<p> Antidepressant-like effects of standardized gypenosides: Involvement of brain-derived</p><p> neurotrophic factor signaling in hippocampus</p><p>Rong-Hao Mua, Xiao-Yan Fangb, Shuang-Shuang Wanga, Cheng-Fu Lic, Shao-Mei Chenc,</p><p>Xue-Mei Chenc, Qing Liu a,d, e,*, Yu-Cheng Lib, Li-Tao Yia,d,e,* a Department of Chemical and Pharmaceutical Engineering, College of Chemical</p><p>Engineering, Huaqiao University, Xiamen 361021, Fujian province, PR China b College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou</p><p>450008, Henan province, PR China c Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361009, Fujian province, PR</p><p>China d Institute of Pharmaceutical Engineering, Huaqiao University, Xiamen 361021, Fujian province, PR China e Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, Xiamen</p><p>361021, Fujian province, PR China</p><p>*Corresponding authors: </p><p>Dr. Li-Tao Yi. Tel./Fax.: 86-592-6162302; E-mail address: [email protected]; [email protected]</p><p>Dr. Qing Liu. Tel./Fax.: 86-592-6162302; E-mail address: [email protected]</p><p>The project was supported by grants from the National Natural Science Foundation of China</p><p>(No. 81202940; 81303278) and the Science Research Foundation of ministry of Health &</p><p>United Fujian Provincial Health and Education Project for Tacking the Key Research (WKJ-</p><p>FJ-31). The authors reported no biomedical financial interests or potential conflicts of interest.</p><p>1 Materials and methods</p><p>Open-field test</p><p>To assess the effect of gypenosides on locomotor activity, mice were administered with gypenosides (25, 50, 100 mg/kg) and then were evaluated in the open-field test 1 h later. The apparatus consisted of a wooden box measuring 40×40×30 cm, with the floor divided into 25 equal squares (8 × 8 cm). The numbers of squares crossed with all paws (crossing) and of standing on the hind legs (rearing) were counted in a 3-min session. The apparatus was cleaned with a detergent and dried after occupancy by each mouse.</p><p>Validation of BDNF antibody</p><p>To assess the validation of BDNF antibody, BDNF recombinant protein (Boster, China) was dissolved in PBS. Four different concentrations of BDNF were set in the validated experiment: the BDNF-25nmol group, the BDNF-50nmol group, the BDNF-100nmol group, the vehicle group. Then the BDNF bands were got in western blotting. The data were shown below (provided as Fig.1S). With the increase of BDNF concentration, there was a corresponding increase of BDNF band value in western blotting. Therefore, the BDNF antibody (Santa Cruz) and the data about BDNF expression in the text of manuscript are</p><p>2 reliable. </p><p>Fig.1S BDNF expression induced by different BDNF treatment.</p><p>Results</p><p>The effects of gypenosides on the open-field test in mice</p><p>As shown in the Fig.2S, one-way ANOVA revealed neither fluoxetine nor gypenosides showed a significant alteration on the crossing number [F(4,35)=1.003, P=0.419] or rearing number [F(4,35)=0.826, P=0.518] of open-field test in mice.</p><p>A B 80 30 r r e e</p><p> b 60 b</p><p> m 20 m u u n</p><p> n</p><p> g 40 g n i n i s r s</p><p> a 10 o e</p><p> r 20 R C</p><p>0 0 e u 5 0 0 e u 5 0 0 l l 2 5 0 l l 2 5 0 ic F - - 1 ic F - - 1 h s s - h s s - e e e s e e e s V id id e V id id e s s id s s id o o s o o s n n o n n o e e n e e n p p e p p e y y p y y p G G y G G y G G</p><p>Fig.2S Effects of gypenosides on the crossing (A) and rearing (B) numbers of mice in open-</p><p>3 field test (n=8).</p><p>4</p>