PUBLIE NUM 2

Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. RESEARCH ARTICLE Received on: 24-06-2016 Accepted on: 11-07-2016 Published on: 09-09-2016 Corresponding Author: Flavien Aristide Alfred TOZE, Faculty of Sciences, Department of Chemistry, University of Douala, PO Box 24157, Douala, Cameroon, Tel.: +237-677-743961, Email: [email protected]. CONFLICT OF INTEREST NONE DECLARED Antioxydant and the urease Inhibition Activities of parinari hypochrysea Mildbr. Exletouzey & F. White (chrysobalanaceae): Twigs. Martial Flora Adjapmoh Essombo1, Flavien Aristide Alfred Toze 1*, Moses K. Langat2, Mehreen Lateef3, Juliette Catherine Vardamides 1, Luc Leonard Mbaze Meva’a1, Muhammad Shaiq Ali 4, Jean Duplex Wansi 1, Alain Francois Kamdem Waffo 1 1-Department of Chemistry, University of Douala, Faculty of Science, 24157 Douala, Cameroon. 2- Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, GU2 7XH, United Kingdom. 3-Pharmaceutical Research Center, Pakistan Council of Scientific & Industrial Research Complex, Karachi, Pakistan. 4-H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan. Abstract A new flavonol, parinariflavone (1) and a new cerebroside, parinarioside (6) together with nine known compounds were isolated from the methanol extract of the twigs of Parinari hypochrysea. Their structures were elucidated by means of spectroscopic analyses including 1D- and 2D-NMR spectroscopy, high resolution mass spectrometric data, chemical reactions, as well as comparison with data from literature. These compounds were screened in vitro for their free radical scavenging activity using DPPH and urease inhibition activity. The radical scavenging activity using DPPH assay gave significantly high activity for parinariflavone(1) with IC50 12.4 ± 0.56 μm compared to the phenolic synthetic antioxidant standard BHA with IC50 44.2 ± 0.15 μm, while urease inhibition activity for the same compound gave moderate antioxidant activity with IC50 33.7 ± 0.11 μm compared to the standard thiourea with IC50 = 21.9 ± 0.63 μm. Keywords: Parinarihypochrysea, Chrysobalanaceae, flavonol, Cerebroside, Antioxidant and urease inhibition activities. Introduction Chrysobalanaceae is a family composed of seventeen genera and about 525 species. In Africa and South America some species have popular indications for various diseases such as malaria, epilepsy, diarrhoea, inflammations and diabetes[1]. Some species of Parinari have been claimed to be effective in the treatment of veneral diseases and erectile dysfunctions [2]. Previous research on the Parinari genus reported the isolation of various flavonol glycoside: myrcetin, quercetin and kampferol [3], fatty acid [4], and diterpenes [5, 6]. A recent study of the methanolic extract of the leaves of this plant described the isolation a new ceramide, parinaramide, a new biflavonoid, sparinaritin, together with kaempferol, quercetin, taxifolin, taxifolin-3-O-rhamnoside, lupeol, betulinic acid, ursolic acid, 2α-hydroxy-ursolic acid, 2,3dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1propanone, and sucrose [7]. This result prompted us to undertake the study of the twigs of Parinarihypochrysea. In this report, we describe the isolation and structural elucidation of two new compounds, parinariflavone(1) and parinarioside(6), together with the antioxidant and the urease compounds. inhibition activitiesof some isolated Material and Methods General Melting point was determined on a Buchi M-560 melting point apparatus, UV spectra were carried out on Evolution 300 BB, IR spectra were run on SHIMAZU FTIR-8900 spectrophotometer, 1H and 13C, 1D and 2D NMR spectra were recorded on a Bruker DRX unitspectrometer operating at 400 MHz (1H), 100 MHz (13C) using TMS as the internal standard. Chemical shifts are reported in δ units and coupling constants (J) in Hz. The EI-MASS was recorded on JEOL MS 600-I mass spectrometer, ESIMASS was on QSTARXL mass spectrometer. Thin-layer chromatography (TLC) was performed on a Merck silica gel precoated plates (GF254, 20 x 20 and 0.20 mm thickness) and column chromatography was carried out on silica gel Kieselgel 60 (230-300 Mesh, 60 Ǻ Mesh), Sephadex LH-20 (Pharmacia) was used for purified column chromatography. Spots were visualized under UV lamp (254 and 365 nm), sprayed with ceric acid solution followed by heating at a temperature of 110°C for 5 minutes. All solvents were distilled before use. © Asian Journal of Ethnopharmacology and Medicinal Foods, 2016. 26 Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. Collection and identification Twigs of P. hypochrysea was collected in the month of May 2014 from DIBAMBA, Littoral region, Cameroon. The sample was identified by Mr. NANA Victor, botanist at the National herbarium of Yaoundé Cameroon, where a deposited voucher specimen (N° 55511 HNC) was compared. Extraction and isolation Dried, ground twigs of P. hypochrysea (0.76 kg) were extracted with methanol (5 L) and evaporated to a crude residue (25.90 g, 3.40%). 23.90 g was successively fractionated on a silica gel VLC with Hex, CH2Cl2, Ethyl Acetate, CH2Cl2: MeOH 9:1 and MeOH to yield fraction A (0.08 g), fraction B (1.50 g), fraction C (2.05 g), fraction D (5.57 g), fraction E (13.05 g) respectively. Fraction B (1.50 g) was subjected to silica gel column chromatography using Hexane and Hex:CH2Cl2 95:5, 90:10, 80:20, 70:30, 50:50, systems and MeOH only gradient to give βsitosterol (10) from (Hex:CH2Cl2 70:30); and 2,3dihydroxypropylhexacosanoate (7) from Hex:CH2Cl2 50:50. The fraction C and D was mixed on the base of TLC to obtain fraction F (7.62 g) and subjected to silica gel column chromatography using CH2Cl2 and the mixtures of CH2Cl2: MeOH 97.5:2.5 to CH2Cl2:MeOH 80:20 and MeOH only gradient to give height subfractions (F1 to F8). kaempferol (2),quercetin (3),taxifolin (4), taxifolin-3O-rhamnoside (5), 2α-hydroxy-ursolic acid (11) were obtained from subfraction F2 (CH2Cl2: MeOH 97.5:2.5). The subfraction F3 and F4 obtained successively in CH2Cl2: MeOH 95:5 and CH2Cl2: MeOH 92.5: 7.5 were separately submitted to gel filtration over sephadex LH-20 eluted with MeOH afforded Quercetin4’-(3”,4”dihydroxybenzoate) (1) (10.00 mg), from CH2Cl2: MeOH 95:5 and 2,3-dihydroxy-1-(4-hydroxy-3,5dimethoxyphenyl)propan-1-one (8), heptadecanoate-βsitosterolglycoside (9), (2R)-N-[(2S,3S,4R,8Z)-1-(β-dglucopyranosyloxy)-3,4-dihydroxytridec-8-en-2-yl]-2hydroxytriacontanamide (6) (35.00 mg) from CH2Cl2: MeOH 92.5:7.5. Methanolysis of compound 6 Compound 6 (15.2 mg) was refluxed with 0.9 M HCl in 82% aq. MeOH (15 mL) for 18 h. The mixture was extracted ten times with n-Hexane and combined organic phase were washed with water and dried over the Na2SO4. Evaporation of solvent gave a colorless compound (10.0 mg) which was chromatographed on sephadex HL-20 (MeOH) to yield fatty acid methyl ester as an amorphous white powder in methanol (6.0 mg), which was analyzed by EI-MASS and 1H and 2D NMR spectroscopy . The aqueous layer was evaporated and the residue was identified as mixture of sphingosine and methylated sugar. The sugar was identified as methyl β-d-glucopyranoside base on the Co-TLC profile (Rf 0.45, (EtOAc/ MeOH/H2O; 5/2/0.5). Quercetin4’-(3”, 4”-dihydroxybenzoate) (1) Amorphous yellow powder (MeOH);Rf = 0.35, silica gel 60 F254, CH2Cl2-methanol (9.5:0.5). - FT-IR (KBr): υmax = 3419, 1700, 1610, 1520, 1452, 1381, 116505 cm−1. - UV (MeOH) λmax: 209, 254, 295, 371 nm - 1H NMR (MeOD, 400 MHz) and 13C NMR (MeOD, 100 MHz) data, see Table 1. - ESI-MS/MS: m/z = 440.7 [M+2H]+, 301.1 [M C15H9O-7]+, 285.1 [M -C15H9O-6]+, 245.1 [M-C13H9O-5]+, 153.0 [M- C7H5O-4]+. - HR-EI-MS: m/z = 438.0580 (calcd. for C22H14O10, 438.0587, [M]+). (2R)-N -[(2S, 3S, 4R, 8Z)-1-(β-d-glucopyranosyloxy)-3, 4dihydroxytridec-8-en-2-yl]-2-hydroxytriacontanamide (6)White powder (MeOH); mp138-139°C;Rf = 0.45, silica gel 60 F254, CH2Cl2-methanol (9.5:0.5). - FT-IR (KBr): υmax = 3404 (broad), 2922, 2852, 1631, 1537, 1464, 1367, 1303, 1258, 1072, 1039, 897, 718 cm−1. - UV (CH2Cl2:MeOH 1:1) λmax: 207, 264, 318, 369 nm. - 1H NMR (MeOD, 400 MHz) and 13C NMR (MeOD, 100 MHz) data, see Table 2. - (+)ESI-MS/MS: m/z = 876.7 [M+ H2O+H] +, 678.7 [M- Glc] +, 338 [M-GlcOCH2HC(NHCOCHOH)(CHOH)2]+, 155 [MHCOH(CH2)3HC=CH(CH2)3Me]+ 83 [M+ HC=CH(CH2)3Me] . - HR-ESI-MS: m/z = 876.7133 (calcd. for C42H98NO11, 876.7140, [M+H2O+H] +). (Methyl 2-hydroxytriacontanoate) 6a 1 H NMR (CDCl3 + CD3OD; 400 MHz), δ: 4.03-4.06 (ddJ = 7.2, 4.4, H-C (2)), 3.24 (s, CH3O-), 1.38-1.10 (br s, - (CH2) n-), 2.08 (t, J = 7.7 Hz, 2H, 2-H), 0.73-0.77 (t, J = 6.4 Hz, Me-C (29)). - EI-MS: m/z = 482.2 (3.1 [M+]) (calcd for C31H62O3, 482.2), 451.1 [M-CH3 (CH2)27CHOHCO]+, 468 (2.8, [M-CH3(CH2)27CHOCOO]+), 90 (19.4, [MCH3OCOCH2OH]+), 76 (15.3, [M-OHCOCH2OH]+, 57 (100, [M-CH3(CH2)3]+). Biological Activities Urease assay and inhibition Reaction mixtures comprising 25 μL of enzyme (Jack bean Urease) solution and 55 μL of buffers containing 100 mM urea were incubated with 5 μL of test compounds (1 mM concentration) at 30oC for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn. Briefly, 45 μL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1 % active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were © Asian Journal of Ethnopharmacology and Medicinal Foods, 2016. 27 Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. processed by using SoftMax Pro software (Molecular Device, USA). All the assays were performed at pH 8.2 (0.01 M K2HPO4.3H2O, 1 mM EDTA and 0.01 M LiCl2). Percentage inhibitions were calculated from the formula 100–(ODtestwell/ODcontrol) x100. Thiourea was used as the standard inhibitor of urease Butrylcholinesterase inhibition activity Butrylcholinesterase inhibition activity was determined by method as described by Ellman[8]. Horse serum butryl cholinesterase enzyme, EC3.1.1.8 (Sigma, USA) was prepared by dissolving the enzyme in phosphate buffer (100 mM, pH 8.0). The enzyme concentration in reaction mixture was adjusted to 0.2 U per well. Sodium phosphate buffer (180 μL, pH 8.0) and buffered Ellman’s Reagent (DTNB, 5, 5-dithiobis [2-nitrobenzoic acid] 0.1 M NaHCO3, 17.85 mmol/L, 10 μL) was added in wells labeled as blank (B substrate and B enzyme), control and test. Test compound solution (of various concentrations of 5-500 μM, 10 μl) was added in each well labeled as test. Then, 20 μL of butrylcholinesterase solution was added in each well including B enzyme,control and test. The contents were mixed and incubated for 15 min at 25°C. The reaction was initiated by the addition of 10 μL substrate solution butrylcholinesterase iodide (10 mM) in each well except B enzyme. The absorbance was measured at 412 nm. The IC50values were determined by monitoring the inhibition effects of various concentrations of under investigation compounds and this was calculated by means of EZ-Fit, Enzyme Kinetics Program. determined in comparison with methanol treated control and IC50was calculated for each compound EZ fit software (Perrella Software, USA) [9, 10]. Results and Discussion The methanolic extract of the twigs of P. hypochrysea was separated by repeated column chromatography and preparative TLC (PTLC) to afford two new and nine known compounds (Fig. 1). The known compounds were identified as kaempferol (2), quercetin (3),taxifolin (4), (5),2,3taxifolin-3-O-rhamnoside dihydroxypropylhexacosanoate (7),2,3-dihydroxy-1-(4hydroxy-3,5dimethoxyphenyl)propan-1-one (8), heptadecanoate-β-sitosterolglycoside (9), β-sitosterol(10), and 2α-hydroxy-ursolic acid (11). The structures were confirmed by spectra comparison with authentic and published values [11, 12]. 5' 6' HO 9 O 2 4' 3' 7 2' 4 5 OH 3 R1 OH 1' R2 HO O OH OH O R OH 6" 5" 2" 3" O O 1" 1. R1 = 4" 7" , R2 = OH 4. R = H O 2. R1 = OH, R2 = H 3. R1 = OH, R2 = OH H3C O (CH2)24CH3 O (CH2)27CH3 OH OH O OH O O 4'' HO HO 6'' 3'' 2'' OH O (CH2)22 3' 2' 30' OH 8 1 28' 9 3 13 2 7 OH 6. O H3C 1'' 29' 27' 4' 1' NH O 5'' 6a. 7. O OH OH OH OH O HO 5. R = O OH OH HO O CH3 OH HO O 8. HO DPPH Radical Scavenging Activity R O (CH ) CH 11. The antioxidant activity was assessed by measurement of O scavenging ability of the isolated compounds on free O 9. R = HO O OH radical 2, 2’-diphenyl-1-picryl hydrazyl OH 10. R = OH (DPPH; C18H12N5O6). The radical DPPH was reduced to Figure 1-Structure of isolated compounds the corresponding colorless hydrazine upon reaction with hydrogen donors. The solution of DPPH was prepared in dimethoxyphenyl)propan-1-one (8), heptadecanoate-βthe concentration of 0.3 mM in ethanol, while serial sitosterolglycoside (9), β-sitosterol(10), and 2α-hydroxydilution of fractions was made in DMSO or methanol ursolic acid (11). The structures were confirmed by depending upon the best solubility and diluted to obtain spectra comparison with authentic and published values [11, final concentrations of 500, 250, 150, 125, 62.5, 31.2, 15.6, 12] . Compound 1 was isolated as amorphous yellow powder 7.8 μM. DPPH solution was added in the volume of 90 μl which gives a positive reaction with ferric chloride test for in each of the well of 96-well plate marked for control and phenolic compound and a positive reaction with Shinoda test compounds. Then, 10 μl of each of the concentrations reagents suggesting that this compound is a flavonoid [13]. of the compound was added to the particular well to start Its molecular formula was determined to be C22H14O10 by the reaction. The contents of the wells were mixed for few HR-EI-MS ([M] +at m/z = 438.0580, calcd. 438.0587). The seconds and the mixture was incubated for 30 minutes at UV spectrum of 1 showed absorption band maximum at 37˚C and the absorbance was measured at 517 λ by 254 (band II) and 371.00 nm (band I) characteristic of microtitre plate reader (Spectra max plus 384 Molecular quercetinnucleus [14,15].The IR spectrum exhibited devices USA). The assay was standardized by characteristic absorption bands for hydroxy group at butylatedhydroxyanisole (BHA) prior to testing the 3419.6 cm-1, a conjugated carbonyl group at 1610.5-1700.0 compounds. Percent radical scavenging activity was © Asian Journal of Ethnopharmacology and Medicinal Foods, 2016. 28 2 15 3 Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. cm-1, an aromatic C=C group at 1519.8 and 1452.3 cm-1. The 1H-NMR spectrum (table 1) displayed a meta-coupled protons at δH6.17 (1H, d, J = 2.0 Hz, H-6) and 6.38 ppm (1H, d, J = 2.0 Hz, H-8), attributed to a tetra-substituted benzene ring A, an ABX system signals of three protons at δH6.89 (1H, d, J = 8.4 Hz, H-5’), 7.73 (1H, d, J = 2.0 Hz, H2’) 7.64 ppm (1H, dd, J = 2.0, 8.4 Hz, H-6’) attributed to a trisubstituted benzene ring B. Attribution 1 H (m, J in Hz) 13 C 2 - 146.2 3 - 137.2 4 - 177.4 5 - 162.5 6 6.17 (d, 2.0) 99.3 7 - 165.7 8 6.38 (d, 2.0) 94.4 9 - 158.3 10 - 104.5 1’ - 124.2 2’ 7.73 (d, 2.0) 116.0 3’ - 148.8 4’ - 149.5 5’ 6.89 (d, 8.4) 116.2 6’ 7.64 (dd, 2.0, 8.4) 121.7 1’’ - 134.9 2’’ 6.94 (d, 2.0) 114.7 3’’ - 148.8 4’’ - 145.3 5’’ 6.77 (d, 8.4) 116.6 6’’ 6.82 (dd, 2.0, 8.4) 118.9 7’’ - 169.8 Table 1-1H (400 MHz) and 13C (100 MHz) NMR assignments for (1) in MeO. aAssignments were based on HMQC, HMBC, COSY and NOESY experiments Above information suggest compound 1 to be a quercetin derivative [10] Furthermore, the 1H NMR spectrum showed a second ABX coupling system at δH 6.77 (1H, d, J = 8.4 Hz, H-5’’), 6.82 (1H, dd, J = 2.0, 8.4 Hz, H-6’’) and 6.94 ppm (1H, d, J = 2.0 Hz, H-2’’) indicated a presence of another trisubstituted aromatic ring. This inference was confirmed by the 13C-NMR data (table 1) and the DEPT experiment, displaying characteristic signals of quercetin and the dihydroxybenzoyl moiety at δC 114.7 (C-2’’), 116.6 (C-5’’), 118.9 (C-6’’), 134.9 (C-1’’), 145.3 (C-4’’), 148.8 (C-3’’) and 169.8 (C-7’’) ppm. These findings clearly indicated that compound 1 has a quercetin skeleton linked to the dihydroxybenzoyl moiety by the ester function. Figure 2 - Selected NOESY correlations of compound 1 To confirm the linkage of the two skeletons, EIMS and NOESY (Fig. 2) experiment were used. The EI-MS, exhibited two important ion fragments at m/z = 153 (C7H5O4) and at m/z = 285 (C15H9NO6) characteristics of dihydroxybenzoate and the quercetin skeleton. In the NOESY spectrum, two important cross peaks between proton H-2’ (δH= 7.73 ppm) and proton H-2’’ (δH = 6.94 ppm), and between proton H-5’ (δH= 6.89 ppm) and proton H-6’’ (δH = 6.82 ppm) suggested that the dihydroxybenzoate moiety is linked to the ring B quercetin skeleton and the two substructures are linked by ester O=C7’’-O-C4’. This compound could result from the intermolecular esterification reaction between 3,4dihydroxybenzoic acid and quercetin.Thus, compound 1 was identified as quercetin 4’-(3”,4”dihydroxybenzoate)named parinariflavone. Compound 6, m.p. 138 - 139°C, was obtained as white powder. The molecular composition was found to be C49H98NO11 by HR-ESI-MS ([M+H2O+H]+ at m/z = 876.7133, calcd. 876.7140).The UV spectrum showed a highly conjugated system with absorption bands characteristic of amide at λmaxat 207, 264, 318, and 369 nm. The IR spectrum showed an absorption band at 3404 cm-1 due to the OH functions, a strong absorption band at 1631 and 1537 cm-1 indicating the presence of a secondary amide group, at 2950, 2900 and 1505 cm-1 (aliphatic) suggesting it to be a fatty acid amide and at 1072 and 1039 cm-1 (glycosidic C-O) [15].The 1H NMR spectrum (table 2) of 6 displayed a downfield doublet at δH = 8.59 ppm (d, J = 9.2 Hz, NH), a very strong aliphatic methylene band at δH = 1.21-1.34 ppm, as well as the signals of six protons at δH = 0.86-0.89 ppm (t, J = 7.2 Hz, H-13and H-30’), three oxymethines at δH = 4.21 (brd, J = 4.4 Hz, H-3), 4.30 (m, H-4) and 4.58 ppm (t, J = 3.2 Hz, H-2’), one oxymethyleneat δH = 4.53 (dd, J = 10.8, 4.4 Hz, H-1a) and 4.72 ppm (dd, J = 10.8, 6.8 Hz, Hb) and one double bond at δH = 5.45-5.58 ppm (m, H-8 and H-9). The 13C NMR (table 2) and DEPT spectral data of 6 were supportive of the above analysis, showing a carbonyl group at δC = 175.7 ppm (C-1’), one double bond at δC = 130.8 (C-8 or C-9) and 130.8 ppm (C-8 or C-9), oxygenated carbons at δC = 75.9 (C-3), 72.4 (C-2’), 71.4 (C-4), 62.9 (C-1), and 51.7 (C- © Asian Journal of Ethnopharmacology and Medicinal Foods, 2016. 29 Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. 2) ppm, aliphatic methylenes at δC = 22.9-30.1 ppm, and two methyls at δC = 14.2 ppm (C-13and C-30’) [16,17]. These findings clearly indicated that compound 6 has a ceramide skeleton. Furthermore, the 1H NMR spectrum indicated the presence of β-d-glucopyranosyl moiety by the anomeric proton at δH = 4.98 ppm (1H, d, J = 8.0 Hz, H1’’) [18]. Thus compound 6 is acerebroside [19, 20]. Attribution 1 H (m, J in Hz) 13 C 1a 4.52-4.56 (dd, 10.8, 4.4) 62.6 1b 4.70-4.75 (dd, 10.8, 6.8) 62.6 2 5.29-5.32 (m) 51.7 3 4.30-4.31 (brd, 4.4) 75.9 4 4.18-4.24 (m) 71.4 5a 2.30-2.32 (m) 33.9 5b 1.90-1.71 (m) 33.9 6 1.77-1.82 (m) 25.8 7 2.19-2.24 (m) 27.9 8 5.45-5.58 (brm) 130.8 9 130.8 10 2.00-2.14 (m) 27.5 11 2.00-2.03 (m) 32.9 12 1.21-1.34 (m) 22.9 13 0.86-0.89 (t, 7.2) 14.2 1’ 175.7 2’ 4.58-4.59 (brt, 3.2) 72.4 3a’ 2.02 (m) 35.5 3b’ 2.21 (m) 4’ 2.06-2.11 (m) 26.7 5’-27’ 1.21-1.34 (m) 29.5-30.1 28’ 1.21-1.34 (m) 32.1 29’ 1.21-1.34 (m) 22.9 30’ 0.86-0.89 (t, 7.2) 14.2 1’’ 4.98 (d, 8.0) 105.4 2’’ 4.00-4.04 (t, 8.0) 75.1 3’’ 4.18-4.24 (m) 78.4 4’’ 4.18-4.24 (m) 71.4 5’’ 3.87-3.89 (m) 78.5 6a’’ 4.37-4.38 (dd, 12.0, 5.2) 62.6 6b’’ 4.49 (brs ) 62.6 NH 8.59 (d, 9.2) Table 2-1H (400 MHz) and 13C (75 MHz) NMR assignments for (6) double bond, and the absolute configuration of 6, the acid methanolysis method of Gaver and Sweeley was used[22]. Methanolysis of compound 6 yielded methyl glucoside, a fatty acid methyl ester (FAME) and a long chain base (LCB)[23]. The fatty acid and sphingosine chain lengths were determined by characteristic fragment-ion peaks observed in the EI-MASS and ESI-MASS spectra (Fig. 3). The FAME was identified as methyl 2hydroxytriacontanoate from EI-MASS, which showed the ion peak at m/z = 437.2 [Me(CH2)26CHOHCO]+. The length of the LCB was also obtained from EI-MS (Fig. 3), which showed significant fragment-ion peak at m/z=422.0 ([Me(CH2)3CH=CH(CH2)4(CHOH)2CHNH2CH2OGlc+H] + ). The location of double bond in the sphingosine chain was elucidated by analysis of HMBC spectrum which showing a strong 3J correlation respectively between olefinic proton H-8 and carbon C-6; H-9 and carbon C-11 which is also correlated with the proton H-13 (Fig. 4). This position was also confirmed by EI-MASS and ESI-MASS spectra, which displayed an intense fragment ion peak at m/z = 83 ([Me(CH2)3CH=CH]+) formed through Mclaffer ty rearrangement Figure 3- Mass fragmentation pattern of compound6 inC5D5Na.aAssignmentswerebased on HMQC, HMBC, COSY and NOESY experiments. The chemical shifts of the allylic methylene carbons in 6 were assigned at δC = 27.5 ppm (C-7 or C-10) and δC = 27.9 ppm (C-7 or C-10) based on the clearly observed HMBC correlations with the olefinic signals at δH = 5.50 ppm (m, H-8 and H-9). Since the chemical shifts of allylic methylene carbons are different when alkene double bonds are cis-oriented (δC<28 ppm) and trans-oriented (δC>30 ppm) [21], the double bond in 6 was assigned as Zconfiguration.In order to determine the lengths of the sphingosine and fatty acid chains, the position of the Figure 4- Selected HMBC correlations of compound6 The chemical shift of the H-2 signal and the C-atom signals of C-1 to C-4, C-1’, and C-2’ of sphingolipids generally allow us to determine the absolute configuration of the phytosphingosine moiety. The H-atom signal at δH = 5.30 ppm (m, H-2) and the C-atom signals at δC = 70.3 (C-1), 75.9 (C-3), 175.7 (C-1’), and 72.4 ppm (C-2’) in 2 were nearly identical to those of previously © Asian Journal of Ethnopharmacology and Medicinal Foods, 2016. 30 Flavien Aristide Alfred TOZE et al.: Asian Journal of Ethnopharmacology and Medicinal Foods, 02 (04), 2016; 26-32. reportedceramides in the literature [24, 25], indicating the same configuration. The structure of 6 was assigned as (2R)-N-[(2S,3S,4R,8Z)-1-(β-d-glucopyranosyloxy)-3,4dihydroxytridec-8-en-2-yl]-2-hydroxytriacontanamide, trivially named as named parinarioside. The antioxidant and Urease Inhibition Activities of isolated compounds (table 3) were evaluated using the radical scavenging activity using DPPH assay and the indophenol method respectively. Compound 1 2 2’ 3 5 6 Antioxidant Activity IC50 (μM) 12.4± 0.56 67.4± 0.12 34.5 ± 0.55 37.1 ± 0.12 22.9 ± 0.21 31.4 ± 0.33 Urease Inhibition ActivityIC50 (μM) 33.7± 0.11 >200 >200 59.5 ± 0.30 29.5 ± 0.32 8 43.5 ± 0.21 56.3 ± 0.91 9 >200 19.4± 0.29 BHA 44.2 ± 0.15 Thiourea 21.9 ± 0.63 Table 3- Antioxidant and Urease Inhibition Activities. BHA butylatedhydroxyanisole (standard of antioxidant), Thiourea (standard inhibitor of urease) The radical scavenging activity using DPPH assay gave moderate activity for the compound 6 with IC50 67.4 ± 0.12 μM compared to the phenolic synthetic antioxidant standard BHA with IC50 44.2 ± 0.15 μM; while compound 6a obtain by methanolysis of compound 6 gave significantly good antioxidant values IC50 34.5 ± 0.55 μM. The radical scavenging activity using DPPH assay gave significantly high activity for the compound 1 with IC50 12.4 ± 0.56 μM compared to the phenolic synthetic antioxidant standard BHA with IC50 44.2 ± 0.15 μM; while Urease Inhibition Activity of compound 1 gave moderate antioxidant values IC50 33.7 ± 0.11 μM compared to the standard Thiourea IC50 21.9 ± 0.63 μM. Conclusion A new flavonol, parinariflavone(1) and a new cerebroside, parinarioside(6) together with nine known compounds were isolated from the methanol extract of the twigss of Parinarihypochrysea.The radical scavenging activity using DPPH assay gave significantly high activity for parinariflavone(1) with IC50 12.4 ± 0.56 μm compared to the phenolic synthetic antioxidant standard BHA with IC50 44.2 ± 0.15 μm, while urease inhibition activity for the same compound gave moderate antioxidant activity with IC50 33.7 ± 0.11 μm compared to the standard thiourea with IC50 = 21.9 ± 0.63 μm.This resultshow that compounds in the plant may react by synergy effect. Conflict of Interest The author declares that they have no conflicts of interest to disclose. Acknowledgements The authors thankful to International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan for research facilities and Third World Academy of Science (TWAS), Italy for financial support. 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