Betulenols from Betula species

Article abstract of DOI:10.1055/s-2000-8591

The essential oils from buds of five Betula species growing in Turkey were investigated by GC-MS. A major component in the essential oils was shown to be 14-hydroxy-β-caryophyllene (6). The structure of β-betulenal (12) which was isolated from Betula essential oils was also confirmed by synthesis. Chemical reactions yielded 14-acetoxy-β-aryophyllene (15), 14- hydroxy-isocaryophyllene (10) and its acetate (14), giving evidence to the natural occurrence in Betula species of the formerly known α-betulenol acetate (3), β-betulenol (2) and β-betulenol acetate (4), respectively. Compounds 6, 9, 10, 12, and 15 were evaluated for antimicrobial activity.

Full text of DOI:10.1055/s-2000-8591

490 Planta Med. 66 (2000)
Letters
acetates (3, 4) and a-betulenal (5) were previously isolated
from the essential oils of several Betula species (Fig.1) (4±8).
Betulenols from Betula Species
Betül Demirci, K. Hüsnü Can BaËser*, Temel Özek,
Fatih Demirci
This study aimed at investigating the essential oil composi-
tions and antimicrobial activity of the main components
isolated from Betula species. Here we report on the occur-
rence and isolation of 14-hydroxy-b-caryophyllene (6), for-
merly named as a-betulenol (1) by Treibs (6), in Betula essen-
tial oils.
Anadolu University, Medicinal and Aromatic Plant and Drug
Research Centre (TBAM), EskiËsehir, Turkey
vision accepted: December 19, 1999; Received: July 26, 1999
Received: July 26, 1999; Accepted: December 19, 1999
Essential oils obtained from buds of five Betula species collec-
ted from Turkey (Table 1) were analyzed by GC-MS. 14-Hy-
droxy-b-caryophyllene (6) was found to be the main constitu-
ent in the oils (Table 2) and was isolated by MPLC. EI-MS anal-
ysis showed a molecular ion peak at m/z 220, indicating a ses-
quiterpene alcohol of C15H24O. 1H- and 13C-NMR spectra
showed doubling of signals indicating two conformers. GC/FT-
IR showed characteristic hydroxy stretchings at 3651 and at
1011 cm±1 of an allylic alcohol. A literature search and com-
parison with spectral data (9) confirmed the structure of 14-
hydroxy-b-caryophyllene (6).
Abstract: The essential oils from buds of five Betula species
growing in Turkey were investigated by GC-MS. A major compo-
nent in the essential oils was shown to be 14-hydroxy-b-caryo-
phyllene (6). The structure of b-betulenal (12) which was isolat-
ed from Betula essential oils was also confirmed by synthesis.
Chemical reactions yielded 14-acetoxy-b-caryophyllene (15),
14-hydroxy-isocaryophyllene (10) and its acetate (14), giving
evidence to the natural occurrence in Betula species of the for-
merly known a-betulenol acetate (3), b-betulenol (2) and b-be-
tulenol acetate (4), respectively. Compounds 6, 9, 10, 12, and
15 were evaluated for antimicrobial activity.
The exocyclic methylene (H-15) and olefinic methylene (H-
14) groups, depending on their stereochemistry as in 6, result
in different conformers with respect to the cyclononene ring
plane; e.g., bb (if in the same direction with H-1) and ba (vica
versa). The presence of two major conformers (ba and bb) was
best seen at room temperature. 1H- and 13C-NMR spectra of 6
recorded at different temperatures resulted in the coalescence
of certain signals at higher temperatures with only one con-
Five Betula (Betulaceae) species, namely B. pendula Roth,
B. browicziana A. Güner (endemic), B. litwinowii Doluch., B. re-
curvata V. Vassil. and B. medwediewii Regel. growing in East-
ern and Northern Turkey were investigated (1), (2). The birch
tree (esp. B. pendula) is medicinally important since ancient
times and has been used in traditional medicine to cure skin
diseases, infections, inflammations, rheumatism, arthritis,
and urinary tract disorders. Birch essential oils were also used
in cosmetic products (3), (4). a- (1) and b-betulenol (2), their
Fig. 2 Structures of compounds 6±15.
Fig.1 Structures of compounds 1±5.
Table 2 13C-NMR data for compound (6) at various temperatures.
Table 1 Collection information of Betula species.
(25 8C)
(40 8C)
(70 8C)
C
ba
bb
56.7
ba
bb
56.7
ba
bb
Betula species
ESSEw Collection
Place
Altitude
Oil Yield*
(%)
1
50.6
29.1
33.9
137.9
128.5
29.7
34.7
153.7
49.5
40.4
32.8
22.1
29.4
59.9
112.8
50.9
29.4
34.0
137.8
128.5
29.8
34.8
153.8
49.1
42.5
32.8
21.9
29.6
59.9
112.8
56.7
30.2
2
3
30.4
29.2
30.5
B. pendula
12527 Erzurum
1800 m
3.82
#
33.5
B. browicziana (E) 12760 Rize: ÞamlihemËsin 1765 m
B. medwediewii 12759 Rize: ÞamlihemËsin 1700 m
4
137.6
129.2
30.4
138.0
128.8
29.8
1.25
#
5
129.1
30.5
B. recurvata
B. litwinowii
12758 Rize: ÞamlihemËsin 1700 m
12757 Artvin 2050 m
6
30.2
6.34
7
40.2
40.4
40.5
w Herbarium of the Faculty of Pharmacy at Anadolu University in EskiËsehir,
Turkey.
8
158.4
49.0
158.2
9
49.4
42.4
±
* Yields are given on dry weight basis.
10
11
12
13
14
15
42.4
# Likens-Nickerson Simultaneous Distillation & Extraction (analytical amount).
(E) Endemic to Turkey.
32.8
21.8
22.0
29.6
61.5
112.1
29.7
29.8
62.0
29.8
Planta Medica 66 (2000) 490±493
ꢀ Georg Thieme Verlag Stuttgart New York
ISSN: 0032-0943
62.0
·
110.1
110.1

Letters
Planta Med. 66 (2000) 491
Table 3 Relative percentages (%) of some Betula components.
Compound
KIP
A
B
C
D
E
b-caryophyllene (9)
b-betulenal (12)
14-acetoxy-b-caryophyllene (15)
14-hydroxy-b-caryophyllene (6)
14-hydroxy-isocaryophyllene (10)
1612
2193
2272
2357
2393
3.9
4.9
5.2
0.8
1.2
3.2
1.3
3.3
4.0
0.8
2.7
0.1
2.0
0.5
0.2
25.3
1.2
28.2
1.0
20.5
0.8
37.5
<0.01
21.9
0.6
A: B. pendula.
B: B. browicziana.
C: B. medwediewii.
D: B. recurvata.
E: B. litwinowii.
KIP: Kovats indices on a polar column
former (Table 2). This phenomenon was previously shown
(10), (11) where the occurrence of 14-hydroxy-9-epi-b-caryo-
phyllene (7) and its acetate (8) (Fig. 2) were reported incor-
rectly. Later, the confusion of compound 7 was corrected and
the structure was reassigned as (1R, 9S)-14-hydroxy-b-caryo-
phyllene (6) (9), (12). Recently, the isolation of 6 was also re-
ported from Pterocaulon serrulatum (13), supporting the pre-
vious data.
it was not possible for us to compare Triebs© (5), (6) betulenols
with our caryophyllenols, based on the literature data, we
propose that 14-hydroxy-b-caryophyllene is synonymous
with a-betulenol and that 14-hydroxyisocaryophyllene is syn-
onymous with b-betulenol in order to stop confusion in no-
menclature.
Antimicrobial activities of the compounds 6, 9, 10, 12 and 15
were evaluated using the micro-dilution broth susceptibility
assay (16). The results are shown in Table 4. Chloramphenicol
was used as reference and moderate activities were observed
on Gram (+)/(±) bacteria. Ketoconazole was used against the
C. glabrata, also moderate activity has been observed. 14-Hy-
droxy-b-caryophyllene was reported to have antibacterial ac-
tivity against B. subtilis and E. coli (13).
In this part of our work, synthetic approaches to obtain 6 and
other derivatives are described based on previous procedures
(9), (12). Thus, b-caryophyllene (9) was treated with SeO2, re-
sulting in the formation of 14-hydroxyisocaryophyllene (10),
isocaryophyllene (11) and its aldehyde (12). Compounds 10
and 12 were isolated from the reaction mixture and their
structures were confirmed by 1H- and 13C-NMR; these com-
pounds were also detected in the Betula essential oils recon-
firming their occurrence in nature. In conclusion, 10 is synon-
ymous with b-betulenol (2), and isocaryophyllen-14-al (12)
with b-betulenal (6). When 12 was subjected to a mild reduc-
tion with NaBH4, compound 10 was obtained. This confirmed
the assignment as b-betulenol (2). The occurrence of (4E)-iso-
caryophyllen-14-al (= b-betulenal) in Cunila spicata was re-
ported (14). Caryophyllen-14-al (= a-betulenal) (13) was pre-
viously reported from lavender oil (15).
Materials and Methods
Separations were performed using a Büchi B-680 MPLC sys-
tem. The column (26 ” 460 mm) was slurry packed with silica
gel 60G using hexane. Gradient elution was performed using
hexane-diethyl ether (0±100% diethyl ether). Compounds
were visualized with UV light (365 and 254 nm) or with vanil-
lin/H2SO4 in EtOH followed by heating. Optical rotations were
measured on an Oriel Pol S-2 polarimeter, IR spectra were
measured using a GC/FT-IR Perkin Elmer Spectrum 2000 spec-
trophotometer, UV spectra were recorded on Shimdazu UV
160A spectrophotometer, 1H- and 13C-NMR spectra were re-
corded on a JEOL JNM-EX90A spectrometer (in CDCl3, TMS) at
90 and 22.4 MHz, respectively. MS were measured using a
Hewlett-Packard G1800A GCD system. An Innowax FSC col-
umn (60 m ” 0.25 mm i.d.) was used with helium (1 ml/min)
as a carrier gas. MS were recorded at 70 eV.
We acetylated 14-hydroxyisocaryophyllene, resulting in the
formation of 14-acetoxyisocaryophyllene (= b-betulenol ace-
tate) (14) giving further support to its assignment. Acetylation
of 6 resulted in the formation of 14-acetoxy-b-caryophyllene
1
(15) (9). Both conformers of 15 were observed in H- and 13C-
NMR experiments at room temperature. This compound was
shown to be naturally present in Betula bud essential oils by
GC-MS (Table 3). Consequently, we suggest, that 14-acetoxy-
b-caryophyllene (15) is synonymous with a-betulenol acetate
(3) which had been reported from B. lenta essential oil (6).
Air dried buds of five Betula species were hydrodistilled for
4 h using a Clevenger-type apparatus or Likens-Nickerson si-
multaneous distillation-extraction method to yield essential
oils. Information about the plant materials is given in Table 1.
We treated the compound 6 with pyridinium chlorochromate
(PCC) to produce the following oxidation products which
were analyzed by GC-MS: caryophylla-4(14),8(15)-dien-5-one
(16) (15), 12 and an unknown compound 17 with relative per-
centages 40, 15 and 25, respectively. All of the compounds
were shown to occur in Betula bud essential oils (Scheme 1).
The present study has shown the occurrence of 14-hydroxy-b-
caryophyllene in all the Betula oils of Turkish origin. Although,
Scheme 1 Oxidation products of 14-hydroxy-b-caryophyllene (6).

492 Planta Med. 66 (2000)
Letters
Table 4 Antimicrobial activity (MIC) results with microtiter dilution method.
Microorganism
6
9
10
12
15
St mg/ml
Escherichia coli
250
250
250
250
125
125
250
250
250
250
125
250
250
250
250
250
125
250
250
250
250
250
250
250
250
250
250
250
125
250
250
7.81
125
62.5
125
62.5*
Staphylococcus aureus
Micrococcus luteus
Pseudomonas aeroginosa
Bacillus cereus
Candida glabrata
6: 14-hydroxy-b-caryophyllene.
9: b-caryophyllene.
10: 14-hydroxy isocaryophyllene
12: b-betulenal
15: 14-acetoxy-b-caryophyllene
St: Chloramphenicol
St*: Ketoconazole
B. pendula bud essential oil (ca. 0.8 g) was used for MPLC sep-
arations of the following compounds: 14-Hydroxy-b-caryo-
phyllene (6): Compound 6 (146 mg) was obtained from the
hexane-diethyl ether (7:3) fraction. [a]D25: ±8.38 (c 4.2, EtOH)
[lit. (13) ±14.2 (c 0.76, CHCl3)]; GC/FT-IR: nmax (cm±1) = 3651,
3073, 1630 and 892 (C=CH2), 1374 [C(CH3)2), 1011; EIMS: m/z
(rel. int.) = 220 (0.5) [M+], 205 (9), 187 (17), 164 (20), 145 (20),
133 (45), 119 (31), 105 (61), 91 (100), 70 (75), 69 (74), 55 (43),
(27), 105 (59), 91 (100), 79 (72), 69 (57), 55 (36), 41 (86). The
spectral data were identical with those reported in the litera-
ture (9).
14-Acetoxyisocaryophyllene (14): Acetylation of the sesquiter-
pene alcohol 10 (15 mg) in pyridine (0.5 ml) with acetic anhy-
dride (0.5 ml) yielded 14 (14 mg). [a]D25: ±14.68 (c 1.3, EtOH);
GC/FT-IR: nmax (cm±1) = 3078, 1761, 1633 (C=C), 1374 (C(CH3)2),
1227 and 1022 (CH3Co2), 887 (C=CH2); The spectral data were
identical with those reported in the literature (9).
1
41 (96). The H- and 13C-NMR data were identical with those
reported in the literature (9).
14-Acetoxy-b-caryophyllene (15): Acetylation of 6 (130 mg) in
pyridine (1 ml) with acetic anhydride (1 ml) yielded 15
(69 mg). [a]2D5: +6.38 (c 2.8, EtOH) [lit (10) +14.0 (c 1.02,
CHCl3)]: GC/FR-IR: nmax (cm±1) = 3076, 1630 and 893 (C=CH2),
1759 (C=O), 1230 and 1024 (CH3CO2); The spectral data were
identical with those reported in the literature (9).
b-Caryophyllene (9): Yields 11 mg. The spectral data were
identical with those reported in the literature (9).
Oxidation of 14-hydroxy-b-caryophyllene with PCC: 6 (5 mg)
was dissolved in dry CH2Cl2 (2 ml). PCC (10 mg) was suspen-
ded in the same solvent (5 ml) and added drop wise to 6. The
mixture was stirred and refluxed for 3 h. Dry diethyl ether
(5 ml) was added to the mixture and filtered through a small
silica gel column. The filtrate was then concentrated in vacuo.
Compounds 12, 16 (15), and 17 were detected by GC-MS.
b-Betulenal (12): Hexane-diethyl ether (9:1) elution of the es-
sential oil yielded the aldehyde 12 (26 mg). [a]D25: ±49.28 (c
2.6, EtOH) [lit. (14) [a]2D0: ±31.88 (c 0.69, CHCl3)]; UV: lmax
=
229.5 (log e 5.45, EtOH); GC/FT-IR: nmax (cm±1) = 3079, 1640
and 891 (C=CH2), 2810, 2709 and 1706 (CHO); The spectral da-
ta were identical with those reported in the literature (14),
(15).
Data for unknown compound 17: GC/FT-IR: nmax (cm±1) = 3079,
1631 and 900 (C=CH2), 1731, 1454; Ei-MS: m/z (rel. int.) = 234
[M+], 219 (3), 205 (5), 191 (5), 187 (5), 177 (8), 161 (7), 149
(26), 135 (28), 121 (27), 107 (46), 93 (65), 91 (69), 79 (84), 77
(51), 69 (59), 55 (69), 41 (100).
Oxidation of b-caryophyllene: b-Caryophyllene (Robertet)
(0.5 g) was dissolved in EtOH (1 ml) and treated with a solu-
tion of SeO2 (0.25 g) in EtOH (12 ml) for 6 h at room tempera-
ture (10), (11). Preparative TLC with hexane-diethyl ether
(7:3) yielded 12 (5 mg) and 10 (8 mg). GC-MS analysis
showed the formation of trace amounts of isocaryophyllene
(11).
Acknowledgements
We would like to thank Dr. Kiymet Güven and Miss Yasemin
ÞekicË for help in performing the antibacterial bioassay. Special
thanks are due to Dr. Simon F. R. Hinkley for constructive re-
marks.
Reduction of b-betulenal (12): The aldehyde 12 (33 mg) was
dissolved in dry MeOH (2 ml) and treated with NaBH4 (7 mg)
in room temperature. Water (10 ml) addition and extraction
with hexane yielded 10 (30 mg).
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Prof. Dr. K. Hüsnü Can BasË er
Anadolu University
Medicinal and Aromatic Plant and Drug Research Centre (TBAM)
26470-EskisË ehir
Turkey
E-mail: khcbaser@anadolu.edu.tr
Fax: +90 222 335 01 27