Two new flavonol glycosides from the leaves of Elaeagnus pungens

Article abstract of DOI:10.1080/10286020.2013.812078

The leaves of Elaeagnus pungens were extracted with 70% ethanol and successively purified by column chromatography. Seven constituents were obtained and characterized, all of which belong to the class of flavonol glycosides. Their structures were elucidat

Full text of DOI:10.1080/10286020.2013.812078

This article was downloaded by: [Ryerson University]
On: 02 December 2014, At: 10:43
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Two new flavonol glycosides from the
leaves of Elaeagnus pungens
Yue-Bin Gea, Meng-Shun Lia, Zhi-Nan Meia & Guang-Zhong Yanga
a College of Pharmacy, South-Central University for Nationalities,
Wuhan, 430074, China
Published online: 04 Jul 2013.
To cite this article: Yue-Bin Ge, Meng-Shun Li, Zhi-Nan Mei & Guang-Zhong Yang (2013) Two
new flavonol glycosides from the leaves of Elaeagnus pungens, Journal of Asian Natural Products
Research, 15:10, 1073-1079, DOI: 10.1080/10286020.2013.812078
To link to this article: http://dx.doi.org/10.1080/10286020.2013.812078
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research, 2013
Vol. 15, No. 10, 1073–1079, http://dx.doi.org/10.1080/10286020.2013.812078
Two new flavonol glycosides from the leaves of Elaeagnus pungens
Yue-Bin Ge, Meng-Shun Li, Zhi-Nan Mei and Guang-Zhong Yang*
College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China
(Received 28 March 2013; final version received 3 June 2013)
The leaves of Elaeagnus pungens were extracted with 70% ethanol and
successively purified by column chromatography. Seven constituents were obtained
and characterized, all of which belong to the class of flavonol glycosides. Their
structures were elucidated on the basis of spectroscopic methods including 1D/2D
NMR and MS analysis techniques. The seven flavonol glycosides were determined as
kaempferol 3-O-b-D-glucopyranosyl-(1 ! 3)-a-L-rhamnopyranosyl-(1 ! 6)-[a-L-
rhamnopyranosyl(1 ! 2)]-b-D-galactopyranoside (1), isorhamnetin 3-O-b-D-gluco-
pyranosyl-(1 ! 3)-a-L-rhamnopyranosyl-(1 ! 6)-b-D-galactopyranoside (2), together
with five known compounds, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliu-m bromide assay in vitro showed that the isolated flavonol
glycosides showed no proliferation activity in the asthma airway smooth muscle cells,
comparing with solvent as the control group.
Keywords: Elaeagnaceae; Elaeagnus pungens; flavonol glycoside; spectroscopic
analysis
1. Introduction
in the past years, including flavonoids [5],
The Elaeagnus is a member of the family
Elaeagnaceae, which comprises three gen-
era and about 80 species that are wide-
spread in subtropical and temperate areas of
East and Southeast Asia [1,2]. Fifty-five
species, including Elaeagnus pungens
Thunb., are found in China. In traditional
Chinese medicine, E. pungens leaf has been
documented as an anti-asthmatic remedy to
treat the severe asthma, chronic bronchitis,
or other respiratory disorders in the early
material medica ‘Bencao Gangmu’ (Ming
dynasty, about 430 years ago). It is also
generally used by Tujia nationality people
in Hubei province to treat shortness of
breath, cough, or bronchitis [3]. It is
nontoxic under oral administration for a
long time in adult without definite IC50
value in mice [4].
lignanoids [6], organic acids [7], and
terpenoids [8]. Our preliminary study has
evaluated the anti-asthmatic, antitussive,
and expectorant activities in vivo of the
ethanolic extract and fractions from the
plant [9]. In general, despite itslong-time
use in human health, literatures about E.
pungens are limited, especially on the
research on chemical components and
effect on airway smooth muscle cells
(ASMCs), the change of which is related
to asthmatic development [10]. Given the
importance of its effect on respiratory
diseases, the chemical composition of E.
pungens leaf was studied in detail and
presented here along with the structures of
the newly obtained flavonol glycosides.
The effect of isolated compounds was
evaluated on the proliferation of ASMCs,
in order to search for the active components
of E. pungens leaf.
Experimentally, the chemical constitu-
ents of E. pungens have been investigated
*Corresponding author. Email: yanggz888@126.com
q 2013 Taylor & Francis

1074
Y.-B. Ge et al.
assigned by HSQC, HMBC, TOCSY, and
2. Results and discussion
1H–1H COSY analyses and sugar moieties
were one b-D-glucose, one b-D-galactose,
and two a-L-rhamnoses. The configur-
ations of glucopyranosyl and galactopyr-
anosyl assigned as b were further
supported by the coupling constants of
the anomeric protons at dH 4.39 (1H, d,
J ¼ 8.0 Hz) and 5.58 (1H, d, J ¼ 7.5 Hz).
The sequence of the sugar moieties was
determined by HMBC correlations between
H-100 at d 5.58 and C-3 at d 134.4; H-1000 at d
4.58 and C-600 at d 67.5; H-1IV at d 4.39 and
C-3000 at d 83.0; H-1V at d 5.24 and C-200
at d 77.4. Thus, compound 1 was deduced
as kaempferol 3-O-b-D-glucopyranosyl-
(1 ! 3)-a-L-rhamnopyranosyl-(1 ! 6)-[a-
L-rhamnopyranosyl(1 ! 2)]-b-D-galacto-
pyranoside, and the structure is shown in
Figure 1.
Compound 1 was obtained as a yellow
powder. Its molecular formula of
C39H50O24 was deduced from the HR-
ESI-MS at m/z 925.2584 [M þ Na]þ.
Acidic hydrolysis of 1 showed the
presence of glucose, rhamnose, and
galactose. The 1H NMR spectrum of
aglycon showed a set of AA0BB0 systems
at dH 8.08 (2H, d, J ¼ 9.0 Hz) and 6.91
(2H, d, J ¼ 9.0 Hz), as well as two meta-
aromatic protons at dH 6.17 (s, 1H) and
6.35 (s, 1H). The 13C NMR spectral data of
the aglycone spectra of 1 revealed the
presence of nine quaternary C-atoms
(including one CvO group at dC 179.3)
and six CH groups. Detailed analysis of
HSQC and HMBC spectra indicated
compound 1 was characterized as being
kaempferol 3-O-glycoside with four sugar
moieties.
Compound 2 was isolated as a yellow
powder. The molecular formula was
established to be C34H42O21 by HR-ESI-
MS at m/z 809.2111 [M þ Na]þ. Acidic
hydrolysis also showed the presence of
glucose, rhamnose, and galactose in
compound 2. By comparing the NMR
data of 2 with those of 1, it was found that
The 1H and 13C NMR data also showed
four glycosyl signals by four anomeric
protons at dH 5.58 (1H, d, J ¼ 7.5 Hz),
4.58 (1H, s), 4.39 (1H, d, J ¼ 8.0 Hz), and
5.24 (1H, s), and the related anomeric C-
atom signals at dC 101.0, 101.7, 105.7, and
102.6. Signals of each sugar unit were
OMe
OH
OH
4'
8
1'
7
9
O
HO
OH
HO
OH
O
O
2
10
6
4
3
5
O
OH
O
OH
O
O
O
H
OH
OH
O
O
OH
OH
O
O
1''
1'''
H
OH
H
H
OH
HO
HO
O
O
H
OH
O
OH
O
O
O
OH
OH
V
OH
OH
IV
1
H
1
H
OH
OH
OH OH
1
2
Figure 1. Key HMBC correlations of compounds 1 and 2.

Journal of Asian Natural Products Research
1075
the structure of the sugar moiety in 2
corresponded to that in 1 except for the
absence of one rhamnosyl group in 2, and
the structure of aglycon of 2 had one more
methoxyl group. The MeO group was
located at C-30 based on the HMBC
correlation of dH 3.89 (3H, s)/C-30 (dC
148.5). Therefore, the aglycon of 2 was
effective components for the ASMCs
proliferation in E. pungens leaf.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured using a
P-1020 digital polarimeter in MeOH
solution from JASCO, Tokyo, Japan. UV
spectra were measured on Shimadzu UV-
260 spectrometer from Shimadzu Co.,
Kyoto, Japan. 1H, 13C, and 2D NMR
spectra were run on a Bruker DRX-500
spectrometer with tetramethylsilane as
internal standard from Ettlingen, Germany.
HR-ESI-MS data were obtained on Bruker
FT-MS Apex III mass spectrometer. GC–
MS data were obtained on Agilent Tech-
nologies 5973 Network Mass Selective
Detector-6890N Network GC System from
Santa Clara, CA, USA. High performance
liquid chromatography (HPLC) was car-
ried out on Ultimate 3000 HPLC system,
Ultimate 3000 pump, Ultimate 3000 Vari-
able Wavelength from Dionex Co., Sunny-
vale, CA, USA and 10 mm £ 250 mm
semi-preparative column from Nacalai
tesque Co., Ltd, Kyoto, Japan. Thin-layer
chromatography was carried out on silica
gel 60 GF254 (Qingdao Haiyang Chemical
Co., Ltd, Qingdao, China), and column
chromatography (CC) was carried out
using silica gel (200–300 mesh) from
Qingdao Haiyang Chemical Group Co.,
macroporous resin D-101 from Anhui
Sanxing Resin Technology Co., Ltd,
Bangbu, China; Sephadex LH-20 from
Shanghai Ai-yan Biological Co., Ltd,
Shanghai, China and C18 reverse-phase
silica gel from YMC Co., Ltd, Kyoto,
Japan. All of the solvents and materials
were reagent grade and purified as required.
identified as isorhamnetin. The 1H and 13
C
NMR data also showed three glycosyl
signals by three anomeric protons at dH
5.12 (1H, d, J ¼ 8.0 Hz), 4.48 (s, 1H), and
4.25 (1H, d, J ¼ 8.0 Hz), and the related
anomeric C-atom signals at dC 105.2,
102.0, and 105.8. In the same way as that
of 1, the sequence of the sugar moieties
was determined by HMBC correlations
(see Figure 1). The HMBC correlation of
H-200 [dH 5.12 (1H, d, J ¼ 8.0 Hz)] with C-
3 (dC 135.6) revealed the sugar chain was
connected to C-3. Therefore, compound 2
was deduced as isorhamnetin 3-O-b-D-
glucopyranosyl-(1 ! 3)-a-L-rhamnopyra-
nosyl-(1 ! 6)-b-D-galactopyranoside, and
the structure is shown in Figure 1.
Five known compounds were identified
as kaempferol 3-O-a-L-rhamnopyranosyl-
(1 ! 6)-[a-L-rhamnopyranosyl(1 ! 2)]-b-
D-galactopyranoside (3) [11], kaempferol
3-O-a-L-rhamnopyranosyl-(1 ! 6)-[a-L-
rhamnopyranosyl(1 ! 2)]-b-D-galactopyr-
anoside-7-O-b-D-glucopyranoside (4) [12],
kaempferol 3-O-a-L-rhamnopyranosyl-
(1 ! 6)-[a-L-rhamnopyranosyl-(1 ! 2)]-
b-D-galactopyranoside-7-O-a-L-rhamno-
pyranoside (5) [12], kaempferol 3-O-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyra-
noside (6) [13], and kaempferol 3-O-a-L-
rhamnopyranosyl-(1 ! 6)-b-D-galactopyra-
noside (7) [14].
The proliferation effects of compounds
1–7 were evaluated on the ASMCs in the
asthma rats. It exhibited that there were no
effects of compounds 1–7 on the prolifer-
ation activity in the ASMCs, compared
with the control group. That suggested the
isolated flavonol glycosides may be not the
3.2 Plant material
The fresh leaves of E. pungens were
collected in June 2010 at She Hill, Wuhan

1076
Y.-B. Ge et al.
city, Hubei province, China. The plant was
authenticated by Dr Dingrong Wan,
Professor in Pharmacognosy at School of
Pharmacy, South-central University for
Nationalities (SCUN). The collected E.
pungens leaf is also consistent with the
standardizedherbalspecimen(No. 42046)in
Hubei Provincial Institute of Drug Control.
The voucher specimen (SCUN08010) has
been deposited in the Herbarium of School
of Pharmacy, SCUN.
HPLC (MeOH–H2O; 24:76, 3 ml/min) to
afford compound 4 (9.3 mg; tR 11.4 min).
Fr. 2.5.2.8.11 (135 mg) was further purified
by CC (Sephadex; MeOH) to get com-
pound 5 (5.1 mg).
The BuOH fraction (62.0 g) was sub-
jected to CC (SiO2; CH2Cl2 –MeOH;
15:1 ! 3:7 gradient system), to give Frs
1–10. Fr. 5 (12 g) was successively
separated by CC (SiO2; CH2Cl2–MeOH;
1:0 ! 4:6) to provide Frs 5.1–5.12. Fr. 5.9
(1.3g) was further subjected to CC (Sepha-
dex; MeOH) to afford Frs 5.9.1–5.9.4. Fr.
5.9.2 (0.2g) was further purified by semi-
preparative HPLC (MeOH–H2O; 49:51,
3 ml/min) to affordcompound6(21.9 mg;tR
9.9 min). Fr. 5.9.3 (120mg) was further
purified by semi-preparative HPLC
(MeOH–H2O; 40:60, 3 ml/min) to afford
compound 7 (11.5 mg; tR 10.6min).
3.3 Extraction and isolation
The collected leaves were dried in shade
and reduced to coarse powder using a
mortar and pestle. The powdered leaves of
E. pungens (4.2 kg) were extracted with
70% EtOH and then successively parti-
tioned with petroleum ether (3 £ 2.0
liters), AcOEt (3 £ 2.0 liters), and BuOH
(2.0 liters). The residual water layer was
concentrated (400 g) and subjected to CC
[macroporous resin; EtOH–H2O; 0:1, 3:7,
6:4, 9:1, 1:0 (v/v)] to give five fractions
(Frs 1–5). Fr. 2 (40.0 g) was subjected to
CC (SiO2; CH2Cl2–MeOH; 15:1 ! 0:1
gradient system) to give five fractions (Frs
2.1–2.5). Fr. 2.4 (2.6 g) was successively
separated by CC (SiO2; CH2Cl2–MeOH;
10:1 ! 0:1) to yield Frs 2.4.1–2.4.6. Fr.
2.4.4 (0.3 g) was further purified by CC
(Sephadex; MeOH) and followed by semi-
preparative HPLC (MeOH–H2O; 35:65,
3 ml/min) to afford compound 1 (10 mg; tR
4.4 min). Fr. 2.5 (13.7 g) was purified by
CC (Sephadex; MeOH) to give Frs 2.5.1–
2.5.10. Fr. 2.5.2 (9.1 g) was subjected to CC
(SiO2; CH2Cl2–MeOH; 9.3:0.7 ! 1:1) to
afford Frs 2.5.2.1–2.5.2.10. Fr. 2.5.2.8
(1.4 g) was subjected to CC (RP-18;
MeOH–H2O; 1:10 ! 3:2) to provide Frs
2.5.2.8.1–2.5.2.8.13. Fr. 2.5.2.8.8 (57 mg)
was further purified by CC (Sephadex;
MeOH) to give compound 2 (3.5 mg) and
compound 3 (9.0 mg). Fr. 2.5.2.8.4 (0.25 g)
was further purified by CC (Sephadex;
MeOH) and followed by semi-preparative
3.4 Acidic hydrolysis
Compounds 1 and 2 (3 mg) were, respect-
ively, added to trifluoroacetic acid (4 N)
solution (6 ml), then heated for 3 h under
908C. After cooled to room temperature,
the hydrolytical solution was extracted
with chloroform (3 £ 6.0 ml). The water
layer was concentrated to 0.5 ml. The
completely concentrated water layer and
reference substances of b-D-glucose, b-D-
galactose, and a-L-rhamnose were derived
by reacting with 1.5 mg hydroxylamine
hydrochloride and 0.5 ml pyridine for 1 h
at 908C. After cooling, 0.9 ml Ac2O was
added and the mixture was heated at 908C
for 1 h. The reaction mixtures were
evaporated under reduced pressure, and
the resulting aldononitrile peracetates
were analyzed by GC–MS. The samples
(10 ml) were injected into
a HP-1
30 m £ 0.2 mm chromatographic column
(Agilent). The injector temperature was
2808C. The helium carrier gas flow was
maintained at 1 ml/min. The oven tem-
perature program was as follows: 1008C
for 2 min, 2008C at 208C/min (remaining

Journal of Asian Natural Products Research
1077
1
Table 1. 13C and H NMR spectral data of compounds 1 and 2.
13C NMR 1H NMR
No.
1
2
1
2
2
3
4
5
6
7
8
158.6
134.4
179.3
163.1
100.6
167.7
95.3
158.9
135.6
179.5
163.0
100.1
166.3
95.0
6.17 (1H, s)
6.35 (1H, s)
6.13 (1H, s)
6.34 (1H, s)
9
158.5
105.4
123.4
132.2
116.2
161.3
116.2
132.2
158.6
105.7
123.0
114.8
148.5
150.9
116.2
123.9
57.0
105.2
73.1
75.1
70.1
75.6
10
10
20
8.08 (1H, d, J ¼ 9.0 Hz)
6.91 (1H, d, J ¼ 9.0 Hz)
7.99 (1H, s)
30
40
50
6.91 (1H, d, J ¼ 9.0 Hz)
8.08 (1H, d, J ¼ 9.0 Hz)
6.82 (1H, d, J ¼ 8.0 Hz)
7.53 (1H, d, J ¼ 8.0 Hz)
3.89 (3H, s)
600
300-OMe
100
101.0
77.4
75.2
70.8
75.7
67.5
101.7
71.3
83.0
72.6
69.5
18.0
105.7
75.4
77.6
71.0
77.7
62.1
102.6
72.4
72.5
74.2
69.9
17.7
5.58 (1H, d, J ¼ 7.5 Hz)
5.12 (1H, d, J ¼ 8.0 Hz)
200
3.85–3.90 (m)
3.72–3.74 (m)
3.72–3.74 (m)
3.47–3.48 (m)
300
400
3.98–4.01 (m)
3.51–3.54 (m)
3.68–3.72 (m)
3.57–3.62 (m)
500
6
67.9
3.73–3.76 (m); 3.40–3.50 (m)
4.58 (1H, s)
3.72–3.74 (m)
3.62–3.67 (m); 3.44–3.48 (m)
4.48 (1H, s)
3.80–3.82 (m)
1000
2000
3000
4000
5000
6000
1IV
2IV
3IV
4IV
5IV
6IV
1V
2V
3V
4V
5V
6V
102.0
71.4
83.2
72.6
69.5
3.50–3.70 (m)
3.94–3.96 (m)
3.39–3.42 (m)
3.45–3.50 (m)
3.32–3.37 (m)
3.47–3.52 (m)
18.1
1.20 (3H, d, J ¼ 6.0 Hz)
4.39 (1H, d, J ¼ 8.0 Hz)
3.68–3.72 (m)
1.05 (3H, d, J ¼ 6.0 Hz)
4.25 (1H, d, J ¼ 8.0 Hz)
3.12–3.17 (m)
105.8
75.4
77.6
70.9
77.7
3.30–3.41 (m)
3.27–3.34 (m)
3.10–3.20 (m)
3.27–3.28 (m)
3.26–3.28 (m)
3.06–3.09 (m)
3.57–3.65 (m)
62.1
3.66–3.80 (m); 3.58–3.64 (m)
5.24 (1H, s)
3.39–3.41 (m)
3.65–3.69 (m)
3.38–3.42 (m)
3.35–3.38 (m)
1.01 (3H, d, J ¼ 6.0 Hz)
3.4.1 Kaempferol 3-O-b-D-
at 2008C for 2 min), then to 2608C at
108C/min (remaining at 3008C for 3 min)
– the total run time was 18 min. The range
of MS was m/z 30–500. The tR values of
b-D-glucose, b-D-galactose, and a-L-
rhamnose derivatives were 11.05, 11.26,
and 8.86 min, respectively.
glucopyranosyl-(1 ! 3)-a-L-
rhamnopyranosyl-(1 ! 6)-[a-L-
rhamnopyranosyl(1 ! 2)]-b-D-
galactopyranoside (1)
Yellow powder; [a ]D 278.3 (c ¼ 0.121,
MeOH); UV(MeOH) lmax 266, 349; 13C

1078
Y.-B. Ge et al.
NMR (125 MHz, MeOH-d4) and 1H
NMR (500 MHz, MeOH-d4) spectral
data are shown in Table 1. HR-ESI-MS:
m/z 925.2584 [M þ Na]þ(calcd for
C39H50O24Na, 925.2571).
BSA at 378C for 40 min. The collected
pellets by centrifuge were cultured with
Dulbecco’s modified eagle medium sup-
plemented with 20% fetal bovine serum,
100 U/ml penicillin and 0.1 mg/ml strep-
tomycin, and subcultured with 0.25%
trypsin–0.02% ethylenediaminetetraacetic
acid disodium salt solution. The cells
between 3 and 8 passages were used for the
experiment.
3.4.2 Isorhamnetin 3-O-b-D-
glucopyranosyl-(1 ! 3)-a-L-
rhamnopyranosyl-(1 ! 6)-b-D-
galactopyranoside (2)
After compounds 1–7 at the concen-
trations of 10, 30, and 100 mM were
cultured with the ASMCs for 48 h, MTT
(5 mg/ml) was added and incubated for 4 h,
then the absorbance at 490 nm was
measured on a microplate reader (Thermo
Electron Co., Waltham, MA, USA).
Yellow powder; [a]D 233.8 (c ¼ 0.106,
MeOH); UV (MeOH) lmax 255, 354 nm;
1
13C NMR (125 MHz, MeOH-d4) and H
NMR (500 MHz, MeOH-d4) spectral
data are shown in Table 1. HR-ESI-MS:
m/z 809.2111 [M þ Na]þ (calcd for
C34H42O21Na, 809.2108).
Acknowledgments
This work was financially supported by the
Natural Science Foundation of China (Grant
No. 31000156) and National ‘Twelfth Five-
Year’ Plan for Science & Technology Support
of China (Grant No. 2012BAI27B06).
3.5 Proliferation experiment
Male Sprague–Dawley rats weighing
180–200 g (Hubei Province Center for
Disease Control and Prevention, Wuhan,
China) were used to make the asthma
model according to the literature [15]. In
brief, rats were actively sensitized on days
1 and 8 using 50 mg/kg21 subcutaneous
injection of ovalbumin in 0.9% sterile
saline containing 200 mg of aluminum
hydroxide. Challenges were carried out
every third day after sensitization, being
exposed to a 2% ovalbumin aerosol mist
produced by an Ultrasonic nebulizer
(YuYue Medical Device Ltd, Jiangsu,
China) for 30 min. After asthma symptoms
appear, rats were killed on day 29 using an
overdose of sodium pentobarbitone
(500 mg kg21; i.p.). Primary ASMCs
were culture by enzymatic method [16]
and the proliferation of ASMCs was
examined by MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazoliu-m bro-
mide) assay. In brief, the airway smooth
muscles from the asthma rats were cut into
small pieces and digested by D-Hanks
solution containing 1 mg/ml type I col-
lagenase, 2 mg/ml papain (Invitrogen,
Grand Island, NY, USA) and 2 mg/ml
References
[1] N.P. Bekker and A.I. Glushenkova, Chem.
Nat. Compd. 37, 97 (2001).
[2] M. Ayaz, M. Riaz, A. Malik, E. Ahmad, I.
Fatima, M. Arif Lodhi, and M.I. Chaudh-
ary, Nat. Prod. Res. 23, 409 (2009).
[3] Z.X. Fang, H. Zhao, and J.H. Zhao,
Yaocai Zhi of Tujia Nationality (China
Science and Technology of Medicine
Press, Beijing, 2006), p. 466.
[4] P.A. Shen, Pharmacological and Clinical
Application of Chinese Material Medica
(People’s Medical Publishing House,
Beijing, 2006), p. 721.
[5] K. Tagahara, J. Koyama, and T. Sugita,
Shoyakugaku Zasshi 35, 340 (1981).
[6] M.J. Guo, H.B. Jiang, X.Q. Tian, N.
Huang, and J. Huang, West China
J. Pharm. Sci. 23, 381 (2008).
[7] X. Zhao, H. Huang, and R.L. Zhu, Chin.
Tradit. Patent Med. 28, 403 (2006).
[8] X. Zhao, R.L. Zhu, B. Jiang, and H.
Huang, China J. Chin. Mater. Med. 31,
472 (2006).
[9] Y.B. Ge, J.Q. Liu, and D.F. Su,
J. Ethnophamacol. 126, 538 (2009).
[10] J.K. Bentley and M.B. Hershenson, Proc.
Am. Thorac. Soc. 5, 89 (2008).

Journal of Asian Natural Products Research
1079
[11] G.C. Kite, C.A. Stoneham, and N.C.
Veitch, Phytochemistry 68, 1407 (2007).
[12] B. Schwarz and T. Hofmann, J. Agric.
Food Chem. 55, 1394 (2007).
[14] H.M. Ho, R.Y. Chen, L.K. Leung, F.L.
Chan, Y. Huang, and Z.Y. Chen, Biomed.
Pharmacother. 56, 289 (2002).
[15] M. Salmon, D.A. Walsh, H. Koto, P.J.
Barnes, and K.F. Chung, Eur. Respir. J.
14, 633 (1999).
[13] H. Wu, S. Dushenkov, C.T. Ho, and S.M.
Sang, Food Chem. 115, 592 (2009).
[16] S.J. Hirst, Eur. Respir. J. 9, 808 (2006).