Anti-osteoporotic and antioxidant activities of chemical constituents of the aerial parts of Ducrosia ismaelis

Article abstract of DOI:10.1016/j.bmcl.2014.05.077

A new pterocarpan glycoside, glycinol-3-O-β-d-glucopyranoside (1), and a new dihydrochalcone glycoside, ismaeloside A (2), were isolated together with 13 known compounds, including several flavonoids (3-8), lignans (9-11), and phenolic compounds (12-15), from the methanol extract of the aerial parts of Ducrosia ismaelis. The chemical structures of these compounds were elucidated from spectroscopic data and by comparison of these data with previously published results. The anti-osteoporotic and antioxidant activities of the isolated compounds were assessed using tartrate-resistant acid phosphatase (TRAP), oxygen radical absorbance capacity (ORAC), and reducing capacity assays. Compound 15 exhibited a dose-dependent inhibition of osteoclastic TRAP activity with a TRAP value of 86.05 ± 6.55% of the control at a concentration of 10 μM. Compounds 1, 3-5, and 8 showed potent peroxyl radical-scavenging capacities with ORAC values of 22.79 ± 0.90, 25.57 ± 0.49, 20.41 ± 0.63, 26.55 ± 0.42, and 24.83 ± 0.12 μM Trolox equivalents (TE) at 10 μM, respectively. Only compound 9 was able to significantly reduce Cu(I) with 23.44 μM TE at a concentration of 10 μM. All of the aforementioned compounds were isolated for the first time from a Ducrosia species.

Full text of DOI:10.1016/j.bmcl.2014.05.077

Accepted Manuscript
Anti-osteoporotic and antioxidant activities of chemical constituents of the aer-
ial parts of Ducrosia ismaelis
Abubaker M. Morgan, Hyun Woo Lee, Sang-Hyun Lee, Chi-Hwan Lim, Hae-
Dong Jang, Young Ho Kim
PII:
S0960-894X(14)00586-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.077
BMCL 21691
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 February 2014
29 April 2014
22 May 2014
Please cite this article as: Morgan, A.M., Lee, H.W., Lee, S-H., Lim, C-H., Jang, H-D., Kim, Y.H., Anti-osteoporotic
and antioxidant activities of chemical constituents of the aerial parts of Ducrosia ismaelis, Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.077
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-osteoporotic and antioxidant activities of chemical constituents of
the aerial parts of Ducrosia ismaelis
Abubaker M. Morgan a,b, Hyun Woo Lee a, Sang-Hyun Lee c, Chi-Hwan Lim b, Hae-Dong Jang c,*
Young Ho Kima,*
a College of Pharmacy, Chungnam National University, Deajeon 305-764, Republic of Korea.
b College of Agriculture and Life Sciences, Chungnam National University, Deajeon 305-764, Republic of
Korea.
c Department of Food and Nutrition, Hannam University, Daejeon 305-811, Republic of Korea.
* To whom correspondence should be addressed. Tel.: +82 42 821 5933; fax: +82 42 823 6566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
College of Pharmacy, Chungnam National University, Deajeon 305-764, Republic of Korea.
E-mail address: haedong@hnu.kr (H.-D. Jang)
Department of Food and Nutrition, Hannam University, Daejeon 305-811, Republic of Korea.

Abstract
A new pterocarpan glycoside, glycinol-3-O-β- -glucopyranoside ( ), and a new dihydrochalcone
1
D
glycoside, ismaeloside A ( ), were isolated together with 13 known compounds, including
2
several flavonoids (
), lignans (
), and phenolic compounds (
), from the methanol
12-15
3–8
9-11
extract of the aerial parts of Ducrosia ismaelis. The chemical structures of these compounds
were elucidated from spectroscopic data and by comparison of these data with previously
published results. The anti-osteoporotic and antioxidant activities of the isolated compounds
were assessed using tartrate-resistant acid phosphatase (TRAP), oxygen radical absorbance
capacity (ORAC), and reducing capacity assays. Compound
exhibited a dose-dependent
15
inhibition of osteoclastic TRAP activity with a TRAP value of 86.05 6.55% of the control at a
concentration of 10 µM. Compounds ,
1 3-5
, and showed potent peroxyl radical-scavenging
8
capacities with ORAC values of 22.79 0.90, 25.57 0.49, 20.41 0.63, 26.55 0.42, and
24.83 0.12 µM Trolox equivalents (TE) at 10 µM, respectively. Only compound was able to
9
significantly reduce Cu (I) with 23.44 µM TE at a concentration of 10 µM. All of the
aforementioned compounds were isolated for the first time from a Ducrosia species.
Keywords: Ducrosia ismaelis , Apiaceae , Antiosteoporosis, antioxidant, TRAP, ORAC.

1
2
Osteoporosis is a chronic, progressive disease of the skeleton characterized by bone fragility due
to a reduction in bone mass and possible alterations in bone architecture which lead to a
propensity for fractures with minimum trauma.1 Bone formation is related to osteoblastic
proliferation, alkaline phosphatase (ALP) activity, and osteocalcin and collage synthesis. Bone
resorption is associated with osteoclast formation and differentiation, and tartrate-resistant acid
phosphatase activity (TRAP).2 Oxidative stress is caused by an imbalance between the
generation of reactive oxygen species (ROS) and the activity of antioxidant defence. Severe
oxidative stress has been implicated in many chronic and degenerative diseases, including
osteoporosis, cancer, ageing, and neurodegenerative diseases such as Alzheimer’s disease,
Parkinson’s disease and amyotrophic lateral sclerosis.3 Some natural flavonoids with potent
antioxidant activity including scopoletin, reveratrol, and baicalein have found to exert anti-
osteoporotic activities through suppressing osteoclast formation and TRAP4-6. Accordingly,
antioxidants have become a topic of increasing interest.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Ducrosia species belong to the Apiaceae family, the members of which typically contain
unusual and/or unique phytochemicals, especially coumarins.7 In traditional medicine, various
Ducrosia species are used as analgesics, pain relievers and cold treatments.8 Antimicrobial,
antimycobacterial, antifungal, central nervous system depressant, and antianxiety effects have
been reported for several Ducrosia species, including D. anethifolia and D. ismaelis.9 D. ismaelis
Asch. (commonly known as Haza or Geshea) is a perennial, herbaceous, and branched plant with
a characteristic aromatic odor.10 The whole herb, especially its aerial parts, has been used in
traditional medicines to treat skin infections and to repel insects and reptiles.11
Despite the use of D. ismaelis in traditional medicines, there is a lack of information on
antioxidant activity which is generally related with prevention of chronic diseases including

24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
osteoporosis and atherosclerosis. In our efforts for the isolation of lead compounds for the
treatment of osteoporosis, we investigated the chemical constituents of the D. ismaelis plant and
evaluated their biological activities. Compounds 1-15 were isolated from the aerial components
of D. ismaelis (Fig. 1). This report details the isolation, structural determination, and
antiosteoporotic and antioxidant activities of these compounds using TRAP, ORAC, and
reducing capacity assays. Dried aerial components of D. ismaelis were extracted with 100%
methanol at room temperature. The crude extract was suspended in water and then successively
partitioned with dichloromethane and ethyl acetate to give CH2Cl2, EtOAc, and water fractions,
respectively. Using various types of column chromatography, two new flavonoid glycosides,
named glycinol-3-O-β-D-glucopyranoside (1)12 and ismaeloside A (2),13 together with 13 known
compounds: daidzin (3),14 daidzein-4'-O-β-D-glucopyranoside (4),15 genistin(5),16 prunetrin (6),17
coumestrol (7),18 isobavachalcone (8),19 4'-hydroxy-3,3',4,5,5'-pentamethoxy-7,9':7',9-
diepoxylignane (9),20 liriodendrin (10),21 pinoresinol-4'-O-β-D-glucopyranoside (11),22 blumenol
C glucoside (12),23 citrusin C (13),24 coniferin (14),25 and (Z)-plicatin B (15),26 were isolated for
the first time from D. ismaelis.27 Their chemical structures (Fig.1) were elucidated based on 1D
and 2D NMR spectra, MS data, and by comparisons with previously published data acquired
from similar compounds (see supplementary information).
Compound 1 was obtained from water fractions as a white amorphous powder (mp 290-295ºC,
[α]2D0 = -171.31 (c 0.1, MeOH), UV λmax (MeOH) nm: 212.0, 283.0, 444.0, 537.0) with a
molecular formula of C21H22O10, determined by a peak in the MicroQ-TOF III mass spectrum
44
(ESI-Q-TOF MS) at m/z 457.1128 [M + Na]+ (calcd for C21H22O10Na, 457.1105). FT-IR (KBr)
absorbance peak were observed at νmax 3350, 2924, 1621, 1499, 1457, 1261, 1171, 1122, 1072,
45
46
1
1037, 967 cm-1. The H NMR spectrum of 1 indicated the presence of an A2-type aliphatic

47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
proton system evidenced by peaks at δH 3.83 (1H, d, J= 11.4 Hz, Hax-6) and 4.02 (1H, d, J=
11.4Hz, Haq-6). Two ABX-type aromatic proton signals also appeared at δH 7.29 (1H, d, J= 8.5
Hz, H-1), 6.71 (1H, dd, J= 8.5, 2.3 Hz, H-2) and 6.52 (1H, d, J= 2.3 Hz, H-4), due to an A-ring
proton, and at δH 7.06 (1H, d, J= 8.1 Hz, H-7), 6.30 (1H, dd, J= 8.1, 2.1 Hz, H-8) and 6.12 (1H, d,
J= 2.1 Hz, H-10) due to a B-ring proton. A singlet proton peak at δH 5.10 (1H, s, H-11a), is
characteristic of pterocarpans. However, the disappearance of a hydrogen atom and the presence
of a hydroxyl group on C-6a (deshielded δC 75.83) indicated that 1 is a 6a-hydroxypterocarpan
derivative. The 1H NMR spectrum of 1 also contained anomeric proton signal at δH 4.80 (1H, d,
J= 7.4 Hz). The coupling constant of the anomeric proton and its doublet multiplicity confirmed
the β–configuration of the glucose moiety. The 13C NMR and distortionless enhancement by
polarization transfer (DEPT) spectra of 1 indicated 21 carbon atoms including two methylene, 12
tertiary, and seven quaternary carbon atoms. These included one anomeric carbon atom at δC
100.66, four oxygenated methine carbon atoms at δC 69.96, 73.49, 76.59 and 76.83, and one
oxygenated methylene carbon atom at δC 61.07 that were assigned to the sugar moiety. The
remaining carbon atoms belonged to the aglycone moiety. The β-glucopyranosyl unit was
positioned at C-3, which is in agreement with correlations observed between the anomeric proton
1
H-1' (δH 4.80) and C-3 (δC 158.81) in the HMBC spectra (see Fig. 2). The H NMR coupling
constant of the anomeric proton was 7.4 Hz with doublet multiplicity, which confirmed the β-
configuration of the glucose moiety. The presence of a D-glucose unit was further confirmed by
acid hydrolysis and gas chromatographic (GC) analyses followed by comparisons of retention
times with authentic samples.28,29 1H-1H COSY spectra showed correlations between H-1 (δH
7.29) and H-2 (δH 6.71), and between H-7 (δH 7.06) and H-8 (δH 6.30) (see Fig. 2). The absolute
configurations were determined as 6a-S and 11a-S by CD spectral analyses (c 0.1, MeOH). A

70
71
72
73
74
negative Cotton effect at 234.2 nm and a positive Cotton effect at 286.4 nm were observed.12
Based on the above data, compound 1 was identified as a new compound, and named glycinol-3-
O-β-D- glucopyranoside (Fig. 1).
Compound 2 was obtained from water fractions as a white amorphous powder (mp 130 -133ºC
[α]2D0 = 21.01 (c 0.1, MeOH), UV λmax (MeOH) nm: 224.0, 278.0, 538.0) with a molecular
75
formula of C21H24O10, as determined by a peak in the MicroQ-TOF III mass spectrum (ESI-Q-
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
TOF MS) at m/z 459.1297 [M + Na]+ (calcd for C21H24O10Na, 459.1267). FT-IR (KBr)
absorbance peaks were observed at νmax 3345, 2924, 1716, 1613, 1514, 1456, 1288, 1259, 1174,
1073, 1038, 800 cm-1. The 1H NMR spectrum of 2 contained methylene proton signals appearing
at δH 3.44 - 3.52 (2H, m, H-β) and an oxymethine proton resonating at δH 5.22 (1H, br.s, H-α).
ABX-type aromatic proton signals were observed at δH 6.41(1H, dd, J= 8.3, 2.2 Hz, H-5'), 6.64
(1H, d, J= 2.2 Hz, H-3'), and 6.98 (1H, d, J= 8.3 Hz, H-6'). AA'BB'-type aromatic proton signals
were observed at δH 6.79 (2H, d, J= 8.4 Hz, H-2,6) and 6.56 (2H, d, J= 8.4 Hz, H-3,5). Proton
signals corresponding to a β-glucopyranosyl group with an anomeric proton signal at δH 4.76 (1H,
d, J= 7.2 Hz) were also observed. The coupling constant of the anomeric proton and its doublet
multiplicity confirmed the β–configuration of the glucose moiety. The 13C NMR and DEPT
spectra of 2 indicated 21 carbon atoms, including 2 methylene, 13 tertiary, and 6 quaternary
carbon atoms. Among these, a peak corresponding to one anomeric carbon atom at δC 102.14,
four oxygenated methine carbon atoms at δC 70.02, 73.51, 75.25 and 76.51, respectively, and one
oxygenated methylene carbon atom at δC 61.28 were assigned to the sugar moiety. The remaining
carbon atoms were assigned to the aglycone moiety. The β-glucopyranosyl unit was positioned at
C-2', which is in agreement with correlations observed between the anomeric proton H-1" (δH
4.76) and C-2' (δC 155.90) in the HMBC spectra (see Fig. 3). The β-configuration of the glucose

93
94
moiety was confirmed by the 7.2-Hz coupling constant of the anomeric proton and by its doublet
multiplicity. The presence of a D-glucose unit was further confirmed by acid hydrolysis and gas
chromatographic (GC) analyses followed by comparisons of retention times with authentic
samples.28,29 1H-1H COSY spectra of 2 showed correlations between H-2 (δH 6.79) and H-3 (δH
6.56), H-5 (δH 6.56) and H-6 (δH 6.79), and between H-5' (δH 6.41) and H-6' (δH 6.98) (see Fig.
3). The absolute configuration was determined as α-R by CD spectral analysis (c 0.1, MeOH)
where a positive Cotton effect at 292.4 nm was observed.30 Based on the above data, compound 2
was determined to be a new compound named ismaeloside A (Fig. 1).
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
1
Compound 15 was identified as (Z)-plicatin B based on a comparison of its H NMR data with
those of (E)-plicatin B. The observed coupling constant of 12.0 Hz between H-1' (δH 6.76) and
H-2' of 15 (δH 5.74) (see Table 3) was compared to the 16.0-Hz coupling constant between H-1'
and H-2' in (E)-plicatin B.26 The 1H and 13C NMR data of 15 have not been published previously.
The anti-osteoporotic activities of compounds 1-15 were determined using TRAP assays (see
supplementary information). TRAP activity was expressed as the percent of the untreated control
(mean one standard deviation, n = 3). RANKL treatments induced osteoclast formation from
RAW 264.7 pre-osteoclast cells and dramatically enhanced TRAP activity up to 170.9%, as
shown in Fig. 4. Compounds 15, 13, 4, and 10 suppressed osteoclast formation in a dose-
dependent manner with TRAP values of 85.05 6.55, 100.93 6.12, 104.77 11.17, and 106.05
7.65 (% of control) at concentrations of 10 µM, respectively. The potent TRAP activity was
observed in compound 3, 4, 8, 10, 12, 13, and 15 at 10 µM when compared to daidzein used as a
positive control. Compound 15 exhibited the highest activity of the tested compounds, as
indicated by the relatively low TRAP value (Fig. 4). This may be due to the higher cell
membrane permeability of 15, resulting from the absence of a glucose unit and the presence of

116
117
118
119
120
121
122
123
124
125
126
127
128
129
two hydrophobic side chains. This agrees with the conclusions of previous work in which
scopoletin was more effective than scopolin in terms of reducing the number and TRAP activity
of multinucleated TRAP-positive cells in a dose-dependent manner.4
According to previous reports, the anti-osteoporotic activities of natural flavonoids as
scopoletin, resveratrol, and baicalein are found to be very closely related with their antioxidant
activities3-6. Thus, the antioxidant activities of compounds 1-15 were investigated in terms of
their peroxyl radical-scavenging and reducing capacity. Based on the chemical reactions
involved, major antioxidant capacity assays can be roughly divided into hydrogen atom transfer-
based and single electron transfer-based assays.31 ORAC assay is based on hydrogen atom
transfer while reducing capacity assay depends upon single electron transfer.31 Figure 5 shows
that the scavenging activities of compounds 1-15 in the presence of peroxyl radicals were dose-
dependent at 1−10 μM (see supplementary information). Compounds (1, 3-5, and 8) showed
potent peroxyl radical-scavenging activities over 20 TE at 10 µM. In particular, at a
concentration of 10 μM, 5 showed the strongest peroxyl radical-scavenging activity with 26.55
130
0.42 Trolox equivalent (TE) against peroxyl radicals generated from 2,2′-azobis(2-
131
132
133
134
135
136
137
138
amidinopropane) dihydrochloride (AAPH). Based on structural analysis of the active isolates (1-
5, 8-9 and 15), the hydroxyl groups on the aromatic ring seem to be responsible for peroxyl
radical-scavenging activity by donating hydrogen atoms to peroxyl radicals.
The ability to transfer single electron was also examined using reducing capacity assay (see
supplementary information). The ability of the isolated compounds 1-15 to stimulate the
reduction of copper ions (Cu2+ to Cu+) by donating electrons was investigated to determine
whether their peroxyl radical-scavenging capacities, which involve the donation of hydrogen
atoms, could be related to their reduction capacities, which involve donating electrons to copper

139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
ions. As shown in Figure 6, only compound 9 was able to significantly reduce Cu2+ ions, with a
reduction value of 23.44 ± 0.45 µM TE at a concentration of 10.0 µM. Present data suggest that
the ability of compound 9 to donate hydrogens or electron atoms to peroxyl radicals and to
convert them into relatively stable compounds may contribute to its peroxyl radical-scavenging
capacity. This data agrees with the conclusions of previous studies.3,4
When compared both anti-osteoporotic and antioxidant activities of 1-15, the potent both
TRAP and peroxyl radical scavenging activities were observed in 3, 4, and 8 among 15 isolated
compounds. In addition, there is no correlation between TARP activity and reducing capacity of
tested 15 compounds. Therefore, these results imply that compound 3, 4, and 8 may augment
antiosteoporotic activity by directly scavenging intracellular reactive oxygen species produced
during RANKL-mediated osteoclast differentiation through donating hydrogen atom. In other
hand, the potent antiosteoporotic activities of compound 10, 12, 13, and 15 not showing strong
antioxidant activities in vitro may be attributed to their indirect antioxidant activities by
activating the endogenous antioxidant defense system. Thus, more research may be needed to
determine whether the potent antiosteoporotic activites compound 10, 12, 13, and 15 result from
indirect antioxidant activity.
Regarding the structure-activity relationships of the isolated compounds, note that aglycones
penetrate cell membranes more readily than do glycosides. For efficient antioxidant activity in
cells, compound must be deglycosylated into its aglycone. Further study using an in vivo animal
model may be required to elucidate the differences in cell-membrane permeability between
glycoside and its aglycone.

160
161
162
163
Acknowledgements
This study was supported by the Priority Research Center Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (2009-0093815), Republic of Korea.

164
165
References and notes
1.
2.
Yang, X.; Ji, C.; Wang, D.; Chen, X. Chinese J. of T. Engi Res. 2012, 16, 15
166
167
Yan-Bin, W.; Cheng-Jian, Z.; Lu-Ping, Q.; Lian-Na, S.; Ting, H.; Lei, J. , Qiao-Yan, Z.;
Jin-Zhong, W. Molecules 2009, 14, 573.
168
169
3.
Yan, X. T.; Lee, S. H.; Li, W.; Sun, Y. N.; Yang, S. Y.; Jang, H. D.; Kim, Y. H. Food
Chem. 2014, 156, 408.
170
4.
5.
Lee, S. H.; Ding Y.; Yan, X. T.; Kim, Y. H.; Jang, H. D. J. Nat. Prod. 2013. 76, 615.
171
172
173
174
175
176
Kim, M. H.; Ryu, S. Y.; Bae, M. A.; Choi, J. S.; Min, Y. K.; Kim, S. H. Food Chem.
Toxicol. 2008, 46, 3375.
6.
7.
He, X.; Andersson, G.; Lindegren, U.; Li, Y. Biochem. Biophys. Res. Commun. 2010, 401,
356.
Karimi, M.; Ebrahimi, A.; Sahraroo, A.; Moosavi, S. A.; Moosavi, F.; Bihamta, M. J.
Food Agric. Environ. 2009; 7, 441.
177
8.
9.
Haghi, G.; Safaei, A.; Safaei, J. Iran. J. Pharm. Res.2004, 3, 90.
178
179
Wamidh, H.; Talib, R. A.; Issa, F. K.; Adel, M. M. British J. of Medicine & Medical Res.
2013, 3, 771.
180
181
10. Al-meshal, I. A.; Taha, I. K.; Mahmoud, M. A. H. Spectroscopy Letters 1985. 18, 491.
11. Al-meshal, I. A. Res. Comm. in Chem Path and Pharm. 54, 1986.
182
183
12. Wei, L.; Kazuo, K.; Yoshihisa, A.; Masao, H.; Hekai, R.; Takafumi, Y.; Tamotsu, N.
Phytochem. 2002, 60, 351.
184
185
13. Kulesh, N. I.; Vasilevskaya, N. A.; Veselova, M. V.; Denisenko, V. A.; Fedoreev, S. A.
Chem. of Nat. Comp., 2008, 44, 6.
186
187
14. Sergey, A. F.; Victor, P., B.; Olga, V. G.; Marina, V., V.; Olga, E. K.; Nadezda, I. K.;
Vladimir, A. D.; Galina, K. T.; Yury, N. Z. J. Agric. F. Chem. 2008, 56, 7023.
188
15. Philip, T. L.; Kristiina, W. Tetrahedron L. 1998, 39, 9559.

189
190
16. Rong, H.; Zhang-Gui, D.; Yun-Feng, L.; Jiang-Yuan, Z.; Ming-Gang, L.; Xiao-Long, C.;
Meng-Liang, W. Chem. of Nat. Comp. 2013, 48, 966.
191
192
17. Drenin, A. A.; Botirov, E. K.; Turov, Y. P. Russian Journal of Bioorganic Chemistry,
2011, 37, 862.
193
194
18. Heung, J. Y.; Jin, H. L.; Marcus, J. C.; Ji, W, L; Kim Y. S.; Hyung, W. R.; Park, C. G.;
Tae-Sook, J.; Park, K. H. Food Chemistry. 2011, 126, 1057.
195
196
19. Guodong, X.; Guowen, L.; Liang, C.; Zijia, Z.; Jun-Jie, Y.; Tao, W.; Zhihong, C.; Xiaohui,
W.; Zhengtao, W. J. of Chrom. A, 2010, 1217, 5470.
197
198
20. Liang, X.; Chenggeng, Z.; Yanru, L.; Ye, T.; Sheng, L.; Shaopeng, Y.; Jinfeng, H.; Qi, H.;
Naihong, C.; Yongchun, Y.; Jiangong, S. J. Nat. Prod. 2011, 74, 1188.
199
21. Zhuwei, W.; Lixia, Z.; Yuqing, S. J. of Chrom Sc. 2005, 43.
200
201
22. Ming-An, O.; Yung-Shung, W.: Zhen-Kun, Z.; Yueh-Hsiung, K. J Agric. Food Chem.
2007, 55, 6460.
202
23. Chen, B; Li, B.; Zhang, G. Acta Botanica Sinica 2002, 44, 344.
203
204
24. Rong, W. T.; De, Z. W.; Yun, S. W.; Yang, L.; Qi, T. Z.; Chong, R., Y. Magn. Reson.
Chem. 2005, 43, 92.
205
206
25. Sheng-Xiong, H.; Xun, L.; Quan-Jiang, N.; Li-Sheng, D.; Shu-Lin, P. Helvetica Chemica
Acta. 2004, 87, 598.
207
208
26. Sanjay, R. M.; Vishal, K. P.; Lester, A. M.; Peter, S.; Segaran, P. Pi.; Delbert, M. S. J.
Nat. Prod. 1999, 62, 102.
209
210
211
212
213
214
27. Dried aerial parts of D. ismaelis Asch. (3.0 kg) were extracted with 100% methanol at
room temperature three times. After removal of the solvent under reduced pressure, the
methanol extract (258 g) was dissolved in 1.0 L of H2O to form a suspension that was
successively partitioned with dichloromethane (CH2Cl2), and ethyl acetate (EtOAc), to
give CH2Cl2 (72.87g), EtOAc (8.45g), and aqueous extract (177.0 g), respectively.
Aqueous extract was chromatographed on a column of highly porous polymer (Diaion

215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
HP-20) and eluted with H2O and MeOH, successively to give four fractions (1a-1d).
Fraction 1a was then separated by column chromatography over silica gel (70–230 mesh),
using CH2Cl2/MeOH/H2O (10:1:0.1 – 1:1:0.1 v/v/v) gradually as eluents, to afford sub-
fraction (2a–2e). Sub-fraction 2d was separated by YMC reverse-phase chromatography,
using MeOH/H2O (0.2:1 v/v) as eluent to give compound 14 (15.0 mg). Fraction 1b was
chromatographed over silica gel, eluting gradually with CHCl3/MeOH/H2O (10:1:0.1 –
1:1:0.1 v/v/v), to provide five sub-fractions (3a-3e). Sub-fraction 3b was separated by
YMC column with MeOH/H2O (0.3:1 v/v) as eluent to obtain 1 (10.0 mg) and 4 (50.0 mg).
Sub-fraction 3c was separated by YMC column, using MeOH/H2O (0.3:1 v/v) as eluent,
and further purified by column chromatography over silica gel, eluting with
CH2Cl2/MeOH/H2O (5:1:0.1 v/v/v) to obtain 2 (10.0 mg), and 10 (12.0 mg). Fraction 1c
was chromatographed over silica gel eluting gradually with CHCl3/MeOH/H2O (12:1:0.1
– 1:1:0.1 v/v/v), to afford five sub-fractions (4a–4e). Sub-fraction 4a was separated by
YMC column, eluting with MeOH/H2O (0.6:1-0.7:1 v/v), afford 9 (37.0 mg). Sub-fraction
4c was separated by YMC column, eluting with MeOH/Me2CO/H2O (0.3:0.3:1 v/v/v),
further by silica gel column with CH2Cl2/MeOH/H2O (10:1:0.1v/v/v) to obtain 6 (3.0 mg),
11 (12.0 mg), and 12 (5.0 mg). Sub-fraction 4d was separated by YMC column, using
MeOH/Me2CO/H2O (0.3:0.3:1 v/v/v), and then by column chromatography over silica gel,
eluting with CH2Cl2/MeOH/H2O (20:1:0.1 v/v/v) to give 3 (15.0 mg), and 5 (20.0 mg).
Compound 13 (20.0 mg) was isolated from sub-fraction 4e using YMC column eluting
with MeOH/Me2CO/H2O (0.3:0.3:1 v/v/v), and further purified by silica gel column, using
CH2Cl2/MeOH/H2O (10:1:0.1 v/v/v). EtOAc extract was subjected to column
chromatography using silica gel (70–230 mesh), eluting with CH2Cl2/MeOH/H2O
(20:1:0.2- 5:1:0.1v/v/v) to give five fractions (5a–5e). Repeated silica gel column
chromatography of fraction 5c with n-hexane/EtOAc (10:1-3:1 v/v), further by YMC
column and eluting with MeOH/H2O (1:1), and (1.5:1 v/v) to give 7 (10.0 mg), and 8
(10.0 mg), respectively. Finally, CH2Cl2 extract was chromatographed over silica gel (70–
230 mesh), using n-hexane/EtOAc/MeOH (20:1:0.1 – 1:1:0.1 v/v/v) gradually as eluents,
to afford sub-fraction (6a–6f). Repeated silica gel column chromatography of fraction 6d
with n-hexane/EtOAc (9:1 v/v), to give 15 (15.0 mg).

245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
28. Acid Hydrolysis and Sugar Identification. Compounds 1 and 2 (2 mg) each was heated in
3 mL of 10% HCl-dioxane (1:1) at 80 °C for 3 h. After the solvent was removed in vacuo,
and the residue was partitioned between EtOAc and H2O to give the aglycone (0.95 mg
for compounds 1 and 2) and the sugar (0.63 mg for sugar 1 and 0.62 mg for sugar 2),
respectively. The sugar components in the aqueous layer were analyzed by silica gel TLC
by comparison with standard sugars. The solvent system was CH2Cl2/MeOH/H2O (2:1:0.2
v/v/v), and spots were visualized by spraying with 95% EtOH/H2SO4/anisaldehyde
(9:0.5:0.5 v/v/v), then heated at 180 °C for 5 min. For sugars, the Rf of glucose by TLC
was 0.30. The results were confirmed by GC analysis as follows. The aqueous layer was
evaporated to dryness using N2 gas. The residue was dissolved in 0.1 mL of dry pyridine,
and then mixed with L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL).
The reaction mixture was heated at 60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole
solution was added, followed by heating at 60 °C for 2 h. The dried product was
partitioned with n-hexane and H2O (0.1 mL, each), and the n-hexane layer was analyzed
by gas liquid chromatography (GC): Column: column DB-5 (0.25 mm x 30 m); detector
µ-ECD, column temp 250 °C, injector temperature 300 °C, detector temperature 280 °C,
carrier gas N2. The absolute configuration of the monosaccharide was confirmed to be -
D
glucose by comparison of the retention time of the monosaccharide derivative (tR 14.83
min) with that of authentic sugar derivative samples prepared in the same manner (D-
glucose derivative tR 14.72 min, L-glucose derivative tR 15.27 min).
265
266
29. Quang, T. H.; Nguyen T. T. N.; Minh, C. V.; Kiem P. V.; Nhiem N. X.; Tai, B. H.; Thao,
N. P.; Luyen B. T. T.; Song, S. B.; Kim,Y. H. Bull. Korean Chem. Soc. 2011, 32, 11.
267
268
30. Alvarez, L.; Delgado, G. Phytochemistry 1999, 50, 681.
31. Dejian, H.; Boxin, O.; Ronald, L. P. J. Agric. Food Chem. 2005, 53, 1841.
269
270
32. Yan, D.; Jeong, A. K.; Yang, S. Y.; Won-Ki, K.; Sang H. L.; Hae D. J.; Kim, Y. H. Bull.
Korean Chem. Soc. 2011, 32, 3493.

R1O
O
O
HO
OH
GlcO
O
4
3
5'
OH
4'
3'
2
3
2
4
5
6
6'
1'
4a
OH
1
6a
6
2'
β
1
α
11b
6b
11a
7
H
OGlc
O
R
8
O
10a
10
OR2
9
2
OH
1
R
H
R1
Glc
H
R2
H
3
4
5
6
H
Glc
H
OH
OH
Glc
Me
Glc
HO
O
O
OH
OMe
OR1
HO
OH
O
O
R
OH
H
H
R
O
7
O
R2O
8
MeO
OGlc
R
R1
H
R2
Me
Glc
Glc
H
H
OGlc
OMe
OMe
H
9
O
Glc
H
10
11
H
OMe
12
13
OH
4''
3''
1''
4
1
5''
HOH2C
5
6
3
2
2''
OGlc
O
1'
OMe
MeO
2'
14
15
271
Figure 1. Chemical structures of isolated compounds.

OH
O
O
O
HO
HO
OH
OH
H
O
HMBC
COSY
OH
272
Figure 2. Selected HMBC and COSY correlation for 1.

HO
OH
OH
OH
O
O
O
HO
HO
HMBC
COSY
OH
273
Figure 3. Selected HMBC and COSY correlation for 2.

1 µM
10 µM
200
180
160
140
120
100
80
60
40
20
0
C
TC
Daidzein
(10 µM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Compounds
274
275
Figure 4. Tartrate-resistant acid phosphatase (% of control) of compounds (1-15). C: control,
which was not treated; TC: treated control, which was treated with RANKL.

30
25
20
15
10
5
1 µM
10 µM
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Compounds
276
277
Figure 5. Peroxyl radical-scavenging activity (Trolox Equivalent, µM) of
compounds (1-15) in ORAC assay.

1 µM
30
25
20
15
10
5
10 µM
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Compounds
278
279
Figure 6. Reducing capacities of compounds 1-15. The results represent the mean SD
of values obtained from three measurements

280
281
Table 1
The 1H and 13C NMR data for 1 in CD3OD (δ in ppm)
a
b
Position
δH (H, mult, J in Hz)
7.29 (1H, d, 8.5)
δC
1
131.79
110.72
158.81
104.28
155.83
2
6.71 (1H, dd,8.5, 2.3)
3
4
6.52 (1H, d, 2.3)
4a
6
3.83 (1H, d, 11.40)
4.02 (1H, d, 11.40)
4.77 (1H, s, OH)
69.57
6a
75.83
6b
119.73
123.83
108.06
160.81
97.61
7
7.06 (1H, d, 8.1)
8
6.30 (1H, dd, 8.1, 2.1)
9
10
6.12 (1H, d, 2.1)
5.10 (1H, s)
10a
11a
11b
3-O-Glc
1'
159.85
84.37
114.69
4.80 (1H, d, 7.4 )
100.66
2'
3.41- 3.41 (3H, m, 2', 3' and 4') 73.49
3'
76.59
69.96
4'
5'
3.27 (1H, d, 8.9)
76.83
6'
3.56 (1H, dd, 12.1, 1.9)
3.75 (1H, dd, 12.1, 1.9)
61.07
282
Assignments were done by HMQC, HMBC, and 1H–1H COSY experiments.
a
283
284
Measured at 600 MHz.
Measured at 150 MHz.
b

285
286
Table 2
The 1H and 13C NMR data for 2 in CD3OD (δ in ppm)
a
b
δC
Position
δH (H, mult, J in Hz)
C=O
208.63
77.00
α
5.22 (1H, br.s)
β
3.44 – 3.52 (2H, m)
43.58
1
125.08
130.40
114.83
156.60
119.22
155.90
103.58
159.27
109.68
130.79
2 and 6
6.79 (2H, d, 8.4)
6.56 (2H, d, 8.4)
3 and 5
4
1'
2'
3'
6.64 (1H, d, 2.2)
4'
5'
6.41 (1H,dd, 2.2, 8.3)
6.98 (1H, d, 8.3)
6'
2'-O-Glc
1''
2''
3''
4''
5''
6''
4.76 (1H, d, 7.2)
102.14
3.32 – 3.36 (3H, m, H2'',3'' and 5'') 73.51
75.25
3.29 (1H, d, 8.9)
70.02
76.51
61.28
3.63 (1H, dd, 6.0, 11.4)
3.83 (1H, dd, 1.9, 11.4)
287
Assignments were done by HMQC, HMBC, and 1H–1H COSY experiments.
a
288
289
Measured at 600 MHz.
Measured at 150 MHz.
b

290
291
Table 3
The 1H and 13C NMR data for 15 in CDCl3 (δ in ppm)
a
δH (H, mult, J in Hz)
b
Position
δC
C=O
1
167.27
126.60
130.15
127.32
156.67
115.44
132.66
143.97
116.33
29.78
2
7.48 (1H, dd, 8.4, 2.4)
3
4
5
6.69 (1H, d, 8.4)
7.35 (1H, d, 1.8)
6.76 (1H, d, 12.0)
5.74 (1H, d, 12.0)
3.27 (2H, t, 7.8)
5.25(1H, m)
6
1'
2'
1''
2''
3''
4''
5''
OCH3
OH
121.63
134.92
17.96
1.71 (3H, s)
1.71 (3H, s)
3.65 (3H, s)
5.69 (1H, br s)
25.88
51.44
292
Assignments were done by HMQC, HMBC, and 1H–1H COSY experiments.
a
293
294
Measured at 600 MHz.
Measured at 150 MHz.
b

The English in this document has been checked by at least two professional editors, both native
speakers of English. For a certificate, please see:
http://www.textcheck.com/certificate/PgbGza

Graphical abstract
Anti-osteoporotic and antioxidant activities of chemical constituents of the
aerial parts of Ducrosia ismaelis
Abubaker M. Morgan, Hyun Woo Lee, Sang-Hyun Lee, Chi-Hwan Lim, Hae-Dong Jang and
Young Ho Kim
GlcO
O
4
1
3
2
6
4a
HO
OH
OH
5'
2'
3
OH
6a
6'
1'
2
1
4'
3'
4
5
11b
6b
11a
6
β
7
α
H
8
O
10a
10
OGlc
O
9
OH
1
2