Exploring Aporphine As Anti-inflammatory And Analgesic .
Supporting Information ic Lead from Dactylicapnos scandensBei Wang‡,§,ǁ, Yin-Jiao Zhao†,ǁ, Yun-Li Zhao†,‡,ǁ, Ya-Ping Liu‡, Xiao-Nian Li ‡, HongBin Zhang†,*, and Xiao-Dong Luo†,‡,*†Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and YunnanProvince, School of Chemical Science and Technology, Yunnan University, Kunming 650091,China‡State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Instituteof Botany, Chinese Academy of Sciences, Kunming, 650201, China§KeyLaboratory of Screening and Processing in New Tibetan Medicine of Gansu Province, Schoolof Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
Contents1. Plant material . 12. Extraction and isolation . 13. Table S1. 1H and 13C NMR Spectral Data ( in ppm) of 1 and 2. . 34. Total synthesis of dactylicapnosine A . 44.1 Sheme S1: Total synthesis of dactylicapnosine A . 44.2 Experimental details and characterization data . 45. Evaluation of anti-inflammatory activity in vitro . 145.1. Effects on cell viability of RAW 264.7 . 14Table S2. Effect of dactylicapnosines on cell viability after 24 h treatment. . 145.2 Effect on the production of inflammatory cytokines in vitro . 15Table S3. Effects of dactylicapnosines on the production of cytokines in LPS-induced RAW264.7 cells . 156. Evaluation of anti-inflammatory and analgesic in vivo . 166.1 Effect on xylene-induced inflammation in mice. . 16Table S4. Effects of dactylicapnosine A on auricle swelling induced by xylene in mice . 176.2 Effect on acetic acid-induced pain in mice. . 17Table S5. The analgesic effect of dactylicapnosine A in mice induced by acetic acid . 186.3 Effect on formaldehyde-induced pain in mice. . 18Table S6. The analgesic effect of dactylicapnosine A in mice induced by formaldehyde . 196.4 Effect on hotplate-induced pain in mice. 19Table S7. The analgesic effect of dactylicapnosine A in mice induced by hotplate . 206.5 Ethical approvals of experimental animals. . 21Reference . 227. Supplementary Figures . 23Figure S01. The HPLC profiles of separation of ( )-1 and (-)-1. . 23Figure S02. The HPLC profiles of separation of ( )-2 and (-)-2. . 24Figure S03. The overlapped experimental CDs of compounds ( )-2 and (-)-2. . 24Figure S04. 1H NMR spectrum of compound 1 (CDCl3/CD3OD) . 25Figure S05. 13C NMR and DEPT spectra of compound 1 (CDCl3/CD3OD). 26Figure S06. HSQC spectrum of compound 1 (CD3OD) . 27Figure S07. HMBC spectrum of compound 1 (CD3OD) . 28Figure S08. HREIMS spectrum of compound 1 . 29Figure S09. ESIMS spectrum of compound 1. 30Figure S10. IR spectrum of compound 1 . 31Figure S11. UV spectrum of compound 1 . 32Figure S12. CD spectrum of compound ( )-1. 33Figure S13. CD spectrum of compound (-)-1 . 35Figure S14. 1H NMR spectrum of compound 2 (CDCl3) . 37Figure S15. 13C NMR and DEPT spectra of compound 2 (CDCl3) . 38
Figure S16. HSQC spectrum of compound 2 (CDCl3) . 39Figure S17. HMBC spectrum of compound 2 (CDCl3) . 40Figure S18. HREIMS spectrum of compound 2 . 41Figure S19. ESIMS spectrum of compound 2. 42Figure S20. IR spectrum of compound 2 . 43Figure S21. UV spectrum of compound 2. 44Figure S22. CD spectrum of compound ( )-2. 45Figure S23. CD spectrum of compound (-)-2 . 47Figure S24. Copies of NMR spectra of compounds in Total synthesis . 498. Crystal data and structure refinement for dactylicapnosine A (1). . 639. ECD Calculation of dactylicapnosine A (1) . 63Table S8 Conformational population of R-1 at MMFF94 force field. . 64Table S9 Standard orientation of R-1 at B3LYP/6-311G* level in gas phase. . 64Table S10 Standard orientation of R-1 at B3LYP/6-311G* level in solvent phase. . 66References . 67
1. Plant materialAir-dried roots of D. scandens were purchased from a market of Chinese medical materialslocated at Zhonghao-Luoshi-Wan of Kunming, Yunnan province, P.R. China, in February 2015.The material was identified by Dr. Ya-Ping Liu. A voucher specimen (No. 201500208) has beendeposited at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.2. Extraction and isolationAir-dried roots of D. scandens (3.9 kg) were extracted with 80.0% MeOH/H2O under refluxconditions, and the solvent was evaporated in vacuo. The residue was dissolved in 0.37% HCl(pH 2-3) and the solution was subsequently basified using 10% ammonia to pH 9-10. The basicsolution was partitioned with EtOAc, affording a two-phase mixture. The EtOAc fraction (104g) was divided into Fr. A-F by using silica gel chromatography (CHCl3/MeOH, 1:0-2:8, v/v).Of which, Fr.C (3.5 g) was separated by RP-18 chromatography (MeOH/H2O, 35:65-100:0) togive six portions, then Fr.C5 (0.65g) was subjected to silica gel chromatography under isocraticconditions (Petroleum ether/Acetone, 10:1, v/v), Sephadex LH-20 (CHCl3/MeOH) andrecrystallized to yield 1 (8.9 mg) and subfractions. Among them, Fr.C5-2 (0.1 g) was elutedthrough silica gel chromatography (Petroleum ether/ Acetone, 5:1, v/v) to afford 2 (8.4 mg).Compounds 1 and 2 were further purified by HPLC equipped with a chiralphase column(Reprosil chiral-AM, 5 µm, 250mm*4.6mm r65am. S2546 (Dr. maisch)), (n-hexane/ethanol,80:20, 1.0 mL/min; n-hexane/ethanol, 70:30, 1.0 mL/min) to give (-)-1 (2.3 mg, tR 25.5min)and ( )-1 (2.2 mg, tR 34.8 min), (-)-2 (2.0 mg, tR 33.9 min) and ( )-2 (2.2 mg, tR 41.8 min),respectively.Dactylicapnosine A (1)light yellow crystalline lumps (CH3OH), mp 171.1-172.3 C, [ ]18D 5.90 (c 0.13, CH3OH).;UV (MeOH) λmax (log ε) nm 211 (4.03), 264 (3.87), 294 (3.59), 394 (3.84); IR (KBr) νmax 3436,2947, 1739, 1709, 1583, 1526, 1463, 1166cm-1; 1H, 13C-NMR spectroscopic data, see Table 1;ESIMS m/z 454 [M Na] ; HRESIMS m/z 431.1591 [M] (calcd for C22H25NO8, 431.1580).1
( )-1a [ ]25D 115 (c 0.10, CHCl3)(-)-1b [ ]25D -118 (c 0.10, CHCl3)Dactylicapnosine B (2)Pale yellow powder, [ ]18D -11 (c 0.1, MeOH); UV (MeOH) λmax (log ε) nm 206 (4.12), 275(3.73), 333 (3.56), 425 (3.60), 529 (3.65); IR (KBr) νmax 3411, 2924, 2853, 1739, 1630, 1528,1384, 1317, 1180cm-1; 1H, 13C-NMR spectroscopic data, see Table 1; ESIMS m/z 440 [M Na] ; HREIMS m/z 417.1423 [M] (calcd for C21H23NO8, 417.1424).( )-2a [ ]24D 42 (c 0.04, CH3OH)(-)-2b [ ]24D -47 (c 0.06, CH3OH).2
3. Table S1. 1H and 13C NMR Spectral Data ( in ppm) of 1 and H (J in Hz)1bδC2cδH (J in Hz)144.1δCδH (J in Hz)143.7δC143.012.6, s7.18, s3.24, t, (6.4)3.58, t, (6.4)6.48, s3.93, s4.02, s3.19, s3.65, s3.54, s3.46, , s3.18, t, (6.5)3.53, overlap6.41, s3.97, s4.01, s3.13, s3.67, s3.53, s3.47, 1.381.1101.9187.6117.461.556.440.353.552.051.6a 1Hand 13C NMR spectra were recorded at 600 and 150 MHz, respectively in CD3OD;b 1Hand 13C NMR data were recorded at 500 and 125 MHz, respectively in CDCl3;c 1Hand 13C NMR data were recorded at 600 and 150 MHz, respectively in CDCl3.36.90, s3.14, t, (6.5)3.57, t, (6.5)6.38, s3.99, s3.21, s3.71, s3.52, s3.51, 0.982.4101.0192.2115.956.440.653.752.251.7
4. Total synthesis of dactylicapnosine A4.1 Sheme S1: Total synthesis of dactylicapnosine A4.2 Experimental details and characterization dataMelting points were measured on a XT-4 melting-point apparatus and were uncorrected. Theinfrared (IR) spectra were recorded on a Nicolet iS10 FTIR spectrometer. Proton nuclear magneticresonance (1H-NMR) spectra were measured on Bruker Avance 300 or 400 spectrometers at 300 or400 MHz. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were recorded on BrukerAvance 300 or 400 spectrometers at 75 or 100 MHz. Chemical shifts are reported as δ values inparts per million (ppm) relative to tetramethylsilane (TMS). High Resolution Mass spectra were4
measured with an Agilent LC/MSD TOF mass spectrometer. For reactions conducted under MWconditions, a SEM DISCOVER SP-D microwave reactor was used. Silica gel (200–300 mesh)for column chromatography and silica GF254 for TLC were produced by Merch Chemicals Co. Ltd.(Shanghai). Toluene and THF used in the reactions were dried by distillation over metallic sodiumand benzophenone. Dichloromethane was distilled from calcium hydride or P2O5. Starting materialsand reagents used in reactions were obtained commercially from Acros, Aldrich, Adamas-beta ,and were used without purification, unless otherwise indicated. All moisture-sensitive reactionswere conducted in oven-dried glassware under a positive pressure of dry nitrogen or argon. Reagentsand starting materials were accordingly transferred via syringe or cannula. Unless otherwise stated,all other reactions were performed under a positive nitrogen atmosphere. Reaction temperaturesrefer to the external oil bath temperature.Compound 5aAllyl ether (3.0 g) in dry DMF (4 mL) in a sealed tube allowed to stir at 190 C for 16 h. Afterthis time, the reaction mixture allowed to cool to room temperature and diluted with water (80mL). The resulting mixture was extracted with EtOAc (4 20 mL), and the combined organicphases were washed with brine (15 mL) and dried over anhydrous MgSO4. After being filteredand concentrated under reduced pressure, the residue was purified by flash chromatography onsilica gel (petroleum ether/ethyl acetate 10:1) to give phenol 5a (2.7g, 93%) as a pale yellowsyrup.The spectral data are consistent with those reported in the literature (Bochicchio, A.; Cefola, R.;Choppin, S.; Colobert, F.; Di Noia, M. A.; Funicello, M.; Hanquet, G.; Pisano, I.; Todisco, S.;Chiummiento, L. Selective Claisen rearrangement and iodination for the synthesis ofpolyoxygenated allyl phenol derivatives. Tetrahedron Lett. 2016, 57, 4053–4055).H NMR (400 MHz, CDCl3): δ 6.44 (s,1H), 6.04-5.94 (m, 1H), 5.49 (s,1H), 5.12-5.05 (m, 2H),3.95 (s,3H), 3.87 (s,3H), 3.80 (s,3H), 3.36 (d, J 6.4 Hz, 2H);15
C NMR (100 MHz, CDCl3): δ 146.4, 140.9, 140.5, 140.3, 136.7, 120.1, 115.7, 108.6, 61.4,61.1, 56.7, 34.1.13Compound 5bTo a solution of phenol 5a (16.95 g, 75.6 mmol) in acetone (200 mL), K2CO3 (52.24 g, 378mmol) was added followed by benzyl bromide (11mL, 90.72 mmol). The resulting mixture wasstirred at 65 C (oil bathed) for 36 h. After being cooled to room temperature, the mixture wasfiltered through a short column of silica gel and washed with ethyl acetate. The filtrate wasconcentrated and the residue was purified by silica gel column chromatography (petroleumether/ethyl acetate 20:1) to give the product (5b, 23.51g, 99%) as a colorless syrup.H NMR (400 MHz, CDCl3): δ 7.47-7.31 (m, 5H), 6.45 (s, 1H), 5.94-5.84 (m, 1H), 5.05 (d, J 13.2 Hz, 2H), 4.95 (s, 2H), 3.95 (s, 3H), 3.90(s, 3H), 3.83 (s, 3H), 3.33(d, J 6.4 Hz, 2H);13C NMR (100 MHz, CDCl3): δ 149.6, 147.4, 144.1, 141.5, 137.9, 137.3, 128.6, 128.4, 128.3,128.1, 116.0, 107.7, 75.5, 61.4, 56.3, 34.3;IR (KBr, thin film, cm 1): 2937, 1637, 1586, 1490, 1461, 1431, 1413, 1374, 1341, 1231, 1191,1127, 1078, 1039, 999, 915, 836, 734, 698;HRMS (ESI ) m/z [M Na] : calcd for C19H22NaO4: 337.1410, found: 337.1410.1Compound 6Olefin 5b (3.0 g) was dissolved in MeOH (2 mL) and CH2Cl2 (20 mL). The solution was thencooled to 78 C. A stream of ozone was passed through the solution (ca. 15 min, progress wasmonitored by TLC). The excess ozone was removed by a stream of oxygen (5 min), anddimethyl sulfide (4 mL) was added. The resulting mixture allowed to stir at room temperature6
for 12 h. After removal of the solvent, the residue was purified by flash chromatography onsilica gel (petroleum ether/ethyl acetate 5:1) to give aldehyde 6 (2.35 g, 78%) as pale- yellowoil.H NMR (400 MHz, CDCl3): δ 9.53 (t, J 2.0 Hz, 1H), 7.40-7.30 (m, 5H), 6.40 (s, 1H), 4.98(s,2H), 3.96 (s, 3H), 3.92(s, 3H), 3.81 (s, 3H), 3.52(d, J 2.0 Hz, 2H);13C NMR (100 MHz, CDCl3): δ 199.6, 149.8, 147.5, 144.5, 142.7, 137.3, 128.6, 128.5, 128.2,120.7, 108.5, 75.4, 61.3, 56.2, 45.2;IR (KBr, thin film, cm 1): υ 2938, 2840, 2720, 1720, 1586, 1491, 1460, 1432, 1415, 1375, 1347,1233, 1127, 1087, 1041, 997, 916, 837, 755, 699, 503;HRMS (ESI ) m/z [M Na] : calcd for C18H20NaO5: 339.1203, found: 339.1201.1Compound 7To a solution of aldehyde 6 (8.0 g, 25.32 mmol) in dichloromethane (150 mL) was added Nbromosuccinimide (NBS, 4.956 g, 27.84 mmol). The resulting mixture was stirred at roomtemperature for 5 min before addition of acetic acid (5 mL). After being stirred for 1h, thereaction was quenched with water (50 mL) and the organic layer was separated. The aqueousphase was then extracted with CH2Cl2 (3 25 mL). The combined organic phases were driedover anhydrous MgSO4. After removal of the solvents under reduced pressure, the residuepurified by flash chromatography on silica gel (petroleum ether/ethyl acetate 10:1) to affordthe bromide (7, 9.52g, 95%) as pale yellow oil.H NMR (300 MHz, CDCl3): δ 9.56 (t, J 1.2 Hz, 1H), 7.40-7.33 (m, 5H), 4.98 (s, 2H), 3.97(s, 3H), 3.94 (s, 3H), 3.88 (s, 3H), 3.83(d, J 1.5 Hz, 2H);13C NMR (75 MHz, CDCl3): δ 198.6, 147.8, 147.7, 147.4, 146.7, 136.8, 128.6, 128.4, 123.0,114.6, 75.7, 61.4, 61.3, 61.0, 45.1;IR (KBr, thin film, cm 1): 2938, 2837, 2718, 1724, 1497, 1466, 1412, 1373, 1347, 1304, 1244,1196, 1118, 1084, 1040, 989, 919, 794, 748, 699;HRMS (ESI ) m/z [M Na] : calcd for C18H19BrNaO5: 417.0308, found: 417.0303.17
Compound 8To a solution of aldehyde 7 (9.52 g, 24.16 mmol) in dichloromethane (200 mL) was added tertbutyl (3,4-dimethoxyphenethyl) carbamate (4a, 6.18 g, 21.96 mmol). After being stirred atroom temperature for 5 min, CF3COOH (8.2 mL, 109.8 mmol) was introduced. The reactionmixture was then stirred for 3 h. After TLC, the reaction mixture was quenched by carefullyaddition of 5% aqueous solution of NaOH, until pH 13-14. Boc anhydride [(Boc)2O, 2.39 g,10.98 mmol] was then added, and the resulting mixture was stirred at ambient temperature for12 h. The resulting mixture was diluted with water (150 mL) and extracted with CH2Cl2 (3 100 mL). The combined organic layer was dried over anhydrous MgSO4. After removal of thesolvents, the residue was purified by flash chromatography on silica gel (petroleum ether/ethylacetate 5:1) to give isoquinoline 8 (12.46 g, 86%) as a syrup.H NMR (400 MHz, CDCl3): δ 7.39-7.18 (m, 5H), 6.71-6.25 (m, 2H), 5.50-5.32 (m, 1H), 5.024.57 (m, 2H), 4.10-4.06 (m, 1H), 3.87-3.61 (m, 14H), 3.40-2.92 (m, 4H), 2.73-2.37 (m, 2H),1.34-1.11 (m, 9H);13C NMR (100 MHz, CDCl3): δ 154.4, 154.2, 148.4, 148.3, 147.8, 147.5, 147.3, 147.1, 146.9,146.6, 146.5, 146.4, 146.2, 137.9, 137.6, 129.4, 129.3, 128.5, 128.0, 127.9, 127.8, 127.5, 126.9,126.6, 120.7, 115.2, 115.1, 112.0, 111.5, 111.4, 111.2, 110.2, 109.9, 79.1, 79.0, 74.8, 74.7, 61.5,61.4, 61.3, 61.0, 55.9, 55.8, 55.7, 53.6, 52.8, 39.0, 36.9, 36.7, 28.5, 28.4, 28.3, 28.2;IR (KBr, thin film, cm 1): 3727, 3629, 2935, 1685, 1654, 1518, 1466, 1412, 1364, 1332, 1244,1227, 1165, 1120, 1098, 1082, 1038, 1001, 937, 859, 766, 699;HRMS (ESI ) m/z [M Na] : calcd for C33H40BrNNaO8: 680.1830, found: 680.1834.18
Compound 9Compound 8 (1.31 g, 2 mmol), palladium acetate (45 mg, 0.2 mmol), PPh3 (534 mg, 2 mmol)and K2CO3 (552 mg, 4 mmol) were put into a tube. N, N-dimethylacetamide (20 mL) was added(operations were conducted in a nitrogen-filled glovebox). After being stirred at roomtemperature for 1h, the sealed vessel was removed from the glovebox, and irradiated in amicrowave reactor (SEM DISCOVER SP-D, 150 W) at 150 C for 1h. The reaction mixtureallowed to cool to room temperature, diluted with water (80 mL) and extracted with ethylacetate (4 50 mL). The combined organic phases were washed with brine (30 mL), dried overanhydrous MgSO4. After being filtered and concentrated under reduced pressure, the residuewas purified by flash chromatography on silica gel (petroleum ether/ethyl acetate 5:1) to giveaporphine 9 (0.9 g, 78%) as pale yellow solid.Melting Point: 161-162 C;1H NMR (400 MHz, CDCl3): δ 7.52-7.29 (m, 5H), 6.67 (s, 1H), 5.05 (d, J 10.0 Hz, 1H), 4.93(d, J 10.8 Hz, 1H), 4.49 (br s, 1H), 4.39 (br s, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.87 (s, 3H),3.72 (s, 3H), 3.64 (s, 3H), 3.32 (dd, J 3.2, 13.6 Hz, 1H), 2.92-2.81 (m, 2H), 2.67-2.61(m, 1H),2.19(t, J 13.6 Hz, 1H), 1.46 (s, 9H);13C NMR (100 MHz, CDCl3): δ 154.8, 151.8, 149.3, 146.8, 145.9, 145.6, 144.7, 137.8, 128.5,128.4, 128.2, 128.0, 127.4, 127.3, 125.4, 120.2, 111.8, 80.1, 75.6, 61.5, 61.4, 61.2, 60.8, 56.1,51.9, 38.9, 30.1, 29.5, 28.5;IR (KBr, thin film, cm 1): 3438, 2945, 1677, 1598, 1463, 1400, 1368, 1324, 1286, 1263, 1168,1121, 1041, 1017, 1001, 975, 847, 824, 772, 755, 700;HRMS (ESI ) m/z [M Na] : calcd for C33H39NNaO8: 600.2568,
Supporting Information for Exploring Aporphine as Anti-inflammatory and Analgesic Lead from Dactylicapnos scandens Bei Wang‡ ,§ǁ, †Yin-Jiao Zhao,ǁ, Yun-Li Zhao†‡, Ya-Ping Liu‡, Xiao-Nian Li ‡, Hong- Bin Zhang †,*, and Xiao-Dong Luo ‡,* †Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan
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Research has shown that lifestyle choices can decrease inflammation to our choiceso; can influence how much inflammation we have in our bodies. Adopting a healthy diet as well as other healthy lifestyle behaviors can have a dramatic effect on inflammation levels. The Anti-Inflammatory Lifestyle Includes Eating anti-inflammatory foods
a powerful non-hormonal anti-inflammatory substance capable of inhibiting cyclooxygenase and reducing the levels of prostaglandins A and E22. This anti-inflammatory action was clearly observed in the evaluation of the analgesic effect of indomethacin and alcoholic green tea extracts on Swiss mice with acetic acid-induced abdominal contortions.
Its effect is dose dependent and can be blocked with naloxone. KEY WORDS: Curcumin, Analgesic, Anti-inflammatory, Formalin test, Hot-plate, Morphine, Naloxone. Please cite this article as follows: Kavousi M, Kazemi S, Hashemi M, Moghadamnia AA. Investigation on the Analgesic and Anti-inflammatory Effects of Curcumin in Mice.
Anti-inflammatory and analgesic potential The extract of stem of S. persica was reported to possess anti-inflammatory activity. ACE-inhibiting ability In vitro screening has reported that S. persica possesses high ACE-inhibiting ability. Anticonvulsant and sedative potential The extracts of S. persica extended sleeping time
The central analgesic action of paracetamol is like aspirin, i.e. it raises pain threshold, but has weak peripheral anti-inflammatory component. Analgesic action of aspirin and Paracetamol is additive. Paracetamol is a good and promptly acting antipyretic. Paracetamol has negligible anti-inflammatory action. It is a poor inhibitor of
2.2.2. Anti-Inflammatory Activities Anti-inflammatory effect of aqueous extract of Ceiba pentandrawas evaluated using two standard methods: the carrageenan induced paw oedema and cotton pellet induced granuloma (chronic inflammation) models[9] [10]. For each method, four groups of 5 rats were used.
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