Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 342352 8 pageshttpdxdoiorg1011552013342352

Research ArticleAntiproliferative Effect and the Isolated Compounds ofPouzolzia indica

Chanyapat Sangsuwon1 Weena Jiratchariyakul1

Yaowalak U-pratya2 and Tanawan Kummalue3

1 Department of Pharmacognosy Faculty of Pharmacy Mahidol University Bangkok 10400 Thailand2Department of Medicine Faculty of Medicine Siriraj Hospital Mahidol University Bangkok 10700 Thailand3Department of Clinical Pathology Faculty of Medicine Siriraj Hospital Mahidol University Bangkok 10700 Thailand

Correspondence should be addressed to Weena Jiratchariyakul weenajirmahidolacth

Received 1 February 2013 Accepted 27 March 2013

Academic Editor Molvibha Vongsakul

Copyright copy 2013 Chanyapat Sangsuwon et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Previous report showed the high potent antiproliferative effect of the methanolic part extracted from the aerial parts of Pouzolziaindica on NB4 and HT93A acute leukemic cell lines with the IC

50values of 285 and 498 120583gmL respectively The bioassay-guided

fractionation of the methanolic part gave 5 fractions that is FFIndashFFV FFII FFIII and FFIV inhibited the above leukemic cell lineswith the IC

50values of 151 (FFII) 144 (FFIII) 321 (FFIV) and 310 (FFII) 97 (FFIII) 105 (FFIV) 120583gmL respectively The com-

pounds in these fractions were isolated using chromatographic technique FFII contained friedelin 1 28-hydroxy-3-friedelanone 2and 7-methoxy-coumarin 3 FFIII contained 6 7-dimethoxy-coumarin 4 scopoletin 5 methyl caffeate 6 FFIV contained sitosterylglucoside 7 and a supposed glycosphingolipid 8 The chemical structures were elucidated by spectroscopic methods

1 Introduction

Pouzolzia indicaGaudichvar angustifoliaWedd (local nameldquoNon tai baihongrdquo) is a Thai medicinal plant in the familyUrticaceae [1 2] It was used as remedy for the ailments infemale infertility cancer and inflammation and as emme-nagogue and insecticide [3] The chemical constituents in Pindica were scarcely reported Only lanceolone an isoflavonecompounds was isolated [4] Previously the antiproliferativeeffect of the methanolic part of this plant was reported [5]It could inhibit the growth of NB4 and HT93A cells with theIC50values of 285plusmn01 and 498plusmn07 120583gmL [5] respectively

The apoptosis of NB4 cells treated with 75120583gmL of thisfraction for 24 hours increased from 32 to 222 whereasHT93A cells underwent apoptosis from 30 to 513 whentreated with the methanolic part at 150 120583gmL [5] The pre-vious results therefore showed high potent antiproliferativeeffect of this methanolic part on these acute leukemic cellsFrom this study the active extract was further fractionatedusing column chromatography The active fractions were

determined by bioassay The compounds in each fractionwere isolated and structurally identified

2 Materials and Methods

21 Cytotoxicity Test

211 Cell Lines and Culture NB4 promyelocytic cell line waskindly provided by Ms Setsuko Miyanishi Tenri Institute ofMedical Research Japan and HT93A promyelocytic cell linewas kindly provided by Dr Kenji Kishi Tokai UniversityJapan Long and short types of PML-RAR120572 have beendetected in NB4 and HT93A cells respectively [6ndash8] Cellswere maintained in RPMI 1640 media supplemented with10 fetal bovine serum (Stem cell Technology VancouverBC Canada)with 1penicillin and streptomycin (Gibco LifeTechnologies Breda the Netherlands) Cells were incubatedat 37∘Cwith 95 humidified atmosphere containing 5 CO

2

[9]

2 Evidence-Based Complementary and Alternative Medicine

Inactive Active

FFI25 g

InactiveActive Active

FFII035g

FFIII330g

FFIV387 g

FFV537g

FFIII-4 FFIII-5019 g 0465g 0146g 006 g 109 g

006 g 019 g 0018 g

(5 mg)(15 mg)(10 mg)

FFIV-1 FFIV-2

(85 mg) (20 mg)Preparative TLC

Hexane

FFIII-1 FFIII-2 FFIII-3

(15 mg) (15 mg) (12 mg) 233 g 1297 g

Active methanolic part (16 g) of P Indica

CC (10 g SiO2)CC (120 g SiO2)

CC (100 g SiO2)

FFII-1 FFII-2 FFII-3

CH2Cl2 MeOH(50 50 2)1 2

3

4 5 6

7 8

CC (320 g SiO2)

Figure 1 Isolation of compounds 1ndash8 from active methanolic part of P indica

212 Cell Viability Assay Cell viability was assessed usingMTT [(345-dimethylthiazol-2-yl)-2-5-diphenyltetrazoliumbromide] assay [10] In brief 1 times 104 cells of NB4 and 1 times 105cells of HT93A were seeded in each well in 96-well plate flatbottom Cells were treated with each fraction isolated fromthis medicinal plant for 48 h at the concentrations varyingfrom 0 to 50 120583gmL After 48 h incubation 50 120583L of 1mgmLofMTT in PBS (SigmaThailand) was added to eachwell andcells were incubated for 4 hr 100120583L of 10 SDS in 001MHClwas added to stop the reaction and was incubated overnightThe absorbance at 590 nm was measured and read using theELISA reader (Biorad USA) Cell viability was calculatedusing the following formula [11]

Cell viability () = (sample ODcontrol OD

) times 100 (1)

213 Statistical Analysis The experiments were performedin triplicate with three independent experiments Data wereexpressed as the mean plusmn standard deviation The 119877-squareequation was used to calculate the IC

50value A 119875 value less

than 005 was considered statistically significance [9]

22 Phytochemistry

221 General 1H and 13C NMR spectra recorded on BrukerDPX-300 Switzerland with deuterated solvents and TMS asa reference APCI-MS atmospheric pressure chemical ioniza-tionmass spectra weremeasured on Perkin Elmermass spec-trometer IR spectra were recorded on FT-IR spectrometerPerkin-Elmer UV spectra were recorded on Hitachi U 320spectrophotometer Melting points were measured on DigitalElectromol 9100 Separation and isolation were performedby column chromatography (CC) using following adsorbentsDiaion HP20 size 250ndash850120583m Mitsubishi Chemical Indus-try silica gel for CC (63ndash200120583mMerck 7734) and low pres-sure column chromatography (LiChroprep Merck) TLCprecoated Kieselgel 60 F

254(Merck) NPPEG or NEU spray

reagent was used to detect flavonoids and coumarins 10

H2SO4in ethanol was used as universal spray reagent and

10FeCl3in ethanol was used to detect phenolic compounds

222 Plant Material The aerial parts of P indica were col-lected from Ratchaburi Province in the central part of Thai-land The plant was identified by the expert from ForestHerbarium-BKF Bangkok Thailand A plant specimen wasdeposited with the voucher number of BKF no 106441 andSN 096588

223 Extraction and Isolation P indica was extracted withethanol and fractionated byDiaionHP20 column elutedwithwater water methanol (1 1) methanol and ethyl acetateThe methanol fraction showed significant antiproliferativeeffect [5] This active methanolic part (16 g) was applied ona silica gel column eluted with gradient solvent systems(ss) of hexane-ethyl acetate and ethyl acetate-methanol toobtain 5 fractions (FFIndashFFV) FFI (25 g ss hexane-EtOAc9 1) appeared as oily liquid FFII (035 g ss hexane-EtOAc1 1) FFIII (330 g ss hexane-EtOAc 1 9) FFIV (387 gss EtOAc-MeOH 8 2) and FFV (537 g ss EtOAc-MeOH7 3) The bioassay-guided fractionation showed the activefractions that is FFIIndashFFIV FFIIndashFFIV were further sep-arated by chromatographic column (Figure 1) FFII waschromatographed on silica gel column eluted with gradientss of hexane-acetone and acetone-methanol to obtain 3subfractions FFII-1-3 FFII-1 (006 g ss hexane-Me

2O 95 5)

appeared as oily liquid FFII-2 (019 g ss hexane-Me2O 8 2)

was recrystallized with Et2O and the white needles 1 (15mg)

was obtained The filtrate was evaporated to dryness andrecrystallized with methanol and 2 (10mg) was obtainedFFII-3 (0018 g) was separated on the preparative TLC usingsolvent system of hexane CH

2Cl2 methanol (50 50 2) giv-

ing four separated bandsThe band with 119877119891value of 075 was

isolated recrystallized in ethyl acetate resulting 3 (5mg)FFIII was chromatographed on silica gel column eluted

with gradient ss of hexane-CH2Cl2and CH

2Cl2-methanol

to obtain 5 subfractions FFIII-1ndash5 FFIII-1 (019 g ss hexane-CH2Cl2 7 3) was recrystallized inmethanol giving 4 (15mg)

Evidence-Based Complementary and Alternative Medicine 3

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

CH2OH

O

O1

2

34

6

7

8 O

5

H3CO

O1

2

34

6

7

8 O

5

H3CO

H3CO

HO

H O

HO

OCH3

12

3

59984006998400

1998400

2998400

39984004998400 H

OH

OH

HO

23

5998400

1998400

2998400

3998400

4998400

45

CH2

H3C

18H3C

HN 1

O

CH2OH

OH

OHOO1998400998400

2998400998400 3998400998400

5998400998400 6

998400998400

4998400998400

1(friedelin) 2 (28-hydroxy-3-fridelanone) 3 (7-methoxy-coumarin)

4 (6-7-dimethoxy-coumarin) 5 (scopoletin) 6 (methyl caffeate)

7 (sitosteryl glucoside) 8 (a supposed glycosphingolipid)

12

3

45

8910

1113

14

17

O

HOOH

12

15

16

18

19

20

21 22

2324 25

26

29

O

OH

67

2728

5998400 19984002998400

39984004998400

6998400

HO

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

O

O1

2

34

6

7

8 O

5H3CO

4a

4a

8a

8a

4a

8a

6998400

(CH2)119899

(CH2)10

Figure 2 Structures of 1ndash8

FFIII-2 (0465 g ss hexane-CH2Cl2 1 1) was recrystallized

with methanol giving 5 (15mg) FFIII-3 (0146 g ss CH2Cl2-

MeOH 95 05) was recrystallized in methanol giving 6(12mg) FFIII-4 (006 g ss CH

2Cl2-MeOH 9 1) was brown

gum FFIII-5 (109 g ss CH2Cl2-MeOH 8 2) appeared as

yellowish gumFFIV was chromatographed on silica gel column

eluted with gradient ss of hexane-acetone and acetone-methanol It produced 2 subfractions that is FFIV-1-2FFIV-1 (233 g ss hexane acetone 7 3) was added withmethanol 7 (85mg) was precipitated as white powderFFIV-2 (1297 g ss acetone methanol 9 1) was addedwith chloroform methanol (95 5) 8 (20mg) was obtainedas white powder Each of the purified compounds 1ndash8

described above possessed the following physicochemicalproperties and the chemical structures were identified usingspectroscopic methods (1H NMR 13C NMR Mass spectraIR spectra and UV-spectra) The structure of compounds1ndash8 (Figure 2) were elucidated as the followings

(1) (friedelin) white needles (Me2O) mp 258ndash260∘C

UV (EtOH)120582max 220 nm IR (CHCl3) ]max 2980 2927

2870 (CH) 1706 (C=O) 1462 1389 (CH) cmminus1 APCI-MS 4274156 [M+H]+ (calc C

30H50O 4267244) 1H

NMR (CDCl3 300MHz) 120575 075 (3H s H-24) 090

(3H s H-23) 098 (3H s H-25) 104 (3H s H-29)104 (3H s H-30) 104 (3H s H-26) 107 (3H s H-27) 120 (3H s H-28) 13-14 (18H complex m H-6

4 Evidence-Based Complementary and Alternative Medicine

7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-810 18) 174 (2H dd J 55 30Hz H-1a H-1b) 22 (1Hm H-4) 234 (1H m H-2b) 240 (1H m H-2a) 13CNMR (CDCl

3 75MHz) 120575 2131(C3) 594 (C10) 582

(C4) 531 (C8) 428 (C18) 420 (C5) 415 (C6) 415(C2) 397 (C13) 392 (C22) 383 (C14) 374 (C9) 360(C16) 356 (C11) 353 (C19) 350 (C29) 328 (C21)324 (C15) 321 (C28) 318 (C30) 305 (C12) 300(C17) 281 (C20) 222 (C1) 202 (C26) 186 (C27)182 (C7) 179 (C25) 146 (C24) 68 (C23) [12ndash14]

(2) (28-hydroxy-3-friedelanone or canophyllol) whiteprisms (MeOH) mp 363-364∘C UV (EtOH) 120582max220 nm IR (CHCl

3) ]max 3200ndash3513 (OH) 1709

(C=O) 2990 2930 2855 (CndashH) 1466 1378 (CndashH) and1039 1116 (CndashO) APCI-MS 4434015 [M+H]+ (calcC30H50O24425038) 1H NMR (CDCl

3 300MHz) 120575

088 (3H s H-24) 091 (3H d J 78Hz H-23) 099(3H s H-25) 102 (3H s H-26) 101 (3H s H-27) 119(3H s H-29) 123 (3H s H-30) 13-14 (18H complexm H-6 7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-8 10 18) 196 (2H dd J 76 37Hz H-1a H-1b)232 (1H m H-2b) 230 (1H m H-4) 240 (1H mH-2a) and 25 (2H m H-28) 371 (1H 28-OH) 13C-NMR (CDCl

3 75MHz) 120575 2131 (C3) 683 (C28) 594

(C10) 582 (C4) 531 (C8) 420 (C5) 417 (C22) 415(C2 C6) 397 (C13) 395 (C18) 383 (C14) 374 (C9)356 (C11) 352 (C17) 344 (C29) 334 (C19) 329(C30) 316 (C21) 313 (C16) 305 (C12) 303 (C20)292 (C15) 234 (C1) 194 (C25) 191 (C26) 182 (C7)181 (C27) 148 (C24) 68 (C23) [13 14]

(3) (7-methoxy-coumarin or herniarin) white needles(MeOH) mp 117-118∘C UV (MeOH) 120582max 254366 nm IR (CHCl

3) ]max 2927 2870 (CH) 1682

(C=O) 1533 (C=C) cmminus1 APCI-MS 1762015 [M]+(calc C

10H8O31761714) 1HNMR (CDCl

3 300MHz)

120575 763 (1H d J 96Hz H-4) 631 (1H d J 96Hz H-3) 709 (1H d J 8Hz H-5) 695 (1H s H-8) 690(1H d J 8Hz H-6) and 399 (3H s 7-OCH

3) 13C

NMR(CDCl375MHz)120575 16202 (C2) 1563 (C7) 1500

(C8a) 1427 (C4) 1211 (C3) 1152 (C4a) 1134 (C6)1114 (C8) 1091 (C5) 563 (OCH

3) [15 16]

(4) (6 7-dimethoxy-coumarin or scoparone) pale yellowneedles (MeOH) mp 146-147∘C UV (MeOH) 120582max345 nm IR (CHCl

3) ]max 2985 2925 2855 (CH) 1684

(C=O) 1535 (C=C) 1462 1389 1363 and 1311 (CH)APCI-MS 2061245 [M]+ (calc C

11H10O42061576)

1H NMR (CDCl3 300MHz) 120575 763 (1H d J 95Hz

H-4) 69 (1H s H-5) 685 (1H s H-8) 625 (1Hd J 95Hz H-3) 390 (3H s 7-OCH

3) 385 (3H

s 6-OCH3) 13C-NMR (CDCl

3 75MHz) 120575 1613

(C2) 1529 (C7) 1501 (C6) 1464 (8a) 1432 (C4)1136 (C3) 1115 (C4a) 10820 (C5) 1001(C8) 5640(2xOCH

3) [15 17]

(5) (6-methoxy-7-hydroxy-coumarin or scopoletin) paleyellow needles (CH

2Cl2MeOH 95 05) mp 203-

204∘C UV (MeOH) 120582max 254 366 nm IR (CHCl3)

]max 3400ndash3550 (OH) 1685 (C=O) 2998 2938 2856

(CH) 1589 1511 (C=C) cmminus1 APCI-MS 1922008[M]+ (calc C

10H8O41921708) 1H NMR (CDCl

3

300MHz) 120575 761 (1H d J 95Hz H-4) 690 (1H s H-5) 685 (1H s H-8) 628 (1H d J 95Hz H-3) and385 (3H s 6-OCH

3) 13C NMR (CDCl

3 75MHz) 120575

1615 (C2) 1502 (C6) 1497 (C7) 1440 (C8a) 1434(C4) 1134 (C3) 1115 (C4a) 1075 (C5) 1032 (C8)564 (OCH

3) [16 17]

(6) ((E)-methyl-3 (3101584041015840-dihydroxy-phenyl) acrylate ormethyl caffeate) pale yellow crystalline powder(MeOH) mp 157ndash159∘C UV (MeOH) 120582max 354 nmIR (CHCl

3) ]max 3530 (OH) 2995 2927 2870 (CH)

1516 1643 (C=C) and 1690 (C=O) APCI-MS 1940623[M]+ (calc C

10H10O41941866) 1H NMR (CDCl

3

300MHz) 120575 750 (1H d J 161 Hz H-3) 717 (1H dJ 2Hz H-21015840) 693 (1H d J 7Hz H-51015840) 688 (1Hdd J 7 2Hz H-61015840) 615 (1H d J 161 Hz H-2) 385(3H s OCH

3) 13C NMR (CDCl

3-CD3OD 75MHz)

120575 1683 (C1) 1474 (C41015840) 1455 (C3) 1448 (C31015840) 1266(C11015840) 1218 (C2) 1151 (C21015840) 1141 (C51015840) 1139 (C61015840)514 (OCH

3) [18 19]

(7) (sitosteryl glucoside) white needles (MeOH) mp258ndash260∘C UV (EtOH) 120582max 220 nm IR (KBr) ]max3200ndash3450 (OH) 2980 2927 2870 (CH) 1560 (C=C)APCI-MS 4124002 [M minus 180]+ (calc C

35H60O6

5768854 1H NMR (CDCl3+CD3OD 300MHz) 120575

059 (3H s H-18) 085 (3H d J 73Hz H-27) 090(3H t J 65 65 Hz H-29) 091 (3H s H-19) 092(3H d J 73Hz H-26) 112 (3H d J 70Hz H-21)130 (2H m H-28) 174 (1H m H-2a) 194 (2H mH-1) 218 (1H m H-2b) 221 (1H m H-4) 328ndash35(5H mH-11015840 H-21015840 H-31015840 H-41015840 H-51015840) 350ndash370 (2Hm H-61015840) 374 (1H m H-3) 531 (1H m H-6) 13CNMR (CDCl

3+CD3OD 75MHz) 120575 1401 (C5) 1219

(C6) 1009 (C11015840) 790 (C3 C31015840) 762 (C51015840) 757 (C21015840)734 (C41015840) 618 (C61015840) 566 (C20) 559 (C17 C24)500 (C9) 456 (C8) 421 (C13) 400 (C12) 399 (C4)375 (C1) 365 (C10) 338 (C7 C22) 317 (C14) 298(C2) 294 (C16) 290 (C25) 280 (C23) 241 (C15)230 (C28) 229 (C11 C21) 209 (C26) 195 (C27) 190(C19) 117 (C29) 116 (C18) [16]

(8) (a supposed glycosphingolipid) white amorphouspowder (CHCl

3 MeOH 3 97) mp 252-254∘C

(MeOH) UV (EtOH) 120582max 236 nm IR (KBr)]max 3230-3450 (OH) 3384 (NH) 2998 29352851(CH) 1642 (C=O)APCI-MS7512596 [M]+(calcC44H81NO87512539) 1H NMR (pyr-d

5 300MHz)

120575 090 (2 times 3H t J6 6 Hz acyl-CH3) 120 (2H m

H-17) 130 (complex m H7-16 (CH2)119899) 18 (6H m

H-6 H-31015840 H-61015840) 24 (2H m H-21015840) 26-28 (1H mOH) 390-450 (7H m H-110158401015840 H-210158401015840 H-310158401015840 H-410158401015840H-510158401015840 H-610158401015840) 424 (1H m H-1a) 466 (1Hm H-1b)470 (1H m H-51015840) 475 (2H m H-2 H-3) 530 (1Hm H-5 H-41015840) 550 (1H dd J66 10 Hz H-4) 6-8(4H br s 4xOH) 859 (1H d J92 Hz NH) 13CNMR (pyr-d

5 75 MHz) 120575 1757 (C11015840) 1309 (C4)

1307 (C41015840) 1238 (C5) 1220 (C51015840) 1056 (C110158401015840) 786(C310158401015840) 784 (C510158401015840) 754 (C210158401015840) 725 (C3) 718 (C410158401015840)

Evidence-Based Complementary and Alternative Medicine 5

FFIFFIIFFIII

FFIVFFV

0

50

100

1 10 100 1000

Cel

l via

bilit

y (

)

log (120583gmL)

(a)C

ell v

iabi

lity

()

0

50

100

1 10 100 1000

FFIFFIIFFIII

FFIVFFV

log (120583gmL)

(b)

Figure 3 Percentage viable cell of FFIndashFFV on leukemic cell lines (a) NB4 and (b) HT93A

FFII IC50 = 151 plusmn 05

FFIII IC50 = 144 plusmn 06

FFIV IC50 = 321 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

FFII IC50 = 310 plusmn 01

FFIII IC50 = 97 plusmn 13

FFIV IC50 = 105 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(b)

Figure 4 Antiproliferative effect of FFIIndashFFIV on leukemic cell lines (a) NB4 and (b) HT93A

705 (C1) 630 (C610158401015840) 577 (C2) 343 (C21015840) 333 (C31015840)322 (C6 C61015840) 296 (C7-C16 C71015840-(CH

2)1015840119899) 229 (C17)

143 (acyl CH3) 141 (acyl CH

3) [ 20-23 ]

3 Results

The antiproliferative effect of FFIndashFFV on human leukemiccell lines was investigated as shown in Figure 3 It was foundthat FFII FFIII and FFIV could inhibit growth of NB4and HT93A (Figure 4) Therefore FFII FFIII and FFIVwere continued to evaluate the IC

50values on these cell

lines at varying concentrations ranging from 0 to 50120583gmL

The results showed that FFII FFIII and FFIV had the IC50

values on NB4 cell line at 151 plusmn 05 120583gmL 144 plusmn 06 and321 plusmn 07 120583gmL respectively whereas the IC

50values of

the HT93A cell line were 310 plusmn 01 97 plusmn 13 and 105 plusmn07 120583gmL respectively as shown in Figure 4 AdditionallyFFIII inhibited growth strongly on both NB4 andHT93A celllines while FFII inhibits growth strongly on NB4 more thanHT93A FFIV showed strong growth inhibition on HT93Amore than NB4

The active fractions FFII FFIII and FFIV were furtherchromatographed on the silica gel columns repeatedly andthe isolated compounds were identified using spectroscopic

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Inactive Active

FFI25 g

InactiveActive Active

FFII035g

FFIII330g

FFIV387 g

FFV537g

FFIII-4 FFIII-5019 g 0465g 0146g 006 g 109 g

006 g 019 g 0018 g

(5 mg)(15 mg)(10 mg)

FFIV-1 FFIV-2

(85 mg) (20 mg)Preparative TLC

Hexane

FFIII-1 FFIII-2 FFIII-3

(15 mg) (15 mg) (12 mg) 233 g 1297 g

Active methanolic part (16 g) of P Indica

CC (10 g SiO2)CC (120 g SiO2)

CC (100 g SiO2)

FFII-1 FFII-2 FFII-3

CH2Cl2 MeOH(50 50 2)1 2

3

4 5 6

7 8

CC (320 g SiO2)

Figure 1 Isolation of compounds 1ndash8 from active methanolic part of P indica

212 Cell Viability Assay Cell viability was assessed usingMTT [(345-dimethylthiazol-2-yl)-2-5-diphenyltetrazoliumbromide] assay [10] In brief 1 times 104 cells of NB4 and 1 times 105cells of HT93A were seeded in each well in 96-well plate flatbottom Cells were treated with each fraction isolated fromthis medicinal plant for 48 h at the concentrations varyingfrom 0 to 50 120583gmL After 48 h incubation 50 120583L of 1mgmLofMTT in PBS (SigmaThailand) was added to eachwell andcells were incubated for 4 hr 100120583L of 10 SDS in 001MHClwas added to stop the reaction and was incubated overnightThe absorbance at 590 nm was measured and read using theELISA reader (Biorad USA) Cell viability was calculatedusing the following formula [11]

Cell viability () = (sample ODcontrol OD

) times 100 (1)

213 Statistical Analysis The experiments were performedin triplicate with three independent experiments Data wereexpressed as the mean plusmn standard deviation The 119877-squareequation was used to calculate the IC

50value A 119875 value less

than 005 was considered statistically significance [9]

22 Phytochemistry

221 General 1H and 13C NMR spectra recorded on BrukerDPX-300 Switzerland with deuterated solvents and TMS asa reference APCI-MS atmospheric pressure chemical ioniza-tionmass spectra weremeasured on Perkin Elmermass spec-trometer IR spectra were recorded on FT-IR spectrometerPerkin-Elmer UV spectra were recorded on Hitachi U 320spectrophotometer Melting points were measured on DigitalElectromol 9100 Separation and isolation were performedby column chromatography (CC) using following adsorbentsDiaion HP20 size 250ndash850120583m Mitsubishi Chemical Indus-try silica gel for CC (63ndash200120583mMerck 7734) and low pres-sure column chromatography (LiChroprep Merck) TLCprecoated Kieselgel 60 F

254(Merck) NPPEG or NEU spray

reagent was used to detect flavonoids and coumarins 10

H2SO4in ethanol was used as universal spray reagent and

10FeCl3in ethanol was used to detect phenolic compounds

222 Plant Material The aerial parts of P indica were col-lected from Ratchaburi Province in the central part of Thai-land The plant was identified by the expert from ForestHerbarium-BKF Bangkok Thailand A plant specimen wasdeposited with the voucher number of BKF no 106441 andSN 096588

223 Extraction and Isolation P indica was extracted withethanol and fractionated byDiaionHP20 column elutedwithwater water methanol (1 1) methanol and ethyl acetateThe methanol fraction showed significant antiproliferativeeffect [5] This active methanolic part (16 g) was applied ona silica gel column eluted with gradient solvent systems(ss) of hexane-ethyl acetate and ethyl acetate-methanol toobtain 5 fractions (FFIndashFFV) FFI (25 g ss hexane-EtOAc9 1) appeared as oily liquid FFII (035 g ss hexane-EtOAc1 1) FFIII (330 g ss hexane-EtOAc 1 9) FFIV (387 gss EtOAc-MeOH 8 2) and FFV (537 g ss EtOAc-MeOH7 3) The bioassay-guided fractionation showed the activefractions that is FFIIndashFFIV FFIIndashFFIV were further sep-arated by chromatographic column (Figure 1) FFII waschromatographed on silica gel column eluted with gradientss of hexane-acetone and acetone-methanol to obtain 3subfractions FFII-1-3 FFII-1 (006 g ss hexane-Me

2O 95 5)

appeared as oily liquid FFII-2 (019 g ss hexane-Me2O 8 2)

was recrystallized with Et2O and the white needles 1 (15mg)

was obtained The filtrate was evaporated to dryness andrecrystallized with methanol and 2 (10mg) was obtainedFFII-3 (0018 g) was separated on the preparative TLC usingsolvent system of hexane CH

2Cl2 methanol (50 50 2) giv-

ing four separated bandsThe band with 119877119891value of 075 was

isolated recrystallized in ethyl acetate resulting 3 (5mg)FFIII was chromatographed on silica gel column eluted

with gradient ss of hexane-CH2Cl2and CH

2Cl2-methanol

to obtain 5 subfractions FFIII-1ndash5 FFIII-1 (019 g ss hexane-CH2Cl2 7 3) was recrystallized inmethanol giving 4 (15mg)

Evidence-Based Complementary and Alternative Medicine 3

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

CH2OH

O

O1

2

34

6

7

8 O

5

H3CO

O1

2

34

6

7

8 O

5

H3CO

H3CO

HO

H O

HO

OCH3

12

3

59984006998400

1998400

2998400

39984004998400 H

OH

OH

HO

23

5998400

1998400

2998400

3998400

4998400

45

CH2

H3C

18H3C

HN 1

O

CH2OH

OH

OHOO1998400998400

2998400998400 3998400998400

5998400998400 6

998400998400

4998400998400

1(friedelin) 2 (28-hydroxy-3-fridelanone) 3 (7-methoxy-coumarin)

4 (6-7-dimethoxy-coumarin) 5 (scopoletin) 6 (methyl caffeate)

7 (sitosteryl glucoside) 8 (a supposed glycosphingolipid)

12

3

45

8910

1113

14

17

O

HOOH

12

15

16

18

19

20

21 22

2324 25

26

29

O

OH

67

2728

5998400 19984002998400

39984004998400

6998400

HO

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

O

O1

2

34

6

7

8 O

5H3CO

4a

4a

8a

8a

4a

8a

6998400

(CH2)119899

(CH2)10

Figure 2 Structures of 1ndash8

FFIII-2 (0465 g ss hexane-CH2Cl2 1 1) was recrystallized

with methanol giving 5 (15mg) FFIII-3 (0146 g ss CH2Cl2-

MeOH 95 05) was recrystallized in methanol giving 6(12mg) FFIII-4 (006 g ss CH

2Cl2-MeOH 9 1) was brown

gum FFIII-5 (109 g ss CH2Cl2-MeOH 8 2) appeared as

yellowish gumFFIV was chromatographed on silica gel column

eluted with gradient ss of hexane-acetone and acetone-methanol It produced 2 subfractions that is FFIV-1-2FFIV-1 (233 g ss hexane acetone 7 3) was added withmethanol 7 (85mg) was precipitated as white powderFFIV-2 (1297 g ss acetone methanol 9 1) was addedwith chloroform methanol (95 5) 8 (20mg) was obtainedas white powder Each of the purified compounds 1ndash8

described above possessed the following physicochemicalproperties and the chemical structures were identified usingspectroscopic methods (1H NMR 13C NMR Mass spectraIR spectra and UV-spectra) The structure of compounds1ndash8 (Figure 2) were elucidated as the followings

(1) (friedelin) white needles (Me2O) mp 258ndash260∘C

UV (EtOH)120582max 220 nm IR (CHCl3) ]max 2980 2927

2870 (CH) 1706 (C=O) 1462 1389 (CH) cmminus1 APCI-MS 4274156 [M+H]+ (calc C

30H50O 4267244) 1H

NMR (CDCl3 300MHz) 120575 075 (3H s H-24) 090

(3H s H-23) 098 (3H s H-25) 104 (3H s H-29)104 (3H s H-30) 104 (3H s H-26) 107 (3H s H-27) 120 (3H s H-28) 13-14 (18H complex m H-6

4 Evidence-Based Complementary and Alternative Medicine

7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-810 18) 174 (2H dd J 55 30Hz H-1a H-1b) 22 (1Hm H-4) 234 (1H m H-2b) 240 (1H m H-2a) 13CNMR (CDCl

3 75MHz) 120575 2131(C3) 594 (C10) 582

(C4) 531 (C8) 428 (C18) 420 (C5) 415 (C6) 415(C2) 397 (C13) 392 (C22) 383 (C14) 374 (C9) 360(C16) 356 (C11) 353 (C19) 350 (C29) 328 (C21)324 (C15) 321 (C28) 318 (C30) 305 (C12) 300(C17) 281 (C20) 222 (C1) 202 (C26) 186 (C27)182 (C7) 179 (C25) 146 (C24) 68 (C23) [12ndash14]

(2) (28-hydroxy-3-friedelanone or canophyllol) whiteprisms (MeOH) mp 363-364∘C UV (EtOH) 120582max220 nm IR (CHCl

3) ]max 3200ndash3513 (OH) 1709

(C=O) 2990 2930 2855 (CndashH) 1466 1378 (CndashH) and1039 1116 (CndashO) APCI-MS 4434015 [M+H]+ (calcC30H50O24425038) 1H NMR (CDCl

3 300MHz) 120575

088 (3H s H-24) 091 (3H d J 78Hz H-23) 099(3H s H-25) 102 (3H s H-26) 101 (3H s H-27) 119(3H s H-29) 123 (3H s H-30) 13-14 (18H complexm H-6 7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-8 10 18) 196 (2H dd J 76 37Hz H-1a H-1b)232 (1H m H-2b) 230 (1H m H-4) 240 (1H mH-2a) and 25 (2H m H-28) 371 (1H 28-OH) 13C-NMR (CDCl

3 75MHz) 120575 2131 (C3) 683 (C28) 594

(C10) 582 (C4) 531 (C8) 420 (C5) 417 (C22) 415(C2 C6) 397 (C13) 395 (C18) 383 (C14) 374 (C9)356 (C11) 352 (C17) 344 (C29) 334 (C19) 329(C30) 316 (C21) 313 (C16) 305 (C12) 303 (C20)292 (C15) 234 (C1) 194 (C25) 191 (C26) 182 (C7)181 (C27) 148 (C24) 68 (C23) [13 14]

(3) (7-methoxy-coumarin or herniarin) white needles(MeOH) mp 117-118∘C UV (MeOH) 120582max 254366 nm IR (CHCl

3) ]max 2927 2870 (CH) 1682

(C=O) 1533 (C=C) cmminus1 APCI-MS 1762015 [M]+(calc C

10H8O31761714) 1HNMR (CDCl

3 300MHz)

120575 763 (1H d J 96Hz H-4) 631 (1H d J 96Hz H-3) 709 (1H d J 8Hz H-5) 695 (1H s H-8) 690(1H d J 8Hz H-6) and 399 (3H s 7-OCH

3) 13C

NMR(CDCl375MHz)120575 16202 (C2) 1563 (C7) 1500

(C8a) 1427 (C4) 1211 (C3) 1152 (C4a) 1134 (C6)1114 (C8) 1091 (C5) 563 (OCH

3) [15 16]

(4) (6 7-dimethoxy-coumarin or scoparone) pale yellowneedles (MeOH) mp 146-147∘C UV (MeOH) 120582max345 nm IR (CHCl

3) ]max 2985 2925 2855 (CH) 1684

(C=O) 1535 (C=C) 1462 1389 1363 and 1311 (CH)APCI-MS 2061245 [M]+ (calc C

11H10O42061576)

1H NMR (CDCl3 300MHz) 120575 763 (1H d J 95Hz

H-4) 69 (1H s H-5) 685 (1H s H-8) 625 (1Hd J 95Hz H-3) 390 (3H s 7-OCH

3) 385 (3H

s 6-OCH3) 13C-NMR (CDCl

3 75MHz) 120575 1613

(C2) 1529 (C7) 1501 (C6) 1464 (8a) 1432 (C4)1136 (C3) 1115 (C4a) 10820 (C5) 1001(C8) 5640(2xOCH

3) [15 17]

(5) (6-methoxy-7-hydroxy-coumarin or scopoletin) paleyellow needles (CH

2Cl2MeOH 95 05) mp 203-

204∘C UV (MeOH) 120582max 254 366 nm IR (CHCl3)

]max 3400ndash3550 (OH) 1685 (C=O) 2998 2938 2856

(CH) 1589 1511 (C=C) cmminus1 APCI-MS 1922008[M]+ (calc C

10H8O41921708) 1H NMR (CDCl

3

300MHz) 120575 761 (1H d J 95Hz H-4) 690 (1H s H-5) 685 (1H s H-8) 628 (1H d J 95Hz H-3) and385 (3H s 6-OCH

3) 13C NMR (CDCl

3 75MHz) 120575

1615 (C2) 1502 (C6) 1497 (C7) 1440 (C8a) 1434(C4) 1134 (C3) 1115 (C4a) 1075 (C5) 1032 (C8)564 (OCH

3) [16 17]

(6) ((E)-methyl-3 (3101584041015840-dihydroxy-phenyl) acrylate ormethyl caffeate) pale yellow crystalline powder(MeOH) mp 157ndash159∘C UV (MeOH) 120582max 354 nmIR (CHCl

3) ]max 3530 (OH) 2995 2927 2870 (CH)

1516 1643 (C=C) and 1690 (C=O) APCI-MS 1940623[M]+ (calc C

10H10O41941866) 1H NMR (CDCl

3

300MHz) 120575 750 (1H d J 161 Hz H-3) 717 (1H dJ 2Hz H-21015840) 693 (1H d J 7Hz H-51015840) 688 (1Hdd J 7 2Hz H-61015840) 615 (1H d J 161 Hz H-2) 385(3H s OCH

3) 13C NMR (CDCl

3-CD3OD 75MHz)

120575 1683 (C1) 1474 (C41015840) 1455 (C3) 1448 (C31015840) 1266(C11015840) 1218 (C2) 1151 (C21015840) 1141 (C51015840) 1139 (C61015840)514 (OCH

3) [18 19]

(7) (sitosteryl glucoside) white needles (MeOH) mp258ndash260∘C UV (EtOH) 120582max 220 nm IR (KBr) ]max3200ndash3450 (OH) 2980 2927 2870 (CH) 1560 (C=C)APCI-MS 4124002 [M minus 180]+ (calc C

35H60O6

5768854 1H NMR (CDCl3+CD3OD 300MHz) 120575

059 (3H s H-18) 085 (3H d J 73Hz H-27) 090(3H t J 65 65 Hz H-29) 091 (3H s H-19) 092(3H d J 73Hz H-26) 112 (3H d J 70Hz H-21)130 (2H m H-28) 174 (1H m H-2a) 194 (2H mH-1) 218 (1H m H-2b) 221 (1H m H-4) 328ndash35(5H mH-11015840 H-21015840 H-31015840 H-41015840 H-51015840) 350ndash370 (2Hm H-61015840) 374 (1H m H-3) 531 (1H m H-6) 13CNMR (CDCl

3+CD3OD 75MHz) 120575 1401 (C5) 1219

(C6) 1009 (C11015840) 790 (C3 C31015840) 762 (C51015840) 757 (C21015840)734 (C41015840) 618 (C61015840) 566 (C20) 559 (C17 C24)500 (C9) 456 (C8) 421 (C13) 400 (C12) 399 (C4)375 (C1) 365 (C10) 338 (C7 C22) 317 (C14) 298(C2) 294 (C16) 290 (C25) 280 (C23) 241 (C15)230 (C28) 229 (C11 C21) 209 (C26) 195 (C27) 190(C19) 117 (C29) 116 (C18) [16]

(8) (a supposed glycosphingolipid) white amorphouspowder (CHCl

3 MeOH 3 97) mp 252-254∘C

(MeOH) UV (EtOH) 120582max 236 nm IR (KBr)]max 3230-3450 (OH) 3384 (NH) 2998 29352851(CH) 1642 (C=O)APCI-MS7512596 [M]+(calcC44H81NO87512539) 1H NMR (pyr-d

5 300MHz)

120575 090 (2 times 3H t J6 6 Hz acyl-CH3) 120 (2H m

H-17) 130 (complex m H7-16 (CH2)119899) 18 (6H m

H-6 H-31015840 H-61015840) 24 (2H m H-21015840) 26-28 (1H mOH) 390-450 (7H m H-110158401015840 H-210158401015840 H-310158401015840 H-410158401015840H-510158401015840 H-610158401015840) 424 (1H m H-1a) 466 (1Hm H-1b)470 (1H m H-51015840) 475 (2H m H-2 H-3) 530 (1Hm H-5 H-41015840) 550 (1H dd J66 10 Hz H-4) 6-8(4H br s 4xOH) 859 (1H d J92 Hz NH) 13CNMR (pyr-d

5 75 MHz) 120575 1757 (C11015840) 1309 (C4)

1307 (C41015840) 1238 (C5) 1220 (C51015840) 1056 (C110158401015840) 786(C310158401015840) 784 (C510158401015840) 754 (C210158401015840) 725 (C3) 718 (C410158401015840)

Evidence-Based Complementary and Alternative Medicine 5

FFIFFIIFFIII

FFIVFFV

0

50

100

1 10 100 1000

Cel

l via

bilit

y (

)

log (120583gmL)

(a)C

ell v

iabi

lity

()

0

50

100

1 10 100 1000

FFIFFIIFFIII

FFIVFFV

log (120583gmL)

(b)

Figure 3 Percentage viable cell of FFIndashFFV on leukemic cell lines (a) NB4 and (b) HT93A

FFII IC50 = 151 plusmn 05

FFIII IC50 = 144 plusmn 06

FFIV IC50 = 321 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

FFII IC50 = 310 plusmn 01

FFIII IC50 = 97 plusmn 13

FFIV IC50 = 105 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(b)

Figure 4 Antiproliferative effect of FFIIndashFFIV on leukemic cell lines (a) NB4 and (b) HT93A

705 (C1) 630 (C610158401015840) 577 (C2) 343 (C21015840) 333 (C31015840)322 (C6 C61015840) 296 (C7-C16 C71015840-(CH

2)1015840119899) 229 (C17)

143 (acyl CH3) 141 (acyl CH

3) [ 20-23 ]

3 Results

The antiproliferative effect of FFIndashFFV on human leukemiccell lines was investigated as shown in Figure 3 It was foundthat FFII FFIII and FFIV could inhibit growth of NB4and HT93A (Figure 4) Therefore FFII FFIII and FFIVwere continued to evaluate the IC

50values on these cell

lines at varying concentrations ranging from 0 to 50120583gmL

The results showed that FFII FFIII and FFIV had the IC50

values on NB4 cell line at 151 plusmn 05 120583gmL 144 plusmn 06 and321 plusmn 07 120583gmL respectively whereas the IC

50values of

the HT93A cell line were 310 plusmn 01 97 plusmn 13 and 105 plusmn07 120583gmL respectively as shown in Figure 4 AdditionallyFFIII inhibited growth strongly on both NB4 andHT93A celllines while FFII inhibits growth strongly on NB4 more thanHT93A FFIV showed strong growth inhibition on HT93Amore than NB4

The active fractions FFII FFIII and FFIV were furtherchromatographed on the silica gel columns repeatedly andthe isolated compounds were identified using spectroscopic

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

CH2OH

O

O1

2

34

6

7

8 O

5

H3CO

O1

2

34

6

7

8 O

5

H3CO

H3CO

HO

H O

HO

OCH3

12

3

59984006998400

1998400

2998400

39984004998400 H

OH

OH

HO

23

5998400

1998400

2998400

3998400

4998400

45

CH2

H3C

18H3C

HN 1

O

CH2OH

OH

OHOO1998400998400

2998400998400 3998400998400

5998400998400 6

998400998400

4998400998400

1(friedelin) 2 (28-hydroxy-3-fridelanone) 3 (7-methoxy-coumarin)

4 (6-7-dimethoxy-coumarin) 5 (scopoletin) 6 (methyl caffeate)

7 (sitosteryl glucoside) 8 (a supposed glycosphingolipid)

12

3

45

8910

1113

14

17

O

HOOH

12

15

16

18

19

20

21 22

2324 25

26

29

O

OH

67

2728

5998400 19984002998400

39984004998400

6998400

HO

12

3

45

67

8

910

1112

1314

1516

1718

19

20

21

22

2324

25 26

27

28

29 30

O

O1

2

34

6

7

8 O

5H3CO

4a

4a

8a

8a

4a

8a

6998400

(CH2)119899

(CH2)10

Figure 2 Structures of 1ndash8

FFIII-2 (0465 g ss hexane-CH2Cl2 1 1) was recrystallized

with methanol giving 5 (15mg) FFIII-3 (0146 g ss CH2Cl2-

MeOH 95 05) was recrystallized in methanol giving 6(12mg) FFIII-4 (006 g ss CH

2Cl2-MeOH 9 1) was brown

gum FFIII-5 (109 g ss CH2Cl2-MeOH 8 2) appeared as

yellowish gumFFIV was chromatographed on silica gel column

eluted with gradient ss of hexane-acetone and acetone-methanol It produced 2 subfractions that is FFIV-1-2FFIV-1 (233 g ss hexane acetone 7 3) was added withmethanol 7 (85mg) was precipitated as white powderFFIV-2 (1297 g ss acetone methanol 9 1) was addedwith chloroform methanol (95 5) 8 (20mg) was obtainedas white powder Each of the purified compounds 1ndash8

described above possessed the following physicochemicalproperties and the chemical structures were identified usingspectroscopic methods (1H NMR 13C NMR Mass spectraIR spectra and UV-spectra) The structure of compounds1ndash8 (Figure 2) were elucidated as the followings

(1) (friedelin) white needles (Me2O) mp 258ndash260∘C

UV (EtOH)120582max 220 nm IR (CHCl3) ]max 2980 2927

2870 (CH) 1706 (C=O) 1462 1389 (CH) cmminus1 APCI-MS 4274156 [M+H]+ (calc C

30H50O 4267244) 1H

NMR (CDCl3 300MHz) 120575 075 (3H s H-24) 090

(3H s H-23) 098 (3H s H-25) 104 (3H s H-29)104 (3H s H-30) 104 (3H s H-26) 107 (3H s H-27) 120 (3H s H-28) 13-14 (18H complex m H-6

4 Evidence-Based Complementary and Alternative Medicine

7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-810 18) 174 (2H dd J 55 30Hz H-1a H-1b) 22 (1Hm H-4) 234 (1H m H-2b) 240 (1H m H-2a) 13CNMR (CDCl

3 75MHz) 120575 2131(C3) 594 (C10) 582

(C4) 531 (C8) 428 (C18) 420 (C5) 415 (C6) 415(C2) 397 (C13) 392 (C22) 383 (C14) 374 (C9) 360(C16) 356 (C11) 353 (C19) 350 (C29) 328 (C21)324 (C15) 321 (C28) 318 (C30) 305 (C12) 300(C17) 281 (C20) 222 (C1) 202 (C26) 186 (C27)182 (C7) 179 (C25) 146 (C24) 68 (C23) [12ndash14]

(2) (28-hydroxy-3-friedelanone or canophyllol) whiteprisms (MeOH) mp 363-364∘C UV (EtOH) 120582max220 nm IR (CHCl

3) ]max 3200ndash3513 (OH) 1709

(C=O) 2990 2930 2855 (CndashH) 1466 1378 (CndashH) and1039 1116 (CndashO) APCI-MS 4434015 [M+H]+ (calcC30H50O24425038) 1H NMR (CDCl

3 300MHz) 120575

088 (3H s H-24) 091 (3H d J 78Hz H-23) 099(3H s H-25) 102 (3H s H-26) 101 (3H s H-27) 119(3H s H-29) 123 (3H s H-30) 13-14 (18H complexm H-6 7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-8 10 18) 196 (2H dd J 76 37Hz H-1a H-1b)232 (1H m H-2b) 230 (1H m H-4) 240 (1H mH-2a) and 25 (2H m H-28) 371 (1H 28-OH) 13C-NMR (CDCl

3 75MHz) 120575 2131 (C3) 683 (C28) 594

(C10) 582 (C4) 531 (C8) 420 (C5) 417 (C22) 415(C2 C6) 397 (C13) 395 (C18) 383 (C14) 374 (C9)356 (C11) 352 (C17) 344 (C29) 334 (C19) 329(C30) 316 (C21) 313 (C16) 305 (C12) 303 (C20)292 (C15) 234 (C1) 194 (C25) 191 (C26) 182 (C7)181 (C27) 148 (C24) 68 (C23) [13 14]

(3) (7-methoxy-coumarin or herniarin) white needles(MeOH) mp 117-118∘C UV (MeOH) 120582max 254366 nm IR (CHCl

3) ]max 2927 2870 (CH) 1682

(C=O) 1533 (C=C) cmminus1 APCI-MS 1762015 [M]+(calc C

10H8O31761714) 1HNMR (CDCl

3 300MHz)

120575 763 (1H d J 96Hz H-4) 631 (1H d J 96Hz H-3) 709 (1H d J 8Hz H-5) 695 (1H s H-8) 690(1H d J 8Hz H-6) and 399 (3H s 7-OCH

3) 13C

NMR(CDCl375MHz)120575 16202 (C2) 1563 (C7) 1500

(C8a) 1427 (C4) 1211 (C3) 1152 (C4a) 1134 (C6)1114 (C8) 1091 (C5) 563 (OCH

3) [15 16]

(4) (6 7-dimethoxy-coumarin or scoparone) pale yellowneedles (MeOH) mp 146-147∘C UV (MeOH) 120582max345 nm IR (CHCl

3) ]max 2985 2925 2855 (CH) 1684

(C=O) 1535 (C=C) 1462 1389 1363 and 1311 (CH)APCI-MS 2061245 [M]+ (calc C

11H10O42061576)

1H NMR (CDCl3 300MHz) 120575 763 (1H d J 95Hz

H-4) 69 (1H s H-5) 685 (1H s H-8) 625 (1Hd J 95Hz H-3) 390 (3H s 7-OCH

3) 385 (3H

s 6-OCH3) 13C-NMR (CDCl

3 75MHz) 120575 1613

(C2) 1529 (C7) 1501 (C6) 1464 (8a) 1432 (C4)1136 (C3) 1115 (C4a) 10820 (C5) 1001(C8) 5640(2xOCH

3) [15 17]

(5) (6-methoxy-7-hydroxy-coumarin or scopoletin) paleyellow needles (CH

2Cl2MeOH 95 05) mp 203-

204∘C UV (MeOH) 120582max 254 366 nm IR (CHCl3)

]max 3400ndash3550 (OH) 1685 (C=O) 2998 2938 2856

(CH) 1589 1511 (C=C) cmminus1 APCI-MS 1922008[M]+ (calc C

10H8O41921708) 1H NMR (CDCl

3

300MHz) 120575 761 (1H d J 95Hz H-4) 690 (1H s H-5) 685 (1H s H-8) 628 (1H d J 95Hz H-3) and385 (3H s 6-OCH

3) 13C NMR (CDCl

3 75MHz) 120575

1615 (C2) 1502 (C6) 1497 (C7) 1440 (C8a) 1434(C4) 1134 (C3) 1115 (C4a) 1075 (C5) 1032 (C8)564 (OCH

3) [16 17]

(6) ((E)-methyl-3 (3101584041015840-dihydroxy-phenyl) acrylate ormethyl caffeate) pale yellow crystalline powder(MeOH) mp 157ndash159∘C UV (MeOH) 120582max 354 nmIR (CHCl

3) ]max 3530 (OH) 2995 2927 2870 (CH)

1516 1643 (C=C) and 1690 (C=O) APCI-MS 1940623[M]+ (calc C

10H10O41941866) 1H NMR (CDCl

3

300MHz) 120575 750 (1H d J 161 Hz H-3) 717 (1H dJ 2Hz H-21015840) 693 (1H d J 7Hz H-51015840) 688 (1Hdd J 7 2Hz H-61015840) 615 (1H d J 161 Hz H-2) 385(3H s OCH

3) 13C NMR (CDCl

3-CD3OD 75MHz)

120575 1683 (C1) 1474 (C41015840) 1455 (C3) 1448 (C31015840) 1266(C11015840) 1218 (C2) 1151 (C21015840) 1141 (C51015840) 1139 (C61015840)514 (OCH

3) [18 19]

(7) (sitosteryl glucoside) white needles (MeOH) mp258ndash260∘C UV (EtOH) 120582max 220 nm IR (KBr) ]max3200ndash3450 (OH) 2980 2927 2870 (CH) 1560 (C=C)APCI-MS 4124002 [M minus 180]+ (calc C

35H60O6

5768854 1H NMR (CDCl3+CD3OD 300MHz) 120575

059 (3H s H-18) 085 (3H d J 73Hz H-27) 090(3H t J 65 65 Hz H-29) 091 (3H s H-19) 092(3H d J 73Hz H-26) 112 (3H d J 70Hz H-21)130 (2H m H-28) 174 (1H m H-2a) 194 (2H mH-1) 218 (1H m H-2b) 221 (1H m H-4) 328ndash35(5H mH-11015840 H-21015840 H-31015840 H-41015840 H-51015840) 350ndash370 (2Hm H-61015840) 374 (1H m H-3) 531 (1H m H-6) 13CNMR (CDCl

3+CD3OD 75MHz) 120575 1401 (C5) 1219

(C6) 1009 (C11015840) 790 (C3 C31015840) 762 (C51015840) 757 (C21015840)734 (C41015840) 618 (C61015840) 566 (C20) 559 (C17 C24)500 (C9) 456 (C8) 421 (C13) 400 (C12) 399 (C4)375 (C1) 365 (C10) 338 (C7 C22) 317 (C14) 298(C2) 294 (C16) 290 (C25) 280 (C23) 241 (C15)230 (C28) 229 (C11 C21) 209 (C26) 195 (C27) 190(C19) 117 (C29) 116 (C18) [16]

(8) (a supposed glycosphingolipid) white amorphouspowder (CHCl

3 MeOH 3 97) mp 252-254∘C

(MeOH) UV (EtOH) 120582max 236 nm IR (KBr)]max 3230-3450 (OH) 3384 (NH) 2998 29352851(CH) 1642 (C=O)APCI-MS7512596 [M]+(calcC44H81NO87512539) 1H NMR (pyr-d

5 300MHz)

120575 090 (2 times 3H t J6 6 Hz acyl-CH3) 120 (2H m

H-17) 130 (complex m H7-16 (CH2)119899) 18 (6H m

H-6 H-31015840 H-61015840) 24 (2H m H-21015840) 26-28 (1H mOH) 390-450 (7H m H-110158401015840 H-210158401015840 H-310158401015840 H-410158401015840H-510158401015840 H-610158401015840) 424 (1H m H-1a) 466 (1Hm H-1b)470 (1H m H-51015840) 475 (2H m H-2 H-3) 530 (1Hm H-5 H-41015840) 550 (1H dd J66 10 Hz H-4) 6-8(4H br s 4xOH) 859 (1H d J92 Hz NH) 13CNMR (pyr-d

5 75 MHz) 120575 1757 (C11015840) 1309 (C4)

1307 (C41015840) 1238 (C5) 1220 (C51015840) 1056 (C110158401015840) 786(C310158401015840) 784 (C510158401015840) 754 (C210158401015840) 725 (C3) 718 (C410158401015840)

Evidence-Based Complementary and Alternative Medicine 5

FFIFFIIFFIII

FFIVFFV

0

50

100

1 10 100 1000

Cel

l via

bilit

y (

)

log (120583gmL)

(a)C

ell v

iabi

lity

()

0

50

100

1 10 100 1000

FFIFFIIFFIII

FFIVFFV

log (120583gmL)

(b)

Figure 3 Percentage viable cell of FFIndashFFV on leukemic cell lines (a) NB4 and (b) HT93A

FFII IC50 = 151 plusmn 05

FFIII IC50 = 144 plusmn 06

FFIV IC50 = 321 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

FFII IC50 = 310 plusmn 01

FFIII IC50 = 97 plusmn 13

FFIV IC50 = 105 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(b)

Figure 4 Antiproliferative effect of FFIIndashFFIV on leukemic cell lines (a) NB4 and (b) HT93A

705 (C1) 630 (C610158401015840) 577 (C2) 343 (C21015840) 333 (C31015840)322 (C6 C61015840) 296 (C7-C16 C71015840-(CH

2)1015840119899) 229 (C17)

143 (acyl CH3) 141 (acyl CH

3) [ 20-23 ]

3 Results

The antiproliferative effect of FFIndashFFV on human leukemiccell lines was investigated as shown in Figure 3 It was foundthat FFII FFIII and FFIV could inhibit growth of NB4and HT93A (Figure 4) Therefore FFII FFIII and FFIVwere continued to evaluate the IC

50values on these cell

lines at varying concentrations ranging from 0 to 50120583gmL

The results showed that FFII FFIII and FFIV had the IC50

values on NB4 cell line at 151 plusmn 05 120583gmL 144 plusmn 06 and321 plusmn 07 120583gmL respectively whereas the IC

50values of

the HT93A cell line were 310 plusmn 01 97 plusmn 13 and 105 plusmn07 120583gmL respectively as shown in Figure 4 AdditionallyFFIII inhibited growth strongly on both NB4 andHT93A celllines while FFII inhibits growth strongly on NB4 more thanHT93A FFIV showed strong growth inhibition on HT93Amore than NB4

The active fractions FFII FFIII and FFIV were furtherchromatographed on the silica gel columns repeatedly andthe isolated compounds were identified using spectroscopic

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-810 18) 174 (2H dd J 55 30Hz H-1a H-1b) 22 (1Hm H-4) 234 (1H m H-2b) 240 (1H m H-2a) 13CNMR (CDCl

3 75MHz) 120575 2131(C3) 594 (C10) 582

(C4) 531 (C8) 428 (C18) 420 (C5) 415 (C6) 415(C2) 397 (C13) 392 (C22) 383 (C14) 374 (C9) 360(C16) 356 (C11) 353 (C19) 350 (C29) 328 (C21)324 (C15) 321 (C28) 318 (C30) 305 (C12) 300(C17) 281 (C20) 222 (C1) 202 (C26) 186 (C27)182 (C7) 179 (C25) 146 (C24) 68 (C23) [12ndash14]

(2) (28-hydroxy-3-friedelanone or canophyllol) whiteprisms (MeOH) mp 363-364∘C UV (EtOH) 120582max220 nm IR (CHCl

3) ]max 3200ndash3513 (OH) 1709

(C=O) 2990 2930 2855 (CndashH) 1466 1378 (CndashH) and1039 1116 (CndashO) APCI-MS 4434015 [M+H]+ (calcC30H50O24425038) 1H NMR (CDCl

3 300MHz) 120575

088 (3H s H-24) 091 (3H d J 78Hz H-23) 099(3H s H-25) 102 (3H s H-26) 101 (3H s H-27) 119(3H s H-29) 123 (3H s H-30) 13-14 (18H complexm H-6 7 11 12 15 16 19 21 22) 14ndash16 (3H complexm H-8 10 18) 196 (2H dd J 76 37Hz H-1a H-1b)232 (1H m H-2b) 230 (1H m H-4) 240 (1H mH-2a) and 25 (2H m H-28) 371 (1H 28-OH) 13C-NMR (CDCl

3 75MHz) 120575 2131 (C3) 683 (C28) 594

(C10) 582 (C4) 531 (C8) 420 (C5) 417 (C22) 415(C2 C6) 397 (C13) 395 (C18) 383 (C14) 374 (C9)356 (C11) 352 (C17) 344 (C29) 334 (C19) 329(C30) 316 (C21) 313 (C16) 305 (C12) 303 (C20)292 (C15) 234 (C1) 194 (C25) 191 (C26) 182 (C7)181 (C27) 148 (C24) 68 (C23) [13 14]

(3) (7-methoxy-coumarin or herniarin) white needles(MeOH) mp 117-118∘C UV (MeOH) 120582max 254366 nm IR (CHCl

3) ]max 2927 2870 (CH) 1682

(C=O) 1533 (C=C) cmminus1 APCI-MS 1762015 [M]+(calc C

10H8O31761714) 1HNMR (CDCl

3 300MHz)

120575 763 (1H d J 96Hz H-4) 631 (1H d J 96Hz H-3) 709 (1H d J 8Hz H-5) 695 (1H s H-8) 690(1H d J 8Hz H-6) and 399 (3H s 7-OCH

3) 13C

NMR(CDCl375MHz)120575 16202 (C2) 1563 (C7) 1500

(C8a) 1427 (C4) 1211 (C3) 1152 (C4a) 1134 (C6)1114 (C8) 1091 (C5) 563 (OCH

3) [15 16]

(4) (6 7-dimethoxy-coumarin or scoparone) pale yellowneedles (MeOH) mp 146-147∘C UV (MeOH) 120582max345 nm IR (CHCl

3) ]max 2985 2925 2855 (CH) 1684

(C=O) 1535 (C=C) 1462 1389 1363 and 1311 (CH)APCI-MS 2061245 [M]+ (calc C

11H10O42061576)

1H NMR (CDCl3 300MHz) 120575 763 (1H d J 95Hz

H-4) 69 (1H s H-5) 685 (1H s H-8) 625 (1Hd J 95Hz H-3) 390 (3H s 7-OCH

3) 385 (3H

s 6-OCH3) 13C-NMR (CDCl

3 75MHz) 120575 1613

(C2) 1529 (C7) 1501 (C6) 1464 (8a) 1432 (C4)1136 (C3) 1115 (C4a) 10820 (C5) 1001(C8) 5640(2xOCH

3) [15 17]

(5) (6-methoxy-7-hydroxy-coumarin or scopoletin) paleyellow needles (CH

2Cl2MeOH 95 05) mp 203-

204∘C UV (MeOH) 120582max 254 366 nm IR (CHCl3)

]max 3400ndash3550 (OH) 1685 (C=O) 2998 2938 2856

(CH) 1589 1511 (C=C) cmminus1 APCI-MS 1922008[M]+ (calc C

10H8O41921708) 1H NMR (CDCl

3

300MHz) 120575 761 (1H d J 95Hz H-4) 690 (1H s H-5) 685 (1H s H-8) 628 (1H d J 95Hz H-3) and385 (3H s 6-OCH

3) 13C NMR (CDCl

3 75MHz) 120575

1615 (C2) 1502 (C6) 1497 (C7) 1440 (C8a) 1434(C4) 1134 (C3) 1115 (C4a) 1075 (C5) 1032 (C8)564 (OCH

3) [16 17]

(6) ((E)-methyl-3 (3101584041015840-dihydroxy-phenyl) acrylate ormethyl caffeate) pale yellow crystalline powder(MeOH) mp 157ndash159∘C UV (MeOH) 120582max 354 nmIR (CHCl

3) ]max 3530 (OH) 2995 2927 2870 (CH)

1516 1643 (C=C) and 1690 (C=O) APCI-MS 1940623[M]+ (calc C

10H10O41941866) 1H NMR (CDCl

3

300MHz) 120575 750 (1H d J 161 Hz H-3) 717 (1H dJ 2Hz H-21015840) 693 (1H d J 7Hz H-51015840) 688 (1Hdd J 7 2Hz H-61015840) 615 (1H d J 161 Hz H-2) 385(3H s OCH

3) 13C NMR (CDCl

3-CD3OD 75MHz)

120575 1683 (C1) 1474 (C41015840) 1455 (C3) 1448 (C31015840) 1266(C11015840) 1218 (C2) 1151 (C21015840) 1141 (C51015840) 1139 (C61015840)514 (OCH

3) [18 19]

(7) (sitosteryl glucoside) white needles (MeOH) mp258ndash260∘C UV (EtOH) 120582max 220 nm IR (KBr) ]max3200ndash3450 (OH) 2980 2927 2870 (CH) 1560 (C=C)APCI-MS 4124002 [M minus 180]+ (calc C

35H60O6

5768854 1H NMR (CDCl3+CD3OD 300MHz) 120575

059 (3H s H-18) 085 (3H d J 73Hz H-27) 090(3H t J 65 65 Hz H-29) 091 (3H s H-19) 092(3H d J 73Hz H-26) 112 (3H d J 70Hz H-21)130 (2H m H-28) 174 (1H m H-2a) 194 (2H mH-1) 218 (1H m H-2b) 221 (1H m H-4) 328ndash35(5H mH-11015840 H-21015840 H-31015840 H-41015840 H-51015840) 350ndash370 (2Hm H-61015840) 374 (1H m H-3) 531 (1H m H-6) 13CNMR (CDCl

3+CD3OD 75MHz) 120575 1401 (C5) 1219

(C6) 1009 (C11015840) 790 (C3 C31015840) 762 (C51015840) 757 (C21015840)734 (C41015840) 618 (C61015840) 566 (C20) 559 (C17 C24)500 (C9) 456 (C8) 421 (C13) 400 (C12) 399 (C4)375 (C1) 365 (C10) 338 (C7 C22) 317 (C14) 298(C2) 294 (C16) 290 (C25) 280 (C23) 241 (C15)230 (C28) 229 (C11 C21) 209 (C26) 195 (C27) 190(C19) 117 (C29) 116 (C18) [16]

(8) (a supposed glycosphingolipid) white amorphouspowder (CHCl

3 MeOH 3 97) mp 252-254∘C

(MeOH) UV (EtOH) 120582max 236 nm IR (KBr)]max 3230-3450 (OH) 3384 (NH) 2998 29352851(CH) 1642 (C=O)APCI-MS7512596 [M]+(calcC44H81NO87512539) 1H NMR (pyr-d

5 300MHz)

120575 090 (2 times 3H t J6 6 Hz acyl-CH3) 120 (2H m

H-17) 130 (complex m H7-16 (CH2)119899) 18 (6H m

H-6 H-31015840 H-61015840) 24 (2H m H-21015840) 26-28 (1H mOH) 390-450 (7H m H-110158401015840 H-210158401015840 H-310158401015840 H-410158401015840H-510158401015840 H-610158401015840) 424 (1H m H-1a) 466 (1Hm H-1b)470 (1H m H-51015840) 475 (2H m H-2 H-3) 530 (1Hm H-5 H-41015840) 550 (1H dd J66 10 Hz H-4) 6-8(4H br s 4xOH) 859 (1H d J92 Hz NH) 13CNMR (pyr-d

5 75 MHz) 120575 1757 (C11015840) 1309 (C4)

1307 (C41015840) 1238 (C5) 1220 (C51015840) 1056 (C110158401015840) 786(C310158401015840) 784 (C510158401015840) 754 (C210158401015840) 725 (C3) 718 (C410158401015840)

Evidence-Based Complementary and Alternative Medicine 5

FFIFFIIFFIII

FFIVFFV

0

50

100

1 10 100 1000

Cel

l via

bilit

y (

)

log (120583gmL)

(a)C

ell v

iabi

lity

()

0

50

100

1 10 100 1000

FFIFFIIFFIII

FFIVFFV

log (120583gmL)

(b)

Figure 3 Percentage viable cell of FFIndashFFV on leukemic cell lines (a) NB4 and (b) HT93A

FFII IC50 = 151 plusmn 05

FFIII IC50 = 144 plusmn 06

FFIV IC50 = 321 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

FFII IC50 = 310 plusmn 01

FFIII IC50 = 97 plusmn 13

FFIV IC50 = 105 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(b)

Figure 4 Antiproliferative effect of FFIIndashFFIV on leukemic cell lines (a) NB4 and (b) HT93A

705 (C1) 630 (C610158401015840) 577 (C2) 343 (C21015840) 333 (C31015840)322 (C6 C61015840) 296 (C7-C16 C71015840-(CH

2)1015840119899) 229 (C17)

143 (acyl CH3) 141 (acyl CH

3) [ 20-23 ]

3 Results

The antiproliferative effect of FFIndashFFV on human leukemiccell lines was investigated as shown in Figure 3 It was foundthat FFII FFIII and FFIV could inhibit growth of NB4and HT93A (Figure 4) Therefore FFII FFIII and FFIVwere continued to evaluate the IC

50values on these cell

lines at varying concentrations ranging from 0 to 50120583gmL

The results showed that FFII FFIII and FFIV had the IC50

values on NB4 cell line at 151 plusmn 05 120583gmL 144 plusmn 06 and321 plusmn 07 120583gmL respectively whereas the IC

50values of

the HT93A cell line were 310 plusmn 01 97 plusmn 13 and 105 plusmn07 120583gmL respectively as shown in Figure 4 AdditionallyFFIII inhibited growth strongly on both NB4 andHT93A celllines while FFII inhibits growth strongly on NB4 more thanHT93A FFIV showed strong growth inhibition on HT93Amore than NB4

The active fractions FFII FFIII and FFIV were furtherchromatographed on the silica gel columns repeatedly andthe isolated compounds were identified using spectroscopic

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

FFIFFIIFFIII

FFIVFFV

0

50

100

1 10 100 1000

Cel

l via

bilit

y (

)

log (120583gmL)

(a)C

ell v

iabi

lity

()

0

50

100

1 10 100 1000

FFIFFIIFFIII

FFIVFFV

log (120583gmL)

(b)

Figure 3 Percentage viable cell of FFIndashFFV on leukemic cell lines (a) NB4 and (b) HT93A

FFII IC50 = 151 plusmn 05

FFIII IC50 = 144 plusmn 06

FFIV IC50 = 321 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

FFII IC50 = 310 plusmn 01

FFIII IC50 = 97 plusmn 13

FFIV IC50 = 105 plusmn 07

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(b)

Figure 4 Antiproliferative effect of FFIIndashFFIV on leukemic cell lines (a) NB4 and (b) HT93A

705 (C1) 630 (C610158401015840) 577 (C2) 343 (C21015840) 333 (C31015840)322 (C6 C61015840) 296 (C7-C16 C71015840-(CH

2)1015840119899) 229 (C17)

143 (acyl CH3) 141 (acyl CH

3) [ 20-23 ]

3 Results

The antiproliferative effect of FFIndashFFV on human leukemiccell lines was investigated as shown in Figure 3 It was foundthat FFII FFIII and FFIV could inhibit growth of NB4and HT93A (Figure 4) Therefore FFII FFIII and FFIVwere continued to evaluate the IC

50values on these cell

lines at varying concentrations ranging from 0 to 50120583gmL

The results showed that FFII FFIII and FFIV had the IC50

values on NB4 cell line at 151 plusmn 05 120583gmL 144 plusmn 06 and321 plusmn 07 120583gmL respectively whereas the IC

50values of

the HT93A cell line were 310 plusmn 01 97 plusmn 13 and 105 plusmn07 120583gmL respectively as shown in Figure 4 AdditionallyFFIII inhibited growth strongly on both NB4 andHT93A celllines while FFII inhibits growth strongly on NB4 more thanHT93A FFIV showed strong growth inhibition on HT93Amore than NB4

The active fractions FFII FFIII and FFIV were furtherchromatographed on the silica gel columns repeatedly andthe isolated compounds were identified using spectroscopic

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

methods FFIIwas composed of friedelin 1 and 28-hydroxy-3-friedelanone 2 and 7-methoxy-coumarin or herniarin 3 FFIIIwas composed of 67-dimethoxy-coumarin or scoparone 4scopoletin 5 and methyl caffeate 6 FFIV was composed ofsitosteryl glucoside 7 and a supposed glycosphingolipid 8Sitosteryl glucoside 7 (85mg) was isolated which was thehighest yield as shown in Figure 1

4 Discussion

P indica which has been long used in Thai traditionalmedicine for treating various diseases including malignan-cies was investigated in this study Based on our previousreport [5] the methanolic part of this plant showed highpotent antiproliferative effect on NB4 and HT93A acutepromyelocytic cell lines Here in this study we demon-strated that eight compounds were isolated from this activemethanolic part It was chromatographed on CC repeatedlyas shown in Figure 1 The bioassay determined the activefractions they were FFII FFIII and FFIV We found thatFFII could inhibit growth on NB4 stronger than HT93Awhile FFIII showed growth inhibition on both NB4 andHT93A Interestingly FFIV exhibited dominantly growthinhibition on HT93A The differences in the antiproliferativeeffects of these fractions might arise from the differentactive compounds themselves and the interactions with theoncoproteins in these acute promyelocytic cell lines that isthe long and short types of PML-RAR120572 in NB4 and HT93Arespectively

The antiproliferative effect of FFII might be caused bythe presence of 2 triterpenes that is friedelin 1 and 28-hydroxy-3-friedelanone 2 and one coumarin 3 namely7-methoxy-coumarin Previously the cytotoxicity of 28-hydroxy-3-friedelanone against A549-human lung cancercell line LLC-mouse Lewis lung carcinoma HL60-humanpromyelocytic cell line and MCF7-human breast cancercell line were demonstrated Hence some triterpenes couldstrongly induce apoptosis by attending the mitochondrialmembrane potential and regulating the expression of Bcl-2different compasses [14 20]The IC

50(120583gmL) of 7-methoxy-

coumarin 3 onHL60 andK562 human chronic leukemia cellswas also demonstratedwith the values of 289 and 193120583gmLrespectively [21 22]

For FFIII the cytotoxic activity of this fraction mightresult from coumarins (4 and 5 namely 67-dimethoxy-coumarin and scopoletin resp) including methyl caffeate6 The previous reports demonstrated that coumarins couldinhibit several human cancer cell lines such as QU-DB largecell lung cancer and human leukemia HL60 cells [22 23]The mechanism of action of coumarins was exerted fromthe inhibition of tubulin polymerization and the induction ofcell cycle arrest at G2M phase [23] The involvement of cellcycle inhibition might be due to the inhibition of the releaseof cyclin D1 an essential enzyme in cell cycle progression[24] Interestingly high concentration of scopoletin can haveantiproliferative effect on lymphoma cell line by inducingapoptosis [25] In additionmethyl caffeate can inhibit growthof human cervical adenocarcinoma cell line (HeLa) [26]Notably methyl caffeate which contains 2 hydroxyl groups

12

3

45

O

OH

1998400

OHN CH2

H

(a)

55 50 45 40

H3

H1a

H1b

173

8

235

0

393

09

157

5

552

1

821

2

558

8

H2998400

(b)

CH1a 424 (1H m)1b 466 (1H m)2 475 (1H m) 5773 475 (1H m) 725

705

1205751H 12057513C

(c)

Figure 5 Amino alcohol part of glycosphingolipid (a) chemicalstructure (b) 1H NMR spectrum and (c) 1H and 13C assignments

on aromatic ring can induce cytotoxic activity via the strongantioxidant activity from these hydroxyl groups [27]

FFIV inhibited HT93A stronger than NB4 cells It con-tained sitosteryl glucoside 7 and a supposed glycosphin-golipid 8The partial structures of 8 included two acyl chainsone of which was palmitic acid 120573-D-glucose and an aminoalcohol 1H-NMR spectrum of 8 showed that the typicalresonances of amino alcohol part of glycosphingolipid wereH-1a at 120575 424 (1H m) H-1b 466 (1H m) and H-2 andH-3 475 (2H m) (Figure 5) [3 28] One acyl chain wasbiosynthetically originated from palmitoyl-CoA which wasshown by the long chain methylene protons of 8 appearingas multiplets at 120575 11ndash13 [29] The presence of sugar protonsas complex multiplets at 120575 390ndash450 ppm (7H m from H-110158401015840

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

to H-610158401015840) was substantiated by carbon signals at 120575 1056(C110158401015840) 754 (C210158401015840) 786 (C310158401015840) 718 (C410158401015840) 784 (C510158401015840) and630 (C610158401015840) The structure of 8 was thus supposed to be aglycosphingolipid The sitosteryl glucoside 7 was previouslyreported to have the antiproliferative effect on human coloncancer cell by inducing the apoptotic pathway [27] Theglycosphingolipid 8 which contains sphingosine can induceapoptosis involving with the ceramide and sphingosine-1-phosphate-mediated pathway [30 31] The result from ourstudy pointed out that coumarins were promising anticanceragent [32]The extract fraction containing mainly coumarinslike FFIII could be developed as a drugmaterial for anticancerphytopharmaceutical

5 Conclusion

The methanolic part of P indica extract inhibited the acutepromyelocytic leukemia cell lines NB4 and HT93A Thebioassay-guided fractionation of the active part got threedifferent active fractions They were FFII FFIII and FFIVThe FFII showed strong growth inhibition on NB4 whereasthe FFIII exhibited strong growth inhibition on bothNB4 andHT93AThe FFIV demonstrated strong growth inhibition onHT93A The active compounds isolated from the FFII con-tained mainly triterpenoids (friedelin 1 and 28-hydroxy-3-friedelanone 2) and some coumarins (7-methoxy-coumarin3) The FFIII contained mainly phenolic compounds (sco-parone 4 scopoletin 5 and methyl caffeate 6) and theFFIV contained mainly glycosides (sitosteryl glucoside 7 andglycosphingolipid 8) P indica was the first report aboutantiproliferative effect on human leukemic cell lines and thestructures of compounds 1ndash8 were elucidated The furtherinvestigation including drug development will be studied onthese fractions especially the FFIII which demonstrated thebest antiproliferative effect on both human leukemic cell lines(NB4 and HT93A)

Conflict of Interests

The authors declare that they do not have conflict of interests

Acknowledgments

This work was supported by the grants from Faculty ofPharmacy Mahidol University The authors especially thankProfessor Patoomratana Tuchinda Faculty of Science Mahi-dol University for the measurements of 1H and 13C NMRspectra

References

[1] T Smitinand ldquoThai Plant Names (Botanical Names VernacularNames)rdquo Royal Forest Department Thailand Bangkok 1980

[2] J J Bennett ldquoPlantae Javanicae Rarioresrdquo vol 1 1838[3] T Srisapoomi W Jiratchariyakul N Partkaittikul and T Kum-

malue ldquoEffect of twoThai herbal remedies on the sensitivity ofchemotherapeutic agents in human cancer cellsrdquo Asian Journalof Traditional Medicines vol 3 no 4 pp 108ndash111 2008

[4] A Sayeed and M A Sattar ldquoA prenylated isoflavone from Pou-zolzia indica its in vitro antimicrobial activity and cytotoxicityevaluationrdquo Oriental Journal of Chemistry vol 19 no 1 pp 35ndash40 2003

[5] Y U-pratya W Jiratchariyakul and T Kummalue ldquoAnti-proli-ferative effects of Pouzolzia indica on acute promyelocytic celllines NB4 and HT93Ardquo Asian Journal of Traditional Medicinesvol 3 no 4 pp 124ndash133 2008

[6] M Lanotte V Martin-Thouvenin S Najman P Balerini FValensi and R Berger ldquoNB4 a maturation inducible cell linewith t(1517)marker isolated from a human acute promyelocyticleukemia (M3)rdquo Blood vol 77 no 5 pp 1080ndash1086 1991

[7] N Nishimura Y Furukawa K Sutheesophon et al ldquoSuppres-sion of ARG kinase activity by STI571 induces cell cycle arrestthrough up-regulation of CDK inhibitor p18INK4crdquoOncogenevol 22 no 26 pp 4074ndash4082 2003

[8] K Kishi K Toba T A Azegami et al ldquoHematopoietic cyto-kine-dependent differentiation to eosinophils and neutrophilsin a newly established acute promyelocytic leukemia cell linewith t(1517)rdquo Experimental Hematology vol 26 no 2 pp 135ndash142 1998

[9] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990

[10] T Mosmann ldquoColorimetric assay MTT based for cell prolif-erationrdquo Journal of Immunological Methods vol 65 pp 55ndash631993

[11] T Kummalue P O-charoenrat W Jiratchariyakul et al ldquoAnti-proliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[12] S B Mahato and A P Kundu ldquo13CNMR spectra of pentacyclictriterpenoidsmdasha compilation and some salient featuresrdquo Phyto-chemistry vol 37 no 6 pp 1517ndash1575 1994

[13] TThiThuy N Huy Cuong and T Van Sung ldquoTriterpenes fromCelastrus Hindsii Benthrdquo Journal of Organic Chemistry vol 45no 3 pp 373ndash376 2007

[14] N T Thao T M Hung M K Lee J C Kim B S Min and KBae ldquoTriterpenoids from Camellia japonica and their cytotoxicactivityrdquo Chemical Pharmaceutical Bulletin vol 58 no 1 pp121ndash124 2010

[15] C H Ma W Ke Z L Sun et al ldquoLarge-scale isolationand purification of scoparone from Herba artemisiae scopariaeby high-speed counter-current chromatographyrdquoChromatogra-phia vol 64 no 1-2 pp 83ndash87 2006

[16] J Intekhab andM Aslam ldquoConstituents from Feronia limoniardquoAnalele Universitii din Bucureti Chimie vol 18 no 2 pp 95ndash1012009

[17] G S Kupriyanova ldquoNMR studies of the electronic structure ofcoumarinsrdquo Journal of Structural Chemistry vol 38 no 3 pp408ndash414 1997

[18] M G De Carvalho G J A De Carvalho and R Braz-FilholdquoChemical constituents from Ouratea floribunda complete 1Hand 13C NMR assignments of atranorin and its new acetylderivativerdquo Journal of the Brazilian Chemical Society vol 11 no2 pp 143ndash147 2000

[19] S P Lee G Jun E J Yoon S Park and C H Yang ldquoInhibitoryeffect of methyl caffeate on Fos-Jun-DNA complex formationand suppression of cancer cell growthrdquo Bulletin of the KoreanChemical Society vol 22 no 10 pp 1131ndash1135 2001

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

[20] T Uto A Sakamoto N H Tung et al ldquoAnti-proliferative activ-itied activities and apoptosis induction by triterpenes derivedfrom Eriobotryo japonica in Human Leucemia cell linesrdquoInternatioanl Journal of Molecular Science vol 14 pp 4106ndash4120 2013

[21] A Lacy ldquoStudies on coumarins and coumarin-related com-pounds to determine their therapeutic role in the treatment ofcancerrdquoCurrent PharmaceuticalDesign vol 10 no 30 pp 3797ndash3811 2004

[22] P N Thanh W Jin G Song K Bae and S S Kang ldquoCytotoxiccoumarins from the root of Angelica dahuricardquo Archives ofPharmacal Research vol 27 no 12 pp 1211ndash1215 2004

[23] K Nardes M Zahra R Mohammad E Nasrollah and GAbbas ldquoUmbelliprenin is cytotoxic agent QU-DB large cell lungcancer cell line but anti-proliferative against A549 adenocarci-noma cellsrdquo DURA Pharmaceutical Sciences vol 20 no 69 pp1ndash6 2012

[24] R Reyes-Chilpa E Estrada-Muniz T Ramırez Apan et alldquoCytotoxic effects of mammea type coumarins from Calophyl-lum brasilienserdquo Life Sciences vol 75 no 13 pp 1635ndash1647 2004

[25] M G Manuele G Ferraro M L Barreiro Arcos P Lopez GCremaschi and C Anesini ldquoComparative immunomodulatoryeffect of scopoletin on tumoral and normal lymphocytesrdquo LifeSciences vol 79 no 21 pp 2043ndash2048 2006

[26] S M Fiuza C Gomes L J Teixeira et al ldquoPhenolic acidderivatives with potential anticancer propertiesmdasha structure-activity relationship studymdashpart 1 methyl propyl and octylesters of caffeic and gallic acidsrdquo Bioorganic and MedicinalChemistry vol 12 no 13 pp 3581ndash3589 2004

[27] S Roussi A Winter F Gosse et al ldquoDifferent apoptotic mech-anisms are involved in the antiproliferative effects of 7120573-hydroxysitosterol and 7120573-hydroxycholesterol in human coloncancer cellsrdquo Cell Death and Differentiation vol 12 no 2 pp128ndash135 2005

[28] W Jiratchariyakul P Moongkarndi H Okabe and A W Fra-hm ldquoInvestigation of anticancer components from Murdannialoriformis (Hassk)rdquoThai Journal of Phytopharm vol 5 no 1 pp10ndash20 1998

[29] H Rimpler and B Arzneistoffe ldquoGeorg Thiemerdquo StuttgartGermany 1990

[30] B Ogretmen and Y A Hannun ldquoBiologically active sphingoli-pids in cancer pathogenesis and treatmentrdquo Nature ReviewsCancer vol 4 no 8 pp 604ndash616 2004

[31] J Woodcock ldquoSphingosine and ceramide signalling in apopto-sisrdquo IUBMB Life vol 58 no 8 pp 462ndash466 2006

[32] M Darla Mark K Rajesh Kumar R Bakthavatchala and RSuresh ldquoDesigning synthesis and characterization of somenovel coumarin derivatives as probable anticancer drugsrdquoMedicinal Chemistry Research 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom