New grayanol diterpenoid and new phenolic glucoside from the flowers of Pieris formosa

Article abstract of DOI:10.1080/10286020903436519

A new grayanol diterpenoid, grayanotoxin XXII (1), and a new phenolic glucoside, benzyl 2-hydroxy-4-O-[-xylopyranosyl(1″→6′)-β- glucopyranosyl]-benzoate (2), were isolated from the flowers of Pieris formosa. Their structures were determined on the basis of spectroscopic analysis and chemical methods.

Full text of DOI:10.1080/10286020903436519

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New grayanol diterpenoid and new
phenolic glucoside from the flowers of
Pieris formosa
Wei-Guang Wang a , Hong-Mei Li a , Hai-Zhou Li a , Zhao-Yuan Wu a
& Rong-Tao Li a
a The College of Life Science and Technology , Kunming University
of Science and Technology , Kunming, 650224, China
Published online: 01 Feb 2010.
To cite this article: Wei-Guang Wang , Hong-Mei Li , Hai-Zhou Li , Zhao-Yuan Wu & Rong-Tao Li
(2010) New grayanol diterpenoid and new phenolic glucoside from the flowers of Pieris formosa ,
Journal of Asian Natural Products Research, 12:1, 70-75, DOI: 10.1080/10286020903436519
To link to this article: http://dx.doi.org/10.1080/10286020903436519
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Journal of Asian Natural Products Research
Vol. 12, No. 1, January 2010, 70–75
ORIGINAL ARTICLE
New grayanol diterpenoid and new phenolic glucoside from
the flowers of Pieris formosa
Wei-Guang Wang, Hong-Mei Li, Hai-Zhou Li, Zhao-Yuan Wu and Rong-Tao Li*
The College of Life Science and Technology, Kunming University of Science and Technology,
Kunming 650224, China
(Received 18 August 2009; final version received 23 October 2009)
A new grayanol diterpenoid, grayanotoxin XXII (1), and a new phenolic glucoside,
benzyl 2-hydroxy-4-O-[b-xylopyranosyl(100 ! 60)-b-glucopyranosyl]-benzoate (2),
were isolated from the flowers of Pieris formosa. Their structures were determined
on the basis of spectroscopic analysis and chemical methods.
Keywords: grayanol diterpenoid; phenolic glucoside; Pieris formosa; Ericaceae
1. Introduction
Pieris formosa (Wall) D. Don, a well-
known poisonous plant, is distributed
mainly in hilly and valley regions of
south and southwest China. Poultry have
been reported to go into coma after
accidentally eating leaves or stems of this
plant. In folk practice, the juice of the fresh
leaves of P. formosa can be used as
insecticide or lotion for the treatment of
ring worm and scabies [7]. In previous
investigations, a number of grayanane and
leucothane diterpenes have been isolated
and identified from this plant [8–12].
Many species of the family Ericaceae
contain diterpenoids possessing several
specialized carbon skeletons with highly
oxygenated functionalities, which results
in the toxicity of such species. These
diterpenoids occur mainly in the genera
Kalmia, Leucothoe, Lyonia, Pieris, and
Rhododendron. Their structures include
four types: (1) the grayanane-type with a
5/7/6/5 ring system; (2) the leucothane-
type with a 6/6/6/5 consecutive carbo-
cycle; (3) the 10/6/5 carbon ring grayanol-
type; and (4) the kalmane-type with a
5/8/5/5 ring system [1]. It seems that all
structural types were biosynthetically
related to each other and derived from
the ent-kaurane diterpenoids [2]. Some of
these diterpenoids possess significant
biological properties, such as potent
acute toxicity in mammals [3,4], antifee-
dant, growth inhibitory, and insecticidal
activities [5,6].
In the course of searching for diter-
penoids with structural diversity and
biological importance, a new grayanol
diterpenoid, grayanotoxin XXII (1), and a
new phenolic glucoside, benzyl 2-
hydroxy-4-O-[b-xylopyranosyl(100 ! 60)-
b-glucopyranosyl]-benzoate (2), were
isolated from the flowers of P. formosa.
The grayanol-type was considered as the
intermediate of all the structural types
*Corresponding author. Email: rongtaolikm@yahoo.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020903436519
http://www.informaworld.com

Journal of Asian Natural Products Research
71
mentioned above [13], and up to the
present, there are only four diterpenoids of
this kind found in natural products. Details
of the isolation and structural elucidation
of these two new compounds are reported
in this paper.
1.16 (t, J ¼ 7.8 Hz); dC 9.0 (q), 28.0 (t),
and 173.6 (s), where the ester carbonyl
carbon at dC 173.6 of the propionyl group
was correlated with H-14 at dH 5.70 (br s)
in the HMBC spectrum, demonstrating the
C-14 location of the propionyl group. This
assignment was in agreement with the
observation of the H-14 signal being
shifted downfield by 1.44 ppm compared
to the signal from 1a (dH 4.26, d,
J ¼ 6.4 Hz). Above deduction was further
verified by 1H–1H COSY, HSQC, and
HMBC (Figure 2) experiments and the
2. Results and discussion
Compound 1 was obtained as a colorless
solid. The FAB-MS indicated a molecular
ion peak at m/z 408, and the HR-ESI-MS at
m/z 407.2432 [M2H]2 supported the
molecular formula of C23H36O6, with six
sites of unsaturation. The IR spectrum
indicated the presence of hydroxyl
(3432 cm21), ester carbonyl (1733 cm21),
unambiguous assignment of all 1H and 13
C
NMR signals (Table 1) was finished.
The relative configurations of the
stereocenters of 1 were assigned the same
as those of 1a on the basis of the similarity
of all the proton and carbon chemical shifts
and proton multiplicities for both com-
pounds. The NOE correlations from H-1 to
H-2b, H-9, and Me-20, as well as, H-7b
correlated with H-9 and Me-19 suggested
that H-9, Me-19 were b-oriented.
Additional NOE correlations from H-3 to
H-6 and Me-18, H-6 to H-2a, H-3, H-14,
and Me-18 indicated that H-3, H-6, H-14,
Me-18 were in a-disposition. Thus, the
relative configuration of compound 1 was
determined as 3(S),6(R),16(R)-trihydroxy-
5-oxo-14(R)-propionyloxy-5,10-seco-ent-
kaur-1(10)-ene and named as grayanotoxin
XXII.
and CvC double bond (1657 cm21
)
groups. The 13C NMR spectrum showed
resonances for 23 carbons differentiated
by DEPT experiment into five methyls, six
methylenes, six methines, and six qua-
ternary carbons, including three oxy-
methine functionalities at dC 82.7, 69.3,
and 81.1, and one oxygenated quaternary
carbon at dC 79.3.
The 1H and 13C NMR spectral data of 1
were quite similar to those of 3(S),
6(R),14(R),16(R)-tetrahydroxy-5-oxo-5,
10-seco-ent-kaur-1(10)-ene (1a, Figure 1),
a known grayanotoxin isolated from
Leucothoe grayana Max. [14]. The only
difference was that the NMR spectra of 1
displayed resonances due to an additional
propionyl group at dH 2.42 (q, J ¼ 7.8 Hz),
Figure 1. Structures of 1 and 2.

72
W.-G. Wang et al.
2,4-dihydroxybenzoate while trichocar-
pine was benzyl 2,5-dihydroxybenzoate.
1
The H NMR spectrum in the aromatic
region exhibited three aromatic protons
(dH 7.31, d, J ¼ 2.5 Hz; 6.60, br d,
J ¼ 8.5 Hz, and 6.79, d, J ¼ 8.0 Hz),
suggesting the existence of 2,4-bisubsti-
tuted phenyl. The 1H NMR signal in
another aromatic region exhibited five
aromatic protons, including two pairs of
symmetrical ones, which appeared at dH
7.49 (2H, d, J ¼ 7.5 Hz), 7.38 (2H, t,
J ¼ 7.5 Hz), 7.29 (1H, m) with the data at
dH 5.37 (2H, br s) could be ascribed to
benzyl alcohol. The HMBC correlations
(Figure 3) of H-14 with C-7, C-8, and C-9,
of H-9 with C-10, C-11, and C-14, and of
H-6 with C-2, C-4, C-5, and C-7 provided
the position of the benzoyl residue.
Figure 2. Key HMBC correlations of
compound 1.
Compound 2 was assigned the mol-
ecular formula of C25H30O13 on the basis
of HR-ESI-MS analysis at m/z 537.1621
[M2H]2. Its UV absorption maximum at
lmax 280 nm was characteristic of phenolic
compounds. At the same time, its IR
spectrum showed specific absorptions at
3442 cm21, which could be ascribed to the
hydroxyl group. The intense IR absorption
band at 1724 cm21 revealed the presence
of ester functionality, while the broad
CZO stretching band in the region of
1076 cm21 suggested its glycosidic nature
[15].
The complete acid hydrolysis of 2
yielded various products, and in the
hydrolysate separated from the aglycone
parts, two sugars identified by the TLC
comparison were found to be glucose and
xylose, respectively. The HMBC corre-
lations from H-60 to C-40, C-50, and C-100,
from H-100 to C-60, and from H-10 to C-4
and C-30, suggested the position of the
glycosidic nature. The signals of the
anomeric doublets at dH 4.90 (H-10) and
dH 4.29 (H-100) presumably belonged to
1
Comparison of the H and 13C NMR
spectral data of aglycone of 2 with those of
trichocarpine [16] showed that the agly-
cone of both compounds was similar
except that the aglycone of 2 was benzyl
Table 1. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectral data (CDCl3) of compound 1 (d in
ppm, J in Hz).
No.
1H
13C
No.
1H
13C
1
5.03 (dd, J ¼ 12.0, 1.5)
2.56 (ddd, J ¼ 15.0, 12.0, 3.0)
2.17 (dd, J ¼ 15.0, 1.5)
3.78 (br s)
128.4
32.7 t
12a
12b
13
14
15
16
17
18
19
20
CH3CH2
CH3CH2
CH3CH2CO
1.91 (m)
1.33 (m)
2.17 (overlap)
5.70 (br s)
1.97 (br s)
27.0 t
2a
2b
3
4
5
52.8 d
81.1 d
57.3 t
79.3 s
22.7 q
21.5 q
27.0 q
16.5 q
9.0 q
82.7 d
48.9 s
215.8
69.3 d
39.7 t
6
4.28 (br s)
1.60 (d, J ¼ 16.0)
1.37 (s)
1.33 (s)
1.32 (s)
1.97 (s)
7a
7b
8
2.71 (dd, J ¼ 16.0, 6.0)
54.4 s
57.8 d
139.9
9
2.25 (d, J ¼ 7.5)
1.16 (t, J ¼ 7.8)
10
11
11b
2.42 (q, J ¼ 7.8)
28.0 t
173.6 s
1.38 (m)
1.80 (m)
22.5 t

Journal of Asian Natural Products Research
73
cisco, CA, USA) column. Column chro-
matography (CC) was carried out on silica
gel (200–300 mesh; Qingdao Marine
Chemical Factory, Qingdao, China),
Lichroprep RP-18 (43–63 mm; Merck,
Darmstadt, Germany), Sephadex LH-20
(Amersham Biosciences AB, Uppsala,
Sweden), and MCI (MCI-gel CHP-20P,
75–150 mm; Mitsubishi Chemical Corpor-
ation, Tokyo, Japan). TLC was performed
on TLC plates (Si gel GF254; Qingdao
Marine Chemical Factory), and detected
by spraying with 5% H2SO4 –EtOH,
followed by heating on a hot plate.
The standard samples of D- and L-glucose,
D- and L-xylose were purchased from the
company of the Sigma (St Louis, MO,
USA).
Figure 3. Key HMBC correlations of
compound 2.
two sugar moieties and the evidence for
the b-configuration of these sugars was
drawn from the coupling constants of 7.3
and 7.4 Hz for H-10 and H-100, respectively.
Therefore, based upon the above
cumulative evidences, compound 2 was
elucidated as benzyl 2-hydroxy-4-O-[b-
xylopyranosyl(100 ! 60)-b-glucopyrano-
syl]-benzoate.
3.2 Plant material
The flowers of P. formosa were collected in
Jindian, Kunming, China, in May 2008.
The sample was identified by Dr Yong-
Peng Ma, Kunming Institute of Botany,
Chinese Academy of Sciences, and a
voucher specimen (KMUST 2008050701)
is deposited at the Laboratory of Phyto-
chemistry, Biotechnology Research
Center, Kunming University of Science
and Technology.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured with a
Jasco DIP-370 digital polarimeter (JASCO
Corporation, Tokyo, Japan). UV spectra
were run on a UV 210A spectrophotometer
(Shimadzu, Kyoto, Japan). IR spectra were
recorded on a Bio-Rad FtS-135 spectro-
photometer with KBr pellets (Bio-Rad
Corporation, Hercules, CA, USA). 1D and
2D NMR spectra were recorded using
Bruker AM-400 and DRX-500 instru-
ments with tetramethylsilane (TMS) as
an internal standard (Bruker BioSpin
Group, Bremen, German). FAB-MS were
measured on a VG Auto Spec-3000
spectrometer (VG PRIMA, Birmingham,
UK), and HR-ESI-MS were taken on an
API Qstar Pulsar instrument (Applied
Biosystem Corporation, Foster City, CA,
USA). Semipreparative HPLC was per-
formed on an Agilent 1200 liquid chro-
matograph with a ZORBAX SB-C18
(5 mm, 9.4 £ 250 mm; Agilent, San Fran-
3.3 Extraction and isolation
The air-dried and powdered plant material
(5.5 kg) was extracted with acetone–water
(7:3, 3£ 25 liters, each 2 days) at room
temperature and filtered. The filtrate was
evaporated in vacuo to afford a residue,
which was dissolved in H2O (2 liters), and
then extracted successively with petroleum
ether (3£ 4 liters), EtOAc (4£ 4 liters),
and n-BuOH (4£ 4 liters), respectively.
The EtOAc extract (450.0 g) was decolor-
ized on MCI gel, eluted with 90% MeOH–
H2O to yield a yellow gum (427.5 g).
The gum was subjected to CC over a silica
gel, eluted with a CHCl3–Me2CO gradient
system (1:0, 9:1, 8:2, 7:3, 6:4, 1:1), to yield

74
W.-G. Wang et al.
six fractions, A–F. Fraction B (15.8 g,
CHCl3 –Me2CO, 8:2) was separated
into three subfractions B1–B3 by a silica
gel column eluting with petroleum
ether–isopropyl alcohol (30:1, 20:1,
10:1). Subfraction B2 (3.7 g, petroleum
ether–isopropyl alcohol 20:1) was purified
by a silica gel column, eluted with
petroleum ether–EtOAc (5:1), and then
followed by Sephadex LH-20 CHCl3–
MeOH (1:1), to afford 1 (7 mg). The n-
BuOH extract (425 g) was purified by
Sephadex LH-20, eluted with MeOH–H2O
(3:7, 6:4, 9:1 gradient system), to give
fractions G1–G3. Fraction G1 (MeOH–
H2O 3:7, 15 g) was subjected to an RP-18
(MeOH–H2O 45:55), semipreparative
HPLC (50% MeOH–H2O, 3 ml/min), to
give 2 (6 mg).
3.3.1 Acid hydrolysis of 2
A solution of 2 (3 mg) in 1 M HCl (0.5 ml)
was heated at 90–1008C in a screw-capped
vial for 5 h. The mixture was partitioned
with EtOAc (0.5 ml), and the EtOAc layer
was compared with the standard samples
of glucose and xylose on TLC (EtOAc–
MeOH–AcOH–H2O 11:2:2:2) by visua-
lizing the spots with Rf ¼ 0.5 and 0.6,
respectively.
3.3.2 Compound 1
28
White powder. ½aꢀD þ2.00 (c ¼ 0.35,
CHCl3); IR (KBr) nmax: 3432, 3095, 2924,
2852, 1733, 1657, 1463, 1421, 1378, 1324,
1276, 1196, 1082, 1053, 1032, 943,
1
888 cm21. H and 13C NMR spectral data,
see Table 1. FAB-MS (neg.): m/z (%) 408
([M]2, 38), 376 ([M–CH3O–H]2, 18), 367
Table 2. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectral data (CD3OD) of compound 2
(d in ppm, J in Hz).
No.
1H
13C
HMBC
1
2
3
4
5
6
7
8
110.8 s
159.9 s
108.1 d
158.2 s
111.7 d
134.3 d
170.0 s
137.4 s
129.5 d
129.2 d
129.2 d
129.2 d
129.5 d
68.2 t
102.7 d
74.8 d
77.8 d
71.2 d
77.6 d
69.7 t
7.31 (d, J ¼ 2.5)
C-1, 2, 4
6.60 (br d, J ¼ 8.5)
6.79 (d, J ¼ 8.0)
C-1, 3, 6
C-2, 4, 5, 7
9
7.49 (d, J ¼ 7.5)
7.38 (t, J ¼ 7.5)
7.29 (m)
7.38 (t, J ¼ 7.5)
7.49 (d, J ¼ 7.5)
5.37 (br s)
C-10, 11, 14
C-8, 9, 11
C-9, 10
10
11
12
13
14
10
20
30
40
50
60
C-8, 11, 13
C-11, 12, 14
C-7, 8, 9
C-4, 30
4.90 (d, J ¼ 7.5)
3.37 (m)
3.25 (br t, J ¼ 8.5)
3.31 (m)
C-10, 40
C-20, 40
C-50, 600
3.61 (ddd, J ¼ 1.5, 6.0, 11.5)
4.08 (dd, J ¼ 1.5, 11.5)
3.73 (dd, J ¼ 6.5, 11.5)
4.29 (d, J ¼ 7.5)
3.10 (d, J ¼ 8.8)
3.43 (br t, J ¼ 7.8)
3.11 (m)
C-10, 300, 60
C-40, 1
C-500, 100
100
200
300
400
500
105.3 d
75.0 d
77.7 d
71.2 d
66.9 t
C-600
C-100, 300, 400
C-2 , 400, 500
C-300
3.81 (dd, J ¼ 6.0, 11.0)
C-100, 300, 400
C-100, 300
3.16 (m)

Journal of Asian Natural Products Research
75
([M–CH2CHCH2]2, 15), 351 ([M–CH3-
CH2CO]2, 5), 346 (10), 326 (5). Negative
HR-ESI-MS: m/z 407.2432 [M2H]2 (calcd
for C23H35O6, 407.2433).
[3] H. Hikino, T. Ohta, M. Ogura, Y.
Ohizumi, C. Konno, and T. Takemoto,
Toxicol. Appl. Pharmacol. 35, 303 (1976).
[4] T. Masutani, I. Seyama, T. Narahashi, and
J. Iwasa, J. Pharmacol. Exp. Ther. 217,
812 (1981).
[5] J.A. Klocke, M.Y. Hu, S.F. Chiu, and
I. Kubo, Phytochemistry 30, 1797 (1991).
[6] M.Y. Hu, J.A. Klocke, S.F. Chiu, and
I. Kubo, J. Econ. Entomol. 86, 706 (1993).
[7] J.S. Chen and S. Zheng, Chinese Poiso-
nous Plants (Science Press, Beijing,
1987), pp. 216–229.
3.3.3 Compound 2
26
White powder. ½aꢀD 265.39 (c ¼ 0.21,
MeOH). IR (KBr) nmax: 3443, 2926, 1724,
1639, 1510, 1453, 1271, 1223, 1076, 991,
1
898, 670 cm21. H and 13C NMR spectral
data, see Table 2. FAB-MS (neg.): m/z (%)
537 [M2H]2, 509 ([M–CO]2, 10), 491
(25), 447 ([M–C7H6]2, 100), 339 (55), 325
(22), 294 ([b-xylopyranosyl(100 ! 60)-b-
glucopyranosyl-18]2, 5), 153 (15). Nega-
tive HR-ESI-MS: m/z 537.1621 [M2H]2
(calcd for C25H29O13, 537.1608).
[8] L.Q. Wang, B.Y. Ding, P. Wang,
W.M. Zhao, and G.W. Qin, Nat. Prod.
Sci. 4, 68 (1998).
[9] L.Q. Wang, S.N. Chen, and G.W. Qin,
J. Nat. Prod. 61, 1473 (1998).
[10] L.Q. Wang, B.Y. Ding, G.W. Qin, G. Lin,
and K.F. Cheng, Phytochemistry 49, 2045
(1998).
[11] L.Q. Wang, S.N. Chen, and G.W. Qin,
Phytochemistry 54, 847 (2000).
Acknowledgements
[12] L.Q. Wang, S.N. Chen, and G.W. Qin,
Fitoterapia 72, 779 (2001).
[13] T. Terai, Nippon Kagaku Zasshi 5, 835
(1988).
We gratefully acknowledge the support for this
research work by the National Natural Science
Foundation of China (20862011), the Fok Ying
Tong Education Foundation (111040), and the
Foundation for the Author of National Excel-
lent Doctoral Dissertation of China (200780).
[14] M. Katai, T. Terai, and J. Katakawa,
Memoirs Osaka Inst. Technol. Ser. A 51, 1
(2006).
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N. Rasool, and M. Zubair, J. Asian Nat.
Prod. Res. 9, 209 (2007).
[16] R.A. Dommisse, L.V. Hoof, and A.J.
Vlietinck, Phytochemistry 25, 1201
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