research article compounds from the fruits of the...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 432829 7 pageshttpdxdoiorg1011552013432829

Research ArticleCompounds from the Fruits of the Popular EuropeanMedicinal Plant Vitex agnus-castus in Chemoprevention viaNADP(H)Quinone Oxidoreductase Type 1 Induction

Shenghong Li1 Shengxiang Qiu2 Ping Yao3 Handong Sun1

Harry H S Fong4 and Hongjie Zhang5

1 State Key Laboratory of Phytochemistry and Plant Resources in West China Kunming Institute of BotanyThe Chinese Academy of Sciences Kunming Yunnan 650204 China

2 South China Botanical Garden Chinese Academy of Sciences 723 Xingke Road Tianhe District Guangzhou 510650 China3Division of Life Science The Hong Kong University of Science and Technology Clear Water Bay Road Hong Kong4Department of Medicinal Chemistry and Pharmacognosy College of Pharmacy University of Illinois at Chicago833 S Wood Street Chicago IL 60612 USA

5 School of Chinese Medicine Hong Kong Baptist University 7 Baptist University Road Kowloon Tong Hong Kong

Correspondence should be addressed to Hongjie Zhang zhanghjhkbueduhk

Received 30 January 2013 Accepted 5 March 2013

Academic Editor Ludger Beerhues

Copyright copy 2013 Shenghong Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants we haveisolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits of Vitex agnus-castus Theseisolates along with our previously obtained novel compound vitexlactam A (1) were evaluated for potential biological effectsincluding cancer chemoprevention Chemically the nitrogenous isolates were found to be two labdane diterpene alkaloids eachcontaining an 120572 120573-unsaturated 120574-lactammoiety Structurally they were elucidated to be 9120572-hydroxy-13(14)-labden-1615-amide (2)and 6120573-acetoxy-9120572-hydroxy-13(14)-labden-1516-amide (3) which were named vitexlactams B and C respectively The 15 knownisolates were identified as vitexilactone (4) rotundifuran (5) 8-epi-manoyl oxide (6) vitetrifolin D (7) spathulenol (8) cis-dihydro-dehydro-diconiferylalcohol-9-O-120573-D-glucoside (9) luteolin-7-O-glucoside (10) 5-hydroxy-36741015840-tetramethoxyflavone(11) casticin (12) artemetin (13) aucubin (14) agnuside (15) 120573-sitosterol (16) p-hydroxybenzoic acid (17) and p-hydroxybenzoicacid glucose ester (18) All compound structures were determinedidentified on the basis of 1D andor 2D NMR and massspectrometry techniques Compounds 6 8 9 and 18 were reported from a Vitex spieces for the first time The cancerchemopreventive potentials of these isolates were evaluated for NADP(H)quinone oxidoreductase type 1 (QR1) induction activityCompound 7 demonstrated promising QR1 induction effect while the new compound vitexlactam (3) was only slightly active

1 Introduction

Botanicals are widely used as either dietary supplements orherbal medicines throughout the world for the preventionand mitigation against various diseases or ailments Amongthese botanicals are plants of the genus Vitex plants Botan-ically this genus was previously placed in the family of Ver-benaceae but was recently revised as belonging to the familyLamiaceae which itself was formerly known as the LabiataeVitex consists of about 250 species distributed worldwide but

mainly in the tropical and temperate zones [1] A number ofspecies (eg V agnus-castus V trifolia V negundo and Vrotundifolia) have been used as traditional medicinal plantsTo date more than 20Vitex species have been investigated forchemical and biological properties with approximately 200compounds mainly flavonoids terpenoids steroids iridoidsand lignans having been isolated and characterized [2]

Vitex agnus-castus Linn is commonly known as thechaste tree grows to a height of 2-3m and is distributedin the Mediterranean Region Central Asia and Southern

2 Evidence-Based Complementary and Alternative Medicine

Europe [3] It is also cultivated in the various regionsincluding the United States [4] The fruits of V agnus-castusare popularly used as a phytomedicine in Europe for thetreatment of female hormonal disorders [5ndash7] The fruitextract is also used as an alternative phytotherapeutic agentin the treatment of mastalgia [8] There has been extensiveresearch conducted on this phytomedicine leading to a largelibrary of published literature on the pharmacognosy tra-ditional uses chemical constituents biologypharmacologyand clinical studies [9] In a previous communication wereported the isolation structure determination and X-raycrystallographic analysis of a novel labdane diterpene lactamfrom the n-hexane extracts of the fruits of this plant [10]Further phytochemical studies of both of the n-hexane andmethanol extracts resulted in the isolation of two additionalnew labdane diterpene lactams (2-3) and fifteen knowncompounds (4ndash18) In this paper we describe the isolationand structure characterization of the two new metabolitesand the identification of the 15 known compounds as wellas evaluating their NADP(H)quinone oxidoreductase type 1(QR1) induction activity potentials

2 Materials and Methods

21 General Experimental Procedures All melting pointswere measured on an XRC-1 micromelting point apparatusand are uncorrected 1D (one-dimensional) and 2D (two-dimensional) NMR (nuclear magnetic resonance) experi-ments were performed either on a Bruker AM-400 or aBruker DRX-500 spectrometer Unless otherwise is specifiedchemical shifts (120575) were expressed in ppm with reference tothe solvent signals FABMS (fast atom bombardment massspectrometry) and HRFABMS (high resolution fast atombombardment mass spectrometry) were taken on a VG AutoSpec-3000 or a Finnigan MAT 90 instrument IR (infrared)spectra were recorded on a Bio-Rad FTS-135 spectrometerwith KBr pellets UV (ultraviolet) spectral data were obtainedon a UV 210A spectrometer Optical rotations were carriedout on a HORIBA SEPA-300 High Sensitive Polarimeteror a Perkin-Elmer model 241 Polarimeter Column chro-matography was performed either on Si gel (silica gel) (200ndash300 mesh Qingdao Marine Chemical Inc China) Si gelH (10ndash40 120583 Qingdao Marine Chemical Inc China) DiaionHP-20 (Shandong Lukang Pharmaceutical Co Ltd China)Chromatorex ODS (Fuji Silysia Chemical Corporation LtdJapan) or LichroprepRp

18gel (40ndash63 120583mMerckDarmstadt

Germany) Fractions were monitored by silica gel TLC(thin layer chromatography) [CHCl

3-Me2CO (chloroform-

acetone) 9 1 8 2 7 3] and spots were visualized by heat-ing silica gel plates sprayed with 10 H

2SO4in EtOH

(ethanol)

22 Plant Material The fruits of V agnus-castus were pur-chased from Frontier Botanicals Norway IA USA (Lot No799 0116)

23 Extraction and Isolation Dried fruits of V agnus-castus(4077 g) were milled and sequentially extracted with n-hexane (3 times 8 L) for 28 h and MeOH (methanol) (4 times 9 L)

for 24 h The n-hexane extract was filtered and concentratedin vacuo to dryness to afford 200 g of a residue (part I)The MeOH extract was filtered concentrated and dilutedwith water (2 L) followed by partitioning with EtOAc (ethylacetate) (4times3 L)The organic layer was evaporated in vacuo todryness to give 60 g of a residue (part II) The water-solublefraction was chromatographed on a column of Diaion HP-20 eluting with aqueous MeOH (30 rarr 80 rarr 100) The80MeOH-H

2O fraction was concentrated in vacuo to yield

48 g of a dry residue (part III)

231 Isolation Part I (200 g) was absorbed on 200 g ofsilica gel and chromatographed on a prepacked (500 g)silica gel column eluting stepwise with n-hexane CHCl

3

CHCl3-Me2CO1 1 and Me

2CO Compound 16 (27mg)

was crystallized from the CHCl3fraction and compound

11 (336mg) was crystallized from the CHCl3-Me2CO1 1

fraction The remaining CHCl3-Me2CO1 1 eluate was fil-

tered (40 g net weight) and subjected to further chromato-graphic separation over a Chromatorex ODS column (eluent80 MeOH-H

2O as eluents) and silica gel columns (using

n-hexane-CHCl31 2 n-hexane-EtOAc3 2 and n-hexane-

Me2CO2 1 as eluents) to provide compounds 1 (40mg) 2

(4mg) 3 (11mg) 4 (25mg) 5 (67mg) 6 (6mg) 7 (14mg) 8(14mg) and 13 (9mg)

Part II (60 g) was absorbed on 100 g of silica gel andchromatographed on a prepacked (300 g) silica gel col-umn eluting with CHCl

3-Me2CO (1 0 9 1 8 2 7 3 0 1)

Compound 12 (1635 g) was crystallized from the CHCl3-

Me2CO1 0-9 1 fraction Part of the CHCl

3-Me2CO8 2

fraction (0810 g) was further chromatographed on RP18

gel(100 g)with 40aqueousMeOHas eluents to give compound17 (125mg)

Part III (48 g) was again chromatographed on a Chroma-torex ODS column eluting with aqueous MeOH (30) andover a silica gel column eluting with CHCl

3-MeOH (3 1)

CHCl3-MeOH-H

2O (4 1 01) and EtOAc-MeOH (12 1) to

yield compounds 9 (108mg) 10 (23mg) 14 (55mg) 15(60mg) and 18 (15mg)

24 Structural Characterization of Novel Isolates

241 Vitexlactam B (2) White crystals mp 162∘CC20H33NO2 [120572]235D + 1875∘ (c 02 CHCl

3) IR (KBr) ]max

3473 3187 3055 2924 2682 1684 1648 1442 1379 12961254 1228 1140 1085 1057 1041 1018 972 962 943 909832 791 777 698 cmminus1 1H NMR (500MHz CDCl

3) 120575 150

(1H dd J = 110 20Hz H-5) 175 (1H m H-8) 178 (1Hm H-11a) 167 (1H m H-11b) 236 (2H br t J = 82HzH2-12) 669 (1H br s H-14) 389 (2H br s H

2-15) 088 (3H

d J = 66Hz H3-17) 085 (3H s H

3-18) 080 (3H s H

3-19)

090 (3H s H3-20) 661 (1H br s NH) 13C NMR data see

Table 1 EIMS (electron impact mass spectrum)mz 319 [M]+(81) 304 (7) 286 (8) 206 (7) 194 (19) 180 (100) 167 (75) 152(11) 138 (47) 123 (17) 110 (81) 96 (86) 82 (58) 69 (72) 55(97) HREIMS mz found 3192509 [M]+ calcd (calculated)3192511

Evidence-Based Complementary and Alternative Medicine 3

Table 1 13C NMR data of compounds 1ndash7 (CDCl3 120575 in ppm)

Carbon 1a 2b 3a 4a 5a 6a 7b

C-1 337 t 325 t 337 t 338 t 339 t 365 t 259 tC-2 188 t 187 t 186 t 189 t 187 t 207 t 194 tC-3 438 t 417 t 436 t 439 t 437 t 423 t 394 tC-4 339 s 333 s 340 s 343 s 348 s 329 s 346 sC-5 475 d 462 d 476 d 480 d 475 d 463 d 1325 sC-6 706 d 217 t 699 d 701 d 703 d 210 t 662 dC-7 363 t 314 t 361 t 364 t 361 t 379 t 727 dC-8 321 d 368 d 319 d 323 d 336 d 741 s 364 dC-9 764 s 768 s 767 s 768 s 768 s 612 d 429 sC-10 440 s 433 s 438 s 441 s 437 s 389 s 1415 sC-11 323 t 320 t 323 t 319 t 318 t 186 t 293 tC-12 217 t 220 t 265 t 257 t 215 t 451 t 386 tC-13 1406 s 1408 s 1636 s 1713 s 1255 s 736 s 730 sC-14 1371 d 1369 d 1212 d 1153 d 1108 d 1461 d 1445 dC-15 466 t 464 t 1753 s 1713 s 1429 d 1111 t 1121 tC-16 1753 s 1758 s 505 t 734 t 1385 d 274 q 278 qC-17 164 q 166 q 160 q 163 q 161 q 320 q 111 qC-18 336 q 337 q 336 q 338 q 336 q 331 q 293 qC-19 237 q 221 q 236 q 239 q 237 q 213 q 281 qC-20 189 q 162 q 190 q 192 q 190 q 247 q 280 qOAc 1705 s 1703 s 1706 s 1707 s 1708(C=O) (2C s)OAc 219 q 218 q 221 q 219 q 214 q(CH3) 209 qaRecorded at 100MHzbRecorded at 125MHz

242 Vitexlactam C (3) White crystals mp 178∘CC22H35NO4 [120572]187D minus 1273

∘ (c 055 CHCl3) IR (KBr) ]max

3364 3297 2925 2867 1711 1670 1465 1426 1383 1362 12711256 1228 1203 1152 1125 1097 1039 1024 977 953 916 849819 cmminus1 1H NMR (400MHz CDCl

3) 120575 131 (1H br d J =

132Hz H-3a) 113 (1H dt J = 27 132Hz H-3b) 158 (1H dJ = 20Hz H-5) 535 (1H br d J = 22Hz H-6) 210 (1H mH-8) 190 (1H m H-11a) 172 (1H m H-11b) 243 (2H mH2-12) 582 (1H br s H-14) 391 (2H br s H

2-16) 087 (3H

d J = 67Hz H3-17) 093 (3H s H

3-18) 097 (3H s H

3-19)

122 (3H s H3-20) 692 (1H br s NH) 203 (3H s 6-OAc)

13C NMR data see Table 1 EIMS mz 377 [M]+ (3) 317 (76)302 (15) 284 (6) 260 (29) 242 (8) 222 (21) 202 (23) 187(48) 167 (60) 150 (28) 133 (41) 119 (64) 110 (68) 96 (97) 83(72) 69 (77) 55 (100) HREIMS mz found 3772547 [M]+calcd 3772566

25 Chemoprevention Evaluation NAD(P)HQuinone Oxi-doreductase Type 1 (QR1) Assay Test compounds wereevaluated for their potential to induce quinone reductasetype 1 (QR1) activity with Hepa 1c1c7 cells The cells wereplated in 96-well plates at a density of 2 times 104 cellsmL in190120583L of 120572-MEM (minimum essential medium) containing100 unitsmL penicillin G sodium 100120583gmL streptomycinsulfate and 250 ngmL amphotericin B supplemented with10 fetal bovine serum at 37∘C in a 5 CO

2atmosphere

After preincubation for 24 h the medium was changed andtest compounds were added to afford a final concentrationrange of 2 to 20120583gmL and then the cells were incubatedfor an additional 48 h The medium was decanted andthe cells were incubated with 50120583L of 08 digitonin and2mM EDTA (ethylenediaminetetraacetic acid) solution (pH78) at 37∘C for 10min Quinone reductase activity wasdetermined by measuring the NAD(P)H-dependent mena-diol mediated reduction of MTT [3-(4 5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide] to a blue formazanCytotoxicity was determined by crystal violet staining assayInduction of QR activity was calculated by comparing theQR specific activity of agent-treated cells with that of vehiclesolvent-treated cells 41015840-Bromoflavone with a CD value of129 nM was used as a positive control CD represents theconcentration of a test compound required to double QRinduction in comparison with the vehicle control

26 Supporting Information Available NMR and MS dataof the known compounds are available as SupplementaryMaterial online at httpdxdoiorg1011552013432829

3 Results and Discussion

31 Plant Extracts and Isolation of Compounds The pur-chased fruits of V agnus-castus were milled and sequentially


15

14

HN

O

16

13

(a)

15

14

HN

16

13O

(b)

Figure 1 Electronic clouds movements of two different conjugated systems in compounds 3 (a) and 1 (b)

extracted with n-hexane and methanolThe n-hexane extractwas successively chromatographed on silica gel and Chro-matorex ODS to afford compounds 1ndash8 11 13 and 16 Themethanol extract was partitioned between EtOAc and waterThe EtOAc layer was chromatographed on silica gel to givecompounds 12 and 17 The water-soluble fraction was chro-matographed on columns of Diaion HP-20 ChromatorexODS and silica gel to yield compounds 9 10 14 15 and 18(Scheme 1)

32 Structure Elucidation and Identification ofIsolated Compounds

321 Vitexlactam B (2) Vitexlactam B (2) was obtained aswhite crystals EI mass spectrum showed strong molecularion peak at mz 319 [M]+ (81 relative intensity) corre-sponding to a molecular formula of C

20H33NO2 which was

confirmed by high resolution EI mass spectrum (found mz3192509 calcd 3192511) The existence of a nitrogen atomwas supported by its odd numbered molecular weight and apositive reaction to the Dragendorff reagent

The 1H and 13C NMR (Table 1) spectra of 2 being verysimilar to those of 1 [10] suggested that 2 is a closelyrelated labdane diterpene alkaloid (Table 1) with an 120572 120573-unsaturated 120574-lactam moiety at the C-9 side chain 2 differedfrom 1 only by the absence of the signals for an acetylgroup and the replacement of an oxygen-bearing methineat 120575C 706 by a methylene signal at 120575C 217 indicating that2 is the 6-deacetoxy derivative of 1 The result was furthersupported by the facts that 1 was 58 atomic mass units lessthan 2 and the lack of an acetoxy group being observedin the IR spectrum of 2 Full assignments of 2 using 2DNMR (including 1H-1H COSY (correlation spectroscopy)HMQC (heteronuclear multiple-quantum correlation spec-troscopy) HMBC (heteronuclear multiple bond correla-tion spectroscopy) and ROESY (rotating-frame Overhauserspectroscopy)) techniques established the structure of 2to be the expected 9120572-hydroxy-13(14)-labden-1615-amideCompound 2 was accordingly identified as the deacetoxyderivative of 1 andwas given the trivial nameof vitexlactamB

322 Vitexlactam C (3) Vitexlactam C (3) was also isolatedas white crystals EI mass spectrum under 70 eV displayeda weak [M]+ ion peak at mz 377 (3) identical with thatof 1 in both the mass charge ratio and the relative intensity[11] In addition a strong fragment ion peak atmz 317 (76)

due to [M-AcOH]+ and a series of fragment ions similarto those for 1 were also observed High resolution EI massspectrum (found mz 3772547 calcd 3772566) establishedthat both compounds have the same molecular formula ofC22H35NO4 Therefore 3 was tentatively identified as an

isomer of 1 Comparison of the 1H and 13C NMR (Table 1)spectra of 3 with those of 1 (Table 1) indicated that the twocompounds were equivalent not only in their skeletons butalso in their oxygenation patterns NMR spectral differencesbetween these two compounds are mainly due to the 120572 120573-unsaturated 120574-lactam moieties in their C-9 side chains Theconjugate functionality occurred in 3 was deduced to betype (a) in contrast to type (b) in 1 (Figure 1) In the formerconjugating system C-13 is in a deshielded position while C-14 and H-14 are in a shielded position On the contrary inthe latter (type (b)) C-13 is in a shielded position while C-14and H-14 are in a deshielded position Accordingly C-13 of 3moved downfield from 120575C 1406 (s) in 1 to 120575C 1636 (s) andC-14H-14 of 3 shifted upfield from 120575CH 1371 (d)671 (1Hbr s) in 1 to 120575CH 1212 (d)582 (1H br s) 2D NMR analysisof 3 revealed that unlike in 1 the 1H-1H COSY correlationbetween H-14 and the nitrogen-bearing methylene at 120575H 391(2H br s) and the 1H-13C interaction (Figure 2) between H

2-

12 [120575H 244 (2H m)] and the lactam carbonyl carbon at 120575C1753 (s) disappeared while 1H-13C interaction between H

2-

12 and the nitrogen-occurring methylene at 120575C 505 (t) wereobserved thus confirming the presence of a type (a) conjugatefunctionality in 3 Other structural correlations includingkey NOEs (nuclear Overhauser effects) (Figure 3) in 3 wereidentical with those in 1

A detailed spectral comparison between 3 and vitexilac-tone (4) [11] was also carried out The molecular weight of3 is lower by 1 mass unit than that of 4 Besides 3 differedfrom 4 (Table 1) mainly by the upfield shifted H

2-16 and C-

16 signals (from 120575HC 477 (2H br d J = 13Hz)734 (t) in 4to 120575HC 394 (2H br s)505 (t) in 3) and the existence of anextra NH proton at 120575H 692 (1H br s) indicating that an 120572120573-unsaturated 120574-lactam moiety in 3 took the place of the 120572120573-unsaturated 120574-lactone in 4 Based on all the abovedescribedspectral features compound 3 was consequently deducedto be 6120573-acetoxy-9120572-hydroxy-13(14)-labden-1516-amide andwas named vitexlactam C

Considering that only mild conditions were employedand that no nitrogen containing solvents and chromato-graphic materials were involved in the entire extraction andseparation procedures we postulate that compounds 1ndash3


O

H

OH

H

O

O

O

R

O

O

H

H

O

15

14

13

16

121120

123 4

56 7

8910

19 18

OH

HR

HN

O

17

12

R = OAcR = H

3

OAcH

OH

O HN15

1413

16

OAcH

OH

OO

4

OH

OAc

OH

OAc

OAc

5 6 7

OH

OH

OH OH

10

GlcO CH3

O

R = HR = OHR = OCH

3

CH3

O

OCH3

OCH3

111213

HO

HO

OR

OGlc

R = H1415

1718R = R = Glc

R = Hp-hydroxybenzoyl

RO

Scheme 1


OH

HN

O

O

OH

Figure 2 Key HMBC correlations of vitexlactam C (3)

O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3

Figure 3 Key NOESY correlations of vitexlactam C (3)

are biogenetic amination products of their correspondinglactones (eg 3 was derived from 4)

323 Identification of Known Compounds Along with thenew compounds fifteen known compounds were also iso-lated in the course of the current study Through comparisonof their 1H and 13C NMR and MS data with those valuesreported in the literature they were identified as threelabdane-type diterpenoids vitexilactone (4) [11] rotundifu-ran (5) [11] and 8-epi-manoyl oxide (6) [12] ([120572]195D minus 118

∘ c=055 CHCl

3) a rearranged labdane (halimane) diterpenoid

vitetrifolin D (7) [13] an aromadendrene-type sesquiter-penoid spathulenol (8) [14 15] a lignan glucoside cis-dihydro-dehydro-diconiferylalcohol-9-O-120573-D-glucoside (9)[16] four flavonoids luteolin-7-O-glucoside (10) [17] 5-hydroxy-36741015840-tetramethoxyflavone (11) [18] casticin (12)[19] and artemetin (13) [20] two iridoid glycosides aucu-bin (14) [21] and agnuside (15) [22] a sterol 120573-sitosterol(16) (comparison with an authentic sample) and twosimple phenolics p-hydroxybenzoic acid (17) [22] and p-hydroxybenzoic acid glucose ester (18) [22] The occurrenceof compounds 7ndash9 and 18 in the genusVitex is being reportedfor the first time

33 Activity Evaluation of the Isolated Compounds on QR1Induction These compounds have been evaluated for theirpotential chemopreventive activity by induction of theubiquitous flavoenzyme NADP(H)quinone oxidoreductase

type 1 (QR1) with cultured Hepa 1c1c7 cells QR1 has beendetermined as an important phase II detoxification enzymethat can protect cells against the harmful effects causedby free radicals and reactive oxygen species by catalyzingthe reduction of quinones to hydroquinones [23] Henceenhanced activity of the enzyme provides protection of cellsfrom potential carcinogenicity Vitetrifolin D (7) was shownto induce QR1 activity with a CD value of 232 120583M Althoughvitexlactam C (3) induced QR1 by 15 times that of the vehiclecontrol at a concentration of 53120583M it was toxic toHepa 1c1c7cells with 57 inhibition of the cells at 265 120583M None of theother compounds demonstrated QR1 induction activity

4 Conclusion

The fruits of Vitex agnus-castus have been popularly usedas a phytomedicine in Europe especially Germany for thetreatment of premenstrual stress syndrome However theevaluation of this herb or its phytochemical constituents forcancer chemoprevention activity has not been reportedThuswe undertook a study of the 18 compounds we isolated fromthe fruits of this plant in a bioassay which have been used forassessing chemoprevention potentialsThe isolates includingseveral novel nitrogen containing labdane diterpenes werethus evaluated for their potentials in the induction of thephase II detoxification enzymeQR1 Results showed that onlythe labdane compounds 3 and 7 demonstratedQR1 inductioneffect We have demonstrated that compounds possessingpotential chemopreventive action do exist in V agnus-castusand that further phytochemical and biological investigationsof this plant material coupled with structure modificationstudies are needed in order to discover additionalmodifiedlabdanes possessing more potent QR1 induction activity andchemopreventive potential

Conflict of Interests

The authors have no conflict of interests with the trademarksincluded in the paper

Acknowledgments

The authors thank the members of the analytical group ofthe State Key Laboratory of Phytochemistry Laboratory andPlant Resources inWest China Kunming Institute of BotanyAcademic Sinica formeasurements of the NMRMS IR UVand ORD spectral data This work was in part supported bythe ldquoHundred Talents Programrdquo of the Chinese Academy ofSciences (awarded to Dr S Li)

References

[1] ldquoDelectis Florae Reipublicae Popularis Sinicae Agendae Acade-miae Sinicae Editardquo in Flora Republicae Popularis Sinicae vol65 pp 131ndash150 Science Press Beijing 1982

[2] S H Li Studies on the chemical and biological constituents ofthree Taxaceae plants Vitex agnus-castus and Isodon xerophilus[PhD thesis] Kunming Institute of Botany the ChineseAcademy of Sciences Kunming Yunnan China 2001


[3] T G Tutin V H Heywood N A Burges D H Valentine SM Walters and D A Webb Flora Europaea vol 3 CambridgeUniversity Press Cambridge UK 1972

[4] United States Department of Agriculture ldquoVitex agnus-castusLrdquo 2013 httpplantsusdagovjavaprofilesymbol=viag

[5] S Y Mills Out of the Earth The Essential Book of HerbalMedicine Viking Arkana London UK 1991

[6] S Y Mills Woman Medicine Vitex Agnus-Castus the HerbAmberwood Christchurch UK 1992

[7] K P Odenthal ldquoVitex agnus castus Lmdashtraditional drug andactual indicationsrdquo Phytotherapy Research vol 12 no 1 supple-ment pp S160ndashS161 1998

[8] A R Carmichael ldquoCanVitex Agnus castus be used for the treat-ment of mastalgia What is the current evidencerdquo Evidence-Based Complementary and Alternative Medicine vol 5 no 3pp 247ndash250 2008

[9] G S Chhabra and K S Kulkarni ldquoVitex agnus castusmdashanoverviewrdquo Journal of Natural Remedies vol 11 no 2 pp 90ndash972011

[10] S H Li H J Zhang S X Qiu et al ldquoVitexlactam A a novellabdane diterpene lactam from the fruits of Vitex agnus-castusrdquoTetrahedron Letters vol 43 no 29 pp 5131ndash5134 2002

[11] H Taguchi ldquoStudies on the constituents of Vitex cannabifoliardquoChemical and Pharmaceutical Bulletin vol 24 no 7 pp 1668ndash1670 1976

[12] Y S Cheng and E von Rudloff ldquoTwo new diterpenoid oxidesfrom the leaf oil of Chamaecyparis nootkatensisrdquo TetrahedronLetters vol 11 no 14 pp 1131ndash1132 1970

[13] M Ono Y Ito and T Nohara ldquoFour new halimane-typediterpenes vitetrifolins D-G from the fruit of Vitex trifoliardquoChemical and Pharmaceutical Bulletin vol 49 no 9 pp 1220ndash1222 2001

[14] R C Bowyer and P R Jefferies ldquoStructure of spathulenolrdquoChemistry amp Industry no 30 pp 1245ndash1246 1963

[15] A Ulubelen G Topcu C Eris et al ldquoTerpenoids from Salviasclareardquo Phytochemistry vol 36 no 4 pp 971ndash974 1994

[16] T Satake T Murakami Y Saiki and C M Chen ldquoChemicaland chemotaxonomic studies of genus Pteris and related genera(Pteridaceae)mdash19 Chemical studies of components of Petris-vittata Lrdquo Chemical amp Pharmaceutical Bulletin vol 26 no 5pp 1619ndash1622 1978

[17] K R Markham B Ternai R Stanley H Geiger and T JMabry ldquoCarbon-13 NMR studies of flavonoids-III Naturallyoccurring flavonoid glycosides and their acylated derivativesrdquoTetrahedron vol 34 no 9 pp 1389ndash1397 1978

[18] K Sachdev andDKKulshreshtha ldquoFlavonoids fromDodonaeaviscosardquo Phytochemistry vol 22 no 5 pp 1253ndash1256 1983

[19] E Wollenweber and K Mann ldquoFlavonols from fruits of Vitexagnus castusrdquo Planta Medica vol 48 no 2 pp 126ndash127 1983

[20] Y Kondo K Sugiyama and S Nozoe ldquoStudies on the con-stituents of Vitex rotundifolia L filrdquo Chemical and Pharmaceu-tical Bulletin vol 34 no 11 pp 4829ndash4832 1986

[21] K Gorler D Oehlke and H Soicke ldquoIridoidglycoside in Vitexagnus-castusrdquo Planta Medica vol 6 pp 530ndash531 1985

[22] M Tabata Y Umetani M Ooya and S Tanaka ldquoGlucosylationof phenolic compounds by plant cell culturesrdquo Phytochemistryvol 27 no 3 pp 809ndash813 1988

[23] R Li M A Bianchet P Talalay and L M Amzel ldquoThe three-dimensional structure of NAD(P)Hquinone reductase a flavo-protein involved in cancer chemoprotection and chemotherapy

mechanism of the two-electron reductionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 92 no 18 pp 8846ndash8850 1995

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Europe [3] It is also cultivated in the various regionsincluding the United States [4] The fruits of V agnus-castusare popularly used as a phytomedicine in Europe for thetreatment of female hormonal disorders [5ndash7] The fruitextract is also used as an alternative phytotherapeutic agentin the treatment of mastalgia [8] There has been extensiveresearch conducted on this phytomedicine leading to a largelibrary of published literature on the pharmacognosy tra-ditional uses chemical constituents biologypharmacologyand clinical studies [9] In a previous communication wereported the isolation structure determination and X-raycrystallographic analysis of a novel labdane diterpene lactamfrom the n-hexane extracts of the fruits of this plant [10]Further phytochemical studies of both of the n-hexane andmethanol extracts resulted in the isolation of two additionalnew labdane diterpene lactams (2-3) and fifteen knowncompounds (4ndash18) In this paper we describe the isolationand structure characterization of the two new metabolitesand the identification of the 15 known compounds as wellas evaluating their NADP(H)quinone oxidoreductase type 1(QR1) induction activity potentials

2 Materials and Methods

21 General Experimental Procedures All melting pointswere measured on an XRC-1 micromelting point apparatusand are uncorrected 1D (one-dimensional) and 2D (two-dimensional) NMR (nuclear magnetic resonance) experi-ments were performed either on a Bruker AM-400 or aBruker DRX-500 spectrometer Unless otherwise is specifiedchemical shifts (120575) were expressed in ppm with reference tothe solvent signals FABMS (fast atom bombardment massspectrometry) and HRFABMS (high resolution fast atombombardment mass spectrometry) were taken on a VG AutoSpec-3000 or a Finnigan MAT 90 instrument IR (infrared)spectra were recorded on a Bio-Rad FTS-135 spectrometerwith KBr pellets UV (ultraviolet) spectral data were obtainedon a UV 210A spectrometer Optical rotations were carriedout on a HORIBA SEPA-300 High Sensitive Polarimeteror a Perkin-Elmer model 241 Polarimeter Column chro-matography was performed either on Si gel (silica gel) (200ndash300 mesh Qingdao Marine Chemical Inc China) Si gelH (10ndash40 120583 Qingdao Marine Chemical Inc China) DiaionHP-20 (Shandong Lukang Pharmaceutical Co Ltd China)Chromatorex ODS (Fuji Silysia Chemical Corporation LtdJapan) or LichroprepRp

18gel (40ndash63 120583mMerckDarmstadt

Germany) Fractions were monitored by silica gel TLC(thin layer chromatography) [CHCl

3-Me2CO (chloroform-

acetone) 9 1 8 2 7 3] and spots were visualized by heat-ing silica gel plates sprayed with 10 H

2SO4in EtOH

(ethanol)

22 Plant Material The fruits of V agnus-castus were pur-chased from Frontier Botanicals Norway IA USA (Lot No799 0116)

23 Extraction and Isolation Dried fruits of V agnus-castus(4077 g) were milled and sequentially extracted with n-hexane (3 times 8 L) for 28 h and MeOH (methanol) (4 times 9 L)

for 24 h The n-hexane extract was filtered and concentratedin vacuo to dryness to afford 200 g of a residue (part I)The MeOH extract was filtered concentrated and dilutedwith water (2 L) followed by partitioning with EtOAc (ethylacetate) (4times3 L)The organic layer was evaporated in vacuo todryness to give 60 g of a residue (part II) The water-solublefraction was chromatographed on a column of Diaion HP-20 eluting with aqueous MeOH (30 rarr 80 rarr 100) The80MeOH-H

2O fraction was concentrated in vacuo to yield

48 g of a dry residue (part III)

231 Isolation Part I (200 g) was absorbed on 200 g ofsilica gel and chromatographed on a prepacked (500 g)silica gel column eluting stepwise with n-hexane CHCl

3

CHCl3-Me2CO1 1 and Me

2CO Compound 16 (27mg)

was crystallized from the CHCl3fraction and compound

11 (336mg) was crystallized from the CHCl3-Me2CO1 1

fraction The remaining CHCl3-Me2CO1 1 eluate was fil-

tered (40 g net weight) and subjected to further chromato-graphic separation over a Chromatorex ODS column (eluent80 MeOH-H

2O as eluents) and silica gel columns (using

n-hexane-CHCl31 2 n-hexane-EtOAc3 2 and n-hexane-

Me2CO2 1 as eluents) to provide compounds 1 (40mg) 2

(4mg) 3 (11mg) 4 (25mg) 5 (67mg) 6 (6mg) 7 (14mg) 8(14mg) and 13 (9mg)

Part II (60 g) was absorbed on 100 g of silica gel andchromatographed on a prepacked (300 g) silica gel col-umn eluting with CHCl

3-Me2CO (1 0 9 1 8 2 7 3 0 1)

Compound 12 (1635 g) was crystallized from the CHCl3-

Me2CO1 0-9 1 fraction Part of the CHCl

3-Me2CO8 2

fraction (0810 g) was further chromatographed on RP18

gel(100 g)with 40aqueousMeOHas eluents to give compound17 (125mg)

Part III (48 g) was again chromatographed on a Chroma-torex ODS column eluting with aqueous MeOH (30) andover a silica gel column eluting with CHCl

3-MeOH (3 1)

CHCl3-MeOH-H

2O (4 1 01) and EtOAc-MeOH (12 1) to

yield compounds 9 (108mg) 10 (23mg) 14 (55mg) 15(60mg) and 18 (15mg)

24 Structural Characterization of Novel Isolates

241 Vitexlactam B (2) White crystals mp 162∘CC20H33NO2 [120572]235D + 1875∘ (c 02 CHCl

3) IR (KBr) ]max

3473 3187 3055 2924 2682 1684 1648 1442 1379 12961254 1228 1140 1085 1057 1041 1018 972 962 943 909832 791 777 698 cmminus1 1H NMR (500MHz CDCl

3) 120575 150

(1H dd J = 110 20Hz H-5) 175 (1H m H-8) 178 (1Hm H-11a) 167 (1H m H-11b) 236 (2H br t J = 82HzH2-12) 669 (1H br s H-14) 389 (2H br s H

2-15) 088 (3H

d J = 66Hz H3-17) 085 (3H s H

3-18) 080 (3H s H

3-19)

090 (3H s H3-20) 661 (1H br s NH) 13C NMR data see

Table 1 EIMS (electron impact mass spectrum)mz 319 [M]+(81) 304 (7) 286 (8) 206 (7) 194 (19) 180 (100) 167 (75) 152(11) 138 (47) 123 (17) 110 (81) 96 (86) 82 (58) 69 (72) 55(97) HREIMS mz found 3192509 [M]+ calcd (calculated)3192511








3) 120575 131 (1H br d J =


2-16) 087 (3H

d J = 67Hz H3-17) 093 (3H s H

3-18) 097 (3H s H

3-19)

122 (3H s H3-20) 692 (1H br s NH) 203 (3H s 6-OAc)



2atmosphere






15

14

HN

O

16

13

(a)

15

14

HN

16

13O

(b)





20H33NO2 which was






2-


2-



2-16 and C-




O

H

OH

H

O

O

O

R

O

O

H

H

O

15

14

13

16

121120

123 4

56 7

8910

19 18

OH

HR

HN

O

17

12

R = OAcR = H

3

OAcH

OH

O HN15

1413

16

OAcH

OH

OO

4

OH

OAc

OH

OAc

OAc

5 6 7

OH

OH

OH OH

10

GlcO CH3

O

R = HR = OHR = OCH

3

CH3

O

OCH3

OCH3

111213

HO

HO

OR

OGlc

R = H1415

1718R = R = Glc


RO

Scheme 1


OH

HN

O

O

OH


O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3




∘ c=055 CHCl





4 Conclusion




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3) 120575 131 (1H br d J =


2-16) 087 (3H

d J = 67Hz H3-17) 093 (3H s H

3-18) 097 (3H s H

3-19)

122 (3H s H3-20) 692 (1H br s NH) 203 (3H s 6-OAc)



2atmosphere






15

14

HN

O

16

13

(a)

15

14

HN

16

13O

(b)





20H33NO2 which was






2-


2-



2-16 and C-




O

H

OH

H

O

O

O

R

O

O

H

H

O

15

14

13

16

121120

123 4

56 7

8910

19 18

OH

HR

HN

O

17

12

R = OAcR = H

3

OAcH

OH

O HN15

1413

16

OAcH

OH

OO

4

OH

OAc

OH

OAc

OAc

5 6 7

OH

OH

OH OH

10

GlcO CH3

O

R = HR = OHR = OCH

3

CH3

O

OCH3

OCH3

111213

HO

HO

OR

OGlc

R = H1415

1718R = R = Glc


RO

Scheme 1


OH

HN

O

O

OH


O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3




∘ c=055 CHCl





4 Conclusion




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















15

14

HN

O

16

13

(a)

15

14

HN

16

13O

(b)





20H33NO2 which was






2-


2-



2-16 and C-




O

H

OH

H

O

O

O

R

O

O

H

H

O

15

14

13

16

121120

123 4

56 7

8910

19 18

OH

HR

HN

O

17

12

R = OAcR = H

3

OAcH

OH

O HN15

1413

16

OAcH

OH

OO

4

OH

OAc

OH

OAc

OAc

5 6 7

OH

OH

OH OH

10

GlcO CH3

O

R = HR = OHR = OCH

3

CH3

O

OCH3

OCH3

111213

HO

HO

OR

OGlc

R = H1415

1718R = R = Glc


RO

Scheme 1


OH

HN

O

O

OH


O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3




∘ c=055 CHCl





4 Conclusion




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

H

OH

H

O

O

O

R

O

O

H

H

O

15

14

13

16

121120

123 4

56 7

8910

19 18

OH

HR

HN

O

17

12

R = OAcR = H

3

OAcH

OH

O HN15

1413

16

OAcH

OH

OO

4

OH

OAc

OH

OAc

OAc

5 6 7

OH

OH

OH OH

10

GlcO CH3

O

R = HR = OHR = OCH

3

CH3

O

OCH3

OCH3

111213

HO

HO

OR

OGlc

R = H1415

1718R = R = Glc


RO

Scheme 1


OH

HN

O

O

OH


O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3




∘ c=055 CHCl





4 Conclusion




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OH

HN

O

O

OH


O

HH

H

HH

CH3

OAc

OHH3

C

NHCH3

CH3




∘ c=055 CHCl





4 Conclusion




Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article compounds from the fruits of the...

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