Diterpenes from the berries of Juniperus excelsa

\ PERGAMON

Phytochemistry 49 "0888# 0084Ð0088

Diterpenes from the berries of Juniperus excelsa Gulacžti Topcžua\\ Ramazan Erenlerb\ Osman C ž akmakb\ Candan Bozok Johanssonc\ c Cennet C ž elik \ Hee!Byung Chaid\ John M[ Pezzutod a

Department of Chemistry\ Marmara Research Centre\ TUBITAK\ P[O[ Box 10\ 30369 Gebze\ Kocaeli\ Turkey b Department of Chemistry\ Faculty of Art + Science\ Gaziosmanpas ža University\ 59099 Tokat\ Turkey c Department of Microbiology\ Faculty of Medicine\ University of Marmara\ Haydarpas ža\ Istanbul\ Turkey d Program for Collaborative Research in the Pharmaceutical Sciences\ College of Pharmacy\ University of IIIinois at Chicago\ Chicago\ IL 59501\ USA Received in revised form 5 October 0887

Abstract From the hexane extract of berries of Juniperus excelsa\ one new and four known diterpenes were isolated besides a known sesquiterpene[ The structures of the known diterpenes were identi_ed as isopimaric\ isocommunic\ "−#ent!trans communic and sandracopimaric acids\ along with the sesquiterpene 3a!hydroxycedrol and the new compound which was elucidated as 2a!ace! toxylabda!7"06#\02"05#\03!trien!08!oic acid "juniperexcelsic acid#[ Cytotoxic activity of the hexane extract was investigated against a panel of cell line and found highyl active against LNCaP\ KB!V "¦VLB# and KB!V "−VLB# cell lines[ Furthermore\ the hexane and methanol extracts\ and the new compound were found to be moderately active against Mycobacterium tuberculosis[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Juniperus excelsa^ Cupressaceae^ Berries^ Diterpenes^ Juniperexcelsic acid^ Sesquiterpene^ Cytotoxic^ Antituberculosis and antibacterial activity

0[ Introduction Although 69 Juniperus species grow throughout the world "Krussmann\ 0859#\ namely in the Balkan Penin! sula\ Saudi Arabia\ Yemen\ Oman\ Iran\ Afghanistan and Crimea "Kerfoot + Lavranos\ 0873#\ only eight spec! ies are found in Turkey "Coode + Cullen\ 0854^ Baytop  zocak\ 0869#[ Juniperus excelsa is widely distributed +O in the Anatolian mountains[ This plant is used locally as a traditional remedy for tuberculosis and jaundice in Saudi Arabia[ In a recent study "Muhammad\ Mossai\ + El!Feraly\ 0881#\ sandracopimaric acid isolated from J[ excelsa was found to exhibit signi_cant activity against Bacillus subtilis\ Staphylococcus aureus and Streptococcus durans[ An alcohol extract of J[ excelsa showed acaricidal activity in plants "Tsitsin\ Kovtunenko\ Polyakov\ + Khaidarov\ 0862#[ Additional literature "Mishra + Agarwal\ 0878# has revealed some pharmacological properties of J[ excelsa extracts\ including CNS depress! ant activity in albino rats[ In this study\ we have isolated four known and one new diterpene in addition to a known sesquiterpene from  Corresponding author[

the berries of J[ excelsa growing in Anatolia[ The new compound had a labdane skeleton which was elucidated as 2a!acetoxy!labda!7"06#\02"05#\03!trien!08!oic acid "0#[ The known diterpenes were identi_ed as iso!

communic acid "1# "Atkinson + Crow\ 0869#\ "−#ent! trans communic acid "2# Blechschmidt + Becker\ 0881#\ sandracopimaric acid "3# "Edwards\ Nicholson\ + Rodger\ 0859^ Wenkert + Buckwalter\ 0861#\ isopimaric acid "4# "De Pascual Teresa\ Barrero\ Muriel\ San Feliciano\ + Grande\ 0868# and the known sesquiterpene was identi_ed as 3a!hydroxycedrol "Kuo\ Wu\ Cheng\ + Wang\ 0889^ Joseph!Nathan\ Santillan\ + Gutierrez\

9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII] S 9 9 2 0 ! 8 3 1 1 " 8 7 # 9 9 5 6 4 ! X

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G[ Topc žu et al[:Phytochemistry 49 "0888# 0084Ð0088

0873# "5#\ by spectral methods including IR\ UV\ MS\ and NMR[ The hexane and methanol extracts and com! pound 0 were tested against Mycobacterium tuberculosis and found to be moderately active[ The hexane extract was evaluated against a number of cell line and found highly active against KB!V "¦VLB# "1[4 mg ml−0#\ KB! V"−VLB# "1[4 mg ml−0#\ and LNCaP "0[2 mg ml−0# while methanol extract and compound 0 were only tested against cultured KB and P!277 cells and the latter indi! cated a week cytotoxic response with KB cells[

1[ Results and discussion The HREIMS of compound 0 gave a molecular ion peak at m:z 259[118885 corresponding to a molecular formula C11H21O3 "calcd 259[129959# The 02C NMR spec! trum displayed 11 signals indicated four methine\ nine methylene\ three methyl and six quaternary carbon atoms by APT experiments[ The signals at d 069[21 and d 071[96 were attributed to the presence of acetyl and acid groups which were supported by the IR spectrum demonstrating absorptions at 0624 and 0149 cm−0 "acetyl#\ 0694 and 1499Ð1599 cm−0 "shoulder# "acid#[ The UV spectrum gave a maximum at 132 nm indicating some conjugation in the molecule[ The presence of one exocyclic and two terminal methylene groups followed from the 02C NMR spectrum observing the signals at d 004[38\ 002[08 and 095[68\ as well as the three pairs of methylene signals in the 0H NMR spectrum at d 3[48\ 3[78 "each 0H\ br s\ H! 06\ H!06?#\ d 3[88\ 4[99 "each 0H\ br s\ H!05\ H!05?# and d 4[95 "0H\ brd\ J09[4 Hz\ H!04#\ 4[10 "0H\ brd\ J06[4 Hz\ H!04?# "Table 0#[ The methyl signals were observed at d 9[59\ 0[10 and 1[98 as singlets\ the latter one was indicative of acetyl methyl protons[ All the 0H NMR data suggested a labdane diterpene structure for 0[ For the location of the acid group in the structure\ most plausible place is C!08\ as expected biogenetically[ As for the acetyl group\ C!0\ C!2\ C!6 and C!01 should be considered based on the multiplicity\ but\ the most signi_cant data obtained from the 02C NMR chemical shift of one of the methyl carbons which was observed at d 12[ In the presence of an a!hydroxyl or acetyl group at C!2\ the a!methyl group at C!3 would have resonated around d 10Ð12 in the 02C NMR spectrum as observed in similar diterpenes isolated from Juniperus communis "Kagawa et al[\ 0882# and isolated from some other plants "Gonzalez et al[\ 0889^ Blechschmidt + Becker\ 0881#[ Otherwise\ only the presence of a substituent at C!3\ either as C!07 or C!08\ the methyl carbon at the same carbon "C!3# would not be shifted down_eld less than d 16[ In labdane diterpenes\ without a C!2 substituent and the presence of a b!carboxyl at C!3\ the a!methyl group of C!3 can be observed at around d 17 while in the case of no substituent either at C!2 or C!3\ the a!methyl "C!07# should be observed at around d 22 "Barrero + Altajeros\

Table 0 0 H and 02C NMR data of 0 in CDCl2 a Position

dH

dC

0 1 2 3 4 5 6 7 8 09 00 01 02 03 04 04? 05 05? 06 06? 07 08 19 CO CH2

Ð Ð 4[18 t "1[4 Hz# Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð 5[25 dd "06[4\ 09[4 Hz# 4[10 br d "06[4 Hz# 4[95 br d "09[4 Hz# 4[99 br s 3[88 br d 3[78 br s 3[48 br s Ð 0[10 s 9[59 s Ð 1[98 s

21[6 t 13[2 tb 62[1 d 36[0 s 49[9 d 14[3 tb 27[4 t 036[5 s 44[5 d 39[0 s 11[5 t 29[2 t 036[9 s 028[9 d 004[4 t Ð 002[1 t Ð 095[7 t Ð 12[7 t 071[0 q 01[3 q 069[2 s 10[1 q

a b

J values are given in parentheses[ Assignments are interchangeable[

0882#[ In fact\ the chemical shifts of the six methylene carbons also indicated the location of the acetyl group[ In the absence of a substituent at C!2\ the chemical shift of C!0 should be observed at d 27Ð30 while C!1 would be at 06Ð19 d\ but the presence of an acetyl group at C!2 caused an b!e}ect on C!1 and a g!e}ect on C!0 thus giving the C!1 signal at d 13[2 and the C!0 signal at d 21[5[ Therefore\ all the 02C NMR assignments of ring A revealed the presence of the acetyl group at C!2 and the narrow triplet at d 4[29 was assigned the a!position of the acetyl group[ The a!orientation of the acetyl group at C!2 was supported by NOE di}erence experiments\ an irradiation at d 4[29 caused an enhancement on the methyl signal at d 0[10 while no NOE enhancement was observed between the d 4[29 and d 9[59 signals[ A HETCOR experiment revealed direct correlations between protons and carbons[ The COLOC experiment con_rmed the location of the acetyl group at C!2 exhi! biting three bond correlation between H!2 and C!08\ as well as H!2 and C!07\ another three bond correlation was also observed between H!2 and C!0\ and a two bond correlation between H!2 and C!1[ Thus\ the COLOC experiment also supported the location of the acid group at C!3[ All the spectral data indicated that compound 0 has the structure 2a!acetoxylabda!7"06#\02"05#\03!trien!

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G[ Topc žu et al[:Phytochemistry 49 "0888# 0084Ð0088 Table 1 Cytotoxic activity results of the extracts and compound 0 "mg ml−0#

Hexane extract Methanol extract Juniperexcelsic acid "0# a

P277

KB

KB!V "¦VLB#

KB!V "−VLB#

LNCaP

BCI

LUI

COL1

ASK8

×4 ×4 ×4

0[8 09[4 ×19

1[4 NTa NT

1[4 NT NT

0[2 NT NT

×19 NT NT

×19 NT NT

4[9 NT NT

¦ NT NT

NT means not tested[

08!oic acid and was given the trivial name juniperexcelsic acid[ The hexane extract was evaluated with a number of cancer cell lines and found to be particularly active against human colon cancer cell line "LNCaP# "ED490[2 mg ml−0 as well as KB!V"¦VLB# "ED491[4 mg ml−0# and KB!V"−VLB# "ED491[4 mg ml−0#\ while compound 0 and methanol extract were only tested with cultured KB and P277 cells\ and the latter indicated a week cytotoxic response with KB cells "ED4909[4 mg ml−0#[ The hexane extract also showed a positive response to ASK 8 glioma cell "Table 1#[ On the other hand\ the hexane and methanol extracts\ as well as the new compound\ juniperexcelsic acid\ were tested against M[ tuberculosis and found to be moderately active with MIC values 04[4\ 06[9 and 03[3 mg ml−0\ respectively "Table 2#[ In the same study\ the known pimarane diterpene sandracopimaric acid "3# and a well known labdane diterpene sclareol were also tested against M[ tuberculosis\ and their MIC values were found as 04[9 and 5[9 mg ml−0\ respectively[ Furthermore\ the hexane and methanol extract were tested against a number of standard bacteria and methanol extract was found to be more or less active against all bacteria cell lines\ par! ticularly B[ subtilis\ S[ aureus and S[ epidermidis while the hexane extract showed some activity against only B[ subtilis and S[ aureus "Table 3#[ 2[ Experimental

2[1[ Plant material The berries of J[ excelsa M[ Bieb[ were collected from Isparta\ south!western Turkey[ A voucher specimen "AEF 08676# was deposited at the Herbarium of the Faculty of Pharmacy\ University of Ankara[ 2[2[ Extraction and isolation Crushed plant material "169 g# was extracted with hexane\ the extract was evapd in vacuo to give 04 g of a residue[ The latter was fractionated on a silica gel column "4[4×69 cm# eluting with hexane\ followed by a gradient of CHCl2 up to )099 and then EtOH[ After TLC appli! cation\ similar frs[ were combined and further separated on smaller silica gel columns and puri_ed on prep[ TLC plates[ The following compounds were obtained] 0 "14 mg#\ 1 "7 mg#\ 2 "5 mg#\ 3 "30 mg#\ 4 "06 mg# and 5 "01 mg#[ 2[2[0[ 2a!acetoxylabda!7"06#\02"05#\03!trien!08!oic acid " juniperexcelsic acid# "0# −0 2 ] 2319\ ðaŁ D14 −10[4>C "MeOH^ c 9[4#[ IR nCHCl max \ cm 1819\ 1499Ð1599 "sh#\ 0624\ 0694\ 0539\ 0484\ 0354\ 0349\ MeOH 0279\ 0149\ 0924\ 899\ 659[ UV lmax "log o# nm] 132"2[6#[ 0 02 H and C NMR] Table 0[ HREIMS m:z] 259[118885 ðMŁ¦ "calcd 259[129959 for C11H21O3#^ EI!MS m:z "rel[int#] 259 ðMŁ¦ "C11H21O3# "9[0#\ 299 ðMÐAcOHŁ¦ "13#\ 174 ðMÐAcOHÐMeŁ¦ "05#\ 144 ð299!COOHŁ¦ "8#\ 010 "29#\ 094 "14#\ 8 "18#\ 76 "32#\ 74"78#\ 72"099#[

2[0[ General 2[3[ Cytotoxic activity test UV] Varian Techtron 574 in EtOH[ IR] Perkin Elmer 879 in CHCl2\ 0H and 02C NMR] Bruker AC 199 L instrument 199 and 49[21 MHz\ respectively with TMS as int standard^ HRMS] VG Zab Spec GCÐMS spectrometer[

The MeOH extract and compound 0 were evaluated with cultured KB cells as described previously "Likhitwitayawuid\ Angerhofer\ Ruangrungsi\ Cordell\

Table 2 Antituberculous activity test against Mycobacterium tuberculosis

Mycobacterium tuberculosis "mg ml−0#

Hexane extract

Methanol extract

Juniperexcelsic acid

Sandracopimaric acid

Sclareol

04[4

06[9

03[3

04[9

5[9

0087

08 16 16 11 11 The results are given in mm as zone diameter by disc!di}usion method[ Hexane extract and sandracopimaric acid were dissolved in chloroform while methanol extract was dissolved in methanol[ b

a

13 14 14 12 13 19 14 11 11 13 06 07 11 19 08 11 14 16 12 19 05 22 19 06 11 12 21 14 02 19 Hexane extract Methanol extract Sandraco!pimaric acid Chloroformb Methanolb

11 21 19 9 12

S[ epidermidis

B!hem[ Streptococcus

Enterococcus

P[ mirabilis

11 16 15 11 11

15 15 12 19 10

2[4[ Antituberculosis activity test

S[ aureus B[ subtilis

Table 3 Antimicrobial activity of the extracts and sandracopimaric acida

+ Pezzuto\ 0882# while the hexane extract was evaluated with BC0\ LU0\ COL!1\ KB!V "¦VLB#\ KB!V "−VLB#\ P277\ LNCaP and ASK cell lines[

E[ coli

Kl[ pneumonia

Ps[ aure`inosa

C[ albicans

G[ Topc žu et al[:Phytochemistry 49 "0888# 0084Ð0088

The broth microdilution method was used "Wallace\ Nash\ + Steele\ 0875^ Cooksey\ Crawford\ Jacobs\ + Shinnick\ 0882#[ M[ tuberculosis H26Rv std[ strain was passaged into screw capped tubes in broth medium 6H8 middlebrook[ They were left at room temperature for 2 weeks[ After observation of the growth\ they were vortexed and the additional suspension was transferred to tubes and adjusted according to Mac Farland turbidity standards with in 85!well middlebrook 6H8 broth medium[ This suspension was used for MIC determinations^ 49 ml H26Rv std[ strain was added to all wells[ Microplates were left for three weeks and MIC values were determined for the lowest concentration where growth was not observed[ The same test was carried out with chloroform as control[ 2[5[ Antimicrobial activity test The disc!di}usion method "Colee\ 0865^ Sleigh + Timburg\ 0870# was used to determine the inhibition zones of the hexane and methanol extracts and san! dracopimaric acid[ The plates were kept overnight in the incubator at 26>C and the sizes of the inhibiton zones were measured[ Samples that had inhibition zones greater than 6 mm compared to control solvent were considered to select for tube dilution tests "Kavanagh\ 0852#\ MeOH and CHCl2 were used as control[ But\ the tube dilution test could not be realized due to an unexpected accident[ Standard bacterial strains S[ aureus ATCC 5427\ Esch! erichia coli ATCC 7628\ Proteus mirabilis ATCC 03042\ Klebsiella pneumonia ATCC 3241\ Pseudomonas aeru! ginosa ATCC 0428\ beta!hemolytic Streptococcus "clini! cal isolate#\ Enterococcus "clinical isolate#\ and a yeast Candida albicans "ATCC 09120# were used[

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