Anti-inflammatory and antitumor phenylpropanoid sucrosides from the seeds of Raphanus sativus

Article abstract of DOI:10.1016/j.bmcl.2014.11.001

A bioassay-guided fractionation and chemical investigation of the MeOH extract of Raphanus sativus seeds resulted in the isolation and identification of eight phenylpropanoid sucrosides (1-8) including two new compounds, named raphasativuside A and B (1-2) from the most active CHCl₃-soluble fraction. The structures of these new compounds were elucidated through spectral analysis, including extensive 2D-NMR data, and chemical reaction experiments. We evaluated the anti-inflammatory effects of 1-8 in lipopolysaccharide (LPS)-stimulated murine microglia BV2 cells. Compounds 2 and 5 exhibited significant inhibitory effect on nitric oxide production in LPS-activated BV-2 cells with IC₅₀ values of 21.63 and 26.96 μM, respectively. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 1-7 showed consistent cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC₅₀ values of 6.71-27.92 μM. Additionally, the free-radical scavenging activity of 1-8 was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay where compounds 1, 3, and 4 scavenged DPPH radical strongly with IC₅₀ values of 23.05, 27.10, and 29.63 μg/mL, respectively.

Full text of DOI:10.1016/j.bmcl.2014.11.001

Accepted Manuscript
Anti-inflammatory and antitumor phenylpropanoid sucrosides from the seeds
of Raphanus sativus
Ki Hyun Kim, Chung Sub Kim, Yong Joo Park, Eunjung Moon, Sang Un Choi,
Jei Hyun Lee, Sun Yeou Kim, Kang Ro Lee
PII:
S0960-894X(14)01172-X
http://dx.doi.org/10.1016/j.bmcl.2014.11.001
BMCL 22156
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
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13 October 2014
1 November 2014
Please cite this article as: Kim, K.H., Kim, C.S., Park, Y.J., Moon, E., Choi, S.U., Lee, J.H., Kim, S.Y., Lee, K.R.,
Anti-inflammatory and antitumor phenylpropanoid sucrosides from the seeds of Raphanus sativus, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.001
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Anti-inflammatory and antitumor phenylpropanoid sucrosides from
the seeds of Raphanus sativus
Ki Hyun Kim a, Chung Sub Kim a, Yong Joo Park a, Eunjung Moon b, Sang Un Choi c,
Jei Hyun Lee d, Sun Yeou Kim b, Kang Ro Lee a,*
aNatural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon
440-746, Korea
bCollege of Pharmacy, Gachon University, Incheon, 406-799, Korea
cKorea Research Institute of Chemical Technology, Deajeon 305-600, Korea
dCollege of Oriental Medicine, Dongguk University, Gyeong Ju 780-714, Republic of
Korea
Correspondence to:
Prof. Dr. KANG RO LEE
Natural Products Laboratory, School of Pharmacy,
Sungkyunkwan University, 300 Chonchon-dong,
Jangan-ku, Suwon 440-746, Korea.
TEL: 82-31-290-7710; FAX: 82-31-290-7730; E-mail: krlee@skku.edu

Abstract
A bioassay-guided fractionation and chemical investigation of the MeOH extract of
Raphanus sativus seeds resulted in the isolation and identification of eight
phenylpropanoid sucrosides (1-8) including two new compounds, named
raphasativuside A and B (1-2) from the most active CHCl3-soluble fraction. The
structures of these new compounds were elucidated through spectral analysis, including
extensive 2D-NMR data, and chemical reaction experiments. We evaluated the anti-
inflammatory effects of 1-8 in lipopolysaccharide (LPS)-stimulated murine microglia
BV2 cells. Compounds 2 and 5 exhibited significant inhibitory effect on nitric oxide
production in LPS-activated BV-2 cells with IC50 values of 21.63 and 26.96 µM,
respectively. All isolates were also evaluated for their antiproliferative activities against
four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds
1–7 showed consistent cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15
cell lines with IC50 values of 6.71–27.92 µM. Additionally, the free-radical scavenging
activity of 1-8 was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay
where compounds 1, 3, and 4 scavenged DPPH radical strongly with IC50 values of
23.05, 27.10, and 29.63 µg/mL, respectively.

Keywords: Raphanus sativus; Brassicaceae; Sucrose derivatives; Anti-inflammation;
Cytotoxicity; Antioxidant activity

Raphanus sativus L. (Brassicaceae), commonly known as radish, is widely consumed as
a vegetable or condiment in human diets throughout the world. Radish has been used in
traditional Chinese herbal medicine for more than 1400 years, since being recorded in
‘Tang Materia Medica’, the first Chinese pharmacopoeia.1 Different parts of the radish,
including the roots, seeds, and leaves, have been known to show various medicinal
properties.2 In particular, the seeds, well-known as Raphani Semen, have long been
used in Korean traditional medicine as a carminative, diuretic, expectorant, laxative, and
contain stomachic, anti-cancer, and anti-inflammatory agents.3-5 Preliminary results
showed that the MeOH extract of R. sativus seeds exhibited significant cytotoxic and
anti-inflammatory effects, which led to the isolation and identification of the active 4-
methylthio-butanyl derivatives that correlated with the cytotoxic and anti-inflammatory
activities in our recent study.6. In the process of our continuing efforts to study this
source, we further isolated eight phenylpropanoid sucrosides (1-8) including two new
compounds, named raphasativuside A and B (1-2) (Fig. 1) from the most active CHCl3-
soluble fraction using a bioassay-guided fractionation method, and evaluated the
antitumor and anti-inflammatory activities of all isolates (1-8).
Compound 1 was isolated as a light yellowish gum. The molecular formula of 1 was
determined to be C44H50O22 by positive mode HR-ESI-MS data at m/z 953.2690 [M +

Na]+ (calcd for C44H50O22Na, 953.2691). The UV and IR spectra displayed absorption
bands associated with hydroxyl and α,β-unsaturated carbonyl ester groups, and aromatic
1
ring functionalities. The H NMR spectrum of 1 (Table 1) showed characteristic signals
attributable to three pairs of olefinic protons [δH 7.71 (d, J = 16.0 Hz, H-7'')/6.46 (d, J =
16.0 Hz, H-8''), 7.48 (d, J = 16.0 Hz, H-7''')/6.26 (d, J = 16.0 Hz, H-8'''), and 7.57 (d, J =
16.0 Hz, H-7'''')/6.48 (d, J = 16.0 Hz, H-8'''')], and a benzyl moiety with an ABX spin
system [δH 7.21 (d, J = 1.5 Hz, H-2''), 7.09 (dd, J = 8.5, 1.5 Hz, H-6''), and 6.77 (d, J =
8.5 Hz, H-5'')], as well as two 1,3,4,5-tetrasubstituted aromatic rings [δH 6.77 (s, H-2'''
and H-6''') and 6.82 (s, H-2'''' and H-6'''')]. Furthermore, the large coupling constants
(16.0 Hz) of the three pairs of olefinic protons suggested the trans-form of the α,β-
unsaturated carbonyl systems in 1.7,8 The C NMR spectrum of 1 (Table 1) exhibited a
13
total of 44 carbons including 27 sp2 carbons between δC 167.8-105.5, whose C NMR
13
chemical shifts were in good agreement with those of two trans-sinapoyl and one trans-
1
feruloyl moieties.7,8 The H and 13C NMR spectrum 1 (Table 1) displayed a
characteristic α-anomeric resonance at δH 5.56 with a small coupling constant (d, J = 4.0
Hz, H-1') due to a gauche conformation, according to the Karplus relation,9 together
with 12 oxygenated carbon signals between δC 103.9-62.6. These date implied that
compound 1 has a disaccharide moiety composed of a pentose and a hexose unit. On the

1
basis of chemical evidence and detailed analysis of H-1H COSY, HMQC, and HMBC
experiments (Fig. 2), as well as comparison of spectroscopic data with authentic
samples,7,8,10 the sugar moiety was confirmed to be sucrose, which were comprised of β-
→1 linkage
D-fructose and α-D-glucose units connected through a 2
(Supplementary
data). According to these results, we concluded that compound 1 is a phenylpropanoid
sucroside acylated by of two trans-sinapoyl and one trans-feruloyl moieties. Upon
inspection of the HMBC specteum, long range correlations were observed between H-3
(δH 5.38)/C-9'' (δC 166.5) and H-4 (δH 5.69)/C-9''' (δC 166.6) in the fructofuranosyl
moiety and between H-6' (δH 4.73 and 4.22)/C-9'''' (δC 167.8) in the glucopyanosyl
moiety (Fig. 2). From these data, the positions of the acyl residues were determined as
shown in Fig. 1. Thus, the structure of 1 was elucidated as (3-O-feruloyl-4-O-sinapoyl)-
→1)
β-D-fructofuranosyl-(2
-(6-O-sinapoyl)-α-D-glucopyranoside and named as
raphasativuside A.
Compound 2 was isolated as a light yellowish gum. The molecular formula was
established as C45H52O23 from the [M + Na]+ peak at m/z 983.2793 (calcd for
C45H52O23Na, 983.2797) in the HR-ESIMS. The UV and IR spectra of 2 were very
1
similar to those of 1. Likewise, the H and 13C NMR data of 2 (Table 1) were quite
similar to those of 1, with a noticeable difference being the chemical shifts assignable to

three trans-sinapoyl moieties rather than two trans-sinapoyl and one trans-feruloyl
moieties in 1.7,8 Moreover, the carbon signals in the β-D-fructose were different, relative
to those of 1, suggesting a change in the linkage position of the acyl residues. Detailed
analysis of the HMBC spectrum of 2 confirmed the presence of two trans-sinapoyl
moieties at C-4 and C-6 on the fructofuranosyl unit and one trans-sinapoyl moiety at C-
6' on the glucopyanosyl unit. Threfore, the structure of 2 was assigned as (3,6-O-
→1)
disinapoyl)-β-D-fructofuranosyl-(2
-(6-O-sinapoyl)-α-D-glucopyranoside
and
named as raphasativuside B.
The six known compounds were identified as (3,4-O-disinapoyl)-β-D-fructofuranosyl-
7
→1)
→1)
(2
(2
-(6-O-sinapoyl)-α-D-glucopyranoside (3), (3-O-sinapoyl)-β-D-fructofuranosyl-
7
10
-(6-O-sinapoyl)-α-D-glucopyranoside (4), tenuifoliside C (5), (3-O-feruloyl)-
8
→1)
β-D-fructofuranosyl-(2
-(6-O-feruloyl)-α-D-glucopyranoside (6), sibiricose A6
(7),11 and sucrose (8),10 by comparison of their spectroscopic data with reported data.
Previously, some studies reported the anti-inflammatory activities of phenylpropanoids
from various plant extracts.12-14 Particularly, among phenylpropanoid glycosides
isolated from Juniperus rigida, citrusin D and sachaliside I induced approximately 50%
inhibition of NO production at the concentration of 100 µM in LPS-stimulated
RAW264.7 macrophage cells.12 Chang et al. also reported the inhibitory effects of

phenylpropanoid sucrosides from Calamus quiquesetinervius on NO production in LPS-
stimulated murine RAW264.7 cell line.15 However, till now the research about the effect
of phenylpropanoid sucrosides from R. sativus on inflammation had not been reported
yet. In this study, to evaluate the anti-inflammatory activities of the isolated compounds
1–8, we treated them in lipopolysaccharide (LPS)-activated murine microglia BV-2 cells,
and then tested for their ability to inhibit the production of the proinflammatory
mediator, nitric oxide (NO) for the first time. As shown in Table 2, all compounds,
except for 8, inhibited NO production (IC50 values < 50 µM). Among them, compounds
2 and 5 significantly inhibited NO levels with IC50 values of 21.63 and 26.96 µM,
respectively, without affecting viability (Supplementary data). At this point, the active
compounds 1-7 did not show the big difference in their ability to inhibit the NO
production (21.63 – 48.92 µM) although they have three different acyl residues at C-3,
C-4 (or C-6), and C-6′ regions. According to the study of Chang et al., the IC50 values of
phenylpropanoid sucrosides which contain trans-coumaroyl moiety at C-4′ region, were
significantly lower than trans-feruloyl moiety at same region.15 However, the fact that
the C-4′ position is significant in mediating the anti-inflammatory activity was not
identified in default of discovery of any compounds with acyl residue at C-4′ position in
this study. On the basis of the result that the sucrose (8) was inactive, it wss deduced

that the presence of acyl residues on the sucrose core in these types of molecules is
essential for exerting the anti-inflammatory effect. Structure–activity relationship study
applying the related diverse derivatives might be needed to elucidate the key moiety in
phenylpropanoid sucrosides for inhibitory activity on NO production.
Next, the cytotoxic activities of the isolates (1–8) were evaluated by determining their
inhibitory effects on human tumor cell lines including A549 (non-small cell lung
carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and
HCT-15 (colon adenocarcinoma) using the SRB bioassay.16 Some phenylpropanoid
sucrosides have been also reported to possess anti-tumor activity in recent studies.17-20
However, no reports are available on the cytotoxic activities of the phenylpropanoid
sucrosides (1–7) isolated from R. sativus in this study against A549, SK-OV-3, SK-
MEL-2, and HCT-15 cells. The results (Table 3) showed that phenylpropanoid
sucrosides 1-7 had consistent cytotoxicity against all of the cell lines tested, with IC50
values ranging from 6.71–27.92 µM. The other compound, sucrose (8) was inactive
(IC50 > 30.0 µM). Recently, it was reported that substitution on phenyl ring of
phenylpropanoid sucrosides did not influence the cytotoxic activity.18,19 This hypothesis
was concluded by the variable activity data for the compounds containing various
phenolic and acetyl groups on the phenyl ring.18,19 This was also identified in this study

where compound 1 and its analog compound 3 showed similar cytotoxicity against the
tested cell lines and compounds 4-6 with different acyl residues at the same position had
similar potency. However, the stronger cytotoxic properties of 1-7 compared to 8 clearly
suggested that acyl residues on the sucrose core in phenylpropanoid sucrosides are
essential for enhancing the cytotoxicity. Interestingly, among the tested compounds,
compound 2 had significantly better cytotoxicity against SK-OV-3 and SK-MEL-2 cells
(IC50 values of 6.71 and 9.50 µM, respectively) than other tested compounds. The fact
that only compound 2 has the acyl residue at C-6 position encouraged us to suggest that
the acyl group attached to C-6 position of the sucrose core plays a positive role in
mediating the cytotoxicity against SK-OV-3 and SK-MEL-2 cells.
On the other hand, it was also reported that phenylpropanoid sucrosides showed
significantly free radical-scavenging activities.15 Thus compounds 1-8 were tested for
radical-scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.21
Compounds 1-7 displayed free-radical scavenging activity (antioxidant activity) in the
DPPH assay, and compounds 1, 3, and 4 had better potencies (IC50 < 30 µg/mL) than
other active compounds. The IC50 values of 1-7 were found to be 23.05, 44,59, 27,10,
29,63, 54,98, 35,33, and 78.20 µg/mL, respectively, compared to 7.63 µg/mL for
ascorbic acid, a well-known standard antioxidant. However, sucrose (8) was inactive.

The free-radical scavenging activity of the tested compounds may be possibly due to a
hydrogen-donating ability (to DPPH) provided by the hydroxy groups of acyl moieties
in the molecule.
In conclusion, this is the first study investigating the anti-inflammatory, anti-tumor, and
antioxidant activities of phenylpropanoid sucrosides isolated from R. sativus. In this
study, compound 2 showed both the most significant anti-inflammatory properties in
murine BV-2 microglia cells, and the highest cytotoxicity against the human tumor cell
lines, SK-OV-3, SK-MEL-2 and HCT-15. Therefore, there is a possibility that it could
be applicable in developing treatments for activated microglia-mediated brain diseases
and tumors. The biological test results also indicated that group of phenylpropanoid
sucrosides might be potentially valuable source for new potent drug candidates.
Acknowledgements
This work was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (2012R1A5A2A28671860).
Supplementary data

Supplementary data associated with this article can be found, in the online version, at
doi:

References and notes
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4. Duke, J. A.; Ayensu, E. S. Medicinal Plants of China; Reference Publ., Inc.:
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5. Chopra, R. N.; Nayar, S. L.; Chopra, I. C. Glossary of Indian Medicinal Plants
(Including the Supplement); Council of Scientific and Industrial Research: New Delhi,
1986.
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Ethnopharmacol. 2014, 151, 503.
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2600.

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Med. Chem. 2012, 27, 875.
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14. Kim, K. H.; Kim, S.; Jung, M. Y.; Ham, I. H.; Whang, W. K. Arch. Pharm. Res.
2009, 32, 7.
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Z. M. A. Eur. J. Med. Chem. 2012, 58, 418.
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Table 1 1H (500 MHz) and 13C NMR (125 MHz) spectral data of 1–2 in CD3OD. (δ in
ppm)a
1
2
Position
δH
δC
δH
δC
63.8
1
3.68 d (12.0)
3.64 d (12.0)
63.8
3.69 d (12.0)
3.65 d (12.0)
2
3
4
5
6
103.9
75.5
75.0
81.4
62.6
103.9
78.2
74.0
80.0
64.7
5.83 d (7.5)
5.69 t (7.5)
4.17 m
5.48 d (7.5)
4.52 t (7.5)
4.20 m
3.98 m
3.92 dd (13.0, 5.0)
5.56 d (4.0)
3.53 dd (10.0, 4.0)
3.73 m
3.32 m
4.54 m
4.52 m
5.51 d (4.0)
3.52 dd (10.0, 4.0)
3.76 m
3.32 m
1′
2′
3′
4′
5′
6′
91.5
71.6
73.5
70.7
71.4
64.6
91.5
71.5
73.6
70.5
71.3
64.6
4.35 dd (11.0, 9.5)
4.73 br d (11.0)
4.22 m
4.32 dd (11.0, 9.5)
4.72 br d (11.0)
4.23 m
1′′ X-3 or X′-3
2′′
3′′
4′′
5′′
6′′
7′′
8′′
126.3
110.6
147.9
149.3
115.0
122.9
146.7
113.3
166.5
55.5
125.3
105.8
147.9
138.4
147.9
105.8
146.7
113.4
166.6
55.5
7.21 d (1.5)
6.89 s
6.77 d (8.5)
7.09 dd (8.5, 1.5)
7.71 d (16.0)
6.46 d (16.0)
6.89 s
7.71 d (16.0)
6.47 d (16.0)
9′′
OCH3
3.87 s
6.77 s
3.87 s
6.77 s
1′′′ X′-4 or X′-6
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
8′′′
124.9
105.5
147.9
138.1
147.9
105.5
146.8
113.2
166.6
55.5
125.3
105.5
147.9
138.2
147.9
105.5
146.5
114.6
168.0
55.5
6.77 s
7.48 d (16.0)
6.26 d (16.0)
6.77 s
7.66 d (16.0)
6.58 d (16.0)
9′′′
OCH3
1′′′′ X′-6′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
7′′′′
8′′′′
3.78 s
6.82 s
3.83 s
6.85 s
125.2
105.6
147.8
138.3
147.8
105.6
145.7
114.6
167.8
55.5
125.2
105.6
148.0
138.3
148.0
105.6
145.8
114.5
167.8
55.5
6.82 s
7.57 d (16.0)
6.48 d (16.0)
6.85 s
7.57 d (16.0)
6.49 d (16.0)
9′′′′
OCH3
3.85 s
3.87 s

aAssignments were based on 2D NMR including HMQC and HMBC. Well-resolved
couplings are expressed with coupling patterns and coupling constants in Hz in
parentheses.

Table 2 Inhibitory effect on NO production of compounds 1-8 in LPS-activated BV-2
cells.
Compound
IC50 (µM)a
Compound
IC50 (µM)a
34.80
21.63
48.92
37.15
26.96
45.50
37.40
>200
18.30
1
2
3
4
5
6
7
8
NMMAb
aIC50 value of each compound was defined as the concentration (µM) that caused 50%
inhibition of NO production in LPS-activated BV-2 cells.
bNMMA as a positive control.

Table 3 Cytotoxicity of compounds 1-8 against four cultured human tumor cell lines
using the SRB bioassay in vitro.
Compound
IC50 (µM)a
SK-OV-3
A549
26.94
20.82
25.87
21.05
26.00
>30.0
23.95
>30.0
0.002
SK-MEL-2
16.70
9.50
HCT-15
18.85
12.37
14.06
17.20
18.30
17.75
26.99
>30.0
0.129
23.79
6.71
1
2
19.25
16.69
24.64
20.03
20.50
>30.0
0.012
18.55
14.15
21.55
27.92
23.88
>30.0
0.001
3
4
5
6
7
8
Doxorubicinb
a
IC50 value of compounds against each tumor cell line, which was defined as the
concentration (µM) that caused 50% inhibition of cell growth in vitro.
b Doxorubicin served as a positive control.

Legends
Figure 1. Chemical structures of 1-8.
1
Figure 2. Key H- H COSY (bold) and HMBCs (
1
→) of
1.

OR4
6'
4'
5'
O
2'
HO
HO
OH
O
1'
1
2
3'
OR2
OH
5
O
3
4
6
OR3
OR1
O
7''
2''
5''
1''
OCH3
X =
9''
3''
8''
R1 R2 R3 R4
6''
4''
OH
1
2
3
4
5
6
7
8
X
X'
H
H
X'
O
X'
X' X'
OCH3
X' =
X' X'
H
H
H
H
H
H
X'
X'
X'
X
OH
X'
X''
X
H
H
H
H
H
OCH3
OCH3
O
X'
H
H
H
X'' =
OCH3
OCH3
Figure 1. Chemical structures of 1-8.

O
H3CO
OH
OCH3
H3CO
HO
O
O
HO
HO
OH
OCH3
OH
O
O
O
O
HO
OH
O
H3CO
O
1
Figure 2. Key H- H COSY (bold) and HMBCs (
1
→) of
1.

Anti-inflammatory and antitumor phenylpropanoid sucrosides from
the seeds of Raphanus sativus
Ki Hyun Kim, Chung Sub Kim, Eunjung Moon, Sang Un Choi, Jei Hyun Lee, Sun
Yeou Kim, Kang Ro Lee*
O
H3CO
OH
OCH3
H3CO
HO
O
O
HO
HO
OH
OCH3
OH
O
O
O
O
HO
OH
O
H3CO
O
A bioassay-guided fractionation and chemical investigation of the MeOH extract of
Raphanus sativus seeds resulted in the isolation and identification of eight
phenylpropanoid sucrosides (1-8) including two new compounds, named
raphasativuside A and B (1-2) from the most active CHCl3-soluble fraction.