New phenolic compounds from Dendrobium capillipes and Dendrobium secundum

Article abstract of DOI:10.1080/10286020.2012.689979

Phytochemical investigation of the stem of Dendrobium capillipes resulted in the isolation of a new flavonol glycoside namely quercetin-3-O-α-l- rhamnopyranosyl-(1 → 2)-β-d-xylopyranoside, along with seven known phenolic compounds which included two flavonol glycosides and five bibenzyls. From the stem of Dendrobium secundum, a new compound named 5-hydroxy-3,4,3,4,5- pentamethoxybibenzyl was characterized, together with two known bibenzyls and three glycosidic flavonoids. Some of the isolated bibenzyls showed cytotoxicity against KB, NCI-H187, and MCF-7 cancer cells, whereas all of the glycosidic flavonoids were devoid of activity.

Full text of DOI:10.1080/10286020.2012.689979

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New phenolic compounds from
Dendrobium capillipes and Dendrobium
secundum
Thanawuth Phechrmeekha a , Boonchoo Sritularak a & Kittisak
Likhitwitayawuid a
a Department of Pharmacognosy and Pharmaceutical Botany ,
Faculty of Pharmaceutical Sciences, Chulalongkorn University ,
Bangkok , 10330 , Thailand
Published online: 14 Jun 2012.
To cite this article: Thanawuth Phechrmeekha , Boonchoo Sritularak & Kittisak Likhitwitayawuid
(2012) New phenolic compounds from Dendrobium capillipes and Dendrobium secundum , Journal of
Asian Natural Products Research, 14:8, 748-754, DOI: 10.1080/10286020.2012.689979
To link to this article: http://dx.doi.org/10.1080/10286020.2012.689979
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Journal of Asian Natural Products Research
Vol. 14, No. 8, August 2012, 748–754
New phenolic compounds from Dendrobium capillipes
and Dendrobium secundum
Thanawuth Phechrmeekha, Boonchoo Sritularak and Kittisak Likhitwitayawuid*
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand
(Received 6 March 2012; final version received 27 April 2012)
Phytochemical investigation of the stem of Dendrobium capillipes resulted in the
isolation of a new flavonol glycoside namely quercetin-3-O-a-L-rhamnopyranosyl-
(1 ! 2)-b-D-xylopyranoside, along with seven known phenolic compounds which
included two flavonol glycosides and five bibenzyls. From the stem of Dendrobium
secundum, a new compound named 5-hydroxy-3,4,30,40,50-pentamethoxybibenzyl was
characterized, together with two known bibenzyls and three glycosidic flavonoids.
Some of the isolated bibenzyls showed cytotoxicity against KB, NCI-H187, and MCF-
7 cancer cells, whereas all of the glycosidic flavonoids were devoid of activity.
Keywords: cytotoxicity; chemical constituents; bibenzyls; flavonoids; Dendrobium
1. Introduction
compound namely quercetin-3-O-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-xylopyra-
noside (1) and two known compounds, i.e.
kaempferol-3-O-a-L-rhamnopyranosyl-
(1 ! 2)-b-D-xylopyranoside or lysima-
chiin (2) [3] and kaempferol-3-O-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyra-
noside (3) [4]. In addition, five bibenzyls
were also isolated and identified as
chrysotobibenzyl (4) [5], crepidatin (5)
[6], gigantol (6) [7], chrysotoxine (7) [8],
and moscatilin (8) [9] (Figures 1 and 2).
From D. secundum, a new compound
Plants of the genus Dendrobium
(Orchidaceae) are widely distributed in
Asia, and Dendrobium capillipes and
Dendrobium secundum are among more
than 90 species of Dendrobium identified
in Thailand [1]. So far, D. capillipes has
not been chemically studied, whereas D.
secundum has been reported to produce
bibenzyls and other phenolic compounds
[2]. As part of our continuing studies on
bioactive compounds of Dendrobium
plants, we conducted investigations on D.
capillipes and D. secundum. In this
communication, we report the isolation
and structural characterization of biben-
zyls and flavonoid glycosides from
D. capillipes and D. secundum, as well as
their cytotoxicity studies.
namely
5-hydroxy-3,4,30,40,50-penta-
methoxybibenzyl (9) was isolated,
together with moscatilin (8) [9], brittonin
A
(10) [10], 4,5,40-trihydroxy-3,30-
dimethoxybibenzyl (11) [2], kaempferol-
3,7-O-di-a-L-rhamnopyranoside (12) [11],
quercetin-3-O-a-L-rhamnopyranoside (13)
[12], and kaempferol-3-O-a-L-rhamnopyr-
anoside (14) [13] (Figure 2).
2. Results and discussion
Three glycosidic flavonoids were isolated
from D. capillipes, including a new
*Corresponding author. Email: kittisak.l@chula.ac.th
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.689979
http://www.tandfonline.com

Journal of Asian Natural Products Research
749
spectrum, suggesting the molecular for-
mula C26H28O15. The UV absorptions at
215, 256, and 346 nm, and the IR peaks at
3349 (br), 1660, and 1040–1361 cm21
were similar to those of 2 [3], indicative of a
glycosidic flavonol structure. The 1H NMR
spectrum of 1 (Table 1) resembled that of 2,
except that in 1 only three aromatic proton
signals were observed for ring B, appearing
at d 7.53 (d, J ¼ 2.0 Hz, H-20), 6.84 (d,
J ¼ 8.5 Hz, H-50), and 7.57 (dd, J ¼ 8.5,
2.0 Hz, H-60). This suggested that the B ring
was oxygenated at C-30 and C-40, and this
was substantiated by the resonances at d
145.0 (C-30, s) and 148.6 (C-40, s) in the 13C
NMR and DEPT spectra (Table 1). From
the above-mentioned NMR properties,
coupled with the molecular mass of 1
being 16 amu higher than that of 2, it could
R
OH
O
O
HO
O
OH
O
HO
HO
O
H3C
HO
O
OH
OH
1
OH
R
2
H
Figure 1. Structures of glycosidic flavonols 1
and 2.
Compound 1,
a yellow-greenish
Table 1. 1H (500 MHz) and 13C NMR
(125 MHz) spectral data of 1 (DMSO-d6).
amorphous solid, exhibited an [M þ Na]þ
ion at m/z 603.1388 in the HR-ESI mass
Position
1H
13C
OMe
R3
2
3
4
5
6
7
8
156.0
132.8
177.1
161.2
98.7
MeO
R1
R4
6.18 d (2.0)
6.39 d (2.0)
164.3
93.5
R2
9
10
156.2
103.8
120.9
115.9
145.0
148.6
115.2
121.5
10
20
7.53 d (2.0)
6.84 d (8.5)
7.57 dd (8.5, 2.0)
R1
R2
R3
R4
30
40
4
OMe
OH
H
OMe
OMe
OH
OMe
OMe
H
50
60
Xylose
5
H
100
5.51 d (7.5)
3.54 t (7.5)
3.32 m
3.34 m
2.95 m, 3.59 dd (4.0, 11.0)
99.4
76.9
76.8
69.6
66.0
6
H
OH
H
200
300
7
OMe
OH
OH
OMe
OMe
OMe
OMe
H
400
500
8
H
OH
Rhamnose
1000
2000
3000
4000
5000
6000
5.08 (br s)
3.48 dd (3.0, 9.0)
3.73 m
3.15 m
3.78 m
0.87 d (6.5)
100.6
70.5
70.6
71.8
68.3
17.4
9
OMe
OMe
OH
OH
OMe
OH
OMe
OMe
OH
10
11
Figure 2. Structures of bibenzyls (4–11).

750
T. Phechrmeekha et al.
be inferred that 1 had the aglycone part
derived from quercetin (3,5,7,30,40-penta-
hydroxyflavone). For the glycosidic part of
1, two anomeric protons were found at d
5.51 (d, J ¼ 7.5 Hz, H-100) and 5.08 (br s,
H-1000), and these protons exhibited HSQC
correlation with the carbons at d 99.4 (C-100)
and 100.6 (C-1000), respectively. This was
reminiscent of an a-L-rhamnopyranosyl-
Compound 9, a white powder, had the
molecular formula of C19H24O6, as indi-
cated by an [M þ Na]þ ion at m/z
371.1469 in the HR-ESI mass spectrum.
The UV absorptions at 215 and 266 nm
suggested a bibenzyl skeleton. The 1H
NMR spectrum (Table 2) showed reson-
ances for two pairs of benzylic protons at d
2.80 (br s, 4H, H2–a and H2–a0), five
methoxy groups at d 3.79 (3H, s), 3.80 (3H,
s), 3.81 (6H, s), and 3.84 (3H, s), and four
aromatic protons at d 6.21 (1H, s, H-2),
6.46 (1H, s, H-6), and 6.35 (2H, s, H-20,60).
From these 1H NMR data, it could be
inferred that 9 was a hexa-oxygenated
bibenzyl, containing a hydroxy and five
methoxy substituents. The 13C NMR
resonances at d 153.0 (C-30 and C-50) and
136.3 (C-40) suggested that ring B of 9 was
tri-methoxylated at C-30, C-40, and C-50
positions, similar to that of brittonin A (10)
[10] (Figure 2). Thus, two methoxy groups
and a hydroxyl group remained to be
placed on ring A. The structure of ring A
was unsymmetrical, as evidenced by the
different chemical shifts of the aromatic
protons at d 6.21 (H-2, 1H, s) and 6.46
(1 ! 2)-b-D-xylopyranosidic
moiety
connecting to the C-3 of the flavonol
skeleton through an ether linkage, as
observed in 2. Additional 13C NMR signals
were observed for the xylose unit at d 76.9
(C-200), 76.8 (C-300), 69.6 (C-400), and 66.0
(C-500), and for the rhamnose at d 70.5
(C-2000), 70.6 (C-3000), 71.8 (C-4000), 68.3
(C-5000), and 17.4 (C-6000). In support of the
proposed structure, C-1000 of the rhamnose
showed HMBC correlation with H-200 (d
3.54, t, J ¼ 8.0 Hz) of the xylose, and H-100
exhibited 3J-correlation with C-3 (d 132.8)
(Figure 3). In addition, acid hydrolysis of 1
yielded D-xylose and L-rhamnose. Based
on the above spectroscopic properties and
chemical evidence, 1 was characterized as
quercetin-3-O-a-L-rhamnopyranosyl-
(1 ! 2)-b-D-xylopyranoside.
Table 2. 1H (300 MHz) and 13C NMR
(75 MHz) spectral data of 9 (CDCl3).
OH
Position
1H
13C
3'
OH
2'
1
2
3
4
5
6
137.8
107.9
149.1
133.7
152.0
104.5
38.1
4'
6.21 s
7
O
O
9
HO
2
10
3
6.46 s
2.80 br s
2.80 br s
O
5
a
a0
38.1
OH
10
137.3
105.5
153.0
136.3
153.0
105.5
–
55.7
60.8
56.0
60.8
4''
5''
O
20
6.35 s
HO
HO
2''
30
1''
40
3''
O
5'''
50
6'''
1'''
60
6.35 s
5.76 s
3.79 s
3.84 s
3.81 s
3.80 s
O
H3C
HO
3'''
2'''
5-OH
3-OMe
4-OMe
30,50-OMe
40-OMe
4'''
OH
OH
Figure 3. Key HMBC correlations observed
for 1 (H ! C).

Journal of Asian Natural Products Research
751
KB and NCI-H187 cells, and moscatilin (8)
was the most potent compound, exhibiting
IC50 values of 2.2 and 10.5 mM, respect-
ively. Moscatilin has been reported from
several species of Dendrobium, and has
recently shown to possess antitumor
activity in mice [14]. When the cytotoxi-
cities against KB and NCI-H187 cancer
cells of 4–11 were compared, the magni-
tudes of the activity were found to be in the
order 8 . 5 . [11 < 7 < 6] . 4. This
implies that both the number and the
position of the OH and OMe groups were
critical for their cytotoxicities. As seen in
moscatilin (8), optimal activity is obtained
when the bibenzyl is penta-oxygenated,
with two OH groups placed at the para-
position and three OMe groups placed at
the meta-position of the aromatic rings.
Any deviation from this structural arrange-
ment seems to result in decrease or loss of
activity.
OMe
3'
H
2'
OMe
OMe
H
2
4'
5'
B
α
1'
α'
MeO
MeO
1
3
6'
A
6
H
4
H
5
HO
Figure 4. Key NOESY correlations observed
for 9 (H $ H).
(H-6, 1H, s). Therefore, the two methoxy
groups should be adjacent and located at
C-30 and C-40 positions, leaving the
hydroxy group to be placed at C-50
position. The proposed structure of 9 was
confirmed by the NOESY correlations
observed between OH-5 and MeO-4, and
between H-2 and MeO-3 protons
(Figure 4). Thus, the structure of 9
was determined as 5-hydroxy-3,4,30,40,50-
pentamethoxybibenzyl.
To conclude, a new glycosidic flavonol
(1) and a new bibenzyl (9) were isolated
from D. capillipes and D. secundum.
Seven known bibenzyls and five flavonoid
glycosides were also identified. Some of
the bibenzyls showed cytotoxicity, but all
of the flavonol glycosides were devoid of
activity.
All of the isolated compounds were
evaluated for cytotoxicity against three
types of cancer cells including KB, NCI-
H187, and MCF-7 (Table 3). The flavonoid
glycosides (1–3, 12–14) were inactive
(showing ,50% inhibition at concen-
tration of 50 mg/ml). Some of the bibenzyls
(Figure 2) showed cytotoxic effects against
Table 3. IC50 values (mM) for cytotoxicity of isolated bibenzyls.
Compound
KB
NCI-H187
MCF-7
Chrysotobibenzyl (4)
Crepidatin (5)
Gigantol (6)
Chrysotoxine (7)
Moscatilin (8)
5-Hydroxy-3,4,30,40,50-pentamethoxybibenzyl (9)
Brittonin A (10)
4,5,40-Trihydroxy-3,30-dimethoxybibenzyl (11)
132.4
14.4
61.9
60.5
2.2
Inactive
Inactive
48.3
1.8
123.7
13.7
71.6
65.6
10.5
87.8
Inactive
63.8
1.4
Inactivea
Inactive
67.8
Inactive
Inactive
Inactive
Inactive
62.6
Ellipticine
Doxorubicin
Tamoxifen
–
11.2
14.0
0.6
–
0.08
–
a Less than 50% inhibition at concentration of 50 mg/ml.

752
T. Phechrmeekha et al.
(DC-082553 and DS/BS-092552) have
3. Experimental
been deposited at the Department of
Pharmacognosy and Pharmaceutical Bot-
any, Faculty of Pharmaceutical Sciences,
Chulalongkorn University.
3.1. General experimental procedures
Optical rotations were measured on a
Perkin Elmer 341 Polarimeter (Perkin-
Elmer, Waltham, MA, USA). UV spectra
were obtained on a Shimadzu UV-160A
UV/VIS spectrophotometer (Shimadzu,
Kyoto, Japan) and IR spectra on a
Perkin-Elmer FT-IR 1760X spectropho-
tometer (Norwalk, Fairfield, CT, USA).
NMR spectra were obtained with a Bruker
Avance DPX-300 FT-NMR spectrometer
(Bruker, Rheinstetten, Germany) or a
JEOL JMN-A 500 NMR spectrometer
(JEOL, Tokyo, Japan). Mass spectra were
recorded on a Bruker microTOF mass
spectrometer (Bruker Daltonics, Bremen,
Germany) or a Micromass LCT mass
spectrometer (Micromass, Manchester,
UK). Diaion HP20SS (Mitsubishi Chemi-
cal, Tokyo, Japan) was used for initial
fractionation. Vacuum liquid column
chromatography (VLC) and flash column
chromatography (FCC) were performed
with silica gel 60 (70–230 mesh, Merck,
Darmstadt, Germany), and silica gel 60
(230–400 mesh, Merck), respectively.
Sephadex LH-20 (25–100 mm, Pharmacia
Fine Chemical, Uppsala, Sweden) was
used for separation of polar constituents.
The authentic sugars comprised L-arabi-
nose (Fluka, Buchs, Switzerland), D-
xylose (Merck), and L-rhamnose (Fluka).
3.3. Extraction and isolation
3.3.1. D. capillipes
The dried and powdered whole plant
(1.7 kg) was macerated with MeOH
(3 £ 5 liter) to give a methanol extract
(236 g) after removal of the solvent. The
extract was then fractionated by VLC
(silica gel; step gradient of n-hexane–
EtOAc–CH2Cl2–MeOH) to yield seven
fractions (I–VII).
Fraction IV (2.6 g) was separated by
VLC (silica gel; gradient of n-hexane–
EtOAc) to give 13 fractions (IV-A to
IV-M). Fraction IV-J (576 mg) was further
separated by FCC (silica gel; petroleum
ether–acetone, 8:2) to yield five fractions
(IV-J1 to IV-J5). Fraction IV-J3 (77 mg)
was purified on Sephadex LH-20 (acetone)
to give 4 (47 mg), whereas fraction IV-J4
(246 mg) was purified on Sephadex LH-20
(CH2Cl2–MeOH, 1:1) to give 5 (209 mg).
Fraction IV-L (97 mg) was purified by
FCC (silica gel; CH2Cl2–acetone, 98:2) to
give 6 (34 mg).
Fraction V (4.7 g) was separated by
VLC (silica gel; gradient of n-hexane–
EtOAc–CH2Cl2–MeOH) to give 11 frac-
tions (V-A to V-K). Fraction V-I (897 mg)
was separated by FCC (silica gel; gradient
of CH2Cl2–acetone) to yield nine fractions
(V-I1 to V-I9). Fraction V-I2 (283 mg) was
separated on Sephadex LH-20 (acetone) to
give 7 (122 mg) and 8 (117 mg).
3.2. Plant material
The fresh whole plants of D. capillipes
Rchb.f. and D. secundum (Blume) Lindl.
were purchased from Jatujak market,
Bangkok, in September 2009. Botanical
identification was done through compari-
son of the specimens with authentic
samples (BKF Nos 114946 and 110498
for D. capillipes and D. secundum,
respectively) at the Department of
National Park, Wildlife and Plant Con-
servation, Ministry of Natural Resources
and Environment. Voucher specimens
Fraction VII (50 g) was separated on
Diaion HP20SS (gradient of H2O–
MeOH–EtOAc) to give 11 fractions
(VII-A to VII-K). Fraction VII-I
(289 mg) was separated on Sephadex LH-
20 (MeOH) to yield four fractions (VII-I1
to VII-I4). Fraction VII-I2 (67 mg) was
purified by FCC (silica gel; EtOAc–
MeOH–H2O, 9.6:0.3:0.1) to give 2

Journal of Asian Natural Products Research
753
(46 mg). Fraction VII-J (212 mg) was
further separated on Sephadex LH-20
(MeOH) to yield six fractions (VII-J1 to
VII-J6). Fraction VII-J4 (24 mg) was
purified by FCC (silica gel; EtOAc–
MeOH–H2O, 9.25:0.5:0.25) to give 3
(9 mg). Compound 1 (4 mg) was obtained
from fraction VII-J6 (16 mg) after purifi-
cation by FCC (silica gel; EtOAc–
MeOH–H2O, 9.4:0.3:0.3).
G4k). Fraction G4e (58 mg) was separated
by repeated FCC over silica gel (n-hexane–
EtOAc, 4:1) to give 11 (5 mg).
Fraction H (30 g) was separated on
Diaion HP20SS (H2O–MeOH and
MeOH–EtOAc gradient) to give 13 frac-
tions (H1–H13). Fraction H7 (469 mg) was
then subjected to FCC over silica gel
(EtOAc–MeOH–H2O, 9.6:0.2:0.2) to give
10 fractions (H7a–H7j). Fraction H7e
(33 mg) was purified on Sephadex LH-20
(MeOH) to afford 12 (8 mg). Fraction H7f
(39 mg) was purified by FCC (silica gel;
CH2Cl2–acetone, 8:2) to give four frac-
tions (H7f1–H7f4). Fraction H7f2 (14 mg)
was further purified on Sephadex LH-20
(MeOH) to give 13 (3 mg). Fraction H8
(39 mg) was subjected to FCC over silica
gel (CH2Cl2–acetone polarity gradient) to
give four fractions (H8a–H8d). Fraction
H8c was purified on Sephadex LH-20
(MeOH) to yield 14 (3 mg).
3.3.2. D. secundum
The dried and powdered whole plant
(1.6 kg) was macerated with MeOH
(3 £ 10 liter). The MeOH extract was
concentrated under vacuum to give a crude
extract (206g), which was subsequently
separated by VLC (silica gel; gradient of n-
hexane–EtOAc and CH2Cl2–MeOH) to
yield eight fractions (A–H).
Fraction F (2.3 g) was subjected to
FCC on silica gel (n-hexane–EtOAc
gradient) to give 11 fractions (F1–F11).
Fraction F3 (230 mg) was further separ-
ated on Sephadex LH-20 (CH2Cl2 –
MeOH, 1:1) to give five fractions (F3a–
F3e). Fraction F3b (184 mg) was further
purified by FCC (silica gel; CH2Cl2–
MeOH, 99.5:0.5) to afford 10 (110 mg).
Fraction F5 (381 mg) was further separ-
ated on Sephadex LH-20 (CH2Cl2 –
MeOH, 1:1) to give five fractions (F5a–
F5e). Fraction F5d (313 mg) was further
subjected to repeated FCC over silica gel
(CH2Cl2–acetone, 98:2) to give 9 (61 mg).
Fraction G (16.8 g) was separated by
VLC over silica gel (CH2Cl2 –MeOH
gradient) to give six fractions (G1–G6).
Fraction G3 (2.7 g) was separated by FCC
over silica gel (n-hexane–EtOAc, 7:3) to
give seven fractions (G3a–G3g). Fraction
G3d (167 mg) was further separated on
Sephadex LH-20 (MeOH) to afford seven
fractions (G3d1–G3d7). Fraction G3d3
(88 mg) was separated by FCC (CH2Cl2) to
yield 8 (37 mg), and fraction G4 (1.3 g) was
separated by FCC over silica gel (CH2Cl2–
acetone, 97:3) to give 11 fractions (G4a–
3.3.2.1. Quercetin-3-O-a-L-rhamnopyr-
anosyl-(1 ! 2)-b-D-xylopyranoside (1).
20
Yellow-greenish amorphous solid; ½aꢀD
267.6 (c 0.037, MeOH); UV (MeOH)
l
max nm (log 1): 215 (4.30), 256 (4.27), and
346 (4.17); IR (KBr) n cm21: 3349, 1660,
1607, 1504, 1361, 1200, 1075, and1040; 1H
and 13C NMR spectral data: see Table 1;
HR-ESI-MS: m/z 603.1388 [M þ Na]þ
(calcd for C26H28O15Na, 603.1326).
3.3.2.2. 5-Hydroxy-3,4,30,40,50-penta-
methoxybibenzyl (9). White powder; UV
(MeOH) lmax nm (log 1): 215 (4.46), 266
(3.19); IR (KBr) n cm21: 3416, 1591,
1461, 1349, and 1238; for 1H and 13C
NMR spectral data, see Table 2. HR-ESI-
MS: m/z 371.1469 [M þ Na]þ (calcd for
C19H24O6Na, 371.1471).
3.4. Acid hydrolysis of 1
Compound 1 (3 mg) was dissolved in 2 N
HCl (2 ml) and refluxed at 1008C for 3 h.
After removal of the solvent, the hydro-

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T. Phechrmeekha et al.
lysate was redissolved in H2O (10 ml) and
extracted with EtOAc (3 £ 10 ml). The
residue obtained from the aqueous layer
after evaporation of the solvent was
redissolved in methanol to give a sample
solution. This sample and standards of the
sugars L-arabinose, D-xylose, and L-
rhamnose were examined on a normal
phase TLC plate, with CHCl3–MeOH–
H2O (5:4:1) as the mobile phase. To
visualize the spots, the TLC plate was
sprayed with 0.5% anisaldehyde solution,
followed by heating at 1008C for 2 min.
The Rf values for L-arabinose, D-xylose,
and L-rhamnose were found to be 0.44,
0.38, and 0.58, respectively.
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Acknowledgments
T.P. is grateful to the Graduate School of
Chulalongkorn University for a research grant.
Thanks are also due to the Research Instrument
Center of Faculty of Pharmaceutical Sciences,
Chulalongkorn University, for providing
research facilities.