Isolation, modification and cytotoxic evaluation of flavonoids from Rhododendron hainanense

Article abstract of DOI:10.1111/j.2042-7158.2012.01560.x

Objectives The aim of this study was to search for antitumour activity of flavonoid compounds. The cytotoxic activity of these compounds in vitro was evaluated against the human leukaemia (HL-60) and human hepatoma (SMMC-7721) cell lines. Methods Eight na

Full text of DOI:10.1111/j.2042-7158.2012.01560.x

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Research Paper
Journal of Pharmacy
And Pharmacology
Isolation, modification and cytotoxic evaluation of
flavonoids from Rhododendron hainanense
Jie Zhaob, Huan-Xing Dinga,b, Deng-Gao Zhaob, Chun-Ming Wangc and Kun Gaoa,b
aSchool of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, bState Key Laboratory of Applied
Organic Chemistry, College of Chemistry and Chemical Engineering and cSchool of Life Sciences, Lanzhou University, Lanzhou, China
Keywords
cytotoxicity; flavonoids; Rhododendron
Abstract
Objectives The aim of this study was to search for antitumour activity of flavonoid
compounds. The cytotoxic activity of these compounds in vitro was evaluated
against the human leukaemia (HL-60) and human hepatoma (SMMC-7721) cell
lines.
Methods Eight natural flavonoids (1–8) were isolated from the aerial parts of
Rhododendron hainanense and a series of modified flavonoid derivatives (9–18) were
obtained from the natural product matteucinol (1),using simple synthetic methods.
Antitumour inhibitory activity of these flavonoids was assessed using the sulfor-
hodamine B method.
Key findings Most of the compounds exhibited good pharmacological activity and
the preliminary structure–activity relationships were described. Within the series
of flavonoid derivatives in this study, compounds 3 (2,3-dihydro-5-hydroxy-7-
methoxy-2-(4-methoxyphenyl)-6,8-dimethyl-4H-1-benzopyran-4-one) and 16
(5-hydroxy-7, 4′-dimethoxy-6, 8-dimethylflavan) exhibited strong inhibitory activ-
ity against the HL-60 cell line with IC50 values (the drug concentration that resulted
in a 50% reduction in cell viability or inhibition of the biological activity) of 15.2 and
13.2 mm, respectively.
hainanense; structure–activity relationship
Correspondence
Kun Gao, School of Biotechnology and
Chemical Engineering, Ningbo Institute of
Technology, Zhejiang University, Ningbo
315000, China.
E-mail: npchem@lzu.edu.cn
Received November 24, 2011
Accepted May 13, 2012
doi: 10.1111/j.2042-7158.2012.01560.x
Conclusions Renewed attention to flavonoid derivatives revealed the possibility
that compounds 3 and 16 could be considered as lead compounds for the develop-
ment of new antitumour agents. Our results have not only enriched the family
of active flavonoids from natural sources, but have encouraged the synthesis of
flavonoid analogues for improving cytotoxic activity.
Introdution
Flavonoids are a large class of plant secondary metabolites
belonging to the wider family of natural polyphenols. They
occur naturally in fruit, vegetables, nuts, seeds, flowers and
bark, and are an integral part of the human diet. Recently,
interest in natural polyphenolic compounds has increased
steadily owing to their antioxidant capacity, and to their
ability to modulate a number of molecular pathways prima-
rily involved in the regulation of cell proliferation, cell func-
tion and inflammatory reactions. Considerable attention is
thus now concentrated on their possible beneficial implica-
tions on human health, such as in the prevention and treat-
ment of cancer, cardiovascular diseases, neurodegenerative
processes and other chronic diseases affecting the liver, gut,
kidney and lung.[1]
Rhododendron hainanense, belonging to the family Eri-
caceae, is distributed throughout southern regions of China.
Some species of the genus Rhododendron have been used as
medicinal plants. The dried leaves of R. dauricum have been
used medicinally as an expectorant and for treatment of
acute-chronic bronchitis.[2,3] Both flowers and fruits of
R. molle have been recorded in ancient and modern mono-
graphs as analgesics in traditional uses. The chemical com-
pound rhodojaponin, isolated from R. molle, has been shown
to have significant blood pressure lowering and heart rate
slowing effects.[4] The ethanolic extract of R. arboretum pos-
sessed a hypotensive effect and respiratory stimulant in cats
and dogs. It was reported also to produce contraction of the
guinea-pig ileum and have an effect on the central nervous
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1785–1792
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Flavonoids from R. hainanense
Jie Zhao et al.
system (CNS).[5] The methanolic extract of the leaves and
twigs of R. dauricum was found to display significant anti-
HIV activity.[6] The characteristic compounds of the genus
Rhododendron are flavonoids, coumarins, and tannins.
During our program of screening for antitumour activity of
flavonoid compounds from natural sources, the alcoholic
(EtOH) extract of the leaves and twigs of R. hainanense led to
the isolation of eight flavonoids (1–8). We found that the
content of matteucinol (1) in R. hainanense was high and
accounted for 0.166%, and had a weak cytotoxic activity in
vitro against the human leukaemia (HL-60) and human
hepatoma (SMMC-7721) cell lines. We therefore modified
the chemical structure of matteucinol in an attempt to
improve its activity. In this study, we report on the isolation,
modification and in-vitro cytotoxicity of a series of natural
and synthetic flavonoid derivatives, as well as the primary
structure-activity relationships.
They were identified by Guo-Liang Zhang, at the School of
Life Science, Lanzhou University China. A voucher specimen
(No. 08-08) has been deposited with the Natural Organic
Academy of Lanzhou University.
Aerial parts of R. hainanense (3 kg) were kept until air
dry. The dry materials were ground into a powder and
extracted three times with 80% EtOH at room temperature,
each for three days. After concentration of the combined
extracts under reduced pressure, the residue (289.9 g) was
suspended in H2O, then extracted with EtOAc and n-BuOH,
respectively. The EtOAc-soluble fraction (93 g) was sub-
jected to a silica gel column chromatography (eluted with
PE–EtOAc, from 40 : 1 to 1 : 1, then with MeOH) to give
nine fractions (fractions 1–9). Further purification of frac-
tion 3 by column chromatography (SiO2; PE–acetone,
15 : 1) yielded compound 3 (10 mg). Fraction 3-1 was sub-
jected to Sephadex LH-20 column chromatography to
afford compound 1 (5 g). Fraction 4 was purified in a
similar procedure (SiO2; PE–acetone, 3 : 1) to give pure
compounds 2 (30 mg), 4 (56 mg) and 6 (10 mg). Fraction
5-2 afforded compound 5 (50 mg) by Sephadex LH-20
column chromatography. Compound 7 (2 mg) was sepa-
rated from fraction 5-3. Fraction 6 was purified by column
chromatography on silica gel eluted with PE–acetone (1 : 1)
to give compound 8 (5 mg).
Materials and Methods
Materials
Acetic acid, anhydrous magnesium sulfate, sodium
cyanoborohydride (NaBH3CN), boron tribromide (BBr3),
anhydrous sodium carbonate, HCl (36~38%) and H2SO4
(95%~98%) were purchased from Lanzhou Aihua Chemical
Reagent Co., Ltd. (China). EtOH, MeOH and CH2Cl2 were
purchased from Tianjing Chemical Reagent Co., Ltd.
(China). All the reagents and chemicals were of analytical
grade. The HL-60 and SMMC-7721 cell lines used for the
bioassays were offered to us by Chun-ming Wang, at the
School of Life Sciences, Lanzhou University, Lanzhou, China.
RPMI-1640 medium was purchased from Gibco Co., Ltd.
(Grand Island, NY, USA). Fetal calf serum (FCS) was pur-
chased from Hangzhou Sijiqing Biological Engineering
Materials Co., Ltd. (China). Trichloroacetic acid (TCA), dim-
ethyl sulfoxide (DMSO), mitomycin and tris(hydroxy-
methyl) aminomethane were purchased from Sigma Aldrich
(St. Louis, MO, USA).
Synthesis
To determine the changes of bioactivity resulting from
different substituents of A, B ring and the type of C ring,
matteucinol (1) was modified to the target compounds
according to the method described in Figure 1. Matteucinol
(1) was oxidized to flavone 9 upon exposure to iodine in
DMSO at 100°C in a 95% yield.[7] The demethylation reaction
of flavone 9 was carried out using HBr in AcOH to obtain the
corresponding 5,7,4′-trihydroxyflavone 10.[8] Matteucinol
was derivatized according to Fujise’s mild acetylation to give
diacetate 12 as colourless needles.[9] Removal of the carbonyl
group was carried out by reduction of 12 with 2 equiv NaBH4
in tetrahydrofuran (THF) and H2O at 0°C for 30 min to give a
90% yield of the monoacetate 17.[10]
Matteucinol with 1.5 equiv (CH3)2SO4/K2CO3 in acetone
afforded 7-O-methylmatteucinol (3), acetylation of com-
pound 3 using Fujise’s conditions gave 13,removal of the car-
bonyl group was carried out by reduction of 13 with 2 equiv
NaBH4 in THF and H2O at 30 min to give an 85% yield of
16, flavan 18 was prepared by deprotection of the methyl
ether using BBr3/CH2Cl2 protocol.[11] Matteucinol with
10 equiv (CH3)2SO4 was refluxed overnight to generate 5,7-
O-dimethylmatteucinol (11). According to the literature,
compound 11 with sodium borohydride in EtOH furnished
the desired compound 14, demethylation of 14 with boron
tribromide furnished compound 15.[12]
General procedures
Column chromatography: silica gel (200–300 mesh;
Qingdao Marine Chemical Factory, China). TLC: silica gel
GF254 plates (10–40 lm; Qingdao Marine Chemical Factory,
1
China). H and 13C NMR spectra: Varian Mercury-300BB
(300 MHz) spectrometer, in CDCl3 and DMSO-d6; d in ppm
rel. to TMS, J in Hz. EI-MS: VG ZAB-HS instrument,
at 70 eV, in m/z. HRESIMS: Bruker APEX-II mass spectro-
meter; in m/z.
Collection and extraction of plant material
Aerial parts of R. hainanense were collected from Jianfeng
town of Hainan Province, China, in the summer of 2008.
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Jie Zhao et al.
Flavonoids from R. hainanense
OMe
b
OMe
a
OH
HO
O
HO
O
O
HO
O
O
OH
O
OH
OH
10
9
Matteucinol
O
OMe
OMe
O
O
O
O
O
O
c
d
Matteucinol
O
O
OH
17
12
OMe
OMe
MeO
OMe
MeO
O
MeO
O
O
h
g
e
f
Matteucinol
OH
3
O
O
O
13
OH
16
O
OH
HO
O
OH
18
OMe
OH
OMe
MeO
O
HO
O
MeO
O
j
k
i
Matteucinol
OMe O
11
OMe OH
OMe OH
15
14
Figure 1 Matteucinol (compound 1) extracted from Rhododendron hainanense was modified to the target compounds according to various methods.
Reagent and conditions: (a) I2, DMSO, H2SO4, 100°C, 95%; (b) HOAC, HBr, reflux, 50%; (c) Ac2O, H2SO4, 0°C, 95%; (d) NaBH4, THF/H2O, 0°C, 90%;
(e) (CH3)2SO4, acetone, K2CO3, reflux, 98%; (f) Ac2O, H2SO4, 0°C, 82%; (g) NaBH4, THF/H2O, 0°C, 85%; (h) BBr3, CH2Cl2, -78°C to room temperature (rt),
60%; (i) (CH3)2SO4, acetone, K2CO3, reflux, 80%; (j) NaBH4, EtOH, rt, 80%; (k) BBr3, CH2Cl2, -78°C to rt, 50%.
1H and 13C NMR spectral data of
compounds 1–18
9.58 (s, 1H, OH), 7.33 (d, J = 7.3 Hz, 2H, Ar-2′-H, Ar-6′-H),
6.80 (d, J = 8.3 Hz, 2H, Ar-3′-H, Ar-5′-H), 5.41 (dd, J = 12.4,
2.4 Hz, 1H, 2-CH), 3.20 (dd, J = 17.0, 12.5 Hz, 1H, 3-CH),
2.75 (dd, J = 17.0, 2.4 Hz, 1H, 3-CH), 1.95 (s, 6H, 6-CH3,
8-CH3); ESI-MS (m/z) 299.1 ([M-H]-).
Matteucinol (1) mp 168–170°C. 1H NMR (DMSO-d6,
300 MHz): d (ppm): 12.37 (s, 1H, 5-OH), 9.67 (s, 1H, OH),
7.44 (d, J = 8.6 Hz, 2H, Ar-2′-H, Ar-6′H), 6.98 (d, J = 8.8 Hz,
2H, Ar-3′-H, Ar-5′-H), 5.48 (dd, J = 12.0, 3.0 Hz, 1H, 2-CH),
3.77 (s, 3H, 4′-CH3), 3.20 (dd, J = 16.7, 12.5 Hz, 1H, 3-CH),
2.78 (dd, J = 16.7, 3.0 Hz, 1H, 3-CH), 1.95 (s, 6H, CH3);
ESI-MS (m/z) 313.1 ([M-H]-).
2,3-Dihydro-5-hydroxy-7-methoxy-2-(4-
methoxyphenyl)-6,8-dimethyl-4H-1-benzopyran-4-one (3)
1H NMR (DMSO-d6, 300 MHz): d (ppm):12.37 (s, 1H,
5-OH), 9.67 (s, 1H, OH), 7.44 (d, J = 8.6 Hz, 2H, Ar-2′-H,
Ar-6′-H),6.98 (d,J = 8.8 Hz,2H,Ar-3′-H,Ar-5′-H),5.48 (dd,
J = 12.0, 3.0 Hz, 1H, 2-CH), 3.77 (s, 3H, 4′-CH3), 3.62 (s, 3H,
7-CH3), 3.20 (dd, J = 16.7, 12.0 Hz, 1H, 3-CH), 2.78 (dd,
Farrerol (2) mp 228–231°C. 1H NMR (DMSO-d6,
300 MHz): d (ppm): 12.39 (s, 1H, 5-OH), 9.67 (s, 1H, OH),
© 2012 The Authors. JPP © 2012
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Jie Zhao et al.
J = 16.7, 3.0 Hz, 1H, 3-CH), 1.95 (s, 6H, CH3). ESI-MS (m/z)
327.1 ([M-H]-).
Syzalterin (10) yellow needles (MeOH); C17H14O5; 13C
NMR (DMSO- d6, 75 MHz): d (ppm): 182.0 (C-4), 163.2
(C-7), 160.9 (C-4′), 159.7 (C-2), 155.9 (C-5), 152.3 (C-9),
128.2 (C-2′/C-6′), 121.5 (C-1′),115.9 (C-3′/C-5′), 106.9
(C-10), 103.4 (C-6), 102.4 (C-3), 101.8 (C-8), 8.2 (CH3-6),
7.9 (CH3-8). ESI-MS (m/z) 297.0 ([M-H]-).
4′-Methylnaringenin (4) 1H NMR (DMSO-d6, 300 MHz):
d (ppm): 12.39 (s, 1H, 5-OH), 9.67 (s, 1H, OH), 9.58 (s, 1H,
OH), 7.33 (d, J = 7.3 Hz, 2H, Ar-2′-H, Ar-6′-H), 6.95 (d,
J = 8.3 Hz, 2H,Ar-3′-H, Ar-5′-H), 5.98 (d, J = 2.5 Hz, 2H,
6-CH, 8-CH), 5.41 (dd, J = 12.4, 2.4 Hz, 1H, 2-CH), 3.77 (s,
3H, 4′-OCH3), 3.20 (dd, J = 17.0, 12.4 Hz, 1H, 3-CH), 2.75
(dd, J = 17.0, 2.4 Hz, 1H, 3-CH). ESI-MS (m/z) 285.1
([M-H]-).
2,3-Dihydro-5,7-dimethoxy-2-(4-methoxyphenyl)-6,8-
1
dimethyl-4H-1-benzopytan-4-one (11) H NMR (DMSO-
d6,300 MHz):d(ppm):12.37(s,1H,5-OH),9.67(s,1H,OH),
7.44 (d, J = 8.6 Hz, 2H,Ar-2′-H,Ar-6′-H), 6.98 (d, J = 8.8 Hz,
2H, Ar-3′-H, Ar-5′-H), 5.48 (dd, J = 12.0, 3.0 Hz, 1H, 2-CH),
3.82 (s, 3H, 5-CH3), 3.80 (s, 3H, 7-CH3), 3.77 (s, 3H, 4′-CH3),
3.20 (dd, J = 16.7, 12.0 Hz, 1H, 3-CH), 2.78 (dd, J = 16.7,
3.0 Hz, 1H, 3-CH), 1.95 (s, 6H, CH3); 13C NMR (DMSO-d6,
125 MHz): d(ppm): 196.6 (C-4), 160.8 (C-7), 159.8 (C-4′),
159.3 (C-5), 157.8 (C-9), 131.0 (C-1′), 127.5 (C-2′/C-6′),
114.1 (C-3′/C-5′), 102.9 (C-10), 102.0 (C-6 and C-8), 78.5
(C-2), 55.4 (OMe-5, OMe-7 and OMe-4′), 43.3 (C-3), 8.9
(CH3-6), 7.6 (CH3-8). ESI-MS (m/z) 341.1 ([M-H]-).
Matteucinol diacetate (12) mp 177–178°C. 1H NMR
(CDCl3, 300 MHz): d(ppm): 7.35–7.41 (m, 2H, Ar-2′-H,
Ar-6′-H), 6.93–6.99 (m, 2H, Ar-3′-H, Ar-5′-H), 5.42 (d,
J = 13.6 Hz, 1H), 3.83 (s, 3 H, OMe), 3.03 (dd, J = 16.6,
13.7 Hz,1 H),2.75 (dd,J = 16.6,2.3 Hz,1H),2.42 (s,3H,Me),
2.03 (s, 3H, Me), 1.95 (s, 3 H, Me);13C NMR (CDCl3,
75 MHz): d(ppm):190.4 (C-4), 169.4 (C-OAc), 167.9
(C-OAc), 159.9 (C-7), 159.1 (C-4′), 153.6 (C-5), 146.5 (C-9),
130.6 (C-1′), 127.5 (C-2′/C-6′), 118.0 (C-3′/C-5′), 117.8
(C-10), 114.2 (C-6), 111.6 (C-8), 79.0 (C-2), 55.4 (OMe-4′),
45.2 (C-3), 21.0 (CH3-OAc), 20.4 (CH3-OAc), 9.6 (CH3-6),
9.2 (CH3-8). ESI-MS (m/z) 397.1 ([M-H]-).
5-Acetoxy-2,3-dihydro-7-dimethoxy-2-(4-methoxyphe-
nyl)-6,8-dimethyl-4H-1-benzopytan-4-one (13) 1H NMR
(CDCl3, 300 MHz): d (ppm): 7.35–7.41 (m, 2 H, Ar-2′-H,
Ar-6′-H), 6.93–6.99 (m, 2 H, Ar-3′-H, Ar-5′-H), 5.42 (d,
J = 13.6 Hz,1 H),3.83 (s,3 H,OMe),3.80 (s,3H,7-CH3),3.03
(dd, J = 16.6, 13.7 Hz, 1 H), 2.75 (dd, J = 16.6, 2.3 Hz, 1 H),
2.42 (s, 3 H, Me), 2.03 (s, 3 H, Me), 1.95 (s, 3 H, Me); 13C NMR
(CDCl3, 75 MHz): d(ppm): 190.4 (C-4), 167.9 (C-OAc),
159.9 (C-7), 159.1 (C-4′), 153.6 (C-5), 146.5 (C-9), 130.6
(C-1′), 127.5 (C-2′/C-6′), 118.0 (C-3′/C-5′), 117.8 (C-10),
114.2 (C-6), 111.6 (C-8), 79.0 (C-2), 55.4 (OMe-4′), 45.2
(C-3), 20.4 (CH3-OAc), 9.6 (CH3-6), 9.2 (CH3-8). ESI-MS
(m/z) 369.1 ([M-H]-).
5, 7, 4′-Trimethoxy-6, 8-dimethylflavan-4-ol (14) 1H NMR
(CDCl3,300 MHz): d (ppm): 7.38 (d,2H,J = 8.7 Hz,Ar-2′-H,
Ar-6′-H), 6.93 (d, 2H, J = 8.7 Hz, Ar-3′-H, Ar-5′-H), 5.25 (t,
1H,J = 8.5 Hz,H-4),4.97 (dd,1H,J = 12.0,1.7 Hz,H-2),3.87
(s, 3H, OMe-5), 3.82 (s, 3H, OMe-7), 3.74 (s, 3H, OMe-4′),
2.48 (ddd, 1H, J = 13.5, 8.5, 1.7 Hz, H-3a), 2.25 (ddd, 1H,
J = 13.5, 12.0, 8.5 Hz, H-3b), 1.95 (s, 6H, 6-CH3, 8-CH3); 13C
NMR (CDCl3, 75 MHz): d(ppm): 160.6 (C-7), 159.6 (C-4′),
159.3 (C-5), 156.7 (C-9), 132.4 (C-1′), 127.7 (C-2′ and C-6′),
Naringenin (5) 1H NMR (DMSO-d6, 300 MHz): d (ppm):
12.39 (s, 1H, 5-OH), 9.67 (s, 1H, OH), 9.58 (s, 1H, OH), 7.33
(d, J = 7.3 Hz, 2H, Ar-2′-H, Ar-6′-H), 6.95 (d, J = 8.3 Hz, 2H,
Ar-3′-H,Ar-5′-H), 5.98 (d, J = 2.5 Hz, 2H, 6-CH, 8-CH), 5.41
(dd, J = 12.4, 2.4 Hz, 1H, 2-CH), 3.20 (dd, J = 17.0, 12.4 Hz,
1H, 3-CH), 2.75 (dd, J = 17.0, 2.4 Hz, 1H, 3-CH). ESI-MS
(m/z) 271.0 ([M-H]-).
(2S)-2,3-Dihydro-5,7-dihydroxy-2-(4-methoxyphenyl)-
8-methyl-4H-1-benzopyran-4-one (6) 1H NMR (DMSO-d6,
300 MHz): d (ppm): 12.37 (s, 1H, 5-OH),9.67 (s, 1H, OH),
7.37 (d, J = 8.6 Hz, 2H,Ar-2′-H,Ar-6′-H), 6.94 (d, J = 8.8 Hz,
2H, Ar-3′-H, Ar-5′-H), 5.97 (s, 8-CH), 5.48 (dd, J = 12.0,
3.0 Hz, 1H, 2-CH), 3.83 (s, 3H, 4′-OCH3), 3.20 (dd, J = 16.7,
12.0 Hz, 1H, 3-CH), 2.78 (dd, J = 16.7, 3.0 Hz, 1H, 3-CH),
17
2.05 (s, 8-CH3). ESI-MS (m/z) 299.1 ([M-H]-). [α]D : -21.0°
(c 0.41, acetone).
(2S)-2,3-Dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-8-
1
methyl-4H-1-benzopyran-4-one (7) H NMR (DMSO-d6,
300 MHz): d (ppm): 12.37 (s, 1H, 5-OH), 9.67 (s, 1H, OH),
7.37 (d, J = 8.6 Hz, 2H,Ar-2′-H,Ar-6′-H), 6.94 (d, J = 8.8 Hz,
2H, Ar-3′-H, Ar-5′-H), 5.97 (s, 8-CH), 5.48 (dd, J = 12.0,
3.0 Hz, 1H, 2-CH), 3.20 (dd, J = 16.7, 12.0 Hz, 1H, 3-CH),
2.78 (dd,J = 16.7,3.0 Hz,1H,3-CH),2.05 (s,8-CH3).ESI-MS
17
(m/z) 285.0 ([M-H]-). [α]D : -22.3° (c 0.41, acetone).
(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-
1
chromene-3,5,7-triol (8) H NMR (CDCl3, 400 MHz): d
(ppm):7.05 (d,1H,J = 1.8 Hz,Ar-6′-H),6.83 (dd,1H,J = 7.8,
1.8 Hz, Ar-2′-H), 6.78 (d, 1H, J = 7.8 Hz, Ar-5′-H), 6.02 (d,
1H,J = 1.8 Hz,H-6),5.92 (d,1H,J = 1.8 Hz,H-8),4.78 (s,1H,
H-2), 4.20 (s, 1H, H-3), 2.89 (dd, 1H, J = 4.8, 17.1 Hz, H-4a),
2.72 (dd, 1H, J = 3.0, 17.1 Hz, H-4b). ESI-MS (m/z) 289.0
([M-H]-). [α]2D2: +14° (c 0.41, acetone).
5,7-Dihydroxy-2-(4-methoxyphenyl)-6,8-dimethyl-4H-
1-benzopyran-4-one (9) 1H NMR (DMSO- d6, 300 MHz): d
(ppm): 12.37 (s, 1H, 5-OH), 9.67 (s, 1H, OH), 7.85 (d,
J = 8.6 Hz, 2H, Ar-2′-H, Ar-6′-H), 7.02 (d, J = 8.8 Hz, 2H,
Ar-3′-H, Ar-5′-H), 6.58 (s, 3-CH), 3.89 (s, 3H, 4′-CH3), 1.95
(s, 6H, CH3); 13C NMR (DMSO- d6, 75 MHz): d (ppm): 182.0
(C-4), 163.2 (C-7), 160.9 (C-4′), 159.7 (C-2), 155.9 (C-5),
152.3 (C-9), 128.2 (C-2′/C-6′), 121.5 (C-1′), 115.9 (C-3′/C-
5′), 106.9 (C-10), 103.4 (C-6), 102.4 (C-3), 101.8 (C-8),
55.1(CH3), 8.2 (CH3-6), 7.9 (CH3-8). ESI-MS (m/z) 311.1
([M-H]-).
© 2012 The Authors. JPP © 2012
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Jie Zhao et al.
Flavonoids from R. hainanense
114.0 (C-3′and C-5′), 107.2 (C-10), 116.0 (C-8), 115.4 (C-6),
76.9 (C-2), 63.5 (C-4), 55.6, 55.3 (OMe-5 and OMe-7), 55.4
(C-4′), 37.6 (C-3). ESI-MS (m/z) 343.1 ([M-H]-).
supplemented with FCS (10%), penicillin (100 U/ml) and
streptomycin (100 mg/ml), being referred to as complete
medium. The cells were grown in a CO2 incubator (Hera Cell,
Heraeus, Germany) at 37°C with 90% humidity and 5% CO2.
All experiments were performed with cells in the logarithmic
growth phase.
7, 4′-Dihydroxy-5-methoxy-6, 8-dimethylflavan-4-ol (15)
1H NMR (CDCl3,300 MHz):d (ppm):7.38 (d,2H,J = 8.7 Hz,
Ar-2′-H,Ar-6′-H), 6.93 (d, 2H, J = 8.7 Hz,Ar-3′-H,Ar-5′-H),
5.25 (t, 1H, J = 8.5 Hz, H-4), 4.97 (dd, 1H, J = 12.0, 1.7 Hz,
H-2), 3.91 (s, 1H, OH), 3.87 (s, 3H, OMe-5), 2.48 (ddd, 1H,
J = 13.5, 8.5, 1.7 Hz, H-3a), 2.25 (ddd, 1H, J = 13.5, 12.0,
8.5 Hz, H-3b), 1.95(s, 6H, 6-CH3, 8-CH3); 13C NMR (CDCl3,
75 MHz): d(ppm): 160.6 (C-7), 159.6 (C-4′), 159.3 (C-5),
156.7 (C-9), 132.4 (C-1′), 127.7 (C-2′and C-6′), 116.0 (C-8),
115.4 (C-6), 114.0 (C-3′and C-5′), 107.2 (C-10), 76.9 (C-2),
63.5 (C-4), 55.6 (OMe-5), 37.6 (C-3). ESI-MS (m/z) 315.1
([M-H]-).
Cytotoxicity assay
The cytotoxicity of compounds 1–18, toward HL-60 and
SMMC-7721 cell lines was determined in 96-well microtitre
plates by the sulforhodamine B method. Briefly, exponen-
tially growing HL-60 and SMMC-7721 cells were harvested
and seeded in 96-well plates with the final volume 100 ml
containing 4 ¥ 103 cells/well.After 24-h incubation,cells were
treated with various concentrations of 1–18 for 48 h. The
cultures were fixed at 4°C for 1 h by addition of ice-cold 50%
TCA to give a final concentration of 10%. Fixed cells were
rinsed five times with de-ionized H2O and stained for 10 min
with 0.4% sulforhodamine B dissolved in 0.1% HOAc. The
wells were washed five times with 0.1% HOAc and left to dry
overnight. The absorbed sulforhodamine B was dissolved in
150 ml unbuffered 1% Tris base (tris(hydroxymethyl) ami-
nomethane) solution in H2O (pH 10.5). The absorbance of
extracted sulforhodamine B at 515 nm was measured on a
microplate reader (Bio-Rad). The experiments were carried
out in triplicate. Each run entailed five or six concentrations
of the compounds being tested. The percentage survival rates
of cells exposed to the compounds were calculated by assum-
ing the survival rate of untreated cells to be 100%.
1
5-Hydroxy-7, 4′-dimethoxy-6, 8-dimethylflavan (16) H
NMR (CDCl3, 300 MHz): d (ppm): 7.41 (d, 2H, J = 8.6 Hz,
Ar-2′-H,Ar-6′-H), 7.09 (d, 2H, J = 8.6 Hz,Ar-3′-H,Ar-5′-H),
4.98 (dd, 1H, J = 10.3, 2.4 Hz, H-2), 3.71 (s, 3H, OCH3), 2.6-
2.7 (m, 2H), 2.2-2.3 (m,1H), 1.9-2.1 (m, 1H). 1.95 (s, 6H,
6-CH3, 8-CH3); 13C NMR (CDCl3, 75 MHz): d(ppm): 159.1
(C-9), 156.4 (C-7), 154.4 (C-4′), 150.1 (C-5), 139.0 (C-1′),
127.1 (C-2′and C-6′), 121.5 (C-3′and C-5′), 107.5 (C-6),
106.9 (C-8), 101.8 (C-10), 77.1 (C-2), 55.3 (OMe-5, 4′), 29.5
(C-4), 21.2 (C-3), 8.9 (CH3-6), 8.8 (CH3-6). ESI-MS (m/z)
313.1 ([M-H]-).
7-Acetoxy-5-hydroxy-4′-methoxy-6,
8-dimethylflavan
1
(17) H NMR (CDCl3, 300 MHz): d (ppm): 7.31 (d, 2H,
J = 8.6 Hz, Ar-2′-H, Ar-6′-H), 6.93 (d, 2H, J = 8.6 Hz,
Ar-3′-H, Ar-5′-H), 4.98 (dd, 1H, J = 10.3, 2.4 Hz, H-2), 3.71
(s, 3H, OCH3), 2.6-2.7 (m 2H), 2.29 (s, 3H, OAc), 2.2-2.3 (m,
1H), 1.9-2.1 (m, 1H), 1.95 (s, 6H, 6-CH3, 8-CH3). 13C NMR
(CDCl3,75 MHz): d(ppm):169.4 (C-OAc),159.1 (C-9),156.4
(C-7), 154.4 (C-4′), 150.1 (C-5), 134.0 (C-1′), 127.1 (C-2′and
C-6′), 113.8 (C-3′and C-5′), 110.4 (C-10), 107.5 (C-6), 106.9
(C-8),77.1 (C-2),55.3(OMe-5,4′),29.5(C-4),21.2(C-3),8.9
(CH3-6), 8.8 (CH3-6). ESI-MS (m/z) 341.1 ([M-H]-).
Statistical analysis
All experiments were carried out three times unless men-
tioned in the procedure. Continuous variables were pre-
sented as mean Ϯ SD. The IC50 was determined as the drug
concentration that resulted in a 50% reduction in cell viabil-
ity or inhibition of the biological activity. IC50 values were
calculated using a four parameter logistic curve (SigmaPlot
11.0). All data were statistically evaluated using Student’s
t-test or the Kruskal–Wallis test (GraphPad Prism 5.01;
GraphPad Software, Inc., San Diego, CA, USA), followed by
Dunn’s post-hoc multiple comparison test when the signifi-
cance value was < 0.05, using the same significance level. The
criterion for statistical significance was generally taken as
P < 0.05.
5, 7, 4′-Trihydroxy-6, 8-dimethylflavan-4-ol (18) 1H NMR
(CDCl3,300 MHz): d (ppm): 7.32 (d,2H,J = 8.6 Hz,Ar-2′-H,
Ar-6′-H),6.93 (d,2H,J = 8.6 Hz,Ar-3′-H,Ar-5′-H),4.98 (dd,
1H, J = 10.3, 2.4 Hz, H-2), 2.6-2.7 (m, 2H), 1.9-2.1 (m, 1H),
2.2-2.3 (m,1H), 1.95 (s, 6H, 6-CH3, 8-CH3); 13C NMR
(CDCl3, 75 MHz): d (ppm): 159.1 (C-9), 156.4 (C-7), 154.4
(C-4′), 150.1 (C-5), 134.0 (C-1′), 127.1 (C-2′and C-6′), 113.8
(C-3′and C-5′), 110.4 (C-10), 107.5 (C-6), 106.9 (C-8), 77.1
(C-2), 29.5 (C-4), 21.2 (C-3), 8.9 (CH3-6), 8.8 (CH3-6).
ESI-MS (m/z) 285.1 ([M-H]-).
Results
The alcoholic extract of the leaves and twigs of R. hainanense
led to the isolation of eight flavonoids (1–8), matteucinol
(1), farrerol (2), 2,3-dihydro-5-hydroxy-7-methoxy-2-(4-
methoxyphenyl)-6,8-dimethyl-4H-1-benzopyran-4-one (3),
4′-methylnaringenin (4), naringenin (5), (2S)-2,3-dihydro-
Cell culture and treatment
Acute promyelocytic leukaemia (HL-60) cell lines and
human hepatoma (SMMC-7721) were grown and main-
tained in RPMI-1640 medium, pH 7.4. The media was
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1785–1792
1789

Flavonoids from R. hainanense
Jie Zhao et al.
3'
2'
1'
4'
5'
1
OH
OH
OMe
OMe
OMe
8
HO
HO
HO
O
MeO
HO
O
2
HO
HO
O
B
7
A
C
3
6'
6
4
5
OH
OH
OH
O
O
OH
OH
O
O
O
3
2
5
1
OMe
OMe
O
O
O
O
O
OH
O
O
OH
6
4
OH
OH
HO
HO
OH
OH
O
O
O
OH
7
O
OH
8
9
O
O
OMe
OMe
OMe
OH
MeO
MeO
O
HO
O
O
O
OMe O
O
OH
10
12
O
11
OMe
OH
OH
MeO
O
HO
O
O
O
OMe OH
OMe OH
O
14
13
15
O
O
OMe
OMe
O
MeO
O
HO
O
OH
17
OH
OH
16
18
Figure 2 The structures of flavonoids 1–18 extracted from the leaves and twigs of Rhododendron hainanense.
5,7-dihydroxy-2-(4-methoxyphenyl)-8-methyl-4H-1-
benzopyran-4-one (6), (2S)-2,3-dihydro-5,7-dihydroxy-2-
(4-hydroxyphenyl)-8-methyl-4H-1-benzopyran-4-one (7)
described in Figure 2. The compounds were assayed for their
inhibitory activity against HL-60 and SMMC-7721 cell lines,
and the results are presented in Table 1. Mitomycin was used
as the positive control in this assay. Compounds 3 and 16
exhibited potent inhibitory activity against HL-60 cells with
lower IC50 values of 15.2 and 13.2 mm, respectively. Com-
pound 8 showed moderate inhibitory activity against HL-60
and SMMC-7721 cells with IC50 values of 61.7 and 78.9 mm,
respectively. Compounds 9, 11, 14 and 15 showed weak
and
(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-
chromene-3,5,7-triol (8).[13–19] To determine the changes of
bioactivity resulting from different substituents of A, B ring
and the type of C ring, matteucinol (1) was modified to the
target compounds according to the methods described
in Figure 1. The structures of all compounds have been
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1785–1792
1790

Jie Zhao et al.
Flavonoids from R. hainanense
Table 1 Cytotoxicity of compounds 1–18 extracted from the leaves and
twigs of Rhododendron hainanense
favourable influence of lipophilicity on cytotoxicity (the
methoxy group at 7 and to a lesser extent at 4′ made com-
pounds more lipophilic; the same applied for an OH at 5
because this OH formed a hydrogen bond with the carbonyl
group, making the structure more lipophilic).When examin-
ing the influence of C-methyl group, compounds 1 and 2,
with the C-methyl group at the 6, 8-position, were more
potent than compounds 6 and 7 with a C-methyl group at the
8-position. Therefore, the existence of the C-methyl substi-
tutents at C-6 and C-8 was also important for antitumour
activity. The carbonyl group on the C ring may not affect the
activity because compound 11 showed slightly higher activity
than compound 14 with a hydroxyl group. These results con-
firmed that methylation of 7, 4′-hydroxyl groups together
with the presence of the C-methyl group at the 6, 8-position
seemed to be the most favourable structural condition for
antitumour activity.
IC50 (mM)
Flavonoids
HL-60
SMMC-7721
Mitomycina
1.67 Ϯ 0.58
160.1 Ϯ 3.8
169.3 Ϯ 3.3
15.2 Ϯ 2.4
186.7 Ϯ 13.3
217.6 Ϯ 4.0
205.0 Ϯ 9.6
NTb
5.53 Ϯ 1.58
131.6 Ϯ 1.2
227.4 Ϯ 27.0
83.5 Ϯ 22.5
304.1 Ϯ 0.3
661.0 Ϯ 16.1
214.0 Ϯ 10.0
NTb
1
2
3
4
5
6
7
8
9
61.7 Ϯ 1.7
93.8 Ϯ 5.1
NDc
78.9 Ϯ 2.3
132.9 Ϯ 25.3
10
11
12
13
14
15
16
17
18
89.4 Ϯ 26.0
NDc
90.0 Ϯ 11.9
135.0 Ϯ 3.7
89.2 Ϯ 16.8
84.4 Ϯ 3.4
13.2 Ϯ 0.9
NDc
247.0 Ϯ 18.3
149.3 Ϯ 17.1
198.8 Ϯ 4.5
103.7 Ϯ 3.1
Conclusions
Flavonoids are an important class of natural compounds with
significant potential to cure, treat and prevent tumour, senes-
cence and cardiovascular diseases. Flavanones have been a
potential source in the search for lead compounds and bio-
logically active components,and have been the focus of much
research and development in the last 30 years.[20] We have iso-
lated and modified the flavonoids from R. hainanense and
evaluated their cytotoxicity. Within the series of flavonoid
derivatives in this study, we found that compounds 3 and 16
were the most active. Compound 3 is a natural product iso-
lated from R. hainanense and to our knowledge, little antitu-
mour activity data has been reported for it. Compound 3
could also be synthesized from matteucinol using a simple
synthetic method. Compound 16 was a new modified com-
pound from the natural product matteucinol. This renewed
attention to flavonoid derivatives revealed the possibility that
compounds 3 and 16 could be considered as lead compounds
for the development of new antitumour agents. Our results
not only enrich the family of active flavonoids from natural
sources, but encourage the synthesis of flavonoid analogues
for improving cytotoxic activity.
264.6 Ϯ 4.2
445.5 Ϯ 25.3
aPositive control. bNot Tested. cND: The IC50 value was not detected
because of poor sample solubility. IC50 value: the drug concentration
that resulted in a 50% reduction in cell viability or inhibition of the bio-
logical activity. The data shown are means Ϯ SD obtained from three
independent experiments.
cytotoxic activity against HL-60 and SMMC-7721 cells, with
IC50 values in the range of 100–200 mm. The IC50 values of
compounds 10, 12 and 17 were not detected because of the
poor sample solubility.
Discussion
The compounds were assayed for their inhibitory activity
against HL-60 and SMMC-7721 cell lines (see Table 1). Com-
pounds 3 and 16 exhibited the strongest inhibitory activity
against HL-60 with lower IC50 values of 15.2 and 13.2 mm,
respectively. Comparison of the activity of compounds 1/2,
4/5, 6/7 and 16/17 suggested that the presence of a methoxy
group instead of the hydroxyl group at C4′ of the B ring
increased the antitumour activity. The loss of this methoxy
substituent reduced the antitumour potency with respect to
the parent compound 3. The fact that the cytotoxicity of fla-
vonoids was enhanced by methoxyl substitution on the B ring
and reduced by hydroxyl substitution led us to believe that
the influence of the substituents was not due to their elec-
tronic properties. In addition, compounds 3 and 11, with
C7-methoxy of the A ring, showed more activity compared
with matteucinol (1). From the compounds of the same skel-
eton, compound 3 was more active than compounds 11 and
13, indicating that the phenolic hydroxyl group at C-5 was
required for the activity. The most striking evidence was the
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
This work was funded by the National Natural Science
Foundation of China (No. 21172196). The bioactivity part
was supported by the Fundamental Research Funds for the
Central Universities (No. lzujbky-2011-37).
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1785–1792
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Jie Zhao et al.
naturally occurring flavanones.
J
simsii. Phytochemistry 2001; 56: 875–
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