全 文 ： 74 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期

Chinese Journal of Natural Medicines 2011, 9(1): 0074−0077
doi: 10.3724/SP.J.1009.2011.00074
Chinese
Journal of
Natural
Medicines







Effects of Persimmon Leaf Total Flavonoid on
Enzyme of Lipoprotein Metabolism and
Antioxidation in Hyperlipidemia Rats
CHEN Li1*, MA Xin-Bo1, LIANG Yu-Hong2, PEI Shi-Cheng1, FENG Yi-Ping1, WEI Min3
1 Department of Pharmacology, Liuzhou Medical College, Liuzhou 545006, China;
2 Liuzhou Worker’s Hospital, Liuzhou 545005, China;
3 Liuzhou People’s Hospital, Liuzhou 545006, China
Available online 20 Jan. 2011
[ABSTRACT] AIM: To explore the effects of persimmon leaf total flavonoid (PLF) on regulating the serum level of lipids, enzymes
of lipoprotein metabolism and antioxidation in the hyperlipidemia rats. METHODS: 50 healthy SD rats were divided into 5 groups
randomly according to the serum TC: hyperlipidemia model group, high dose group of PLF, medium dose group of PLF, low dose
group of PLF, and lovastatin treatment positive control group. Serum lipoprotein level, lipoprotein lipase (LPL), hepatic lipase (HL),
superoxide dismutase (SOD) and malondialdehyde (MDA) were tested after treated with corresponding drugs once per day for 4
weeks. RESULTS: Compared with the model group, the levels of the serum TC, TG, LDL-C, and MDA decreased significantly, while
HDL-C, LPL, HL and SOD increased significantly (P < 0.05 or P < 0.01). CONCLUSION: The lipid metabolic disorder in hyperlipi-
demic rats was improved notably through the treatment with PLF, indicating that the PLF plays an important role in decreasing lipids.
[KEY WORDS] Persimmon leaf; Total flavonoid; Hyperlipidemia; Lipoprotein metabolism; Antioxidation
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2011)01-0074-04

1 Introduction
Long-lasting high-fat diet causes the reduction of the ac-
tivity of hepatic lipase (HL) and lipoprotein lipase (LPL) and
other lipid metabolic enzymes, leads to lipid metabolism
disturbance, and hyperlipidemia, and atherosclerosis, which
isthe main reason for coronary heart disease, hypertension
and cerebrovascular disease. Persimmon (Diospyros kaki L.)
is a member of the Ebenaceae family. Accumulating re-
searches indicated that water extract and alcohol extract of
persimmon leaf could soften the blood vessel, lower blood
pressure, and anti-oxidize, prevent bleeding, and so on. Main
effective components of persimmon leaf are flavonoid com-
pound [1]. In this research, comparing with positive control,
lovastatin, persimmon leaf flavonoid (PLF) was extractedto
investigate the effect on lipoprotein metabolism in the hyper-
lipidemia rats, and explore the relative mechanism.

[Received on] 20-June-2010
[*Corresponding author] CHEN Li: Lecturer, Tel: 86-772-2613842,
E-mail: chenlilzyz@163.com
These authors have no any conflict of interest to declare.
2 Materials
2.1 Drugs
The dried leaves of persimmon were purchased from
Medicine Station of Nanning (Nanning, China), and identi-
fied by researcher WEI Fa-Nan, Guangxi Institute of Botany,
Chinese Academy of Sciences. Ebenaceae Diospyros kaki L.,
PLF was extracted from persimmon leaves powder in 70%
EtOH .To reduce the pressure and the condense of the extrac-
tion liquid, Sherwood oil was used to remove chlorophyll
with 20% NaOH to adjust the pH to 9 or 10. DiscardSher-
wood oil phase, adjust the pH of water phase to 5 or 6 by
15% HCl, repeat the extraction by ethyl acetate, dispose wa-
ter phase, and obtain PLF after deep dry ethyl acetate phase.
Normalized by the standard product, the rutin, the percentage
of total PLF is 65.84%, and obtain probability is 1.08%.
Cholesterol and pig bile salt were purchased from Sinopharm
Chemical Reagent Co., Ltd., (Shanghai, China, standard: 25
g/bottle, biochemical reagents, batch numbers: F20081110,
F20090517); Lovastatin was purchased from Yangtze River
Pharmaceutical Group, (Taizhou, China, standard: 20mg/tablet,
batch number: 09122401); Propylthiouracil was purchased
from Shanghai Fuxing Zhaohui Pharmaceutical Co., Ltd.
CHEN Li, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 74−77
2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 75

(Shanghai, China, batch number: 20090202); SOD, MDA,
total lipase (LPL+HL) assay kits and Coomassie breiliant
blue protein quantification kit were purchased from Nanjing
Jiancheng Bioengineering Institute (Nanjing, China, batch
Nos.: 20091018, 20091019, 20091113, 20091020).
2.2 Equipments
Centrifuge (LDZ4-0.4 self-balancing micro-centrifuge,
Beijing Centrifuge Machine Factory); High-speed refriger-
ated centrifuge (TGL-16G-A, Shanghai Anting Scientific
Instruments Factory); 722S Visible Spectrophotometer
(Shanghai Precision & Scientific Instrument Co., Ltd.);
RE-52 rotary evaporator (Shanghai Anting Experimental
Apparatus Co., Ltd.); FJ-200 high-speed dispersion homoge-
neous machine (Shanghai Specimen Model Production Fac-
tory); Automatic biochemical analyzer (Japan OLYMPUS
AU600).
2.3 Animals
30 male and 30 female SD rats (180-200g) were pro-
vided by Experimental Animal Center Guangxi Medical
University (Test Animal Production License: SCXK Gui
2008-0003, experimental animal permit license: SYXK Gui
2008-0005).
3 Method
3.1 Indicators and detection methods
3.1.1 Serum TC, TG, HDL-C, LDL-C levels
At the end of week 4 and week 6, 1 ml blood was
drawled 12h after fast. To get the serum, after 10 min siting
the blood was centrifuged at low temperature at 3 000 r·min−1
for 10 min, prepare the serum. Automatic biochemical ana-
lyzer was used to test the levels of TC, TG, HDL-C and
LDL-C.
3.1.2 Hepatic tissue protein content
Hepatic tissue protein content was measured according
to the instruction of Coomassie breiliant blue assay kit. 10%
liver homogenate was diluted to 1% withsaline. To get the
linear relationship, take 0.05 mL 10% liver homogenization
and adjustthe protein concentration lower 1.3 g·L−1. The
concentration of protein was detected by 722S visible spec-
trophotometer with wavelength at 595 nm.
3.1.3 Activity of SOD in hepatic tissue
Determination of SOD activity was conducted according
to the instruction of the assay kit manual. By trail, 17 µL 1%
liver homogenization was performed under the inhibition rate
within 15%-55% (the curve shows in linear). 722S visible
spectrophotometer was used to determine the absorbance
(OD) of each tube at wavelength 550 nm, and the SOD activ-
ity of hepatic tissue was calculated.
3.1.4 MDA content in hepatic tissue
According to the instruction of MDA assay kit,0.1mL
10% liver homogenization was incubated at 95 °C for 80 min,
use 722S visible spectrophotometer was used to determine
the absorbance of each tube at wavelength 532 nm and e the
MDA content in hepatic tissue Was calculated.
3.1.5 Activity of LPL and HL of hepatic tissue
According to the instruction of total lipase (LPL+HL)
assay kit, 10% liver homogenization was used and the ab-
sorbance of LPL and HL in each tube was detected by 722S
visible spectrophotometer to calculate the activities of LPL
and HL.
3.2 Hyperlipodemia rats model set up and separation
5 male and 5 female SD rats were selected randomly by
TC levels in serum and fed with basal animal food. Other 50
rats are fed by hyper-fatty animal food (Formula: 83.6% basis
diet, 1% cholesterol, 0.2% pig bile salt, 0.2% propylthiouracil,
12% lard, 3% yolk powder [4-5]. Mixture of cholesterol, pig
bile salt, propylthiouracil and yolk powder together was fed
for 6 weeks to make hyperlipidemia rats. After 12 h rats’
fasting, 1mL blood was drawled from inner canthus to make
serum. Levels of TC, TG, HDL-C, and LDL-C were detected
to ensure the model is set up successfully (Table 1), and ran-
domly divided these rats into 5 groups according to TC le-
velwith half male and half female: hyperlipidemia model
group, high, middle, low dose PLF group and lovastatin
treatment positive control group.

Table 1 Comparison of blood lipid in blank control group and hyperlipidemia model group ( x ± s)
n TC TG HDL-C LDL-C (mmol·L−1)
Blank control group 10 2.99 ± 0.36 0.53 ± 0.08 1.46 ± 0.14 2.60 ± 0.30
Hyperlipidemia model group 50 5.85 ± 0.65** 0.74 ± 0.07** 0.51 ± 0.06** 5.62 ± 0.59**
**P < 0.01 vs blank control group

3.3 Preparation of reagent
3.3.1 Preparation of PLF intragastric liquid
5, 2.5 and 1.25 g PLF were grinded in 5mL Tween 80.
With additional 500 mL distilled water added, three different
concentrations of intragastric liquid (0.01, 0.005 and 0.002 5
g·mL−1) were made.
3.3.2 Preparation of lovastatin intragastric liquid
Grinded 6 lovastatin tablets (20 mg per tablet) in 2 mL
Tween 80, added distill water to 300 mL to make 0.4
mg·mL−1 of lovastatin intragastric liquid.
3.3.3 Preparation of blank intragastric liquid
Mixture of 1 mL Tween 80 and 99 mL distill water as
blank intragastric liquid.
3.4 Rat groups and drug administration
Lovastatin tablets were the positive control. The blank
control group was fed with basis diet; other groups were fed
with hyper-fatty diet. The blank control group and hyperlipi-
demia group were intragastrically administered blank intra-
CHEN Li, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 74−77
76 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期

gastric liquid (15 mL·kg−1·d−1), gave the certain PLF intra-
gastric liquid to each group once a day, for 4 weeks, details
see Table 2.
3.5 Statistical analysis
Statistical software SPSS13.0 was used to analyze all data.
All results were presented as x ± s and compared each two

Table 2 Rat groups and drug administration (n = 10)
Group Drug Dose
Blank control Blank intragastric liquid 15 mL·kg−1·d−1
Hyperlipidemia model Blank intragastric liquid 15 mL·kg−1·d−1
Lovastatin positive
control
Lovastatinintragastric
liquid 3.0 mg·kg
−1·d−1
High PLF dose PLF 0.1 g·kg−1·d−1
Middle PLF dose PLF 0.05 g·kg−1·d−1
Low PLF dose PLF 0.025 g·kg−1·d−1
data by One-Way ANOVA and Pared - Sample t Test. The P <
0.05 or P < 0.01 means statistical significance.
4 Results
4.1 Effects of PLF on serum TC, TG, HDL-C, and LDL-C
levels in hyperlipidemia rats
After 4 weeks treatment, rats were weighted after 12h
fasting, and then anesthetized by 20% urethane solution1 mL
blood was drawled from abdominal aorta, stored at 4 °C for
10 min, and centrifuged at 3 000 r·min−1 for 15min, to make
serum. As shown in Table 3, compared with the hyperlipide-
mia model group, the levels of the serum TC, TG and LDL-C
decreased significantly in all the three PLF treatment groups
(P < 0.01), while HDL-C increased significantly (P < 0.01).
The high-dose treatment prevented these developments in
hyperlipidemia rats.

Table 3 Changes of serum TC, TG, HDL-C and LDL-C levels after 4-week treatment in each group ( x ± s, n = 10)
Group TC TG HDL-C LDL-C (mmol·L−1)
Blank control 3.01 ± 0.36# # 0.55 ± 0.08# # 1.45 ± 0.15# # 2.60 ± 0.30# #
Hyperlipidemia model 6.11 ± 0.28** 0.84 ± 0.04** 0.47 ± 0.04** 6.01 ± 0.34**
Lovastatin positive control 3.49 ± 0.41**# # 0.51 ± 0.03# # 0.98 ± 0.07**# # 3.13 ± 0.44**# #
High PLF dose 3.78 ± 0.28**# # 0.57 ± 0.04# #△ 0.93 ± 0.07**# # 3.73 ± 0.39**# #△△
Middle PLF dose 4.25 ± 0.23**# #△△ 0.59 ± 0.03# #△△ 0.89 ± 0.06**# #△△ 4.19 ± 0.19**# #△△
Low PLF dose 4.99 ± 0.69**# #△△ 0.65 ± 0.09# #△△ 0.86 ± 0.04**# #△△ 4.39 ± 0.33**# #△△
**P < 0.01 vs blank control group; # #P < 0.01 vs hyperlipoidemia model group; △P < 0.05, △△P < 0.01 vs lovastatin group

4.2 Effects of PLF on LPL and HL activities in hyperlipi-
demia rats
Rat’s liver was removed immediately after taking blood
from abdominal aorta, and rinsed with ice-cold saline. Fol-
lowing the instruction of assay kit, hepatic tissue homogeni-
zation was prepared to test the activities of LPL and HL. As
listed in Table 4, compared with the hyperlipidemia model
group, LPL and HL activities significantly increased in all the
three PLF treatment groups (P < 0.01) dose-dependently. In
addition, all three PLF treatments were better to alleviate the
disorder than lovastatin treatment.
4.3 Effect of PLF on liver tissue SOD and MDA in hyper-
lipidemia rats
As listed in Table 5, all the three PLF treatment signifi-
cantly increased SOD activity compared with the hy-
Table 4 The comparison of LPL and HL in liver tissue after
4-week treatment in each group ( x ± s, n = 10)
Group LPL HL (U·mgprot−1)
Blank control 1.74 ± 0.07# # 1.32 ± 0.07# #
Hyperlipidemia model 0.77 ± 0.10** 0.41 ± 0.05**
Lovastatin positive control 1.13 ± 0.05**# # 0.86 ± 0.04**# #
High PLF dose 1.32 ± 0.06**# #△△ 1.08 ± 0.05**# #△△
Middle PLF dose 1.15 ± 0.05**# # 0.83 ± 0.04**# #
Low PLF dose 1.11 ± 0.06**# # 0.82 ± 0.03**# #△
**P < 0.01 vs blank control group; # #P < 0.01 vs hyperlipoidemia
model group; △P < 0.05, △△P < 0.01 vs lovastatin group
perlipidemia model group (P < 0.01), while the MDA levels
decreased significantly (P < 0.01). All three PLF treatment
were better to alleviate the disorder than lovastatin treatment.

Table 5 Comparison of SOD and MDA in liver tissue after
4-week treatment in each group ( x ± s, n = 10)
Group SOD (U/mgprot) MDA (nmol/mgprot)
Blank control 252.24 ± 22.03# # 1.76 ± 0.15# #
Hyperlipidemia model 167.25 ± 5.74** 8.36 ± 0.46**
Lovastatin positive
control 207.34 ± 4.67**
# # 3.55 ± 0.22**# #
High PLF dose 213.70 ± 7.75**# # 3.43 ± 0.18**# #
Middle PLF dose 201.33 ± 4.43**# # 4.07 ± 0.68**# #△△
Low PLF dose 200.53 ± 3.44**# # 4.19 ± 0.44**# #△△
**P < 0.01 vs blank control group; # #P < 0.01 vs hyperlipoidemia
model group; △△P < 0.01 vs lovastatin group
5 Discussion
Both LPL and HL are key enzymes of lipoprotein me-
tabolism and play critical roles. Presently, controlling the
activity of lipoprotein metabolism enzyme is the principle
treatment to hyperlipidemia[2]. In our research, the capability
of increasing HL activity in high dose PLF group is notably
stronger than lovastatin, which means PLF could benefit the
body lipoprotein metabolism by increasing the activities of
LPL and HL in hepatic tissues of hyperlipidemia rats.
CHEN Li, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 74−77
2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 77

Usually, hyperlipidemia is associated with changes in li-
pid peroxidation and lipid peroxide (LPO), such as MDA.
MDA could injury the activity of the enzyme mitochondrial
membrane, stop the activation of CoA in mitochondrial
membrane, disturb fatty acid oxidation, and then initial the
fat stays in liver [3]. SOD plays a vital role in balancing body
oxidation and antioxidation. In hyperlipodemic rats, the de-
crease of antioxidative enzyme activities and the ability of
scavenging free radical were observed with significant in-
crease of MDA content. As a result, oxidative stress is effec-
tively increased when hyperlipidemia happens. Our study
proved this by the decreased SOD activity and increased
MDA level in hepatic tissue from hyperlipidemia rats. After 4
weeks treatment, all the three PLF treatment groups showed
significantly higher SOD activity than the hyperlipidemia
model group (P < 0.01), and the high dose PLF is best to
increase SOD activity. The levels of MDA were decreased
significantly in all three PLF treatment groups, especially in
the high dose PLF group. Results indicated that the lipid
peroxidation of hyperlipidemia rats can be alleviated by PLF,
and rising antioxidation capacity may be one of the mecha-
nisms to prevent lipid disorder in hyperlipidemia rat.
Currently, most of the medicines for hyperlipoidemia are
chemical compounds and pharmacology is not clear. In addi-
tion, those medicines have wide side effects on liver and
kidney for. High price leads limited clinical application [4-5].
As an option treatment, traditional Chinese medicine has
been emphasized with stable effect, limited toxicity and
side-effect, affluent resource and low economy. This research
showed PLF significantly decreased TC, TG, LDL-C, MDA,
and increased HDL-C, LPL, HL and SOD, the lipid meta-
bolic disorder of hyperlipidemia rats, suggesting the value of
PLF in clinic in future.
References
[1] Lin JF, Lin HT, Xie LQ, et al. A review of chemical constitu-
ents, medicinal function, clinical application of persimmon
leaves and their development and utilization [J]. Food Ferment
Ind, 2005, 31(7): 90-96.
[2] Zhang JS, Peng B, Cui C. Effects of olive antihyperlipidemia
capsule on enzyme of lipoprotein metabolism in rats [J]. Chin J
Exp Tradit Med Form, 2007, 13(5): 49-50.
[3] Tur Mari JA. The quality of fat: olive oil [J]. Arch Latinoam
Nutr, 2004, 54(2): 59-64.
[4] Wang XH. Overview about the regulatory function of Chinese
Medicinal Herb on serum lipids [J]. J Pharm Pract, 2007,
25(2): 73-77.
[5] Liu HR. The summaries on the effect of bio-active components
from natural products therapy for hyperlipidemia [J]. Hunan
Guiding J Tradit Chin Med Pharmacol, 2002, 8(12): 742-745.


柿叶黄酮对高脂血症大鼠肝脂蛋白代谢相关酶活性及抗氧化能
力的影响
陈 丽 1*, 马新博 1, 梁宇红 2, 裴世成 1, 冯艺萍 1, 韦 敏 3
1柳州医学高等专科学校 柳州 545006;
2柳州市工人医院 柳州 545005;
3柳州市人民医院 柳州 545006
【摘 要】 目的：研究柿叶总黄酮(PLF)对饮食性高脂血症大鼠血脂及肝脏脂质代谢的影响。方法：建立饮食性高脂血症大
鼠模型, 将高脂血症大鼠按照血清 TC 水平随机分成 5 组 (n = 10), 每组雌雄各半, 分别为：高脂模型组、PLF 高剂量组、PLF
中剂量组、PLF 低剂量组和洛伐他汀组。连续给药 4 周后检测各组大鼠脂蛋白酯酶 (LPL)、肝酯酶 (HL)、超氧化物歧化酶 (SOD)、
丙二醛 (MDA) 和血脂水平等各项指标。结果：与模型组比较, 各用药组 TC、TG、LDL-C、MDA 水平降低, HDL-C、LPL、
HL 和 SOD 水平升高均具有显著性差异(P < 0.01 或 P < 0.05)。结论：PLF 对高脂血症大鼠的脂质代谢紊乱有显著的调节作用。
【关键词】 柿叶; 总黄酮; 高脂血症; 脂蛋白代谢; 抗氧化