. NC; normal control group, HF; high-fat fed group, PL;
6 high-fat with powdered persimmon leaf fed group.
7
10
11
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Accepted Manuscript

Supplementation of whole persimmon leaf improves lipid profiles and sup -

To appear in: Food and Chemical Toxicology

Received Date: 8 February 2006

1 Supplementation of whole persimmon leaf improves lipid profiles and

2 suppresses body weight gain in rats fed high-fat diet

4 J. S. Lee1, M. K. Lee2, T. Y. Ha3, S. H. Bok4, H. M. Park5, K. S. Jeong6, M. N. Woo7, M. S.

9 Korea, 3Korea Food Research Institute, Seongnam-si, Gyeonggi-do, 463-746, 4Bionutrigen,

11 702, 6Department of Veterinary Medicine, 7Department of Food Science and Nutrition,

12 Kyungpook National University, Daegu, 702-701, South Korea

15 Myung-Sook Choi, Ph.D

16 Department of Food Science and Nutrition,

18 E-mail: mschoi@knu.ac.kr, Tel: +82-53-950-6232, Fax: +82-53-950-6229

20 Running title : Persimmon leaf supplement & lipid metabolism

1 (63.48 g/100 g) content in the powdered persimmon leaf.

5 cardiovascular diseases incidence (McNamara, 2000). Plant polyphenols exert

8 diseases (Yugarani et al., 1992; Hartog et al., 1993).

9 In the leaf of persimmon diospyros kaki, flavonoid oligomers, tannins, phenols,

12 beneficial effects on haemostasis, constipation, hypertension, apoplexy and atherosclerosis,

14 food on lipid lowering metabolisms in rats fed a high-fat diet.

16 Materials and methods

17 Preparation of powdered persimmon leaf and analysis of general composition

4 AOAC (1995) method and the Prosky-AOAC (1988) method, respectively.

6 Determination of total phenolic contents in powdered whole persimmon leaf

7 Total phenolic content was analyzed spectrophotometrically by a modified Folin-

14 were added to a test tube, followed by addition of 0.125 mL of Folin-Diocalteu reagent.

18 nm using a spectrophotometer (UVIDEC-50, JAS-CO Corporation, Tokyo, Japan). The

6 Animals and diets

2 <Insert table 1 here>

14 University for animal studies.

16 Plasma, hepatic, and fecal lipids

17 Plasma total cholesterol (Total-C) and high-density lipoprotein (HDL)-cholesterol

18 concentrations were determined using a commercial kit (Sigma) based on a modification of

8 concentrations of 5 g/L and 3 mmol/L, respectively. The cholesterol and triglyceride

11 enzymatically with 3-α-hydroxysteroid dehydrogenase (Crowell and Macdonald, 1980).

13 Hepatic and fatty morphology

17 were viewed using an optical microscope with a magnifying power of ×200.

1 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and acyl-CoA:cholesterol

2 acyltransferase (ACAT) activities

8 min at 4°C. The resulting microsomal pellets were then redissolved in 1 mL of a

10 HMG-CoA reductase and ACAT activities.

11 The HMG-CoA reductase activities were determined as described by Shapiro et al.

14 of NADPH (dissolved in a reaction buffer containing 0.1 mol/L of triethanolamine and 10

15 mmol/L of EDTA) was preincubated at 37°C for 5 min. Next, 10 µL of 50 nmol of

8 synthesized per min per mg of protein.

11 cholesterol substrate, 6 mg of cholesterol and 600 mg of Tyloxapol (Triton WR-1339, Sigma)

14 µg of cholesterol/mL. Next, reaction mixtures containing 20 µL of the cholesterol solution

15 (6 µg of cholesterol), 20 µL of a 1 mol/L of potassium phosphate buffer (pH 7.4), 10 µL of 0.6

2 was stopped by the addition of 500 µL of isopropanol–heptane (4:1, vol/vol), 300 µL of

6 picomoles of cholesteryl oleate synthesized per min per mg of protein.

8 Plasma leptin concentration

10 Systems Laboratories Inc., USA).

16 defined as p< 0.05.

7 <Insert table 2 here>

9 Food intakes, weight gains and food efficiency ratio

14 significantly lower in the PL group than in the HF.

15 <Insert Table 3 here>

5 <Insert Fig. 1 here>

14 brown adipose tissue compared to the HF group (Table 4).

15 <Insert Table 4 here>

17 Plasma and hepatic lipids

18 Concentrations of plasma and hepatic lipids are shown in Table 5. The