Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 214 New sesquiterpenoids from the rhizomes of Acorus tatarinowii Xiao-Lin Feng, a Yang Yu, *a Hao Gao, a Zhen-Qiang Mu, a Xiao-Rui Cheng, b Wen-Xia Zhou, b and Xin-Sheng Yao *a a Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 51632, P. R. China. E-mail: tyaoxs@gmail.com; 118yuyang@163.com; Tel (Fax): +86-2-85221559. b Beijing Institute of Pharmacology and Toxicology, Tai Ping Road 27, Beijing 85, P. R. China. List of Supporting Information 1 AChE and BACE1 activity assays... 1 2 The 1D and 2D NMR spectra of 4-epi-2-hydroxyacorenone (1)... 3 3 The 1D and 2D NMR spectra of 4-epi-2-acetoxyacorenone (2)... 6 4 The 1D and 2D NMR spectra of acotatarone A (3)... 9 5 The 1D and 2D NMR spectra of acotatarone B (4)... 12 6 The 1D and 2D NMR spectra of tatarinowin C (5)... 16 7 The 1D and 2D NMR spectra of acotatarone C (6)... 19
1 AChE and BACE1 activity assays. 1.1 Chemicals and reagents Lyophilized powder of acetylcholinesterase (AChE) from electric eel source, acetylthiocholine iodide (ATCH) and 5,5 -dithiobis [2-nitrobenzoic acid] (DTNB) were purchased from Sigma-Aldrich (USA). TruPoint TM beta-secretase assay kit 384 was obtained from PerkinElmer (USA). Recombinant human beta-secretase (BACE1) was purchased from Invitrogen (USA). DMSO was obtained from ACROS (USA). Huperzine A and Ⅵa were synthesized by the Beijing institute of Pharmacology and Toxicology..1M phosphate buffer (PB) with ph of 7.4 was used as a buffer throughout the AChE inhibitory assay. 1.2 In Vitro AChE Activity Assay The AChE activity was measured using a modified 96-well microplate assay based on Ellman s method. 1 The enzyme hydrolyses the substrate (ATCH) resulting in the formation of thiocholine which reacts with Ellman s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 45 nm. The AChE stock solution ( U/mL) was kept at -2 C. The further enzyme-dilution was done in deionized water..7 mm DTNB and 3 mm ATCH were dissolved in the PB, respectively. The reaction mixture contained: 2 μl of.4 U/mL of AChE, 4 μl of test compound dissolved in PB (DMSO<1%, which was no influence on AChE activity) and 2 μl of ATCH, which incubated for 3 min at 37 C. Then ice bath for 3 s, 1 μl of 1. N HCl was added to terminate reaction. After the addition of 12 μl of.7 mm DTNB, the absorbance was measured at 45 nm (EnSpire 23, PerkinElmer) in 5 min. Huperzine A was used as positive control, with IC 5 value of 22.6 nm. 1-12 did not show any significantly inhibition of AChE activity, even at the concentration 1 1-3 M. 1.3 In Vitro BACE1 Activity Assay The assay was carried out using a homogeneous time-resolved fluorescence technique and a TruPoint TM beta-secretase assay kit 384 according to the manufacturer s protocol. Briefly, the test compounds in DMSO and recombinant human beta-secretase (BACE1) were first incubated in a 384-well black OptiPlate TM for 3 S1
min at 2-25 C. Then, the fluorescent Eu-CEVNLDAEFK-Qsy7 substrate was added to the wells and incubated for 6 h at 2-25 C, and the reaction was terminated by adding stop solution. Relative fluorescence units (RFU) at an excitation of 34 nm and an emission of 615 nm and kinetics were measured using a micro-plate reader Envision (PerkinElmer). Ⅵa was used as a positive control, 2 with IC 5 value of nm. Most of the sesquiterpenoids did not show any percentage of fluorescence reduction, except that 4-epi-2-acetoxyacorenone (2) showed week inhibitory rate (I %) at the concentration 1 1-3 M (33.6 %). 1.4 Statistics All statistical analyses were performed using Graph Pad 5.3 for Windows. Data are shown as means ± S.D. The percent inhibition of AChE activity was calculated as follows: I % = (E S) / E %, where E and S were the respective enzyme without and with the test sample, respectively. The percentage of inhibition of BACE1 activity was calculated using the following equation: I % = [1 (S B) / (N B)] %, where N is the activity of enzyme without test sample, S is the activity of enzyme with test sample and B is the background without enzyme and test sample. For the determination of IC 5 concentrations, the mean % inhibition dose-response curves were fitted to the dose-response-inhibition [log (inhibitor) vs. response-variable slope (four parameters)]. The dose-response equation: Y = Bottom + (Top Bottom) / [1 + 1(logIC 5 X) Hillslope], where X is the compound concentration, Y is the I %, top and bottom are the plateaus in the units of the y-axis, Hillslope describes the steepness of the family of curves. Reference: [1] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Feather-Stone, Biochem. Pharmacol. 1961, 7, 88-95. [2] X. R. Cheng, Y. Zhou, W. Gu, J. Wu, A. H. Nie, J. P. Cheng, J. W. Zhou, W. X. Zhou, Y. X. Zhang, Journal of Alzheimer s Disease, 213, 37, 823-834. S2
2 The 1D and 2D NMR spectra of 4-epi-2-hydroxyacorenone (1) 7.26 88 85 8 77 74 4.243 4.233 4.223 2.74 2.624 2.621 2.611 86 82 75 69 62 2.188 2.135 1.97 1.874 1.819 1.738 1.34 1.35 1.6.874.857 8 75 7 65 6 55 5 45 4 35 3 25 15 5 1..98.92.86.96 1.93.9 1. 2.91.94 2.99 2.88.99 2.89-5 7. 6. 5. 4. 3. 2. 1..5 1 H NMR spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3.8 144.2 13 77.3 77. 76.7 73.6 61.1 48.5 46.9 43. 41.2 34.6 25.1 23.2 18.4 15.6 24 2 18 16 14 1 8 6 4 23 22 21 19 18 17 16 15 14 13 12 11 9 8 7 6 5 4 3 2 1 13 C NMR spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3 S3
1 2 3 4 5 6 7 8 9 11 12 13 14 15 6. 5. 4. f2 (ppm) 3. 2. 1. HSQC spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3 1. 2. 3. 4. 5. 6. 6. 5. 4. f2 (ppm) 3. 2. 1. 1 H- 1 H COSY spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3 S4
2 4 6 8 12 14 16 18 7. 6. 5. 4. f2 (ppm) 3. 2. 1. HMBC spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3 NOESY spectrum for 4-epi-2-hydroxyacorenone (1) in CDCl 3 S5
3 The 1D and 2D NMR spectra of 4-epi-2-acetoxyacorenone (2) 7.2599 6.6283 6.6251 6.6191 6.6145 6.685 5.1883 5.1863 5.1771 5.1657 5.1639 2.758 2.712 2.68 2.6735 2.6672 2.6319 2.6255 2.6189 749 78 359 2.2513 32 1.954 1.9184 1.8899 1.8482 1.7634 1.441 1.3618 1.2545 1.797.919.8946.8785.869 9 8 7 6 5 4 3.99 1..88.95.95.98 4.6 2.17 3.16 1.27 1.9.53 2.88 3.8 3.3 7. 6. 5. 4. 3. 2. 1..5 1 H NMR spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 18 17 16 15 14 13 1 1 9 8 7 6 5 4 3 22 21 19 18 17 16 15 14 13 12 11 9 8 7 6 5 4 3 2 1 13 C NMR spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 S6
HSQC spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 1 H- 1 H COSY spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 S7
HMBC spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 NOESY spectrum for 4-epi-2-acetoxyacorenone (2) in CDCl 3 S8
4 The 1D and 2D NMR spectra of acotatarone A (3) 7.265 6.7786 6.7737 3.282 2.9963 2.61 2.4317 2.3421 2.211 1.9225 1.7695 1.7151 1.1898 1.1686.9343.778.7479 12 11 9 8 7 6 5 4 3 2 1 1..97 1.8 2.11 1.11 2.82 1.25 1.23 2.99 2.98 3.4-1 7. 6. 5. 4. 3. 2. 1..5 1 H NMR spectrum for acotatarone A (3) in CDCl 3 13 C NMR spectrum for acotatarone A (3) in CDCl 3 S9
HSQC spectrum for acotatarone A (3) in CDCl 3 1 H- 1 H COSY spectrum for acotatarone A (3) in CDCl 3 S1
2 4 6 8 12 14 16 18 7. 6. 5. 4. f2 (ppm) 3. 2. 1..5 HMBC spectrum for acotatarone A (3) in CDCl 3.5 1. 2. 3. 4. 3. 2. f2 (ppm) 1..5 NOESY spectrum for acotatarone A (3) in CDCl 3 S11
5 The 1D and 2D NMR spectra of acotatarone B (4) 17 16 15 14 13 1 1 9 8 7 6 5 4 3-7. 6. 5. 4. 3. 2. 1..5. 1 H NMR spectrum for acotatarone B (4) in CDCl 3 9 8 7 6 5 4 3-23 22 21 19 18 17 16 15 14 13 12 11 9 8 7 6 5 4 3 2 1 13 C NMR spectrum for acotatarone B (4) in CDCl 3 S12
2 1 7. 6. 5. 4. 3. 2. 1..5 1 H NMR and 1D-NOE spectra for acotatarone B (4) in CDCl 3 1 2 3 4 5 6 7 8 9 11 12 13 14 15 7. 6. 5. 4. f2 (ppm) 3. 2. 1..5. HSQC spectrum for acotatarone B (4) in CDCl 3 S13
1 2 3 4 5 6 7 7. 6. 5. 4. 3. f2 (ppm) 2. 1..5. -.5 1 H- 1 H COSY spectrum for acotatarone B (4) in CDCl 3 2 4 6 8 12 14 16 18 22 8. 7. 6. 5. 4. f2 (ppm) 3. 2. 1..5. -.5 HMBC spectrum for acotatarone B (4) in CDCl 3 S14
NOESY spectrum for acotatarone B (4) in CDCl 3 S15
6 The 1D and 2D NMR spectra of tatarinowin C (5) 65 6 55 5 45 4 35 3 25 15 5-5 7. 6. 5. 4. 3. 2. 1..5 1 H NMR spectrum for tatarinowin C (5) in CDCl 3 1E+5 1E+5 1E+5 9 8 7 6 5 4 3-23 22 21 19 18 17 16 15 14 13 12 11 9 8 7 6 5 4 3 2 1 13 C NMR spectrum for tatarinowin C (5) in CDCl 3 S16
HSQC spectrum for tatarinowin C (5) in CDCl 3 1 H- 1 H COSY spectrum for tatarinowin C (5) in CDCl 3 S17
HMBC spectrum for tatarinowin C (5) in CDCl 3 NOESY spectrum for tatarinowin C (5) in CDCl 3 S18
7 The 1D and 2D NMR spectra of acotatarone C (6) 6 55 5 45 4 35 3 25 15 5-5 7. 6. 5. 4. 3. 2. 1..5 1 H NMR spectrum for acotatarone C (6) in CDCl 3 4 4 38 36 34 3 3 28 26 24 2 18 16 14 1 8 6 4 21 19 18 17 16 15 14 13 12 11 9 8 7 6 5 4 3 2 1 13 C NMR spectrum for acotatarone C (6) in CDCl 3 S19
2 1 5.8 5.6 5.4 5.2 5. 4.8 4.6 4.4 4.2 4. 3.8 3.6 3.4 3.2 3. 2.8 2.6 2.4 2.2 2. 1.8 1.6 1.4 1.2 1..8 1 H NMR and 1D-NOE spectra for acotatarone C (6) in DMSO-d 6 1 2 3 4 5 6 7 8 9 11 12 13 14 15 6. 5. 4. 3. f2 (ppm) 2. 1. HSQC spectrum for acotatarone C (6) in CDCl 3 S2
1 H- 1 H COSY spectrum for acotatarone C (6) in CDCl 3 2 4 6 8 12 14 16 18 7. 6. 5. 4. f2 (ppm) 3. 2. 1..5 HMBC spectrum for acotatarone C (6) in CDCl 3 S21