A Triterpene from Musa errans

Abstract

The dichloromethane extract of the corm of Musa errans, an endemic form of wild banana afforded 31-norcyclolaudenone (1), squalene, and a mixture of stimasterol and sitosterol. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy. Antimicrobial tests on 1 indicated that it has low activity against C. albicans, E. coli, P. aeruginosa, and T. mentagrophytes; and inactive against B. subtilis, S. aureus and A. niger.

Full text

Philippine Journal of Science
136 (2): 167-171, December 2007
ISSN 0031 - 7683
Research Note
Consolacion Y. Ragasa1,2*, Aillen T. Martinez1,
Jennifer Elizabeth Y. Chua,1 and John A. Rideout3
Key Words: Musa errans, Musaceae, 31-norcyclolaudenone
*Corresponding author: ragasac@dlsu.edu.ph
1Chemistry Department, De La Salle University, Taft Avenue, Manila
2Center for Natural Sciences and Ecological Research,
De La Salle University, Taft Avenune, Manila
3School of Chemical and Biomedical Sciences,
Central Queensland University, Queensland, Australia
167
A Triterpene from Musa errans
INTRODUCTION
Musa errans, a form of wild banana is endemic to the
Philippines. It is also known as Musa troglodytarium
Blanco. The leaves are used as topicals for chest
pains. The juice of the corms is used as antituberculars.
The sap is vulnerary and it is used in the treatment of
gonorrhoea (Quisumbing 1978). A new subspecies of
Musa acuminata has been described and named M.
acuminata ssp. Errans (Valmayor 2001). There are
no reported studies on Musa errans, but a number of
studies were reported on its congener Musa acuminata
and Musa sapientum. Musa acuminata afforded
oxabenzochrysenones (Opitz et al. 2002), 4'-hydroxy-
anigorootin, 4, 4'-dihydroxy-anigorootin, 3,3'-bishydroxy
anigorufone (Otalvaro et al. 2002), and (S)-(+)-6-
methoxy-α-methyl-2-naphthaleneacetic acid, which is
an anti-inflammatory drug (Abad et al. 2000). Musa
sapientum afforded 6 triterpenes: cyclomusalenol,
cyclomusalenone, 24-methylenecycloartanol, stigmast-
7-methylenecycloartanol, stigmast-7-en-3-ol, lanosterol,
The dichloromethane extract of the corm of Musa errans, an endemic form of wild banana
afforded 31-norcyclolaudenone (1), squalene, and a mixture of stigmasterol and sitosterol.
The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy. Antimicrobial
tests on 1 indicated that it has low activity against C. albicans; E. coli, P. aeruginosa, and T.
mentagrophytes; and inactive against B. subtilis, S. aureus, and A. niger.
and β-amyrin and eight flavonoids: quercetin and its
3-O-galactoside, 3-O-glucoside, and 3-O-rhamnosyl
glucoside (Zeid and Abou 1999), 4-epicycloeucalenone,
and 4-epicyclomusalenone (Akihisa et al. 1997), (24S)-
14α,24-dimethyl-9β,19-cyclo-5α-cholest-25-en-3β-ol,
(24S)-24-methyl-∆25-sterols and their 24-methylene
isomers, 4,4-dimethyl-, 4α-methyl-, and 4-demethyl
sterols, 3-oxo-4α-methylsteroids (Akihisa et al. 1986)
and 31-norcyclolaudenone (Knapp and Nicholson
1970; Desai et al. 1982). The corm of banana was
reported to have the following constituents: α-pinene,
β-pinene, β-myrcene, limonene, α-cubebene, α-copaene,
α-cedrene, β-caryophyllene, and α-humulene (Ndiege
at al. 1991). The dichloromethane extracts of banana
“dwarf Cavendish” afforded by GC-MS fatty acids and
sterols as major compounds and aromatic compounds,
fatty alcohols, and alkanes as minor compounds (Oliveira
et al. 2006).
We now report the isolation, structure elucidation,
and antimicrobial test results of 1 from the corm of Musa
errans.

Ragasa et al.: A Triterpene from Musa errans Philippine Journal of Science
Vol. 136 No. 2, December 2007
168
MATERIALS AND METHODS
General Experimental Procedures
NMR spectra were recorded on a Bruker Avance 400
in CDCl3 at 400 MHz for 1H and 100 MHz for 13C.
Column chromatography was performed with silica gel 60
(70-230 mesh), while the TLC was performed with plastic
backed plates coated with silica gel F254. The plates were
visualized with vanillin-H2SO4 and warming.
Sample Collection
The corm part of Musa errans was collected in August
2005 in Negros Occidental. It was identified as Musa
errans (Blco.) Teod. Var. botoan Teod. with control number
058 by Danilo N. Tandang and noted by Dr. Domingo A.
Madulid of the Philippine National Museum.
Isolation
About 1 kg of the corm of Musa errans was cut and ground
in an osterizer, then air-dried. The air-dried corm (286 g)
were ground in an osterizer, soaked in dichloromethane
for 3 days, then filtered. The filtrate was concentrated
under vacuum to afford a crude extract (2 g), which was
chromatographed in increasing proportions of acetone in
dichloromethane at 10 % increment. The dichloromethane
fraction was rechromatographed in 5 % ethyl acetate in
petroleum ether, then petroleum ether to afford squalene
(18 mg). The 10 % acetone in dichloromethane fraction
was rechromatographed in 10 % ethyl acetate in petroleum
ether, then 2.5 % ethyl acetate in petroleum ether to afford a
1 (10 mg). The 30 % acetone in dichloromethane fractions
were rechromatographed (2x) in 15 % ethyl acetate in
petroleum ether to afford a mixture of stigmasterol and
sitosterol (24 mg).
Antimicrobial Tests
The microorganisms used in these tests were obtained
from the University of the Philippines Culture Collection
(UPCC). These are Aspergillus niger UPCC 4219,
Candida albicans UPCC 2168, Bacillus subtilis UPCC
1295, Pseudomonas aeruginosa UPCC 1244, Escherichia
coli UPCC 1195, Staphylococcus aureus UPCC 1143,
and Trichophyton mentagrophyte UPCC 4193. The
test compound was dissolved in 95% ethanol. The
antimicrobial assay procedure reported in the literature
(Guevara and Recio 1985) was employed. The activity
index was computed by subtracting the diameter of the
well from the diameter of the clearing zone divided by
the diameter of the well.
O
CH
3
H
CH
3
CH
3
CH
3
H
3
C
H
H
CH
3
1
1
4
10 8
11
14
17
20 25
27
28
21
18
Figure 1. Structure of 31-norcyclolaudenone (1) from Musa errans
RESULTS AND DISCUSSION
The 1H NMR spectrum of 1 (Figure 1) indicated resonances
for geminal olefinic protons at δ 4.67 (d, J = 1.6 Hz) and
4.68 (d, J = 1.6 Hz), a cyclopropyl at δ 0.39 (d, J = 4.0
Hz) and 0.61 (d, J = 4.0 Hz), an allylic methyl at δ 1.65
(s), and 5 other methyl groups at δ 0.87 (d, J = 6.8 Hz),
0.89 (s), 0.99 (d, J = 6.4 Hz), 1.00 (s), and 1.01 (d, J = 7.6
Hz). The 13C NMR spectrum of 1 showed resonances for
30 carbons (Table 1) with the following functionalities:
a ketone carbonyl at δ 213.33 and olefinic carbons at δ
109.36 and 150.18. The other resonances were attributed
to methyl, methylene, and methine carbons. These
resonances indicated a triterpene with a cyclopropyl, a
ketone carbonyl, and an olefin functionalities.
The COSY spectrum indicated 6 isolated spin systems
as follows. H2-1/H2-2; H3-30/H-4/H-5/H2-6/H2-7/H-8;
H2-11/H2-12; H2-15/H2-16/H-17/H-20/H3-21,H2-22/H2-
23/H-24/H3-28; H2-19; H3-26/H2-27 (Fig. 2).
The 1H and 13C connectivities in 1 were verified by
HMQC. The structure of 1 was elucidated by analysis of
the HMBC 2D NMR data with key HMBC correlations
shown in Figure 3. The carbonyl was placed at C-3 due
to long-range correlation between the carbonyl and the
α-methylene protons at C-2, α-methine protons at C-4,
and methyl protons at C-30. The cyclopropylene protons
(H2-19) were attached to C-9 and C-10 since long-range
correlations were observed between these protons and C-9
and C-10. The double bond was assigned to C-25 and
C-27 due to long-range correlation between these carbons
and the allylic methyl C-26. All long-range correlations
observed were consistent with the structure of 1.
The relative stereochemistry of 1 was deduced from
NOESY and is as shown in Fig. 3. The C-30 methyl was
close in space to H-5, which was in turn close to C-29

Ragasa et al.: A Triterpene from Musa errans Philippine Journal of Science
Vol. 136 No. 2, December 2007
169
18
21
28
27
25
20
17
14
11
8
10
4
1
1
O
CH
3
H
CH
3
CH
3
CH
3
H
3
C
H
CH
3
H
: 1H- 1H COSY
: HM BC
Figure 2. 1H-1H COSY and Key 1H-13C long-rang range correlations
for 1
18
21
28
27
2520
17
14
11
8
10
4
1
O
CH3
H
CH3
CH3
CH3
H3C
H
H
CH3
Figure 3. Key NOESY correlations for 1
Table 1. 400 MHz 1H NMR and 100MHz 13C NMR of 1 in CDCl3
Position δC 1 δH mult. (J Hz) 1
1 32.77 1.60, 1.89
2 40.97 2.41 (2H)
3 213.33 ---
4 50.00 2.24
5 46.06 1.57
6 25.86 0.73 (2H)
7 25.18 1.32, 1.70
8 47.07 1.63
9 24.96 ---
10 29.69 ---
11 27.20 1.25, 2.05
12 32.85 1.65 (2H)
13 45.33 ---
14 48.78 ---
15 35.58 1.29 (2H)
16 28.01 1.32, 1.90
17 52.21 1.60
18 17.88 1.00 (s, Me)
19 26.94 0.39 d (4.0)
0.61 d (4.0)
20 36.00 1.36
21 18.33 0.87 d (6.8, Me)
22 33.88 1.35 (2H)
23 31.46 1.45 (2H)
24 41.60 2.10
25 150.18 ---
26 18.64 1.65 (s, Me)
27 109.36 4.67 d (1.6)
4.68 d (1.6)
28 20.16 1.01 d (7.6, Me)
29 19.15 0.89 (s, Me)
30 10.73 0.99 d (6.4, Me)
methyl, which was close to H-17, which was in turn close
to C-21 methyl, which was finally close to C-28 methyl.
Thus, they are on the same face of the molecule. On the
opposite face of the molecule are the cyclopropylene
protons that were close to H-8, which was in turn close
to C-18 methyl. Literature search revealed that 1 is
31-norcyclolaudenone, which was reported to have been
previously isolated from Musa sapientum (Knapp et al.
1970, Desai et al. 1982).
As part of our continuing search for possible
antimicrobial compounds, 1 was tested for its antimicrobial
potential against the following bacteria: Bacillus subtilis
and Staphylococcus aureus (gram-positive), Pseudomonas
aeruginosa, and Escherichia coli (gram-negative)
and fungi: Candida albicans (yeast), Trichophyton
mentagrophytes (parasitic), and Aspergillus niger (mold).
The agar well method was employed. The microorganisms
tested were chosen based on their availability in the culture
collection of the University of the Philippines-Natural
Sciences Research Institute (UP-NSRI).
Results of the antimicrobial tests (Table 2) indicated
that 1 at a concentration of 30 µg was slightly
active against the following bacteria: E. coli with an
activity index (AI) of 0.1, while the standard antibiotic
chloramphenicol indicated an AI of 2.8; P. aeruginosa
with an AI of 0.1, while the standard antibiotic showed
an AI of 1.3 and fungi: C. albicans with an AI of 0.2,
while the standard antibiotic Canesten indicated an AI
of 0.8; and T. mentagrophytes with an AI of 0.1, while
the standard antibiotic chlotrimazole gave an AI of 4.5.
Compound 1 was inactive against B. subtilis, S. aureus,
and A. niger.

Ragasa et al.: A Triterpene from Musa errans Philippine Journal of Science
Vol. 136 No. 2, December 2007
170
Table 2. Antimicrobial Test Results on 1
Organism Sample
(30 µg)
Clearing Zone (mm) Antimicrobial
Index (AI)
Replicate 1 Replicate 2 Replicate 3
E. coli 112 11 11 0.1
Chloramphenicol 23 2.8
P. aeruginosa 111 11 11 0.1
Chloramphenicol 14 1.3
S. aureus 1- - - 0
Chloramphenicol 25 3.2
B. subtilis 1- - - 0
Chloramphenicol 20 2.3
C. albicans 112 12 12 0.2
Canesten, 0.2 ga 18 0.8
T. mentagrophytes 112 11 11 0.1
Canesten, 0.2 ga 55 4.5
A. niger 1- - - 0
Canesten, 0.2 ga 23 1.3
a Contains 1% chlotrimazole
ACKNOWLEDGEMENTS
The antimicrobial tests were conducted at the University of
the Philippines-Natural Sciences Research Institute (UP-
NSRI). A research grant from the Science Foundation,
and the University Research Coordination Office of De
La Salle University is gratefully acknowledged.
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