Guajavadimer A, a dimeric caryophyllene-derived meroterpenoid with a new carbon skeleton from the leaves of Psidium guajava.

Transcription

1 Guajavadimer A, a dimeric caryophyllene-derived meroterpenoid with a new carbon skeleton from the leaves of Psidium guajava. Chuang-Jun Li, Jie Ma, Hua Sun, Dan Zhang, and Dong-Ming Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , People s Republic of China. *Corresponding author. zhangdm@imm.ac.cn; Fax/Tel:

2 Supporting Information Table of contents 1. General experimental procedures 2. Plant material 3. Extraction and isolation 4. Physico-chemical constant of guajavadimer A (1) 5. Single crystal X-ray data and structure of guajavadimer A (1) Table S1 X-ray crystallographic data for guajavadimer A (1) Figure S1. X-ray crystal structure of guajavadimer A (1). 6. Hepatoprotective activity assay Table S2 Hepatoprotective effect of compound 1 (10 μm) against APAP-induced toxicity in HepG2 cell. 7. PTP1B activity assay Figure S2. The UV spectrum of guajavadimer A (1) in CH 3 OH. Figure S3. The IR spectrum of guajavadimer A (1). Figure S4. The HR-ESI-MS spectrum of guajavadimer A (1) Figure S5. The HR-ESI-MS spectrum of guajavadimer A (1) Figure S6. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S7. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S8. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S9. The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S10 The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S11 The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S12. The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S13 The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S14. The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz). Figure S15 The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S16. The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S17. The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

3 Figure S18. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S19. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S20. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S21. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S22. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S23. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S24. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S25. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S26. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S27. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S28. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S29. The NOESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S30. The NOESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S31. The TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S32. The TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S33. The ROESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S34. The ROESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S35. The ROESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz). Figure S36. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S37. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S38. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S39. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) Figure S40. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz)

4 1. General experimental produces. Melting point was measured on an XT melting instrument and uncorrected. Optical rotation was measured on a JASCO P-2000 polarimeter. UV spectrum was measured on a JASCO V650 spectrophotometer. IR spectrum was recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). NMR spectra were acquired with VNS-600 spectrometers. HRESIMS spectrum was collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector, using an YMC-Pack ODS-A column ( mm, 5 μm). Column chromatography was performed with silica gel ( mesh, Qingdao Marine Chemical Inc., Qingdao, People s Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried out on glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. 2. Plant material. The leaves of Psidium guajava L. were collected in Guangxi, China, in September A voucher specimen (No. FL ) was identified by Professor Lin Ma from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College and was deposited at the herbarium of the Institute of Material Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. 3. Extraction and isolation. Air-dried leaves of Psidium guajava L. (20 kg) were extracted with 80% ethanol (200 L 2h 2). After evaporation of EtOH in vacuo, the aqueous residue was diluted with water and then partitioned with CHCl 3 (30 L 3). The CHCl 3 extract (800 g) was subjected to column chromatography on silica gel with CHCl 3 MeOH (100:0 5:1) to afford 10 fractions (A 1 A 10 ). Fraction A 1 (102 g) was separated by a silica gel column ( mesh, cm) eluted with cyclohexane EtOAc (100:0 50:50, three time column volume for each gradient) to afford 20 fractions

5 (B 1 B 20 ). Subfraction B 3 (13 g) was separated by a silica gel column (5 50cm) using cyclohexane EtOAc (95:5-70:30, three times of column volume for each gradient) as eluent to afford 10 fractions (C 1 C 10 ). Fraction C 3 (2.5g) was passed over an RP-18 column ( cm) with MeOH water (80 100%, three times column volume for each gradient) to give 5 subfractions C 3 a- C 3 e - The fraction C 3 d was purified by preparative HPLC (8 ml/min, t R 48.5 min, 98% MeOH, detected at 280 nm; YMC-Pack ODS-A column mm, 5 μm) to give 1 (15 mg). 4. Physico-chemical constant of guajavadimer A (1) Guajavadimer A (1). colorless crystal (in MeOH: CHCl 3 ), mp ºC, [α] 25 D 1.3 (CHCl 3 ; c 0.10); UV (MeOH) λ max (log ): 203 (3.67), 235 (3.34), 299 (3.30), 352 (2.64) nm; IR (KBr) ν max 3279, 2938, 1628, 1493, 1445, 1287, 1126, 1025, 774, 630 cm -1 ; HR-ESI-MS m/z ([M - H] -, calcd for C 61 H 70 O 8 : ). 5. X-ray Crystallographic Analysis of guajavadimer A (1) (See Table SI) Colorless crystal of 1 was obtained from MeOH: CHCl 3. Crystal data was acquired on a Agilent Xcalibur Eos Gemini diffractometer employing graphite monochromated Cu-Kα radiation (λ = Å). The structure was solved by direct methods using SHELXS-97. The crystal structure of 1 was solved by direct methods (SHELXS-97), expanded using a difference Fourier technique, and refined by the SHELXS-97 program and the full-matrix least-squares calculations. All non-hydrogen atoms were refined anisotropically, and all of the hydrogen atoms were fixed at calculated positions. Crystallographic data (excluding structure factor tables) for 1 have been deposited at the Cambridge Crystallographic Data Center as supplementary publication (CCDC ). Copies of the data can be obtained free of charge by application to CCDC, 12, Union Road, Cambridge CB21EZ, UK [Fax: (+44) ; deposit@ccdc.cam.ac.uk]. Table S1 X-ray crystallographic data for guajavadimer A (1) Empirical formula C 61 H 70 O 8 CHCl 3 Formula weight Temperature/K Crystal system orthorhombic

6 Space group P a/å (4) b/å (8) c/å (13) α/ β/ γ/ Volume/Å (4) Z 4 ρ calc g/cm μ/mm F(000) Crystal size/mm Radiation CuKα (λ = ) 2Θ range for data collection/ 5.94 to Index ranges -8 h 12, -20 k 18, -37 l 35 Reflections collected Independent reflections [R int = , R sigma = ] Data/restraints/parameters 10218/0/668 Goodness-of-fit on F Final R indexes [I>=2σ (I)] R 1 = , wr 2 = Final R indexes [all data] R 1 = , wr 2 = Largest diff. peak/hole / e Å /-0.50 Flack parameter 0.013(15)

7 Figure S1. X-ray crystal structure of Hepatoprotective activity assay Human HepG2 hepatoma cells were cultured in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/ml streptomycin at 37 C in a humidified atmosphere of 5% CO 2 +95% air. The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA. The MTT assay was used to assess the cytotoxicity of test samples. The cells were seeded in 96-well multiplates. After an overnight incubation at 37 C with 5% CO 2, 10 μm test samples and APAP (final concentration of 8 mm) were added into the wells and incubated for another 48 h. Then 100 μl of 0.5 mg/ml MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 μl of DMSO after aspiration of the culture medium. The plates were placed on a plate shaker for 30 min and read immediately at 570 nm using a microplate reader [10]. Table S2 Hepatoprotective effect of compound 1 (10 μm) against APAP-induced toxicity in HepG2 cell. Compounds OD (mean ± SD) Cell survival rate (% of normal) Control 2.376± APAP 8mM 1.451±0.114 *** ±0.094 ## Bicyclol a 1.650±0.190 # a Positive control substance. *** P< 0.001, compared with control. ## P< 0.01, compared with model (APAP) # P< 0.05, compared with model (APAP). 7. PTP1B activity assay [11] Recombinant human GST-PTP1B protein was over expressed by hgst-ptp1b-bl21 E. coli and purified by GST affinity chromatography. The reagent pnpp was used as substrate for the measurement of PTP1B activity. Compounds were pre-incubated with the enzyme at room temperature for 5 min. Assay was performed in final volume of 100

8 μl in the active system containing 50 Mm HEPES, 5 mm DTT, 150 mm NaCl, 2 mm EDTA, and 2 mm pnpp (ph 7.0), incubated at 30 C for 10 min, stopped by addition of 50 μl 3 M NaOH. Then, the absorbance was determined at 405 nm wavelength. The similar system without GST-PTP1B protein was used as blank. The effects of compound 1 on PTP1B activity were measured, compound 1 not exhibited inhibit PTPIB activity, with a inhibition values of 1.3% at 10 μm.

9 Figure S2. The UV spectrum of guajavadimer A (1).

10 Figure S3. The IR spectrum of guajavadimer A (1).

11 Figure S4. The HR-ESI-MS spectrum of guajavadimer A (1)

12 Figure S5. The HR-ESI-MS spectrum of guajavadimer A (1)

13 Figure S6. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

14 Figure S7. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

15 Figure S8. The 1 H NMR spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

16 Figure S9 The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

17 Figure S10 The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

18 Figure S11 The 13 C NMR spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

19 Figure S12. The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

20 Figure S13. The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

21 Figure S14. The DEPT spectrum of guajavadimer A (1) in CDCl 3 (150 MHz).

22 Figure S15 The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

23 Figure S16. The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

24 Figure S17. The 1 H- 1 H COSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

25 Figure S18. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

26 Figure S19. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

27 Figure S20. The HSQC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

28 Figure S 21. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

29 Figure S22. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

30 Figure S23. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

31 Figure S24. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

32 Figure S25. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

33 Figure S26. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

34 Figure S27. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

35 Figure S28. The HMBC spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

36 Figure S29. The NOESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

37 Figure S30. The NOESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

38 Figure S31. The TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz)

39 Figure S32. The TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

40 Figure S13. The ROESY spectrum of guajavadimer A (1) in CDCl 3.

41 Figure S34. The ROESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz)

42 Figure S35. The ROESY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz).

43 Figure S36. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) H-3B H-10B H-10A H-7,3B

44 Figure S37. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) H-3B H-3B H-7B H-7B H-2B

45 Figure S38. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) H-7, 3B H-5A H-7B H-5B H-3B

46 Figure S39. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) H-10A H-10A H-9A H-3A

47 Figure S40. The 1D-TOCSY spectrum of guajavadimer A (1) in CDCl 3 (600 MHz) H-10B H-9B H-1B H-10B