Jiang, Yunnan Province, China, in September 2018 and identified by the corresponding author Haifeng Tang. The voucher sample (No. 20180903) was deposited inside the Division of Chinese Materia Medica and Organic Medicines, School of Pharmacy, Air Force Health-related University, Xi’an, China. three.three. Extraction and Isolation The dried rhizomes of Paris mairei (1.0 kg) were chopped and refluxed with 70 ethanol (ten.0 L) thrice (every two h). The ethanol resolution was mixed and condensed having a vacuum rotary evaporator to acquire a syrupy residue (584.0 g). The extraction was PX-478 Autophagy suspended in water (three.0 L) and extracted with identical volume of petroleum ether and water saturated n-BuOH 3 instances, successively. The water saturated inside the n-BuOH layer was vacuum evaporated to offer a gummy residue (132.0 g). The crude extraction was separated by silica gel column chromatography and eluted by gradient eluent of CH2 Cl2 MeOH-H2 O (100:0:0, 50:1:0, 20:1:0, eight:1:0.1, 6:1:0.1, eight:2:0.2, 7:2.five:0.1, and six.five:3.5:0.1) to offer you 13 fractions (Fr.13) depending on the TLC evaluation. Fr.13 was separated by silica gel column chromatography and eluted by a gradient eluent of CH2 Cl2 -MeOH-H2 O (8:1:0.1, eight:two:0.2, 7:two.five:0.1, and 6:three:0.1) to get Fr.13-1 (1.1 g) and Fr.13-2 (830 mg). Fr.13-1 was eluted by MeOH on a Sephadex LH-20 to get rid of pigmentum and separated to Fr.13-1-1 (64 mg), Fr.13-1-2 (57 mg), and Fr-13-1-3 (145 mg) on ODS silica gel. Then, Fr.13-1-1 and Fr.13-1-3 were isolated by semi-preparative HPLC using MeCN-H2 O (35:65, 40:60) because the mobile phase at a flow price of eight.0 mL/min to afford compound 1 (9.1 mg, tR = 24.3 min) and four (eight.8 mg, tR = 48.6 min), respectively. Fr.11 was eluted by MeOH on a Sephadex LH-20 to remove pigmentum to acquire Fr.11-1 (4.two g), Fr.11-2 (five.0 g), and Fr.11-3 (430 mg). Fr.11-2 wasMolecules 2021, 26,13 ofJPH203 In Vivo subjected to ODS silica gel and purified by a semi-preparative HPLC utilizing MeCN-H2 O (50:50) because the mobile phase at a flow price of 8.0 mL/min to afford compound three (5.7 mg, tR = 44.1 min) and compound 7 (7.6 mg, tR = 40.2 min). Fr.12 was eluted by CH2 Cl2 -MeOH (20:80) on a Sephadex LH-20 to take away pigmentum and subjected to ODS silica gel to obtain Fr.12-1 (125 mg) and Fr.12-2 (670 mg). Then, compound two (26.7 mg, tR = 21.0 min) was supplied by semi-preparative HPLC utilizing MeCN-H2 O (60:40) because the mobile phase at a flow price of 8.0 mL/min. Fr.9 was purified by MeOH on a Sephadex LH-20 and separated on ODS silica gel to receive Fr.9-1 (231 mg), Fr.9-2 (102 mg), and Fr.9-3 (193 mg). The three collections have been successively purified by semi-preparative HPLC applying MeCN-H2 O (50:50, 40:60, 40:60) as the mobile phase at a flow price of 8.0 mL/min to obtain compounds 5 (11.5 mg, tR = 35.three min), 8 (five.5 mg, tR = 38.4 min), and 9 (four.six mg, tR = 28.7 min). Fr.10 was eluted by CH2 Cl2 -MeOH (50:50) on a Sephadex LH-20 to get rid of pigmentum and subjected to ODS silica gel to acquire Fr.10-1 (75 mg) and Fr.10-2 (one hundred mg). Fr.10-1 and Fr.10-2 were isolated by semi-preparative HPLC applying MeCN-H2 O (75:25) because the mobile phase at a flow price of 8.0 mL/min to afford compounds ten (7.five mg, tR = 18.three min) and six (24.4 mg, tR = 21.6 min), respectively. The purity of all compounds was assessed by HPLC as becoming a lot more than 95 . 3.four. Compound Characterization Data Pamaiosides A (1): white amorphous solid, []22D – 95.0 (c 0.05, MeOH); IR (KBr) max (cm-1 ): 3420, 2930, 1080, 990, and 840; constructive ESI-MS m/z 995.13 [M Na] , negative ESI-MS m/z 971.28 [M – H]- ; positive HR-ESI-MS m/z 995.4824.