/ Essent. Oil Res., 10, 9-19 (Jan/Feb 1998)

RESEARCH REPORT

Chemical Composition of the Essential Oils from the Aerial Parts of Bupleurum gibraltarium Lam. Arturo Velasco-Negueruela,* Maria J o s e P e r e z - A l o n s o a n d J e s u s Pala-Paul Departamento de Biologia Vegetal I (Botdnica), Facultad de Biologia Universidad Complutense, 28040-Madrid, Spain Ana C a m a c h o , Ana Maria F e r n a n d e z Ocafia, Carlos F e r n a n d e z Lopez a n d J o a q u i n Altarejos Facultad de Ciencias Experimentales, Universidad de Jaen, 23071 Jaen, Spain Area de Industrias

Maria C o n c e p c i o n Garcia Vallejo Forestales del CIFOR-INIA, Apartado 8111, 28080-Madrid,

Spain

Abstract The oils from the umbel rays, stems, leaves, fruits and the whole aerial parts of Bupleurum gibraltarium Lam. have been examined by means of GC/MS and retention indices. The dominant constituents in the oil from the whole aerial parts were found to be: oc-pinene (5-9%), sabinene (9.2%), limonene (6.5%), 2,3,4trimethylbenzaldehyde (17.2%), terpinen4-ol (5.1%), /rans-chrysanthenyl acetate (4.7%), elemol (4.3%) and guaiol (5-3%). The major c o m p o n e n t s of the umbel rays oil were: a - p i n e n e (17.6%), sabinene (33.8%), limonene (7.3%), and 2,3,4-trimethyIbenz'aldehyde (8.4%). The stem oil was characterized by the presence of a-pinene (6.0%), sabinene (21.8%), limonene (7.2%), 2,3,4-trimethylbenzaldehyde (10.7%), guaiol isomer (5.1%), elemol (5.5%) and guaiol (5.4%). The leaf oil had as main c o m p o n e n t s a - p i n e n e (13.0%), sabinene (50%) and limonene (10.0%), whereas in the oil from the fruits, a-pinene (42.7%), sabinene (28.3%), and limonene (9.0%) were found to b e the most important ones.

Key Word index Bupleurum gibraltarium, Apiaceae, essential oil c o m p o s i t i o n , sabinene, limonene, 2,3,4-tnmethylbenzaldehyde.

a-pinene,

Introduction The genus Bupleurum L. belongs to section Coriacea Godron of the carrot family, Apiaceae. Section Coriacea comprises three species endemic to the Mediterranean basin and North Africa. Bupleurum foliosum Salzm. ex DC. from Morocco and S.W. Spain; Bupleurum fruticosum from South Europe and North Africa and Bupleurum gibraltarium Lam. (B. verticale Ortega) from Morocco and C and S of Spain (1). These species are shrubs with evergreen leaves and covered with a continous network of secretory ducts (2). 'Address for c o r r e s p o n d e n c e

Received:June Revised: January

1996 1997

1041-2905/9K/0OO1 -0009501.00/0-—©199H Allured Publishing Corp.

1

10

VELASCO-NEGUEKUELA ET AL.

Abundance 650000-1

600000 -

330000

500000

450000 -

Figure 1. Total ion chromatogram of the oil from the whole aerial parts of Bupleurum gibrallarium Lam. (Pan one)

400000

350000 -

300000

250000-

200000

150000-

100000

50000 -

Time ->

5.00

10.00

15.00

The oils from B. fruticosum have b e e n the subject of c o n s i d e r a b l e w o r k (2-6). Bohlmann et al, (7) isolated three acetylenic c o m p o u n d s , two m o n o t e r p e n e s . (Z)-(3-ocimene and (E)-[3-ocimene, two isomeric m o n o t e r p e n e a l d e h y d e esters, ferulol a n d isoferulol a n g e l a t e s from the roots extracts of B. gibraltarium. Flavonoids and cinnamic acids have also b e e n found in the aerial parts of B. gibrallarium (8). More recently, the flower oil of B. gibraltarium was studied by TLC and GC/ MS by Cabo et al. (9,10). The major constituents found were P-pinene/S-3-carene (37.2%), a-pinene (10.4%), limonene (8.2%), myrcene/a-phellandrene (7.3%), terpinen-4-ol (8.9%) and 1,8-cineole (7.6%). The subject of this paper is the GC/MS analysis of the oils obtained from the aerial parts ofB. gibraltarium.

Experimental Plant Material B. gibrallarium was gathered in Puente de la Sierra, N o v e m b e r 1995, Jaen, Spain. Voucher specimens were deposited in the Herbarium of the Department of Animal Biology, Plant Biology and Ecology, University ofjaen, Spain. Plant material was determined by Prof. Carlos Fernandez from the University o f j a e n . The plant material consisted in the whole aerial parts (sample BG), the umbel rays (sample BGU). the stems (sample BGS), the leaves (sample BGL) and the fruits (sample BGF). These samples were hydrodistilled in a Clevenger-type apparatus during 8 li The oils were dried

BUPLEURUM

GtBR.
over anhydrous sodium sulfate and submitted to GC/MS analysis. One single analysis of each type of oil was performed. Yields based on dry weight of sample (mL%) were as follows: sample DG 1.3%; BGU 2.0%; BGS 0.4%; BGL 1.6% and BGF 2.2%. GO Analytical GC was d o n e on a Varian 3300 gas chromatograph fitted with a silicone DB-1 capillary column (30 m x 0.25 mm), Carrier gas N2, flow rate 1.5 mL/min. Split mode. Temperature programmed from 80°-225°C at 4°C/min. Injector temperature 280°C. FID detector, temperature 300°C. Injection volume for all samples 0.1 p.L. GC/MS: Analyses were carried out o n a Hewlet-Packard 5890 gas c h r o m a t o g r a p h fitted with a phase bonded poly (5% diphenyl 95% dimethylsiloxane) silicone PTE5 capillary column (30 m x 0.25 mm, 0.25 |-lrn film thickness). Carrier gas He. Flow rate 1.5 mL/min. Temperature program regimen was 70°C (2 min) and then programmed to 250°C at 2°C/min. Injector temperature 250°C. The chromatograph was coupled to a HP 5971A mass selctive detector at 70 eV Most constituents were identified by comparison of their mass fragmentation patterns with those found in the built-in spectrometer libraries Wiley.L and NBS54K.L. Retention indices and authentic standards were also used for further identification. When authentic standards were not available, retention indices compared with the literature values were used (11-13)- For those constituents where only the mass spectra were recorded and the fragmentation pattern was compared with bibliographic data (14), tentative identification was reported.

Results and Discussion We have examined the composition of the oils obtained from the aerial parts of B. gibrallarinm. The chemical composition of the oils can be seen in Table I. Components are listed in order of their elution from a fused silica capillar}' column (DB-1 and PTE5). Peak number, c o m p o n e n t s , retention times for both columns, retention indices for DB-1 column, percentages and identification methods were also listed. Component concentration were calculated from GC DB-1 column peak areas without correction factor. Several c o m p o n e n t s were best determined in the PET5 column. Therefore, their peak areas were calculated in this column as the relative percentages in both columns were maintained. Examination of the oil from the whole aerial parts indicated the presence of at least 136 components, (Figures 1 and 2) 103 of which were identified accounting for 9 5 9 % of the oil. The main constituents found were: a-pinene (5.9%), sabinene (9-2%o), limonene (6.5%), terpinen-4-ol (5.1%), trans-chrysanthenyl acetate (4.7%), 2,3,4-trimethylbenzaldehyde (17.2%), elemol (4.3%) and guaiol (5.3%). It is worth noting that the major constituent of the oil, 2,3,4-trimethylbenzaldehyde (peak 64) and its isomer 2,3,6-trimethylbenzaldehyde (peak 68) have to be considered (15-18) artefacts produced during steam distillation from ferulol and isoferulol esters by saponification and proton-catalyzed rearrangements. A comparison of an ether extract and the steam distilled oil from the leaves and fruits of B. gibrallarium has finally confirmed this assumption. C o m p o u n d s 64 and 68 were not detected in the ether extract. These c o m p o n e n t s have been found in the volatiles from numerous Apiaceae (19-20). When the trace and minor constituents of the high boiling fraction of the oil were examined by MS. we found several peaks with a mass fragmentation pattern in accordance with the spectra of ferulol and isoferulol esters. Peak numbers 117, 120, 121, 122. 123. 126, 127, 130, 131. 132 and 136 (Figure 2) had always ni/c 83(100) and m/e 55. Therefore, according to the published data (21-24) these components have to be considered as isomeric esters of ferulol/isoferulol of angelic, tiglic or 3-methylcrotonic acids, Angelates and tiglates give characteristic fragments at m/e 83 and 55. Mass spectra for angelates [11 and 12] have been reported by Bohlmann et al. (7,24), and the cis-configuration was determined by 'H-NMR. Their mass spectra were similar to those reported in Figures 3 and 4. Owing to the steric hindrance of the methyl group at the C-2 position in the isoferulyl c o m p o n e n t [2]. this c o m p o n e n t must elute before its ferulyl isomer [11; where the methyl group is at the C-6 position. As a result, we believe that the mass spectrum reported in Figure 4 is the cis-2-methylbut-2-en-l-oic acid, 2,4,4-trimethyl-3-carboxaldehyde cyclohexa-2,5-dien-l-yl ester (isoferulyl angelate) [21, and that the one in Figure 3 is the cis-2-methylbut-

12

VELASCO-NEGLTERUELA ET AX.

Abundance

TIC:

BUP.D

650000

eooooo -

500000

450000 -

400000

Figure 2. Total ion chromatogram of the oil from the whole aerial parts of Bupleumm gibraharium I.am. (Part two)

350000

300000 -

250000 -

200000

150000

100000

CiJL^

50000

VJW 0

Time

->22.00

24.00

2S.00

28.00

30.00

32.00

34.00

36.00

38.00

2-en-l-oic acid, 4,4,6-trimethyl-3-carboxaldehyde cycIohexa-2,5-dien-l-yl ester (ferulyl angelate) [1], The mass spectra of the two isomers reported in Figures 5 a n d 6 can also be differentiated as ferulyl and isoferulyl esters for the same reason as discussed a b o v e with the ester moiety isovalerate or 2-methylbutyrate measuring the ratios at m/e 57/85, always higher in the 2-methylbutirates (2.4+0.2) while in isovalerates it is equal to 1.1±0.2. In the present ease, this ratio is equal to 1.2 and as a result we believethat the mass spectaim reported in Figure 5 is the 3-methylbutan-l-oic acid, 2,4,4-trimethyl-3-carboxaldehycle cyclohexa-2.5 dien-1-yl ester (isoferulyl isovalerate) [4] and that the one in Figure 6 is the 3-methylbuian-l oic

BUPLEURUM

Abundanc' 60000

13

GIBRALTARIUM

Scan 2550

(34.817 m i n ) : BUP.D

S5000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000

133

147

166 219

1 61 (

0 80

100

120

140

160

180

233

•T 220

200

240

246 260

Figure 3. Mass spectrum of isoferulyl angelate [2] Abungggc

Scan 2705

(36.847 mini:

BUP.D

50000 45000 40000 35000

2500C 20000 15000 10000 121 3

115

5

133

147

177

rUl, 30

100

120

140

160

180

208

189 200

2 9

)

233

220

248

240

Figure 4. Mass spectrum of ferulyl angelate [11 acid, 4,4,6-trimethyl-3-carboxaldehyde cyclohexa-2,5-dien-l-yl ester (ferulyl isovalerate) [3]. With respect to the other isomers (peaks 117, 121, 122, 123, 126, 127, 130 132 and 136), the fragmentation pattern of the ferulol/isoferulol moiety can be recognized in their mass spectra. We think it is worth mentioning'that the fragmentation pattern of ferulol is: Ferulol: m/e(rel.int.) l66[Ml(47), 151(M + -CH 3 )(17), 121(M + -CH 3 -CH 2 O)(20), 137(M*-CHO)(100), 148(M*-H 2 0)(12), 133(M*-H 2 OCH 3 )(8), 105(M + -H 2 O-CH 3 -CO)(26), 91 (tropylium)(22). The oil from the umbel rays was found to contain at least 111 c o m p o n e n t s of which 101 amounting to 95.8% of the oil were identified. The major c o m p o u n d s were a - p i n e n e (17.6%), sabinene (338%), limonene (7.3%) and 2,3,4-trimethylbenzaldehyde (8.4%). The oil from the stems was characterized by the presence of a-pinene (6.0%), sabinene (21.8%), limonene (7.2%), 2,3,4-trimethylbenzaldehyde (10.7%), guaiol isomer ( 5 1 % ) guaiol (5.4%) and elemol (5-5%). Eighty o n e out of 90 constituents were identified representing 96.8% of the oil. The leaf oil was characterized by the presence of a - p i n e n e (13.0%), sabinene (50.0%), limonene (10.0%) and 2,3,4-trimethylbenzaldehyde (3.4%) as major constituents. Eighty seven out of 91compo-

14

VELASCO-NEGUERUELA ET AL.

1/ Table I. Percentage composition of Bupleurum gibraltanum Lam. oils Peak "

1 2 3 4 5 6 (7; 8 9 10 11 12 13 14 15 16 17

)£. !

19

20 21 22 •23"

24 25 26 27 28 29 30 31 32 33 34 35 36 37

&) 39 40 41 42 43 44 45 46 47 48 49 50

Component

valeric acid 2-methylhexanoic acid t (Z)-3-hexenol hexanoi 2-methylcrotonic acid** a-thujene a-pinene camphene dehydrosabinene"* sabinene P-pinene myrcene trimethylbenzene 1 "* a-phellandrene 5-3-carene a-terpinene trimethylbenzene 1 '* p-cymene limonene 1,8-cineole (Z)-p-ocimene (E)-P-ocimene y-terpinene unknown cis-sabinene-hydrate cis-linalool oxide (furanoid) trans-linalool oxide (furanoid) terpinolene trans-sabinene hydrate cis-p-menth-2-en-1 -ol trans-p-menth-2-en-1 -ol cis-verbenol trans-verbenol unknown trans-chrysanthenol safranal unknown terpinen-4-ol 2,3-dimethylbenzaldehyde 1 p-cymen-8-oi a-terpineoi myrtenai cis-piperitol verbenone unknown trans-piperitol unknown trans-carveol myrtenyl acetate citronellol

Rt1

Rt2

Rl

\BG!

3.01 3.16 3.23 3.38 4.14 4.58 4.78 5.10 5.22 5.74 5.82 6.12 6.22 6.63 6.73 6.91 7.08 7.14 7.35 7.39 7.54 7.88 8.30 8.42 8.56

2.41 2.48 2.66 2.73 2.87 3.41 3.53 3.74 3.74 3.99 4.07 4.17 4.17 4.45 4.58 4.65 4.69 4.69 4.87 4.87 4.87 5.13 5.37 5.37 5.49

795 804 828 838 856 915 923 938 938 955 966 968 968 987 997 1001 1003 1003 1012 1012 1012 1025 1036 1036 1.42

0.2 0.1 t t 0.6 0.2

8.72

5.58

1047

9.13 9.34 9.68 10.57 11.28 11.34 11.51 11.64 11.94 12.06 12.48 12.94

5.93 5.99 6.11 6.64 7.02 7.02 7.13 7.13 7.13 7.32 7.66 7.96

1064 1067 1073 1099 1113 1113 1117 1117 1117 1124 1137 1148

•"ST)

13.06 13.13 13.39 13.52 13.60 13.73 13.92 14.09 14.37 14.56 14.71 15.01

8.18 8 18 8 26 8 36 8 36 8 45 8 45 8 52 8 74 8 81 9 02 9 29

1157 1157 1160 1163 1163 1167 1167 1169 1178 1 180 1188 1198

t 0.1 0.4 t t 0.1 t 0.1 0.1 0.1 0.2 1.3

C5.9.3S

t t 5.2'<

0.4 0.5 0.3 0.1 t 1.1 0.2 0.3

'5Jr "6.1 1.1 0.1 2.3 t 0.2

BGU

BGS

BGL

BGF

0.1 0.1 t .0.1 t t t 0.1 0.1 0.5 0.3 0.7 0.5 42.7 6.0 1.3.0 17.6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 28.3 50.0 21.8 33.8 .2.00.7 , 1 2 , 0.7 1.6 .3.2 1.1 1.6 0.8 0.8 0.6 ' 1.6 0.1 0.1 0.1 0.1 0.2 t t 0.6 1.4 1.1 1.3 0.2 0.2 0.2 0.2 0.1 0.5 0.3 0.1 9.0 7.2 10.0 7.3 0.1 ~ 5ZZZ3IIHZJL1 1.0 1.0 '1.5 1.2 0,1 t 2.0 2.5 1.1 1.6

Methods

GC'.MS MS GC 2 ,MS GC\MS MS GC\MS GC\MS GC'.MS MS GC\MS GC'.MS GC\MS MS GC\MS GC'.MS GC'.MS MS GC\MS GC'.MS GC'.MS GC'.MS GC'.MS GC'.MS

-

-

-

-

0.3

0.1

0.1

0.2

GC 2 ,MS

0.1

0.1

-

t

t

GC 2 ,MS

0.1 0.5 0.2 0.3 0.3 0.1 0.8 0.1 t 0.2 0.8

0.1 0.4 0.2 0.2 0.3 0.1 0.8 t 0.1 0.1 0.5

-

-

-

0.4 0.1 0.1 0.1 0.1 0.3 t 0.1 0.2 0.7

0.2 0.1 0.1 0.1 0.1 0.3 t 0.1

GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC'.MS GC'.MS

2jL_

0.1 0.2 0.1 0.1 0.1 t 0.2 t 0.1 0.2 0.1

0.4 0.1 0.1 0.2 t 0.3 t t 0.1 0.7 2.5

0.1

2^a_._ 1 J 2 _

-

t

t

-

0.2

0.2

-

-

-

0.1

-

t t

t t t

GC 2 ,MS GC 2 ,MS

t

t

-

GC'.MS MS GC 2 ,MS GC'.MS GC'.MS GC 2 ,MS GC'.MS GC 2 ,MS GC'.MS GC 2 ,MS GC'.MS

iwi

m BUPLEURUM

15

GJBRALTARIUM

Table I. Continued Peak1

51 52 53 54 55 56)

Component

methyl thymol cuminaldehyde carvone methyl carvacrol unknown trans-chrysanthenyl acetate 57 cis-chrysanthenyl acetate 58 cis-verbenyl acetate unknown 59 60 sabinyl acetate* 61 trans-verbenyl acetate 62 unknown 63 trans-pinocarvyl acetate -,. ' 6 4 ) 2,3,4-trimethylbenzaldehyde 65 1,2,3,4-tetramethylbenzene1" 66 a-cubebene 6 7 \ \ citronellyl acetate 68 \ 2,3,6-trimethylbenzaldehyde 69 methyl 2,3,6-trimethylbenzoate 1 70 cx-copaene 71 geranyl acetate 72 P-cubebene 73 P-elemene 74 a-gurjunene 75 p-caryophyllene a-guaiene 76 77 aromadendrene 78 c-cadinene 79 a-humulene 80 a!-o-aromadendrene 81 y-muurolene 82 germacrene D 83 cadinene isomer* unknown 84 unknown 85 86 epibicyclosesquiphellandrene 87 p-dihydroagarofuran 88 germacrene-B 89 dihydroagarofuran isomer1"* 90 p-bisabo!ene 91 y-cadinene 92 8-cadinene 93 cadina-1,4-diene 94 elemol

Rt1

Rt2

Rl

,
15.24 15.38 15.57 15.65 16.00

9.29 9.48 9.67 9.67 9.92

1198 1205 1212 1212 1221

0.1 t t t 0.1

0.1 t t 0.1

16.50

10.21

1231

4.7 :,

16.74 17.41 17.50 17.77 17.85 17.96

10.41 10.84 10.84 10.90 10.90 10.90

1238 1254 1254 1256 1256 1256

0.2 0.3 t 0.5 0.1 t

18.07

11.01

1260

19.06

11.50

20.15 20.33 20.45

BGS

BGL

BGF

Methods

. -

. -

-

t t

t t

-

t t t

GC 2 ,MS GC 2 ,MS GC 1 ,MS GC 2 ,MS

-

2.0

1.2

1.4

1.9

GC 2 ,MS

0.2 0.2

-

0.1 0.1

t 0.2

GC 2 ,MS GC 2 ,MS

-

GC 2 ,MS GC 2 ,MS

BGU f

-

0.4

-

-

-

-

0.4

0.3

0.4

0.3

0.1

GC 2 ,MS

1277

nD

8.4

10.7

3.4

4.5

GC 2 ,MS

12.03 12.40 12.58 .

1296 1308 1314

0.3

0.4 0.1 1.0

-

-

t 1.3

t t

-

GC 2 ,MS GC 2 ,MS GC\MS

20.57

12.58

1314

1 8

1.0

1.3

t

-

GC 2 ,MS

21.04 21.43 21.72 22.05 22.12 22.86 23.29 24.08 24.26 24.58 24.71 25.00 25.57 25.88 25.69 26.00 26.21

12.88 13.21 13.21 14.01 14.37 15.03 15.25 15.84 15.84 16.11 16.24 16.46 16.81 16.81 17.00 17.00 17.00

1324 1334 1331 1360 1372 1393 1399 1418 1418 1427 1431 1438 1449 1449 1455 1455 1455

0.7 0.3 0.2 0.1 0.3 t 0.5 0.1 t 0.2 0.4 0.2 0.3 0.1 1.0 t t

0.2 0.1 0.1 0.1 0.1 t 0.2 t t 0.1 0.2 0.1 0.1 t 0.3

0.5 0.2 0.1 0.1 0.3 t 0.5 0.1 t 0.1 0.4 0.1 0.2 0.1 0.9

0.2 0.1 t 0.1 0.1 t 0.2 t

0.3 t t t 0.1

0.1 0.1 0.1 0.1 t 0.5

t t 0.1

MS GC 2 ,MS GC\MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC\MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC\MS GC 1 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS

-

-

-

-

26.32 26.47 26.55

17.30 17.48 17.48

1464 1470 1470

0.5 0.3 0.7

0.2 0.1 0.2

0.3 0.3 0.5

0.1 0.1 0.2

0.1 t t

GC 2 ,MS GC 2 ,MS GC 2 ,MS

26.66 27.04 27.32 27.69 27.99 28.80

17.48 17.77 18.05 18.23 18.50 18.83

1470 1479 1487 1493 1502 1513

0.3 0.2 0.4 2.0 0.4 4.3

0.1 0.1 0.1 1.0 0.3 0.6

0.3 0.1 0.1 1.7 0.3 5.5

0.1 t t 0.5 0.1 0.5

t

MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS

0.3 0.1

0J TJB

•

, /

t t

-

0.1

0.1

-

0.2 t 0.1

16

VELASCO-NEGUERUELA ET AL.

Table I. C o n t i n u e d Peak8 Component (E)-nerolidol trans-sesquisabinen hydrate 97 dodecanoic acid 98 spathulenol 99 caryophyllene oxide unknown 100 guaiol isomer1"* 101 (10T) guaiol unknown 103 104 unknown 105 lO-epi-y-eudesmol unknown 106 107 1-epi-cubenol y-eudesmol 108 109 cubenol 110 a-muurolol 111 p-eudesmol 112 valerianol 113 guaiol isomer1"* 114 " bulnesol 115 guaiol isomer1"* 116 isoferulyl isovalerate 1 " 117 unknown 118 unknown unknown 119 120 isoferulyl angelate1" 121 unknown unknown 122 123 unknown 124 farnesol isomer"1* 125 ferulyl isovalerate1" unknown 126 127 unknown 128 unknown 129 P-selinene epoxide1"* 130 unknown 131 ferulyl angelate1" 132 unknown 133 guaiyl acetate1"* 134 unknown 135 unknown 136 unknown 95 96

Rt1

Rt2

Rl

(BG'

BGU

BGS

BGL

29.27

19.32

1530

0.5

0.1

0.1

-

GC\MS

29.59 29.74 29.81 30.02 30.40 30.70 30.78 30.90 31.07 31.28 31.60 31.83 31.99 32.38 32.55 32.69 32.82 32.97 33.41 33.72 33.85 34.08 34.24 34.73 34.82 34.98 35.13 35.22 35.39 35.48 35.64 35.81 36.15 36.20 36.69 36.86 37.09 37.40 38.25 38.56 38.86

19.44 19.63 19.63 19.79 19.95 20.16 20.31 20.40 20.50 20.75 20.91 21.24 21.24 21.50 21.70 21.70 21.85 21.85 22.23 22.49 22.49 22.68 22.87 23.03 23.03 23.03 23.29 23.29 23.48 23.48 23.83 23.83 24.03 24.03 24.44 24.44 24.44 25.19 25.88 26.50 27.08

1534 1540 1540 1546 1551 1559 1564 1567 1570 1579 1584 1596 1596 1604 1611 1611 1617 1617 1630 1639 1639 1645 1652 1657 1657 1657 1666 1666 1673 1673 1685 1685 1692 1692 1706 1706 1706 1734 1759 1782 1803

0.3 0.2 0.3 0.2 0.2 0.6 5.3 N t t 0.8 0.3 0.8 0.9 0.5 0.3 0.6 1.0 0,6 2.1 0.5 0.2 0.1 0.1 t 0.4 t 0.1 t 0.1 0.1 t t t 0.1

t 0.1 0.2 0.2

-

0.1

0.1 0.1 0.1

-

GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 1 ,MS

-

t 0.3 t

t 01 t 0.1

-

M

BGF

0.6 0.6

J/5.\ 5,4

0.3 0r3

t 0.1

-

-

-

-

0.1 0.1 0.2 0.2 t t 0.1 0.2 0.1 0.3 0.1 t t t

0.4 0.1 1.0 1.2 0.5 0.3 0.6 1.7 0.8 3.4 0.4 0.3 0.1 0.1

0.1

-

t 0.1 t 0.1 t t

-

-

-

t

-

-

t

0.1 t 0.1

-

t t t t

-

0.2 t

-

-

- I

Methods

MS GC 2 ,MS

GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC Z ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS GC 2 ,MS MS

MS

GC\MS MS

MS

MS

MS

-

-

• Order of eluhon on DB-1 and PTE5; Rt1=Retention time on PTE5; Rt2=Retention time on DB-1; Rl = Retention Index on DB-1 and according to n-paraffins; BG = Bupleurum gibraltarium whole aereal plant oil; BGU = B. gibraltarium umbel rays oil; BGS = B. gibraltarium stem oil; BGL = B. gibraltarium leaf oil; BGF = B. gibraltarium fruit oil; MS = mass spectrum; G C = Retention inaex according to authentic standars; GC 2 = Kovats index according to bibliography; ' tentatively identified according only to mass spectrum; * correct isomer not found; guaiol = guaia-1(5)-en-11-ol; bulnesol = guaia-1(10)-en-11-ol; valerianol = 4(JH,5a-eremophil-1(10)-en-11 -ol; safrana! = 2,6,6-trimethyM ,3-cyclo-hexadien-1 -carboxaldehyde; t _ trace (<0.05%)

BUPLEVRUM

GIBRALTAJUUM

Scan 2476 ( 3 3 . 48 m i n i : BUP.D 1 7 14000 -

12000

57

10000

8000

6000 -1 i 1000 3! 2000

0

1

221

^4 iiliIU

235

250

Vi M Ml O.L, JLU iik-nL "V160 180 220

240

Figure 5. Mass spectrum of isoferulyl isovalerate [4] Scan

Abur.dar.ee

^bU2

(35.493

mini:

BUP.D

7000

5"

5000

sC'JO

3000

11

1 2000 -

1000

1 113 l[ '. 1 ll

1

20B 0X / Z ->

__ 40

Lii

!

189 60

80

100

120

140

160

luJj r

180

200

221 220

240

260

Figure 6. Mass spectrum of ferulyl isovalerate 131 nents were identified representing 98.3% of the oil. In the fruit oil, 55 out of 58 c o m p o n e n t s were identified accounting for 98.9% of the oil. The major constituents found were a-pinene (42.7%), sabinene (28.3%), limonene (9-0%) and 2,3,4-trimethylbenzaldehyde (4.5%). Recently Gil et al. (25) r e p o r t e d the c o m p o s i t i o n of the oils o b t a i n e d from the aerial parts of B. gihraltarium collected at three different localities in Spain. Twenty five constituents w e r e identified. The main components found were a-pinene (9.0-22.4%), 8-3-carene/P-pinene (21.1-37.4%), m y r c e n e / cc-phellandrene (3-4-13.6%) limonene (8.6-12.5%), 1,8-cineole (3.9-8.4%), terpinen-4-ol (5.6-9.2%). Our results are different in that 89 c o m p o u n d s were positively identified and sabinene (9.2-50.0%). and 2,3,4trimethylbenzaldehyde (3.4-17.2%) were also found as major constituents of the oils. It is interesting to note that the percentage of guaiane derivatives in the stem oil (151%)) may justify the high anti-inflammatory activity of the oil reported in the literature (25,26). Because of this, the oil obtained from the entire aerial parts of B. gihraltarium may b e very useful in a r o m a t h e r a p y and perfumery/cosmetics.

18

VELASCO-NEGUERUELA ET AL.

Table II. Mass spectra data of unknown and tentatively identified constituents of Bupleurum gibraltarium oils Peak Rl 2 5 9 13 17 24 34 37 39

804 856 938 968 1003 1036 1117 1137 1157

45 1167 47 1178 55 1221 59 1254 62 1256 65 1296 69

1324

84 85 89

1455 1455 1470

100 101 103 104 106 113 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136

1557 1559 1567 1570 1584 1617 1639 1639 1645 1652 1657 1657 1657 1666 1666 1673 1673 1685 1685 1692 1692 1706 1706 1706 1734 1759 1782 1803

Component 2-methylhexanoic acic 2-methylcrotonic acid dehydrosabinene trimethylbenzene trimethylbenzene unknown unknown unknown 2,3-dimethylbenzaldehyde unknown unknown unknown unknown unknown 1,2,3,4-tetramethylbenzene methyl 2,3,6-trimethyl benzoate unknown unknown dihydro-agarofuran isomer unknown guaiol isomer unknown unknown unknown guaiol isomer guaiol isomer isoferulyl isovalerate unknown unknown unknown isoferulyl angelate unknown unknown unknown famesol isomer ferulyl isovalerate unknown unknown unknown P-selinene epoxide unknown ferulyl angelate unknown guaiylacetate unknown unknown unknown

m/e (rei.int) 130[M + ](0) 100[M + ](100) 134[M + ](6) 120[M + ](52) 120[M + ](52) 150[M + ](28) 152[M + ](6) 180[M + ](24)

74(100) 55(78) 91(100) 105(100) 105(100) 121(100) 79(100) 121(100)

87(30) 39(48) 92(48) 77(20) 77(18) 105(60) 94(90) 105(48)

60(25) 85(46) 119(23) 91(16) 91(12) 91(58) 59(80) 91(28)

45(14) 54(38) 77(16) 106(16) 106(8) 77(30) 77(40) 79(26)

57(12) 45(20) 39(15) 65(8) 65(8) 107(30) 43(35) 133(25)

75(4) 69(10) 65(10) 39(8) 39(8) 35(10) 137(12) 165(8)

134[M + ]{85) 154[M f ](0) 152[M+)(0) 152[M + ](6) [M + ]? 194[M + ](5)

133(100) 95(100) 107(100) 109(100) 43(100) 107(100)

79(90) 70(66) 91(35) 119(80) 121(50) 43(82)

105(78) 41(54) 137(15) 91(50) 91(46) 150(54)

77(40) 55(36) 79(15) 43(40) 135(40) 91(34)

93(15) 83(24) 121(12) 55(30) 41(30) 119(22)

119(25) 139(18) 41(10) 137(24) 105(28) 134(15)

134[M + ](10)

119(100) 43(20)

91(18)

77(12)

65(6)

105(5)

178[M + ](54) 220[M + ](60) 204[M~](5)

147(100) 146(86) 149(100) 205(84) 104(100) 105(35)

119(64) 177(68) 132(30)

91(38) 43(52) 91(27)

77(24) 91(48) 57(20)

163(10) 105(44) 79(15)

161(58) 93(55) 93(70) 81(40) 137(72) 161(60) 59(70) 189(50) 85(68) 55(17) 105(30) 117(17) 105(18) 55(30) 107(30) 105(40) 135(50) 85(56) 55(42) 41(40) 117(18) 91(45) 105(20) 105(15) 43(31) 93(80) 43(64) 83(28) 105(18)

41(50) 107(50) 119(65) 107(35) 93(63) 121(40) 189(68) 204(48) 121(46) 105(17) 41(28) 91(10) 121(16) 105(30) 121(28) 55(24) 81(39) 121(50) 105(41) 43(36) 91(17) 119(30) 121(18) 121(10) 105(36) 43(70) 57(64) 117(28) 166(15)

189(40) 161(40) 59(45) 93(30) 43(58) 91(30) 91(50) 59(42) 166(46) 159(14) 121(20) 77(5) 166(6) 166(25) 55(24) 166(24) 107(30) 166(23) 147(40) 55(36) 55(12) 55(25) 166(10) 166(8) 166(28) 204(56) 71(56) 147(25) 91(15)

119(35) 204(15) 133(25) 43(20) 81(58) 59(20) 79(45) 189(42) 235(10) 137(12) 159(15) 53(5) 233(3) 233(5) 149(20) 233(5) 121(20) 208(10) 208(20) 208(20) 41(10) 159(15) 208(5) 233(5) 208(20) 189(40) 147(48) 189(5) 147(10)

222[M + ](10) 222[M*](0) 222[M + ](8) 222[M + ](4) 222[M + ](4) 222[M + ](5) 222[M + ](4) 222[M + ](2) 250[M + ](2) 248[M + ](4) 222[M + ](14) 222[M + ](4) 248[M + ](3) 248[M + ](0) 248[M + ](0) 248[M + ](0) 222[M + ](0) 250[M + ](2) 248[M + ](0) 248[M + ](0) 222[M + ](2) 220[M + ](18) 248[M + ](0) 248[M + ](2) 248[M + ](0) 264[M + ](0) 222[M*](0) 222[M + ](0) 248[M*](0)

207(100) 43(100) 161(100) 149(100) 149(100) 57(100) 204(100) 161(100) 137(100) 83(100) 132(100) 132(100) 83(100) 83(100) 83(100) 83(100) 69(100) 137(100) 83(100) 83(100) 132(100) 107(100) 83(100) 83(100) 83(100) 161(100) 133(100) 132(100) 83(100)

137(62) 121(60) 107(75) 59(50) 59(76) 105(70) 161(80) 107(60) 57(77) 43(21) 133(50) 133(30) 55(28) 121(40) 135(32) 121(48) 41(58) 57(65) 43(48) 135(48) 133(54) 43(50) 55(20) 55(25) 55(32) 105(90) 132(80) 133(45) 55(20)

'•J

BUPLEURUM

GIBRALTARIUM

19

References 1. T. G.Tutin, BupleurumL., In: FloraEuropaea. Edits., T. G.Tutin, etal,, pp345-350, Cambridge University Press, Cambridge (1976). 2. A. Manunta, B. Tiriilini and D. Fraternale, Secretory tissues and essential oil composition of Bupleurum fruticosum L.]. Essent. Oil Res., 4, 461-466 (1992). 3. A. Manunta, Bupleurum fruticosum L. (fam. Umbelliferae L.J arbuste inressant de la flore du Maroc septentrional. I" Colloque Inter. Plantes Arom. et Medicinales du Maroc, Rabat, May (1984), 4. V. Pizzi, A. Atzei and A. Manunta, About the content in essential oil in the officinal species of Sardinia. The essential oils in the Umbelliferae of Sardinia. 1° Morphosystematics and chemical notes on Bupleurum fruticosum Linnaeus. Rivista Ital. EPPOS, 56, 239-244 (1974). 5. A. Manunta, I. Morelli and V. Pizzi, L'huile essent idle du Bupleurum fruticosum I Plantes Medicin. et Phytoth, 2 1 , 20-25 (1987). 6. L. Peyron and M. Roubaud, Sur une essence de Bupleurum fruticosum L. Plante Medicin, Phytoth., 4, 172-175 (1970). 7. F. Bohlmann, C, Zdero and M. Grenz, Notiziibcr einen Weiteren TerpenaldehydeEsteraus Umbelliferen, Chem. Ber., 108, 2822-2823 (1975). 8. J. Carbonnier and A.-M. Cauwet-Marc, Constituants du genre Bupleurum L. (Umbelliferae): mise aupoint des connaissance actuetles. Bull, Mus. Nat. His. Nat. section B, 3, 213-263 (1979) 9. J. Cabo, M. M. Cabo, J. Jimenez and M. A. Ocete, Essence de Bupleurum gibraltaricum Lam. (Ombelliferes). I - Eludespreliminaires. Plante Medicin. Phytoth. 20, 168-173 (1986). 10. J. Cabo, M. M. Cabo, J. Jimenez, C, Navarro and M. A. Ocete, Essence de Bupleurum gibraltaricum Lam. (Ombelliferes). Ill Etudes par GC et GC/MS. Plante Medicin, Phytoth, 20, 178-182 (1986). 11. A. Swigar and R. M. Silverstein, Monolerpenes. Aldrich Chem. Co., Milwaukee ( 1981). 12. W. Jennings and T. Shibamoto, Qualitative Analysis of Flavour and Fragance Volatiles by Capillary: GC. Academic Press, New York (1980). 13- L. M. Libbcy, A paradox database for GC/MS Data on components of essentia! oils and other volatiles. J. Essent. Oil Res., 3, 192-194 (1991). 14 R. P. Adams, Identification of Essentia! Oils Components by Gas Cbromatogrpby'Mass Spectroscopy Allured Publ. Corp., Carol Stream, IL (1995). 15. K. C. Wong, M. C. Feng. T. W. Sam and G. L. Tang, Composition of the leaves and roo/ oils of Eryngiiunfoelidurn L. J. Essent. Oil Res., 6, 369-374 (1994). 16. F. Bohlmann and C. Zdero, Uber weilerelerpenaldehydesterauseryngium-arten. Chem, Ber., 104,1957-1961 (1971). 17. F. Bohlmann and G. Weickgenannt, Synthese der Ferula-Terpene. Chem. Ber.. 107, 1769-1772 (1974). 18. K.-H. Kubezcka and 1. L'llmann, Terpenoids of the essential oil from Molopospennum peloponnesiacum roots. Phytochemistry, 20, 828-830 (1981). 19. K.-H. Kubezcka,'A. Bartsch and I. Ullmann, Recent Investigation of Essential Oils of Apiaceae. In: Alherische Ole. Edit. K.-H. Kubezcka, p p 158-187, Georg Thieme Verlag Sttutgart, New York (1982). 20. F. Bohlmann and M. Greny., Uber u>eitere terpen-deiivale aus Umbelliferen. Tetrahedron Letters, 17, 1453-1456 (1970) 21. J. Lemmich, P. A. Pedersen, M, S. Sood and B. E. Nielsen, Constituents of umbelliferous plants. XL Terpenoids from the roots of Selinum carvifolium L. Acta Chem Scand., 25, 995-999 (1971). 22. F. Bohlmann and M. Grenz, KeuelerpenaldehydeslerausPeucedanum luxurians Tamamsch. Chem. Ber., 109, 788-790 (1976). 23. D. Drake and J. Lam, Seseli acetylene from Etyngium bourgatli. Phytochemistry, 11, 2651-2652 (1972). 24. F. Bohlmann and C. Zdero. Uber ierpenderivate aus Ferula hispanica. Chem Ber , 102. 221 1-2215 (1969). 25- M. L. Gil. J. Jimenez, M. A. Ocete, A. Zarzuelo and M. M. Cabo, Comparative study of different essential oils of Bupleurum gibraltaricum Lam. Phannazic, 44, 284-287 (1989) 26. M. A. Ocete, S. Risco, A. Zarzuelo and J. Jimenez, Pharmacological activity of the essential oil of Bupleurum gibraltaricum Antiinflammatory activity and affects on isolated rat uteri |. Ft i m o p harm., 25, 305-313 (1989).