Bioorganic Chemistry Volume 66 Issue 2016 [Doi 10.1016_j.bioorg.2016.04.001] Eom, Hee Jeong; Kang, Hee Rae; Kim, Ho Kyong; Jung, Eun Bee; Par -- Bioactivity-guided Isolation of Antioxidant Triterpen

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  Bioactivity-guided isolation of antioxidant triterpenoids from  Betula platyphylla  var.  japonica  bark Hee Jeong Eom a , Hee Rae Kang a , Ho Kyong Kim a , Eun Bee Jung b , Hyun Bong Park c , Ki Sung Kang b, ⇑ ,Ki Hyun Kim a, ⇑ a School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea b College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea c Department of Chemistry, Yale University, New Haven, CT 06520, United States a r t i c l e i n f o  Article history: Received 9 January 2016Revised 17 March 2016Accepted 1 April 2016Available online 1 April 2016 Keywords:Betula platyphylla  var.  japonica BetulaceaeTriterpenoidsAntioxidantDPPH radical scavenging a b s t r a c t The bark of   Betula platyphylla  var.  japonica  (Betulaceae) has been used to treat pneumonia, choloplania,nephritis, and chronic bronchitis. This study aimed to investigate the bioactive chemical constituentsof the bark of   B. platyphylla  var.  japonica . A bioassay-guided fractionation and chemical investigation of the bark of   B. platyphylla  var.  japonica  resulted in the isolation and identification of a new lupane-typetriterpene, 27-hydroxybetunolic acid ( 1 ), along with 18 known triterpenoids ( 2–19 ). The structure of thenewcompound( 1 ) waselucidatedonthebasisof 1Dand2DNMRspectroscopic dataanalysis aswellas HR-ESIMS. Among the known compounds, chilianthin B ( 17 ), chilianthin C ( 18 ), and chilianthin A ( 19 )weretriterpene-lignanesters,whicharerarelyfoundinnature.Compounds 4 , 6 , 7 , 17 , 18 ,and 19 showedsignificant antioxidant activities with IC 50  values in the range 4.48–43.02 l M in a DPPH radical-scavenging assay. However, no compound showed significant inhibition of acetylcholine esterase(AChE). Unfortunately, the new compound ( 1 ) exhibited no significance in both biological activities.This study strongly suggests that  B. platyphylla  var.  japonica  bark is a potential source of natural antiox-idants for use in pharmaceuticals and functional foods.   2016 Elsevier Inc. All rights reserved. 1. Introduction Betula platyphylla  var.  japonica (Miquel)Hara(Betulaceae),well-knownas‘‘Asianwhitebirch”,iswidelydistributedinJapan,main-land China, Korea, and eastern Siberia, and its bark has been usedin Chinese traditional medicine for the treatment of variousinflammatory diseases, including pneumonia, choloplania, nephri-tis, and chronic bronchitis [1]. Regarding phytochemical studies of thisplant,itisarichsourceoftriterpenes,includingbetulin,whichare reported to display significant activity against multidrug-resistant cancer cells (KB-C2 or K562/Adr) [2,3]. The presence of betulin and related triterpenes in the outer bark of   B. platyphylla var.  japonica  has been determined, while isolation of diarylhep-tanoids and arylbutanoids from its inner bark and dammarane-type triterpenes from its leaves, root bark, and pollen has beenreported [2–9]. A recent study showed that  B. platyphylla  barkexhibited cognitive-enhancing activity in a scopolamine-inducedmodel, and the major diarylheptanoids were identified as theactive compounds [10].As part of a continuing search for bioactive constituents fromKorean medicinal plant sources, an EtOH extract of the bark of   B. platyphylla  var.  japonica  was found to exhibit antioxidant activityin a DPPH radical-scavenging assay, with an IC 50  value of 9.85 l g/mL, which was also identified by other groups [11,12].Bioassay-guided fractionation and repeated chromatography of the EtOH extract resulted in isolation of a new lupane-type triter-pene ( 1 ), together with 18 known triterpenoids ( 2–19 ) from theactive fractions, the EtOAc-soluble and  n -BuOH-soluble fractions(Fig.1).Toourknowledge,thetriterpene-lignanesters,compounds( 17–19 )areuncommonnaturalproducts,andthisisthefirstreportof their isolation from the family Betulaceae. We report herein theisolation, structural characterization, and biological activities of constituents  1–19  (Fig. 1). 2. Experimental  2.1. General experimental procedures Optical rotations were measured on a Jasco P-1020 polarimeter(Jasco, Easton, MD, USA). IR spectra were recorded on a Bruker   2016 Elsevier Inc. All rights reserved. ⇑ Corresponding authors. E-mail addresses: (K.S. Kang), Kim).Bioorganic Chemistry 66 (2016) 97–101 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage:  and filtered. The filtrate was evaporated under reduced pressureusing a rotavapor to obtain the EtOH extract (351g), which wassuspended in distilled H 2 O (2L) and successively solvent-partitioned with CHCl 3 , EtOAc, and  n -BuOH, yielding 274g, 25g,and 30g of residue, respectively. Each fraction was evaluated forantioxidant activity using DPPH radical-scavenging assay. TheEtOAc-soluble and  n -BuOH-soluble fractions showed significantradical-scavenging capacities with IC 50  values of 10.21 and8.79 l g/mL, respectively, while the CHCl 3  soluble fraction exhib-ited weak activity, with an IC 50  value of 78.26 l g/mL. To identifypotential antioxidant components, the active fractions, the EtOAcand  n -BuOH soluble fractions, were further investigated. TheEtOAc-soluble fraction (25g) was separated by silica gel columnchromatography using a solvent system of CH 2 Cl 2 -MeOH-H 2 O(9:3:0.1) to provide seven fractions (I-VII). Fraction II (5.3g) wasfractionated by reversed-phase (RP)-C18 column chromatographywith 70% MeOH and 100% MeOH to give six sub-fractions(II-1–II-6). Fraction II-2 (0.7g) was subjected to a silicacolumn chromatography using a gradient solvent system of CH 2 Cl 2 -MeOH (15:1, 3:1, 1:1), and 100% MeOH to obtain 17sub-fractions (II-2a–II-2q). Fraction II-2d (15mg) was purified bysemi-preparative RP HPLC with a solvent system of CH 3 CN-H 2 O(56:44, flowrate: 2mL/min) usinga PhenomenexLuna C18(2) col-umn (250mm   10mm i.d., 10 l m) to yield compounds  2 (0.5mg),  3  (0.8mg), and  5  (1.4mg). Fraction II-2e (21mg) waspurified by semi-preparative RP HPLC using a solvent system of CH 3 CN-H 2 O (56:44) to afford compounds  4  (0.3mg),  6  (0.6mg), 7  (0.4mg), and  8  (0.3mg). The  n -BuOH-soluble fraction (30g)was applied to silica column chromatography using a gradient sol-vent system of CH 2 Cl 2 -MeOH (10:1), CH 2 Cl 2 -MeOH-H 2 O (7:4:1),and 100% MeOH to give two fractions (B1 – B2). Fraction B1(4.6g) was separated by silica column chromatography with agradient solvent system of CH 2 Cl 2 -MeOH-H 2 O (7:3:0.5 and 7:4:1)to obtain five fractions (B1a – B1e). Fraction B1a (78.7mg) waspurified by semi-preparative RP HPLC with a solvent system of MeOH-H 2 O (97:3) to obtain compound  9  (2.0mg). Fraction B1b(486.3mg) was separated by preparative RP HPLC with a gradientsolvent system of MeOH-H 2 O (50% MeOH- 100% MeOH, flow rate:5mL/min) using an Agilent Eclipse XDB-C18 column (250mm  21.2mmi.d., 7 l m) to yield eight fractions (B1b1– B1b8). FractionB1b2 (20.6mg) was purified by semi-preparative RP HPLC with asolvent system of MeOH-H 2 O (83:17) to give compound  13 (2.6mg) and fraction B1b4 (20.6mg) was purified by semi-preparative RP HPLC with a gradient solvent system of MeOH-H 2 O (93% MeOH - 85% MeOH in 30min) to obtain compounds  1 (0.8mg),  10  (0.8mg),  15  (0.6mg), and  16  (1.8mg). Fraction B1b6(31.2mg) was purified by semi-preparative RP HPLC with 85%MeOH to afford compounds  11  (7.0mg), and  14  (3.7mg). FractionB1e(646.2mg)wasseparatedoverpreparativeRPHPLCwithasol-vent system of MeOH-H 2 O (88:12) to yield six fractions (B1e1 –B1e6). Fraction B1e3 (10.3mg) was purified by semi-preparativeRP HPLC with a solvent system of MeOH-H 2 O (83:17) to furnishcompound  19  (1.3mg). Fractions B1e4 (4.4mg) and B1e6(9.4mg)werepurifiedbysemi-preparativeRPHPLCwithasolventsystem of MeOH-H 2 O (78:22) to give compound  18  (0.8mg) andcompound  17  (2.7mg), respectively. Fraction B2 (7.87g) was sub- jected to silica column chromatography using a gradient solventsystem of CH 2 Cl 2 -MeOH (5:1), CH 2 Cl 2 -MeOH-H 2 O (7:3:0.1, 7:4:1),and 80% MeOH to give four fractions (B2a – B2d). Fraction B2b(210mg) was subjectedtopreparativeRPHPLCwitha solventsys-tem of MeOH-H 2 O (9:1) to obtain nine sub-fractions (B2b1–B2b9).Fraction B2b8 (9.7mg) was purified by semi-preparative RP HPLCwith a solvent system of MeOH-H 2 O (88:12) to furnish compound 12  (0.9mg).  2.3.1. 27-Hydroxybetunolic acid ( 1 ) White powder,  ½ a  25D  +2.86 ( c   0.07, MeOH);  ½ a  25D  +13.33 ( c   0.02,CHCl 3 ); IR (KBr)  m max : 3300, 2939, 2828, 1720, 1454, 1031cm  1 ; 1 H (700MHz) and  13 C (175MHz) NMR spectroscopic data, seeTable 1; ESI-MS (positive mode):  m/z   471 [M+H] + ; HR-ESI-MS(positive mode):  m/z   471.3462 [M+H] + (calculated for C 30 H 47 O 4 ,471.3474).  2.4. DPPH radical-scavenging assay Antioxidant activities of compounds  1–19  were evaluated interms of their free radical-scavenging capacities by DPPH assay[13]. In microwells, 100 l L of an aqueous solution of completelydissolved sample (control: 100 l L of distilled water) were addedtoanethanolicsolutionofDPPH(100 l L,60 l M).Thefinalconcen-trations of the tested samples in the assayed solutions were 5, 10,25and50 l M. VitaminCwas usedas thestandardfor comparison.The ability to scavenge DPPH radicals was calculated in terms of percentage inhibition according to the following equation: % inhi-bition=[(A 0  A 1 )/A 0    100], whereA 0  istheabsorbanceofthecon-trol (without sample) and A 1  is the absorbance in the presence of the sample.  2.5. Acetylcholine esterase (AChE) inhibition assay Inhibition of AChE activity by compounds  1–19  was evaluatedby a spectrophotometric method described previously [14] withminor modification. Briefly, in 96-well plates, a reaction mixtureof 25 l L of 15mM acetylthiocholine iodide in water, 125 l l of 3mM 5,5 0 -dithiobis [2-nitrobenzoic acid] in buffer and 25 l L of   Table 1 1 H and  13 C NMR data of   1  in CDCl 3 . ( d  in ppm, 700 MHz for  1 H and 175 MHz for  13 C). a Position  1 d H  d C 1 1.43m, 1.90m 40.0 t2 2.44m, 2.47m 34.2 t3 218.3s4 47.4s5 1.41m 55.1 d6 1.47m, 1.75m 19.8 t7 1.42m, 1.56m 35.3 t8 41.5s9 1.44m 51.5 d10 37.3s11 1.24m, 1.44m 21.8 t12 0.80m, 1.72m 25.3 t13 2.35m 39.3 d14 46.9s15 1.37m, 1.86m 23.4 t16 1.35m, 2.34m 33.2 t17 56.2s18 1.77m 19.7 d19 3.01m 46.5 d20 150.3s21 1.41m, 1.99m 30.6 t22 1.43m, 2.00m 37.0 t23 1.07s 27.0 d24 1.02s 21.1 d25 0.94s 16.7 d26 0.99s 16.2 d27 3.83 d (12.5); 4.24 d (12.5) 61.1 t28 180.1s29 4.62s, 4.74s 110.1 t30 1.69s 19.7 d a  J   values are in parentheses and reported in Hz; the assignments were based on 1 H- 1 H COSY, HSQC, and HMBC experiments. H.J. Eom et al./Bioorganic Chemistry 66 (2016) 97–101  99  the compounds (25, 50, 100, and 200 l M) were added, and theabsorbance at 405nm was measured. Thereafter, 25 l L of AChEsolution (0.22U/mL) were added to the wells and the microplatewasreadagainatthesamewavelength10timesat1minintervals.Thepercentageinhibitionof eachtest solutionwas thencalculatedusing the following equation: % inhibition=1   (A sample /A control )  100, where A control  is the absorbance of the control (without sam-ple) and A sample  is the absorbance in the presence of the sample. 3. Results and discussion  3.1. Bioactivity-guided isolation of isolated compounds The EtOH extract of   B. platyphylla  var.  japonica  barks displayedsignificant antioxidant activity in the DPPH radical-scavengingassay, with an IC 50  value of 9.85 l g/mL. Based on the bioactivity-guided isolation principle, the EtOH extract was fractionated toyield CHCl 3 , EtOAc, and  n -BuOH soluble fractions. Among thederived soluble fractions, the EtOAc and  n -BuOH soluble fractionsshowed potent radical-scavenging capacities, with IC 50  values of 10.21 and 8.79 l g/mL, respectively, while the CHCl 3  soluble frac-tionexhibitedweakradical scavengingcapacity, withanIC 50  valueof78.26 l g/mL.TheseresultsledustoinvestigatetheEtOAcand n -BuOH soluble fractions for antioxidant compounds. Further frac-tionation of the EtOAc soluble fraction using repeated columnchromatography afforded seven known triterpenoids ( 2–8 ). Simi-larly, chemical investigation of the active  n -BuOH soluble fractionresulted in the isolation and identification of a new triterpene ( 1 ),together with 11 known triterpenoids ( 9–19 ).  3.2. Structure elucidation of isolated compounds Compound ( 1 ) was isolated as a white powder. The molecularformula was determined to be C 30 H 46 O 4  from the molecular ionpeak [M+H] + at  m/z   471.3462 (calculated for C 30 H 47 O 4 ,471.3474) in the positive mode HR-ESIMS (Fig. S1, Sup. material)and NMR spectroscopic data (Table 1). The IR spectrum exhibitedabsorptions of hydroxy (3300cm  1 ) and carbonyl (1720cm  1 )groups. The  1 H NMR spectrum (Table 1) showed the presence of signals due to five tertiary methyls at  d H  0.94 (s), 0.99 (s), 1.02(s), 1.07 (s), and 1.69 (s), two oxygenated proton signals at  d H 3.83 (d,  J   =12.5Hz) and  d H  4.24 (d,  J   =12.5Hz), and two olefinicproton signals at  d H  4.62 (s) and 4.74 (s) (Fig. S2, Sup. material).The characteristic NMR data of vinylic methyl at  d H  1.69 (s) andolefinic proton signals at  d H  4.62 (s) and 4.74 (s) suggested thepresence of an isopropenyl group in compound  1 . The  13 C NMR spectrum (Table 1) showed 30 carbon signals (Fig. S3, Sup. mate-rial), which were attributed to 5 methyl, 12 methylene, and 5methine groups, as well as 8 quaternary carbons, including twoolefinic carbons [ d C  150.3 and 110.1], an oxygenated carbon [ d C 61.1], and two carbonyl groups [ d C  218.3 and 180.1] deduced byanalysis of HSQC (Fig. S4, Sup. material) and HMBC spectra(Fig. S5, Sup. material). These data suggested that compound  1  isa lupane-type triterpenoid, which was also implied by comparisonof its data with those of betunolic acid ( 14 ) [15]. The  1 H and  13 CNMRspectraof   1  weresimilartothoseof   14 , withanapparent dif-ference that a tertiary methyl group in  14  was replaced by an oxy-genated methylene group [ d H  3.83 (d,  J   =12.5Hz) and  d H  4.24 (d,  J   =12.5Hz);  d C  61.1] in  1 . The location of this oxygenated methy-lene group was confirmed to be C-27 by the HMBC correlationsof H-26/C-8, H-26/C-14, H-27/C-8, H-27/C-13, H-27/C-14, and H-27/C-15 (Fig. 2), suggesting that compound  1  was a C-27-hydroxylatedproductof  14 .Thegrossstructureof  1  wassupportedby the cross peaks in the  1 H- 1 H COSY (Fig. S6, Sup. material) andHMBC spectra (Fig. 2). The absolute stereochemistry of   1  wasestablished to be identical to  14  by analysis of the NOESY data(Fig. S7, Sup. material), and by comparing the coupling constants,chemical shifts, and specific rotation value with those of betunolicacid( 14 ).IntheNOESYspectrumof  1 ,keyNOESYcorrelationsofH-5/H-9, H-9/H-27, H-13/H-26, H-19/H-13, H-18/H-27, H-23/H-5, H-23/H-9,H-24/H-25,andH-25/H-26wereobserved,andthespecificrotation value of   1  was positive,  ½ a  25D  +13.33 in CHCl 3 , which wascomparable to the published specific rotation data of   14  ( ½ a  25D +45.0 in CHCl 3 , positive) [15]. Thus, the structure of   1  was eluci-dated to be 27-hydroxybetunolic acid.The known compounds were identified as cylicodiscic acid ( 2 )[16], methyl 27- O - trans -caffeoylcylicodiscate ( 3 ) [17], 27- O - trans -caffeoylcylicodiscic acid ( 4 ) [18], myricerol ( 5 ) [19], uncarinic acidE ( 6 ) [20], myriceric acid B ( 7 ) [21], obtusilinin ( 8 ) [22], 3 b -acetyloleanolicacid( 9 )[23],oleanolicacid( 10 )[24],oleanonicacid( 11 ) [25], oleanolic acid acetate methyl ester ( 12 ) [26], winchicacid ( 13 ) [27], betunolic acid ( 14 ) [15], betulin ( 15 ) [28], betulinic acid ( 16 ) [28], chilianthin B ( 17 ) [29], chilianthin C ( 18 ) [29], andchilianthin A ( 19 ) [29] by comparing their obtained spectroscopicdata with values reported previously. To our knowledge, com-pounds( 17–19 ) aretriterpene-lignanesters, whichareuncommontypes of natural product, and this is the first report of their isola-tion from the family Betulaceae.  3.3. DPPH radical-scavenging assay TheDPPH-scavengingactivitiesofisolatedcompounds 1–19 areshown in Table 2. Of these compounds,  4 ,  6 ,  7 ,  17 ,  18 , and  19 showed high radical-scavenging activities with IC 50  values in therange 4.48–43.02 l M (Table 2). Particularly, compounds  4 (IC 50  =6.23 l M),  7  (IC 50  =4.48 l M), and  19  (IC 50  =6.54 l M) werestrong antioxidants when compared to the reference radical scav-enger, ascorbic acid (IC 50  =3.02 l M). The other compoundsshowed no radical-scavenging activities within the concentrationrange tested (IC 50  >50 l M).Interestingly,theactivecompounds 4 , 6 , 7 , 17 , 18 ,and 19 haveaphenylpropanoid moiety, in particular compounds  17 ,  18 , and  19 are triterpene-lignan esters which have a lignan unit, a phenyl-propanoid dimer. This suggested that the phenylpropanoid moietyis an essential functional group for the antioxidant activity of theisolated triterpene. Phenylpropanoids are secondary metaboliteswidely distributed in plants and that have therapeutic activityagainst hypertension, viral infections, fungal infections, tumorsandcancer, as well asanimmunomodulatoryeffect [30–33]. These effects are associated with the antioxidant and free radical-scavenging capacities of their structural components, such as thenumber and location of hydroxyl groups on the aromatic ringand the side-chain structure [34]. Indeed, many studies havedemonstrated that phenylpropanoids are potent antioxidants and COOHOHHHOH   H 3542 3225 2627282930 Fig. 2.  1 H- 1 H COSY ( ) and key HMBC ( ) correlations of   1 .100  H.J. Eom et al./Bioorganic Chemistry 66 (2016) 97–101
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