3136 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Ye et al.
1
1014.0, 969.7, 944.0, 911.9, 879.7, 827.7, 807.9; H NMR (300
MHz, DMSO-d6) δ 12.69 (s, 1H, 5-Ar-OH), 10.93 (s, 1H, 7-
Ar-OH), 9.77 (s, 1H, 40-Ar-OH), 9.28 (s, 1H, 30-Ar-OH), 7.63
(d, 2H, J = 7.4, 60-Ar-H), 6.92 (d, 1H, J = 8.6 Hz, 50-Ar-H),
6.47 (s, 1H, 8-Ar-H), 6.28 (d, 1H, 6-Ar-H), 5.43 (d, 1H, J = 6.3
Hz, 100-H), 4.47 (d, 1H, J = 9.3 Hz, 1000-H), 1.07 (d, 3H, J = 5.9
Hz, 6000-H); 13C NMR (75 MHz, DMSO-d6) δ 177.37 (C-4),
164.05 (C-7), 161.22 (C-5),156.42 (C-2), 156.60 (C-9), 148.40 (C-
40), 144.74 (C-30), 133.31 (C-3), 121.58 (C-10), 121.18 (C-60),
116.27 (C-50), 115.22 (C-20), 103.97 (C-10), 101.19 (C-100),
100.73 (C-1000), 98.66 (C-6), 93.57 (C-8), 76.46 (C-300), 75.91(C-
500), 74.07 (C-200), 71.85 (C-4000), 70.56(C-400), 70.37(C-2000),
70.01(C-3000), 68.23 (C-5000), 66.99 (C-600), 17.71 (C-6000). It was
confirmed that compounds c and f were rutin, 4H-1-benzopyran-
4-one, 3-[(6-O-(6-deoxy-R-
L
-mannopyranosyl)-β-D-glucopyranosyl]-
oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy, assigned as com-
pound c.
Compound d was blue in ferric chloride (FeCl3) reaction:
yellow powder (MeOH/H2O), mp 207-209 °C; ESI-MS (m/z)
354 [M þ 1]; Anal. Calcd16H18O9; UV λmax (MeOH) 328.5 nm; IR
v (cm-1) 3389.5 (OH stretching vibration), 2956.0, 2928.8, 2857.1
(C;H stretching vibration), 2626.4, 1685.6 (CdO stretching
vibration), 1638.9, 1614.3, 1528.7, 1517.7, 1442.8, 1384.1 (aro-
matic ring skelton vibration), 1289.4, 1252.4, 1190.4, 1158.8,
1133.6, 1114.1, 1086.2, 1038.1, 970.2, 818.4; 1H NMR (300
MHz, H2O-d6) δ 7.47 (d, 1H, J = 15.9 Hz, R-H), 7.03 (s, 1H,
20-H), 6.96 (d, 1H, J = 8.3 Hz, 60-H), 6.83 (d, 1H, J = 8.3 Hz, 50-
H), 6.17 (d, 1H, J = 15.9 Hz, β-H), 5.25-5.18 (m, 1H, 3-H), 4.19
(d, J = 3.0 Hz, 5-H), 3.80 (dd, 1H, J = 8.5 Hz, 4-H), 2.22-2.00
(m, 4H, 2-H, 6-H); 13C NMR (75 MHz, H2O-d6) δ 1176.57 (C-70),
168.13 (C-9), 146.60 (C-4), 145.71 (C-3,), 143.72 (C-7), 126.39 (C-
1), 122.24 (C-6), 115.65 (C-5), 114.65 (C-2), 113.78 (C-8), 74.40
(C-10), 70.93 (C-40), 70.13 (C-30), 68.70 (C-50), 36.08 (C-60), 35.95
(C-20). It was confirmed that compound d was chlorogenic
acid, (1S,3R,4R,5R)-3-[[3-(3,4-dihydroxyphenyl)-1-oxo-2-pro-
penyl]oxy]-1,4,5-trihydroxycyclohexanecarboxylic acid.
The structures of compounds a-d are shown in Figure 1.
3.4. Quantification of Four Active Components in Hawthorn
Fruit Extract. The HPLC chromatogram of the ethanol extract of
hawthorn fruit is shown in Figure2. There are more than 10 peaks
in the extract, which accounted for about 4%. In 100 g of
dried extract, chlorogenic acid (d) accounted for 0.95 g; rutin
(c), 1.42 g; hyperoside (b), 0.32 g; and quercetin (a), 0.16 g. In
addition, ursolic acid accounted for 0.32 g; oleaniolic, 0.08 g; and
maslinic acid, 0.08 g. The total content of compounds a-d was
about 2.85 g in 100 g of dried extract of hawthorn fruit.
3.5. IR of Four Monomers and Their Mixture to HMGR.
According to the ratio of the four monomers in crude extract of
hawthorn fruit, quercetin (a), hyperoside (b), rutin (c), and
chlorogenic acid (d) were adjusted to concentrations of 0.16,
0.32, 1.42, and 0.95 mg/mL with PPB (pH 6.8), respectively. Then
their IR to the activity of HMGR was analyzed by HPLC. The IR
values of compounds a-d at 1 mg/mL were 6.28, 9.64, 23.53, and
10.56%, respectively. Their total IR was about 50.01% (Table3).
According to their ratio in hawthorn fruit extract, the mixture of
compounds a-d was matched with the concentration of 2.85 mg/
mL. The IR of the matched mixture was up to 79.5%, much
higher than that of the sum of four monomers.
Figure 1. Structures of compounds a-d: (a) quercetin; (b) hyperoside;
(c) rutin; (d) chlorogenic acid.
period. There were no significant differences in the body weights
among different groups during treatment, suggesting the mixture
or compounds a-d was safe and well tolerated in the mice.
The mice were fed a high-fat and -cholesterol (HFHC) diet for
2 months. Compared to the normal diet group, the levels of TC,
TG, and LDL-C increased (p < 0.01) and that of HDL-C
decreased (p < 0.05) in the hyperlipidemic group. Then, hyperli-
pidemic mice were treated with compounds a-d or matched
mixture from hawthorn fruit orally for 6 weeks or left untreated.
The levels of TC, TG, and LDL-C in therapy groups decreased in
different degrees and that of HDL-C increased in the blood of the
hyperlipidemic mice (Table 4). Among these monomers, querce-
tin (a) showed the highest lipid-lowering effect (p < 0.05). The IR
values of hyperoside, rutin, and chlorogenic acid decreased in
order, which was similar to the result of their inhibitory activity to
HMGR. In general, the lipid-lowering efficacy of compounds
a-d was not perfect; the content of TC, TG, and LDL-C showed
no difference from that of the hyperlipidemic control. However,
the mixture of compounds a-d could significantly reduce the
levels of TC, TG, and LDL-C by 46.5, 49.6, and 58.1%,
respectively (p < 0.01). This confirmed that there was a positive
synergetic effect on lowering lipid among these four compounds,
similar to that of HMGR.
3.7. Structure-Activity Relationship of Compounds a-c to
HMGR and Lipid-Lowering Efficacy. As seen in the structures of
compounds a-c in Figure 1, they contained a quercetin glycone.
Hyperoside (b) is a monoglycoside of compound a, linked with a
β-
glycoside of compound a, linked with a disaccharide of 6-O-
rhamnosyl- -glucose at the 3-OH of quercetin. In general, the
D-galatose on the 3-OH of quercetin. Rutin (c) is a disaccharide
L-
D
introduction of the glycosyl group increased the hydrophilic
ability of a compound. The greater the number of the glycosyl
group is, the stronger is the hydrophilic ability. From the data of
Table 5, the IR values of quercetin, hyperoside, and rutin to
HMGR were 39.6, 30.4, and 16.5%, respectively, which gradually
decreased with the increase of glycosyl group number. Similarly,
the efficacy of quercetin, hyperoside, and rutin in lowering TC,
TG, and LDL-C was decreased with the increase of glycosyl
group number (Table 4). Quercetin (a), without a glycosyl group,
showed the highest inhibitory activity and lipid-lowering effect.
This indicated that the activities of inhibition to HMGR and
lowering lipid were related to its hydrophilic ability. The weak
3.6. Effects of Four Monomers and Their Mixture on Lipid-
Lowering Efficacy in Mice. As mixture of compounds a-d
showed an improved IR to HMGR in vitro, the effects of four
monomers and their mixture on lipid-lowering efficacy in vivo
were investigated.
After a 6 week treatment, all mice showed good health status,
and no mortality was recorded during the whole experimental