Hypocholesterolemic activity of hesperetin derivatives

Article abstract of DOI:10.1016/S0960-894X(03)00574-2

Hesperetin ester and ether derivatives possessing a long alkyl chain were synthesized for examining their hypocholesterolemic activities in high cholesterol-fed mice. Hesperetin 7-O-lauryl ether (4b) and hesperetin 7-O-oleyl ether (4e) exhibited strong cholesterol-lowering effects.

Full text of DOI:10.1016/S0960-894X(03)00574-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2663–2665
Hypocholesterolemic Activity of Hesperetin Derivatives
Tae-Sook Jeong,a Eun Eai Kim,b Chul-Ho Lee,a Jung-Hoon Oh,a Surk-Sik Moon,c
Woo Song Lee,a Goo-Taeg Oh,a Sangku Leea,* and Song-Hae Boka,b
aKorea Research Institute of Bioscience and Biotechnology, 52 Oun, Yusong, Taejon 305-333, Republic of Korea
bBionutrigen Company, Ltd., 52 Oun, Taejon 305-333, Republic of Korea
cDepartment of Chemistry, Kongju National University, Kongju 314-701, Republic of Korea
Received 24 March 2003; accepted 29 May 2003
Abstract—Hesperetin ester and ether derivatives possessing a long alkyl chain were synthesized for examining their hypocholestero-
lemic activities in high cholesterol-fed mice. Hesperetin 7-O-lauryl ether (4b) and hesperetin 7-O-oleyl ether (4e) exhibited strong
cholesterol-lowering effects.
# 2003 Elsevier Ltd. All rights reserved.
Flavonoids represent an important class of natural pro-
ducts found in fruits, vegetables, tea, and wine. The
daily human intake of flavonoids was estimated to be
about 2–3 g/day with a high dietary intake of herbs.1
Flavonoids have shown a broad range of biological
activities, including antiallergic,2 antibacterial,3 anti-
inflammatory,4 antimutagenic,5 antioxidant,6 and anti-
cancer7 effects. In the course of screening several flavo-
noids for the development of a hypocholesterolemic
agent, hesperetin (1), aglycon of hesperidin, was found
to show a significant cholesterol-lowering ability in
cholesterol fed mice.8 Our efforts toward the develop-
ment of a potent hypocholesterolemic agent have
focused on examining structure–activity relationship.
We envisioned that introduction of a lipophilic group
into hesperetin (1) may lead to a chance for exploring a
new compound with increased activities. Herein, we
describe the synthesis and biological evaluation of
hesperetin derivatives possessing a long alkyl chain
linked to the 7-hydroxyl position of hesperetin.
with hesperetin 30-O-ester compounds 3 as minors. On
the other hand, ether derivatives of hesperetin were pre-
pared by alkylation with alkyl bromide in N,N-dimethyl-
formamide utilizing sodium carbonate as a base.
Hesperetin 7-O-ether compounds 4 were yielded pre-
dominantly. The alkyl halides were formed by mesyla-
tion of the corresponding alcohol with methanesulfonyl
chloride, followed by replacement of the mesyl group
with sodium bromide.9
Several analogues of hesperetin were prepared for
studying the relationship between the structure and the
hypocholesterolemic activity, particularly the impor-
tance of the length of the lipophilic chain linked to the
7-hydroxy position of hesperetin. The synthesized com-
pounds were tested for their cholesterol lowering activ-
ities in high cholesterol-fed C59BL/6J mice. The plasma
total cholesterol levels were measured after feeding a
high cholesterol diet supplemented with 0.05% (wt/wt
in diet) of the test compounds for 10 days (Table 1).10
All synthesized compounds except 2a and 4a, showed
the increased cholesterol-lowering effects in comparison
with hesperetin (1). The introduction of a lipophilic
chain at the 7-hydroxyl position of hesperetin strongly
affected the cholesterol lowering activity, and C-12 was
the best length for the activity. The hesperetin deriva-
tives possessing a short alkyl chain, 2a and 4a, showed a
weak activity as compared to those possessing a long
alkyl chain. In general, ether derivatives 4 of hesperetin
(1) showed more potent activities than ester derivatives
2. Interestingly, it was found that the presence of unsa-
turation in a lipophilic chain enhanced significantly the
Ester or ether derivatives of hesperetin differing in
length of the lipophilic chain connected to the 7-
hydroxyl position of hesperetin (1) were synthesized.
Ester derivatives were prepared by acylation of hesperetin
with appropriate acid chlorides in the presence of tri-
ethylamine as shown in Scheme 1. Hesperetin 7-O-ester
derivatives 2 were obtained as major products together
*Corresponding author. Tel.: +82-42-860-4552; fax: +82-42-861-
2675; e-mail: sangku@kribb.re.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00574-2

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T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2663–2665
Scheme 1. Reagents and reaction conditions: (i) R1COCl, Et3N, 0 ꢁC; (ii) R2Br, Na2CO3, DMF.
References and Notes
Table 1. Effects of hesperetin derivatives on plasma total cholesterols
in high cholesterol-fed mice
1. Hertog, M. G. L.; Hollman, P. C. H.; Venema, D. P. J.
Agric. Food Chem. 1992, 40, 1591.
2. Matsuo, N.; Yamada, K.; Yamashita, K.; Shoji, K.; Mori,
M.; Sugano, M. In Vitro Cell Dev. Biol. 1996, 32, 340.
3. Hamiltonmiller, J. M. T. Antimicrob. Agent Chemother.
1995, 39, 2375.
4. Shivji, G. M.; Zielinska, E.; Kondo, S.; Mukhtar, H.; Sander,
D. N. J. Invest. Dermatol. 1996, 106, 787.
5. Yamada, J.; Tomita, Y. Biosci. Biotech. Biochem. 1994, 58,
2197.
6. Miura, S.; Watanabe, J.; Sano, M.; Tomita, T.; Osawa, T.;
Hara, Y.; Tomita, I. Biol. Pharm. Bull. 1995, 18, 1.
7. Han, C. Cancer Lett. 1997, 114, 153.
8. (a) Hesperetin (1) was reported to show cholesterol-low-
ering effects in rats fed a high cholesterol diet: Lee, S. H.;
Park, Y. B.; Kwon, Y. K.; Choi, M. S.; Bok, S. H.; Jeong, T. S.
Nutr. Res. 1999, 19, 1245. (b) Kim, H. K.; Jeong, T. S.; Lee,
M. K.; Park, Y. B.; Choi, M. S. Clin. Chimi. Acta 2003, 327,
129.
Group
N
Body weight (g)a
0 day 10 day
Total cholesterol (mg/dl)b
0 day
10 day
Control 10 24.8ꢀ1.7 24.9ꢀ1.5
98ꢀ10
103ꢀ7
96ꢀ11
95ꢀ4
223ꢀ29
216ꢀ13
220ꢀ10
192ꢀ15
209ꢀ11
214ꢀ14
196ꢀ10
218ꢀ15
189ꢀ16
202ꢀ15
208ꢀ20
192ꢀ18
1
6
6
6
6
6
6
6
6
6
6
6
25.4ꢀ1.5 25.6ꢀ2.3
23.4ꢀ0.6 23.7ꢀ0.5
22.4ꢀ2.4 22.4ꢀ3.4
21.7ꢀ1.5 23.4ꢀ1.2
22.3ꢀ0.3 23.7ꢀ1.2
22.3ꢀ0.3 23.8ꢀ0.6
21.9ꢀ1.0 22.5ꢀ2.9
22.0ꢀ2.1 22.4ꢀ2.6
22.5ꢀ0.7 23.0ꢀ1.5
23.1ꢀ1.0 24.4ꢀ1.0
23.4ꢀ0.6 23.7ꢀ0.5
2a
2b
2c
2d
2e
4a
4b
4c
4d
4e
97ꢀ11
95ꢀ6
96ꢀ7
102ꢀ5
95ꢀ5
93ꢀ8
101ꢀ4
96ꢀ11
aAll values are expressed as meanꢀSD.
bMeanꢀSD; all values are significantly different (p<0.05) from con-
trol group.
9. All newly synthesized compounds gave satisfactory spectral
data.
10. The hypocholesterolemic effects of the synthesized com-
pounds were investigated in male C57BL/6J mice maintained
at Korea Research Institute of Bioscience and Biotechnology
(Taejon, Korea). The mice were housed in a room with con-
trolled temperature (22ꢀ2 ꢁC), relative humidity (55ꢀ5%),
and lighting (alternating 12 h cycle of light and dark). At 8
weeks of age, six animals were randomly assigned to a group,
and fed a high cholesterol diet (CRF-1 supplemented with
15% fat, 1.25% cholesterol, and 0.5% Na-cholate, Oriental
Yeast Co. Ltd., Japan) without additional supplement (con-
trol), or a high cholesterol diet supplemented with 0.05% (wt/
wt in diet) of the test compounds (experimental group). The
diet and water were given ad libitum. After treating the test
compounds for 10 days, the mice were anesthetized with ethyl
ether, and the blood was obtained from the retro-orbital sinus
using a heparinized capillary tube. Then, the blood was cen-
trifuged at 8000g for 10 min, and the plasma was collected.
The concentration of plasma total cholesterol was measured
with an automatic blood chemical analyzer (CIBA Corning,
OH, USA). To evaluate statistical significance between control
and experimental groups, Student’s t-test was performed, and
activity as shown in compounds 2e and 4e (2d vs 2e, 4d
vs 4e). Among the tested compounds, hesperetin 7-O-
ether analogues having lauryl and oleyl moieties (4b11
and 4e12) exhibited strong cholesterol-lowering activ-
ities.
In conclusion, hesperetin 7-O-ester and hesperetin 7-O-
ether derivatives possessing a long alkyl chain were
synthesized and their hypocholesterolemic activities
were evaluated. The lipophilic group linked at the 7-
hydroxyl group of hesperetin influenced the activities
and the length of C-12 showed the most potent activ-
ities. Further detailed evaluations of hesperetin 7-O-
lauryl ether (4b) and hesperetin 7-O-oleyl ether (4e)
showing strong hypocholesterolemic activities are in
progress.
a p value of 0.05 was considered to be statistically significant.
1
Acknowledgements
11. 4b: H NMR(400 MHz, CDCl ) d 12.01 (s, 1H), 7.04 (d,
3
J=2.0 Hz, 1H), 6.93 (dd, J=8.4, 2.0 Hz, 1H), 6.88 (d, J=8.0
Hz, 1H), 6.05 (d, J=2.0 Hz, 1H), 6.03 (d, J=2.8 Hz, 1H), 5.32
(dd, J=12.8, 2.8 Hz, 1H), 3.95 (t, J=6.4 Hz, 2H), 3.91 (s, 3H),
This work was supported by grants from the Ministry of
Science and Technology (M1-0015-00-0012), Korea.

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2665
3.07 (dd, J=17.2, 2.8 Hz, 1H), 2.77 (dd, J=17.2, 2.8 Hz, 1H),
1.75 (m, 2H), 1.26 (m, 18H), 0.88 (t, J=6.4 Hz, 3H); 13C
Hz, 1H), 6.03 (d, J=2.2 Hz, 1H), 6.01 (d, J=2.2 Hz, 1H), 5.70
(s, 1H), 5.32 (m, 3H), 3.93 (t, J=6.6 Hz, 2H), 3.90 (s, 3H),
3.05 (dd, J=17.1, 3.0 Hz, 1H), 2.76 (dd, J=17.1, 3.0 Hz, 1H),
1.98 (m, 3H), 1.72 (m, 2H), 1.27 (m, 23H), 0.86 (t, J=6.8 Hz,
3H); 13C NMR(100 MHz, CDCl 3) d 195.8, 167.6, 164.0, 162.8,
147.0, 145.9, 131.6, 130.0, 129.8, 118.1, 112.6, 110.6, 103.0,
95.5, 94.6, 78.9, 68.5, 56.0, 43.2, 31.9, 29.8, 29.7, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 28.8, 27.2, 27.1, 25.8, 22.6, 14.1.
NMR(100 MHz, CDCl ) d 196.1, 167.8, 164.3, 163.0, 147.2,
3
146.1, 131.8, 118.4, 112.9, 110.8, 103.2, 95.8, 94.8, 79.1, 68.8,
56.3, 43.4, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.1, 26.1,
22.9, 14.4.
1
12. 4e: H NMR(400 MHz, CDCl ) d 12.00 (s, 1H), 7.02 (d,
3
J=2.0 Hz, 1H), 6.91 (dd, J=8.3, 2.0 Hz, 1H), 6.89 (d, J=8.3