A novel 10-hydroxycamptothecin-glucoside from the fruit of Camptotheca acuminata

Article abstract of DOI:10.1080/14786419.2015.1107057

Glycosides were isolated from the fruit of Camptotheca acuminata and identified using NMR, MS, UV and IR spectrometries. 10-O-(1-β-D-glycosyl) camptothecin (1) was identified for the first time in a natural material. In addition, compounds 2-4 were firstly reported from the fruits of C. acuminata and indentified as syringaresinol-4, 4′-O-bis-β-D-glucoside (2), hyperoside (3) and pumiloside (4), respectively. Two known compounds, vincoside-lactam (5) and strictosidinic acid (6), were also obtained.

Full text of DOI:10.1080/14786419.2015.1107057

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A novel 10-hydroxycamptothecin-glucoside from
the fruit of Camptotheca acuminata
Qun Guo & Qiaoyu Yuan
To cite this article: Qun Guo & Qiaoyu Yuan (2015): A novel 10-hydroxycamptothecin-
glucoside from the fruit of Camptotheca acuminata, Natural Product Research, DOI:
10.1080/14786419.2015.1107057
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Date: 28 November 2015, At: 00:38

Natural Product research, 2015
ꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2015.1107057
ꢀ
A novel 10-hydroxycamptothecin-glucoside from the fruit of
Camptotheca acuminata
Qun Guo and Qiaoyu Yuan
dꢅpꢆꢄꢁmꢅnꢁ ꢃf Pꢀꢆꢄmꢆꢇꢅꢈꢁiꢇꢆꢉ tꢅꢇꢀnꢃꢉꢃgy, cꢃꢉꢉꢅgꢅ ꢃf Biꢃꢉꢃgiꢇꢆꢉ enginꢅꢅꢄing, Wꢈꢀꢆn Pꢃꢉyꢁꢅꢇꢀniꢇ,
Wꢈꢀꢆn, cꢀinꢆ
ABSTRACT
ARTICLE HISTORY
rꢅꢇꢅivꢅꢂ 14 apꢄiꢉ 2015
aꢇꢇꢅpꢁꢅꢂ 2 oꢇꢁꢃbꢅꢄ 2015
Glycosides were isolated from the fruit of Camptotheca acuminata
and identified using NMR, MS, UV and IR spectrometries. 10-O-(1-β-D-
glycosyl) camptothecin (1) was identified for the first time in a natural
material. In addition, compounds 2–4 were firstly reported from the
fruits of C. acuminata and indentified as syringaresinol-4, 4′-O-bis-β-
D-glucoside (2), hyperoside (3) and pumiloside (4), respectively. Two
known compounds, vincoside-lactam (5) and strictosidinic acid (6),
were also obtained.
KEYWORDS
Camptotheca acuminata;
gꢉyꢇꢃꢊiꢂꢅ; 10-o-(1-β-d-
gꢉyꢇꢃꢊyꢉ) ꢇꢆmpꢁꢃꢁꢀꢅꢇin;
ꢊyꢄingꢆꢄꢅꢊinꢃꢉ-4, 4′-o-biꢊ-β-d-
gꢉꢈꢇꢃꢊiꢂꢅ
1
. Introduction
Camptotheca acuminata Decne, a plant found only in China, is a deciduous tree of the
Nyssaceae family. This plant is the source of a natural quinoline alkaloid, 10-hydroxycamp-
tothecin (HCPT). It has been reported that 10-hydroxycamptothecin has antitumour activity
(
inhibition of Topo I) with clinical applications in cancer therapeutics (Huang et al. 2003;
Zhang 2005). 10-hydroxycamptothecin is also the precursor for the synthesis of several
other anticancer drugs, such as topotecan (Kingsbury & Boehm 1991), 9-nitrocamptothecin
(
1
9-NC) and 9-aminocamptothecin (9-AC) (Cabri et al. 1995). Because only a trace amount of
0-hydroxycamptothecin is present in C. acuminata (Wang & Lin 2005), there is a need for
additional sources of 10-hydroxycamptothecin.
In this study, six glycosides were obtained from the fruits of C. acuminata and were identi-
1
13
fied using NMR (including H NMR, C NMR, DEPT, HSQC, HMBC, COSY and NOESY), MS, UV and
IR spectrometries. A glucose derivative of 10-hydroxycamptothecin, 10-O-(1-β-D-glycosyl)
CONTACT Qꢈn Gꢈꢃ
gꢈꢃqꢈn19@163.ꢇꢃm
sꢈppꢉꢅmꢅnꢁꢆꢉ ꢂꢆꢁꢆ fꢃꢄ ꢁꢀiꢊ ꢆꢄꢁiꢇꢉꢅ ꢇꢆn bꢅ ꢆꢇꢇꢅꢊꢊꢅꢂ ꢆꢁ ꢀꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2015.1107057.
©
2015 tꢆyꢉꢃꢄ & Fꢄꢆnꢇiꢊ

2
Q. GUO AND Q. YUAN
camptothecin (compound 1), was isolated and identified for the first time from nature. This
represents a potential new source of 10-hydroxycamptothecin. This finding is potential to
increase the production of 10-hydroxycamptothecin in C. acuminata if a simple hydrolysis
reaction is used. In addition, we identified syringaresinol-4,4′-O-bis-β-D-glucoside, hypero-
side and pumiloside (compounds 2–4, respectively). Syringaresinol-4,4′-O-bis-β-D-glucoside
has never been reported in extracts from C. acuminata. Hyperoside and pumiloside have not
been previously found in its fruit. Two known compounds, vincoside-lactam (compound 5)
and strictosidinic acid (compound 6), were also obtained from the extract (Figure 1).
2
. Results and discussion
Compound 1 was obtained as a white flocculent crystal. The ESI-MS showed a quasimolecular
+
ion peak at m/z 527.0 [M + H] , indicating a molecular weight of 526. The fragment ion at m/z
+
3
65.0 [M-162 + H] suggested that compound 1 is a glycoside of hydroxycamptothecin (molec-
ular weight of 364 and formula of C H N O ) with a six-carbon glycosyl unit (C H O ). The
2
0
16
2
5
6
10
5
+
fragment ion at m/z 321.1 [M + H-162–44] was generated by the loss of 44 (CO ) atomic mass
2
units, implying a lactone ring structure. The HR-ESI-MS of compound 1 exhibited a molecular
+
ion peak at m/z 527.173[M + H] , supporting a molecular formula of C H N O {calculated for
26
26
2
10
+
−1
[
M (C H N O )+H ]: 527.167}. IR spectroscopy showed absorption at 3392 cm for hydroxyl
groups, 1740 cm for the carbonyl of a lactone ring, 1655 cm for the carbonyl of lactam, 1590
and 1504 cm for an aromatic ring. The C NMR spectrum showed 26 carbon signals, of which
were assigned to glucose and the remaining 20 were assigned to the aglycone. Except for the
glucose signals, the C NMR spectrum of compound 1 is similar to that of 10-hydroxycamp-
26 26 2 10
−1
−1
−1
13
6
13
1
tothecin (Xu et al. 2005). Detailed analyses of the H NMR spectrum showed that compound
1
does not have a C10-OH signal, which is at δ 10.34 in 10-hydroxycamptothecin. Apart from
1
the glucose residue signals, the other H NMR data for compound 1 were very close to those
of 10-hydroxycamptothecin (Xu et al. 2005). The glucosyl unit was placed at C-10 based on an
HMBC correlation from the anomeric proton (H-1′, δ 5.13) to the C-10 (δ 156.4). The magnitude
of the coupling constant of the H-1′ signal (J = 7.8 Hz, in CD OD) defined the anomeric carbon
3
1
1
figuration for glucose as β(Podlasek et al. 1995). HSQC, HMBC, H- H COSY and NOESY correlations
1
13
allowed for the complete assignments of the H and C-NMR signals of compound 1 (10-O-(1-β-
D-glycosyl) camptothecin). The retention time of the peak in the HPLC of the derivative of sugar
obtained after acid hydrolysis of compound 1 coincided with the derivative of D-glucose. The
13
MP, UV and C NMR spectroscopic data for 10-O-(1-β-D-glycosyl) camptothecin are reported
herein for the first time, and the H NMR spectrum signals were assigned according to HSQC
1
and HMBC. Compound 1 is determined as 10-O-(1-β-D-glycosyl) camptothecin. It was obtained
from nature for the first time.
Compound 2 was obtained as a white powder. The ESI-MS showed a quasimolecular
+
2
ion peak at m/z 765 [M + Na] , indicating a molecular weight of 742. The +MS (765) had
+
3
a fragment ion at m/z 603 [M-162 + Na] and + MS (765→603) had a fragment ion at m/z
41 [M-2 × 162 + Na] , implying that compound 2 contained two hexose units. The aglucon
weight of compound 2 is 418. The C NMR spectrum showed only 14 carbon signals, indicat-
ing that compound 2 has a highly symmetrical structure. According to the H and C NMR
spectroscopic data, the molecular formula of 2 was determined to be C H O . The structure
+
4
13
1
13
34 46 18
was identified as syringaresinol-4,4′-O-bis-β-D-glucoside by comparing the spectroscopic

NATURAL PRODUCT RESEARCH
3

4
Q. GUO AND Q. YUAN
data with the values in the literature (Gu et al. 2005; Zhang et al. 2008). Syringaresinol-4,4′-
O-bis-β-D-glucoside has not been previously isolated from C. acuminata.
+
Compound 3 was obtained as a yellowish powder. The ESI-MS m/z 487 (M + Na) and 325
+
(
M-162 + Na) indicated a molecular weight of 464 and a glycoside. The structure of 3 was
elucidated by IR, MS, and NMR spectroscopies, and the spectroscopic data were compared
with data from the literature (Dai et al. 2004; Wang et al. 2004). In addition, the HPLC reten-
tion time of 3 was consistent with that of an authentic hyperoside sample. Compound 3
was thus identified as hyperoside.
Compound 4 was obtained as a white flocculent crystal. It has a molecular weight of 512,
+
as determined by the ESI-MS at m/z: 511.7[M-H] . The structure of 4 was elucidated by IR, MS
and NMR spectroscopies, and the spectroscopic data were compared with data reported in
the literature (Carte’et al. 1990). Compound 4 was identified as pumiloside.
Compound 5 was elucidated by IR, MS, NMR spectrometries and comparison of its spec-
troscopic data with those reported in the literature (Yin & Hu 2005). Compound 5 was iden-
tified as vincoside-lactam.
Compound 6 was elucidated by IR, MS, NMR spectrometries. The assignments of C-2
and C-19 were corrected base on DEPT-experiment comparing with those reported in the
literatures (Hamzah et al. 1994). Compound 6 was identified as strictosidinic acid.
3
. Experimental
3
.1. Plant material
The fruits of C. acuminata Decne were collected from the mountainous area of Enshi autono-
mous prefecture, located in the southwest of Hubei province in China. The research materials
came from cultivated plants. The specimens were carefully identified by Professor Chen Keli
(
Hubei University of Chinese Medicine, China), and the voucher specimens (C200910) were
deposited in College of Biological Engineering, Wuhan Polytechnic.
3
.2. Extraction and isolation
The dried fruits of C. acuminata Decne (1.9 kg) were crushed into coarse powder and extracted
with 50% ethanol at 80 °C. After removing the alcohol with a rotary evaporator, the con-
centrated extract were loaded onto X-5 styrene type non-polar macroporous resin column
2
−1
(
surface area is 500–600 m g ) and eluted with discontinuous gradients of 30, 50 and 70%
ethanol. The 50% ethanol elution fraction was then loaded onto a CT-4S polystyrene-type
macroporous adsorption resin column (particle size 0.3–1.2 mm, Nankai Hecheng, China),
washed with 30% ethanol to remove water-insoluble impurities, and eluted with 50% ethanol
in 6 fractions of 500 mL. After thin layer chromatography, the fractions containing similar
spots were combined. After removing alcohol with a rotary evaporator, the concentrated
eluates were purified using methanol–acetone precipitation. Compound 2 (approximately
5
0 mg) and 3 (approximately 60 mg) were directly obtained. The purities of compounds 2
and 3 were estimated to be > 94% by HPLC-UV analysis. Compounds 4–6 (approximately
5 mg) and 1 (approximately 38 mg) were obtained by further separation and purification
4
by preparative HPLC. The purities of the four compounds were estimated to be > 97% based
on analytical HPLC.

NATURAL PRODUCT RESEARCH
5
3
.3. Chromatographic and spectroscopic methods
Preparative HPLC was carried out on a P270 preparative HPLC system (Chinese Dalian Elite)
with ZORBAX Eclipse XOB-C18 Prep HT 21.2 × 150 mm 5 μm column (Agilent). HPLC was
®
carried out on a LC-20AT HPLC (Shimadzu) with an Inertsil ODS-SP 4.6 × 150 mm 5 μm
column (Shimadzu).
Melting points were determined on a microscopic melting point determination apparatus
(
Reichert). UV spectra were recorded on a UV-1800 spectrophotometer (Shimadzu), and IR
spectra were recorded on a VERTEX70 FT-IR spectrometer (Bruker, Germany). MS and HRMS
were obtained with 1100 LC/MSD Trap XCT (Agilent, USA) and a 4800 Plus MALDI TOF/TOF™
1
13
Analyzer. H and C NMR spectra, HSQC and HMBC were recorded on a Unity-Inova 600 or
Mercury VX-300 superconducting magnetic resonance spectrometer (Varian, USA). 1H-1H
COSY and NOESY were recorded on Bruker Avance III 600 NMR spectrometer equipped with
a TCI cryoprobe.
3
.4. Characterisation
1
0-O-(1-β-D-glycosyl) camptothecin (1): white flocculent crystal, mp 228–232 °C,
−
1
tR = 4.2 min (HPLC, CH CN/H O, 18:82, 1 mL min , 256 nm); ESI-MS m/z:+MS: 527.0
3
2
+
+
+
+
[
M + H] , 365.0 [M-162 + H] , 321.1 [M-162-CO2 + H] , 1052.8 [2 M + H] ; HR-ESI-MS:
m/z = 527.173; UV (MeOH) λmax: 221 nm, 263 nm, 293 nm, 311 nm, 328 nm, 364 nm,
−
1
3
1
78 nm; IR (KBr) υmax (cm )=3392 (broad), 2926, 1740, 1655, 1622, 1590, 1504, 1370,
1
240, 1212, 1164, 1075, 1047; H NMR (600 MHz, DMSO-d6): δ 5.26 (2H, s, H-5), 8.53 (1H,
s, H-7), 7.64 (1H, s, H-9), 7.57 (1H, d, J = 9 Hz, H-11), 8.08 (1H, d, J = 9 Hz, H-12), 7.28 (1H,
s, H-14), 5.42 (2H, s, H-17), 0.88 (3H, t, J = 7.2 Hz, H-18), 1.86 (2H, m, H-19), 5.13 (1H, d,
J = 4.2 Hz, Glc-1′), 3.50–3.24 (4H, m, Glc-2′, 3′, 4′, 5′), 3.75(1H, m) and 3.52(1H, m) (Glc-
6
′), 6.54 (1H, s, 20-OH), 4.659 (1H, t, J = 5.4, 6′-OH), 5.484 (1H, s), 5.199 (1H, s) and 5.127
1
13
(
(
1H, s) (2′,3′,4′-OH); H NMR (600 MHz, CD OD): δ 5.11 (1H, d, J = 7.8 Hz, Glc-1′); C NMR
600 MHz, DMSO-d6): δ 151.12 (C-2), 146.03 (C-3), 50.67 (C-5), 130.59(C-6), 130.85 (C-7),
3
1
29.47 (C-8), 110.79 (C-9), 156.38 (C-10), 123.45 (C-11), 130.79 (C-12), 144.66 (C-13), 96.67
(
C-14), 150.44 (C-15), 118.93 (C-16), 65.66 (C-17), 8.21 (C-18), 30.64(C-19), 72.82 (C-20),
1
7
72.99 (C-21), 157.24 (C-22), 100.57 (Glc-1′), 73.63 (Glc-2′), 77.62 (Glc-3′), 70.04 (Glc-4′),
7.07 (Glc-5′), 61.05 (Glc-6′).
Syringaresinol-4,4′-O-bis-β-D-glucoside (2): White powder (methanol-acetone), mp 255–
−1
2
57 °C. tR = 4.18 min (HPLC, CH CN/H O, 18:82, 1 mL min , 256 nm), ESI-MS m/z:+ MS:765
3 2
+
2
+
3
+
[
[
M + Na] , + MS (765): 603 [M-162 + Na] , + MS (765→603): 441 [M-2 × 162 + Na] , 185
+ − 2 -
162 + Na] , 235;-MS:777 [M + Cl] , -MS (777): 579 [M-H-162] , 417 [M-2 × 162-H]. UV (MeOH)
−1
λmax: 212 nm, 272 nm; IR (KBr) υmax (cm ): 3393 (-OH), 2923 (CH , CH ), 2864, 1595, 1508,
2
3
1
1464, 1422, 1379, 1336, 1237, 1132, 1074, 1024, 993. H NMR (600 MHz, D O): δ 6.69 (4H, s,
2
H-2, 2′, 6, 6′), 4.78 (2H, s, H-7, 7′), 3.22 (2H, m, H-8, 8′), 3.88 (2H,d, J = 7.8 Hz, H-9a, 9′a), 4.22
(
(
5
1
2H, dd, J = 7.2,7.2 Hz, H-9e, 9′e), 3.76 (12H, s, OCH ), 4.90 (2H, d, J = 7.2 Hz, Glc-1″, 1‴), 3.18
3
2H, s), 3.36 (2H, dd) and 3.43 (4H, m) (Glc-2″, 2‴, 3″, 3‴, 4″, 4‴, 5″, 5‴), 3.58 (2H, dd, J = 12.6,
1
3
.4 Hz, Glc-6″, 6‴), 3.69 (2H,d, J = 12 Hz, Glc-6″, 6‴). C NMR (600 MHz, D O): δ 138.5 (C-1,
2
′), 104.5 (C-2, 2′, 6, 6′), 153.0 (C-3, 3′, 5, 5′), 133.5 (C-4, 4′), 86.1 (C-7, 7′), 53.6 (C-8, 8′), 72.1
(
5
C-9, 9′), 56.8 (OCH ), 103.4 (C-1’ ’, 1‴), 74.1 (C-2″, 2‴), 76.7 (C-3″, 3‴), 69.8 (C-4″, 4‴), 76.1 (C-5″,
3
‴), 61.0 (C-6″, 6‴).

6
Q. GUO AND Q. YUAN
The characterisation of hyperoside (3), pumiloside (4), vincoside-lactam (5) and stricto-
sidinic acid (6) are reported in the supplementary material.
3
.5. Acid hydrolysis and derivatisation of sugar
−1
Compound 1 (2 mg) was dissolved in 2 mL of CF COOH (4 mol L ) and was reacted for 3 h
3
at 95 °C. The reaction solution was extracted with CH Cl three times to remove impurities,
2
2
and the aqueous layer was blown dry. The hydrolysate (from the aqueous layer), D-glucose
and D-galactose (every 2 mg) were, respectively, put into three small flasks, and were added
pyridine (0.5 mL) and L-cysteine methyl ester hydrochloride (1 mg), respectively. These three
samples were heated at 60 °C for 60 min. Then, o-tolyl isothiocyanate (5 μl) was added to the
reaction mixtures and further reacted at 60 °C for 60 min. Then, the reaction mixture was
−1
analysed by C HPLC (acetonitrile–water 25–75, 0.8 mL min , 35 °C) and detected by a UV
1
8
detector (at 250 nm) (Takashi et al. 2007). The reaction mixture of compound 1 exhibited
a peak at tR = 10.1 min and D-glucose and D-galactose derivatives at tR = 10.1 min and
tR = 9.1 min.
4
. Conclusion
In order to find new valuable compounds from the plant C. acuminata, we used a macropo-
rous resin chromatography column and preparative HPLC, and the spectroscopy techniques
of NMR, MS, IR and UV, to isolate and identify six glycosides from the fruit of C. acuminate.
10-O-(1-β-D-glycosyl) camptothecin was isolated from nature for the first time and represents
a new potential source for 10-hydroxycamptothecin, which is an important compound with
anticancer activity. Syringaresinol-4,4′-O-bis-β-D-glucoside was extracted from this plant
for the first time. Hyperoside and pumiloside were also extracted from the fruit of this plant
for the first time.
Disclosure statement
No potential conflict of interest was reported by the authors.
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