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).