Key words
Rheum undulatum
- Polygonaceae - methoxylated stilbene - antihyperlipidemic effect - high-cholesterol
diet
Abbreviations
ALT:
alanine aminotransferase
AST:
aspartate aminotransferase
HCD:
high-cholesterol diet
HDL-C:
high-density lipoprotein cholesterol
HE:
hematoxylin and eosin
LDL-C:
low-density lipoprotein cholesterol
RHA:
rhapontin
RHG:
rhapontigenin
TC:
total cholesterol
TG:
triglyceride
VLDL-C:
very low-density lipoprotein cholesterol
Introduction
Hyperlipidemia, an increase in the blood lipid level, is the leading cause of cardiovascular
disease and death worldwide. Reducing the LDL-C level in individuals with hyperlipidemia
decreases the risk of cardiovascular disease [1]. By contrast, HDL-C lowers the risk of cardiovascular diseases by reversing cholesterol
transport, inhibiting LDL-C oxidation, and decreasing platelet aggregability [2], [3]. Statins are widely prescribed as lipid lowering agents; however, they can elicit
serious side
effects such as myopathy and hepatocytotoxicity [4], [5]. Therefore, the development of new agents for lowering blood lipid levels is needed.
Stilbenes are well-studied compounds that possess anticancer, antioxidative, and anti-inflammatory
activities [6], [7], [8], and methoxylated stilbenes enhance those bioactivities [9], [10]. Rhapontigenin (3,3′,5-trihydroxy-4′-methoxystilbene) is an aglycone of rhapontin
(3,3′,5-trihydroxy-4′-methoxystilbene-3-O-glucoside), which is isolated from the roots of Rheum undulatum L. from the family Polygonaceae ([Fig. 1]) [11]. Rhapontigenin is a methoxylated stilbene with anticancer, antioxidative, and anti-inflammatory
effects [12], [13]. Rhapontigenin is much more potent than rhapontin in inhibiting thrombosis and allergic
reactions [14]. To increase the bioactivity of rhapontin isolated from a methanol extract from
the roots of Rheum undulatum
[15], [16], rhapontigenin is prepared from rhapontin by an enzymatic transformation [17]. In this study, RHA and RHG were investigated for their antihyperlipidemic activity,
and the results were compared with simvastatin, a well-known hypolipidemic drug.
Fig. 1 Structures of rhapontin (A) and rhapontigenin (B).
Results and Discussion
RHA, RHG, or simvastatin (positive control) was orally administered to HCD-fed rats
for four weeks (Table 1S, Supporting Information). The percent changes (%Δ) in body weight were not significantly
reduced except in HCD-fed rats treated with RHG at 5 mg/kg/day ([Table 1]). The %Δ body weight significantly (p < 0.05) decreased in HCD-fed rats treated
with RHG at 5 mg/kg/day compared with the HCD-fed control. The relative liver weight
increased in HCD-fed rats compared with the water-treated control; however, it decreased
in HCD-fed rats
treated with RHA and RHG compared with rats fed the normal diet. Daily food consumption
was similar in all groups, indicating that the test compounds did not suppress appetite.
Table 1 Effects of the high-cholesterol diet and simvastatin, rhapontin, or rhapontigenin
treatment on body weight, relative liver weight, and average daily food consumption
in rats.
|
Treatment group
|
% ΔWeight on day 28
|
Relative liver weight
|
Average daily diet consumption (g/day)
|
|
Group I, normal diet + 10 mL/kg/day distilled water; group II, HCD; group III, HCD
+ 1 mg/kg/day simvastatin; group IV, HCD + 1 mg/kg/day RHA; group V, HCD + 2.5 mg/kg/day
RHA; group VI, HCD + 5 mg/kg/day RHA; group VII, HCD + 1 mg/kg/day RHG; group VIII,
HCD + 2.5 mg/kg/day RHG; and group IX, HCD + 5 mg/kg/day RHG; a, b, c, d Means in the same column not sharing a common superscript are significantly (p < 0.05)
different between groups
|
|
I
|
141.7 ± 14.5a, b
|
2.8 ± 0.3a
|
15.3 ± 1.4
|
|
II
|
155.8 ± 17.0b
|
4.4 ± 0.8c, d
|
15.9 ± 2.9
|
|
III
|
147.4 ± 14.8a, b
|
4.0 ± 0.7d
|
15.5 ± 2.6
|
|
IV
|
143.4 ± 10.7a, b
|
3.7 ± 0.3b, c, d
|
17.2 ± 3.1
|
|
V
|
148.8 ± 9.3a, b
|
4.2 ± 0.4b, c, d
|
16.0 ± 2.8
|
|
VI
|
143.4 ± 12.0a, b
|
4.1 ± 0.4c, d
|
17.2 ± 2.5
|
|
VII
|
137.9 ± 6.5a, b
|
3.9 ± 0.4b, c
|
15.7 ± 2.7
|
|
VIII
|
143.1 ± 21.1a, b
|
3.5 ± 0.3b
|
16.5 ± 2.8
|
|
IX
|
130.5 ± 7.5a
|
3.9 ± 0.6b, c, d
|
16.5 ± 2.1
|
To investigate the antihyperlipidemic effect of RHA and RHG, the serum lipid level
was measured in HCD-induced hyperlipidemic rats after treatment. The TC and LDL-C
levels in HCD-fed rats increased 2.0- and 5.8-fold, respectively, compared with the
water-treated control; however, the serum lipid level decreased in HCD-fed rats treated
with RHA or RHG dose-dependently, indicating the efficacy of RHA and RHG in preventing
the elevation of the serum lipid level under experimentally-induced hyperlipidemia
([Table 2]). Although there was a slight increase
in the HDL-C level in HCD-fed rats treated with RHG, the significant difference in
the HDL-C level was not shown after RHG treatment. The serum TC level was significantly
(p < 0.05) lower in HCD-fed rats treated with RHG than that in the HCD-fed control.
The serum TC levels in HCD-fed rats treated with RHA or RHG at 5 mg/kg/day were reduced
by 21.0 % and 33.6 %, respectively, compared with the HCD-fed control. Furthermore,
although there was an increase in the HDL-C level in HCD-fed rats treated with RHG
at 5 mg/kg/day, the serum lipid level was similar to that of the
HCD-fed rats treated with simvastatin at 1 mg/kg/day. High levels of LDL-C, TC, and
VLDL-C contribute to the development of cardiovascular disease. In the present study,
although the HDL-C level in HCD-fed rats treated with RHG increased slightly, there
was no significant change in the HDL-C level in all HCD-fed rats. This observation
indicates that methoxylated stilbenes, particularly RHG, may possess cardioprotective
potential. The results from serum lipid analysis in HCD-fed rats treated with methoxylated
stilbenes indicate that the RHG aglycone was more effective than its
glycosylated RHA counterpart in the reduction of serum lipid levels.
Table 2 Effect of the high-cholesterol diet and simvastatin, rhapontin, or rhapontigenin
treatment on the serum lipid level in rats.
|
Treatment group
|
U/L
|
Levels (mg/dL)
|
|
|
AST
|
ALT
|
TC
|
HDL
|
LDL
|
VLDL
|
TG
|
|
Group I, normal diet + 10 mL/kg/day distilled water; group II, HCD; group III, HCD
+ 1 mg/kg/day simvastatin; group IV, HCD + 1 mg/kg/day RHA; group V, HCD + 2.5 mg/kg/day
RHA; group VI, HCD + 5 mg/kg/day RHA; group VII, HCD + 1 mg/kg/day RHG; group VIII,
HCD + 2.5 mg/kg/day RHG; and group IX, HCD + 5 mg/kg/day RHG; a, b, c, d, e Means in the same column not sharing a common superscript are significantly (p < 0.05)
different between groups
|
|
I
|
86.7 ± 11.0
|
40.5 ± 8.1
|
75.8 ± 10.5a
|
40.8 ± 4.8b
|
19.3 ± 3.4a
|
15.7 ± 2.6e
|
78.4 ± 13. 0e
|
|
II
|
92.5 ± 7.0
|
39.3 ± 4.3
|
154.8 ± 18.6c
|
28.6 ± 4.8a
|
112.1 ± 22.8d
|
14.1 ± 2.2d, e
|
70.5 ± 10.9d, e
|
|
III
|
101.3 ± 21.1
|
40.3 ± 3.8
|
99.3 ± 28.7a, b
|
26.5 ± 2.9a
|
61.8 ± 26.3b
|
11.0 ± 1.5b, c
|
54.8 ± 9.6a, b, c
|
|
IV
|
90.4 ± 6.8
|
42.5 ± 4.3
|
161.5 ± 39.9c
|
31.8 ± 3.6a
|
102.0 ± 29.1c, d
|
15.4 ± 1.4e
|
76.8 ± 7.2e
|
|
V
|
88.2 ± 12.4
|
38.0 ± 6.9
|
131.8 ± 8.0b, c
|
30.5 ± 6.9a
|
86.0 ± 16.5b, c, d
|
12.8 ± 1.0c, d
|
64.0 ± 5.1c, d
|
|
VI
|
85.8 ± 6.1
|
35.2 ± 3.6
|
122.3 ± 33.2b
|
27.7 ± 4.7a
|
73.8 ± 26.3b, c
|
12.0 ± 2.4b, c, d
|
59.8 ± 11.8c, d, e
|
|
VII
|
84.5 ± 6.9
|
41.2 ± 1.5
|
115.3 ± 24.9b
|
30.5 ± 4.7a
|
74.8 ± 23.6b, c
|
10.1 ± 1.2a, b
|
50.3 ± 5.8a, b
|
|
VIII
|
87.8 ± 7.0
|
39.5 ± 4.2
|
114.0 ± 25.2b
|
30.2 ± 5.1a
|
68.0 ± 23.4b
|
10.1 ± 1.9a, b
|
50.3 ± 9.5a, b
|
|
IX
|
89.0 ± 14.1
|
38.3 ± 5.3
|
102.8 ± 14.5a, b
|
33.7 ± 8.1a
|
60.4 ± 12.5b
|
8.8 ± 1.9a
|
44.0 ± 9.5a
|
The oral administration of rhaponticin (rhapontin) (125 mg/kg) from rhubarb rhizomes
reduced the serum lipid level in KK/Ay diabetic mice [18]. Compared to the previous report, the oral administration of remarkably low concentrations
of rhapontin (2.5 and 5 mg/kg) from R. undulatum showed a hypolipidemic effect in HCD-fed rats. A polyphenolic stilbenoid, resveratrol,
shows beneficial effects on the prevention of dyslipidemia by activating cholesterol
7α-hydroxylase and increasing the bile acid pool size [19]. Furthermore, pterostilbene (3,5-dimethoxy-4′-hydroxystilbene) lowers serum lipoprotein
and cholesterol levels in hypercholesterolemic hamsters by acting as a peroxisome
proliferator-activated receptor α (PPARα) that plays a central role in lipid homeostasis [20]. Pterostilbene also shows a higher hypolipidemic effect than resveratrol. RHA and
RHG showed inhibitory activities against 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase and squalene synthase, which are key enzymes in the cholesterol biosynthesis
pathway
[21]. In addition, although RHG, a methoxylated stilbene aglycone, contains more hydroxyl
groups and one less methoxyl group than pterostilbene, it may show the same antihyperlipidemic
mechanism. Further studies are needed to clarify the mechanism of action of RHG on
antihyperlipidemia.
The liver from HCD-fed rats treated with RHA or RHG was examined for histopathological
changes. Liver cells from HCD-fed rats were altered compared with those of rats fed
a normal diet, which did not show abnormal morphology ([Fig. 2]). Numerous lipid droplets were observed in the cytoplasm of centrilobular hepatocytes
in livers from HCD-fed rats compared with those from rats fed a normal diet ([Fig. 2 A] and [B]). By contrast, the number of lipid droplets significantly decreased in HCD-fed
rats treated with simvastatin ([Fig. 1 C]), RHA, or RHG, dose-dependently ([Fig. 2 D–I]). Lipid droplets in liver cells from HCD-fed rats treated with RHA or RHG at 5 mg/kg/day
were hardly observed ([Fig. 2 F] and [I]). The results indicate that methoxylated stilbenes, especially RHG, can reverse
HCD-induced liver cell degeneration.
Fig. 2 Histological analysis of the liver from hyperlipidemic rats who were fed a high-cholesterol
diet and treated with rhapontin or rhapontigenin. A normal diet; B HCD; C HCD + 1 mg/kg/day simvastatin; D HCD + 1 mg/kg/day RHA; E HCD + 2.5 mg/kg/day RHA; F HCD + 5 mg/kg/day RHA; G HCD + 1 mg/kg/day RHG; H HCD + 2.5 mg/kg/day RHG; I HCD + 5 mg/kg/day RHG. All images are shown at 200× magnification. (Color figure
available online only.)
In conclusion, RHA and RHG treatment lowered the serum lipid level dose-dependently
and reversed hyperlipidemia in HCD-fed rats. A histological study also showed that
RHA and RHG decreased the number of lipid droplets in liver cells from HCD-fed rats,
consistent with the serum lipid analysis results. RHG showed a higher preventive effect
against hyperlipidemia than RHA, illustrating that the aglycone was more effective
to reduce the risk for hyperlipidemia than the glycosylated counterpart.
Materials and Methods
Chemicals
RHA (3,3′,5-trihydroxy-4′-methoxystilbene-3-O-glucoside) was isolated from a methanol extract of Rheum undulatum roots, and RHG (3,3′,5-trihydroxy-4′-methoxystilbene) was produced from RHA via an
enzymatic transformation using Pectinex® (Novozymes) as previously described [17]. The purity of RHA and RHG was over 95 %. Simvastatin (purity ≥ 97 %) and DMSO were
purchased from Sigma Chemical Co.
Experimental animals and diets
Sprague Dawley rats (n = 54), five weeks old and weighing approximately 100–110 g,
were purchased from Koatech. They received a normal diet for one week to allow for
adaptation. Rats were housed in cages under strict standard conditions (22 ± 1 °C;
55 ± 5 % humidity; 12-h light and 12-h dark cycles). The animals had free access to
water and a normal diet or an experimental HCD purchased from Feedlab. The composition
of diets is shown in Table 1S, Supporting Information. All experimental procedures were approved by the Korea University
Institutional Animal Care and Use
Committee (approval No. KUIACUC-2012–100) and were followed according to the Guide
for the Care and Use of Laboratory Animals (NIH publication No. 85–23, 1996).
Induction of hyperlipidemia with a high-cholesterol diet
After adaptation, rats were randomly divided into nine groups, with each group consisting
of six rats. RHA or RHG was orally administrated to rats daily for four weeks. Animals
in group I (negative control) were fed a normal diet and orally treated with distilled
water at 10 mL/kg/day for four weeks. Animals in group II (experimental control) were
fed the HCD instead of the normal diet (Table 1S, Supporting Information). Animals in group III (positive control) were fed the HCD
and orally treated with simvastatin at 1 mg/kg/day in 10 mL/kg DMSO and distilled
water
(1 : 9, v/v) for four weeks. Animals in groups IV, V, and VI were fed the HCD and
orally treated with RHA at 1, 2.5, and 5 mg/kg/day in 10 mL/kg DMSO and distilled
water (1 : 9, v/v), respectively. Animals in groups VII, VIII, and IX were fed the
HCD and orally treated with RHG at 1, 2.5, and 5 mg/kg/day in 10 mL/kg DMSO and distilled
water (1 : 9, v/v), respectively.
Measurement of daily food intake, body weight, and relative liver weight
Daily food intake was assessed by subtracting the weight of any leftover food from
the weight of the total amount of food provided, divided by six for each group. The
body weight of each rat was obtained weekly. The absolute liver weight was obtained,
and the relative liver weight per 100 g of total body weight was calculated as follows:
Blood collection and biochemical assays
After treatment for four weeks, blood was collected from the heart of overnight fasted
rats under diethyl ether anesthesia and allowed to clot at room temperature. Sera
were obtained by centrifugation at 10 000 × g at 4 °C for 20 min. Serum TG, TC, HDL-C, AST, and ALT levels were measured using
the FUSI DRI-CHEM SLIDE kit and a FUSI DRI-CHEM 4000 analyzer (Fujifilm). The serum
LDL-C level was calculated by applying Frieldwannʼs equation:
The serum VLDL-C level was calculated by applying the following equation:
Hepatic morphology
For histological analysis, liver specimens were fixed in 10 % formalin and embedded
in paraffin. Paraffin blocks were cut at 5 µm, and sections were stained with hematoxylin
and eosin and then mounted with Canada balsam. The slides were observed under an optical
microscope at 200× magnification [22].
Statistical analysis
All data are presented as means ± standard error. The data were analyzed using the
SPSS statistical analysis program. Significant differences between groups were determined
by one-way analysis of variance. Post hoc Duncanʼs multiple-range tests were performed
when between-group differences were identified. Results were considered to be statistically
significant at p < 0.05.
Supporting information
Details about the composition of the experimental diets are available as Supporting
Information.
Acknowledgments
This research was supported by the High Value-added Food Technology Development Program,
Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
