Food Chemistry 135 (2012) 2904–2909

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Food Chemistry
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Secondary metabolites from the unripe pulp of Persea americana and their
antimycobacterial activities
Ying-Chen Lu a,1, Hsun-Shuo Chang a,1, Chien-Fang Peng b, Chu-Hung Lin c, Ih-Sheng Chen a,c,⇑
a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 807, Taiwan, ROC
b
Department of Biomedical Laboratory Science and Biotechnology, College of Health Science, Kaohsiung Medical University, Kaohsiung, Taiwan 807, Taiwan, ROC
c
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 807, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: The fruits of Persea americana (Avocado) are nowadays used as healthy fruits in the world. Bioassay-
Received 3 April 2012 guided fractionation of the active ethyl acetate soluble fraction has led to the isolation of five new fatty
Received in revised form 8 June 2012 alcohol derivatives, avocadenols A–D (1–4) and avocadoin (5) from the unripe pulp of P. americana, along
Accepted 12 July 2012
with 12 known compounds (6–17). These structures were elucidated by spectroscopic analysis. Among
Available online 20 July 2012
the isolates, avocadenol A (1), avocadenol B (2), (2R,4R)-1,2,4-trihydroxynonadecane (6), and (2R,4R)-
1,2,4-trihydroxyheptadec-16-ene (7) showed antimycobacterial activity against Mycobacterium tubercu-
Keywords:
losis H37RV in vitro, with MIC values of 24.0, 33.8, 24.9, and 35.7 lg/ml, respectively.
Persea americana
Avocado
Ó 2012 Elsevier Ltd. All rights reserved.
Lauraceae
Pulp
Fatty alcohol
Antimycobacterial activity

1. Introduction
Persea americana Mill. (Lauraceae) is an evergreen tree, distrib-
uted in tropical and subtropical regions around the world. Its fruit,
avocado, is edible and now wildly cultivated in tropical and sub-
tropical regions throughout the world (Bailey, 1970). Previous
studies of this plant have identified various classes of chemical
constituents, such as monoterpenoids (Pino, Rosado, & Aguero,
2000), sesquiterpenoids (Scora & Scora, 1998), triterpenoids (Wer-
man, Mokady, & Neeman, 1990), flavonoids (De Almeida et al.,
1998), alkaloids (Nagaraj et al., 2010), steroids (Sciancalepore &
Dorbessan, 1982), carotenoids (Gross, Gabai, Lifshitz, & Sklarz,
1974), and long-chain fatty alcohol derivatives (Kashman, Neeman,
& Lifshitz, 1969). Numerous bioactivities, such as cytotoxicity
(Oberlies, Rogers, Martin, & McLaughlin, 1998), antifungal (Adika-
ram, Ewing, Karunaratne, & Wijeratne, 1992), antibacterial (Nee-
man, Lifshitz, & Kashman, 1970), antiviral (Miranda et al., 1997),
nitric oxide and superoxide generation inhibition (Kim et al.,
2000), antioxidant (Terasawa, Sakakibara, & Murata, 2006), anti-
cardiovascular disease (Adeboye, Fajonyomi, Makinde, & Taiwo,
1999), acetyl-CoA carboxylase inhibition (Hashimura, Ueda,
⇑ Corresponding author at: School of Pharmacy, College of Pharmacy, Kaohsiung
Medical University, Kaohsiung, Taiwan 807, Taiwan, ROC. Tel.: +886 7
3121101x2191; fax: +886 7 321 0683.
E-mail address: m635013@kmu.edu.tw (I.-S. Chen).
1
These authors contributed equally to this work.
Kawabata, & Kasai, 2001), skin lysyl oxidase inhibition (Werman
et al., 1990), insecticidal (Rodriguez-Saona, Millar, & Trumble,
1997), and liver injury suppressing (Kawagishi et al., 2001) effects
have also been demonstrated, and suggest avocado fruits are a
healthy natural product to consume. Recently, about 1400 species
of Formosan plants have been screened in our laboratory for anti-
mycobacterial activity against Mycobacterium tuberculosis H37RV
in vitro. The methanolic extract of the unripe pulp of P. americana
showed in vitro antimycobacterial activities against the M. tubercu-
losis strain H37RV with a MIC value of 15 lg/ml and was found to be
one of the active leads. The methanolic extract was partitioned into
an ethyl acetate soluble fraction and an H2O-soluble fraction. Only
the ethyl acetate fraction showed the MIC value in vitro as 12 lg/
ml. Bioassay-guided fractionation of the active ethyl acetate solu-
ble fraction from the unripe pulp of this plant led to the isolation
of five new compounds (1–5), together with 12 known isolates
(6–17) (Fig. 1). The isolation and structure elucidation of these
compounds, together with an assessment of their in vitro antimy-
cobacterial activity, are described herein.
2. Materials and methods
2.1. General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital
polarimeter. IR spectra (KBr or neat) were taken on a Perkin–Elmer

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.073
 Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909
R3
R4
1 R 1= R2= R 3= OH, R 4= H, Δ6
3 R 1= R2= R 3= R 4= OH
7 R 1= R2= R 3= OH, R 4= H
10 R 1= OAc, R 2= R3= OH, R4= H
12 R 1= R3= OAc, R2= OH, R4= H
4 Δ16
6
9 Δ6
OH OH
O H3CO
7 7 OH HO
13 14
Fig. 1. Structures of compounds 1–17.
System 2000 FT-IR spectrometer. 1D (1H, 13C, DEPT) and 2D (COSY,
NOESY, HSQC, HMBC) NMR spectra using CDCl3 as the solvent were
recorded on Varian Gemini-2000 (200 MHz for 1H NMR, 50 MHz
for 13C NMR), Varian Unity Plus 400 (400 MHz for 1H NMR,
100 MHz for 13C NMR), and Varian VNMRS-600 (600 MHz for 1H
NMR, 150 MHz for 13C NMR) spectrometers. Chemical shifts were
referenced internally to the solvent signals in CDCl3 (1H, d 7.26;
13
C, d 77.0), with TMS as the internal standard. Low-resolution
ESIMS were obtained on an API 3000 mass spectrometer (Applied
Biosystems) and high-resolution ESIMS on a Bruker Daltonics APEX
II 30e mass spectrometer. Silica gel (70–230, 230–400 mesh)
(Merck) was used for column chromatography. A spherical C18
100A reversed-phase silica gel (RP-18) (20–40 lM) (Silicycle) was
used for medium-pressure liquid chromatography.
2.2. Plant material
The unripe fruits of P. americana were collected from the cam-
pus of Kaohsiung Medical University, Kaohsiung, Taiwan, in July,
2009, and identified by one of the authors (I.-S.Chen). A voucher
specimen (Chen 5035) has been deposited in the Herbarium of
the School of Pharmacy, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung, Taiwan, ROC.
2.3. Extraction and isolation
The unripe fruits (11.9 kg) of P. americana were sliced and dried
by constant temperature oven (50 °C) to obtain the dried unripe
pulp (2.3 kg; 19.3%). The dried unripe pulp was extracted with cold
MeOH (10 l) at room temperature three times. Upon concentration,
the MeOH extract was partitioned between EtOAc–H2O (1:1) to ob-
tain an EtOAc-soluble fraction (280 g) and an H2O-soluble fraction
(283 g). An aliquot of the active EtOAc-soluble fraction (100 g) was
2905
R2 OH OH
R1 R1
2 R1= OH, Δ6
8 R1= OH
11 R1= OAc
OH OH
OH
O
O
5
H3CO
HO O O
O O HO
15 16
17 Δ 22
applied to a silica gel column, eluting with a gradient of n-hexane–
EtOAc, to give 16 fractions (A-1–A-16). Fractions A-1, A-2, A-4, A-6,
A-7, A-9, and A-11–A-14 were active. Fraction A-12 (10.5 g) was
recrystallized from n-hexane to give crystals (A-12-c) and the
mother liquor (A-12-m). Fraction A-12-c (332 mg) was subjected
to a RP-C18 column, eluting with MeOH–H2O (4:1), to obtain 11
fractions (A-12-c-1–A-12-c-11). Fraction A-12-c-10 (10.2 mg) was
chromatographed on a RP-C18 column, eluting with acetone–H2O
(3:1) to afford 4 (0.8 mg). Fraction A-12-m (10 g) was submitted
to a column containing silica gel, eluting with a gradient of n-
hexane–acetone, to yield seven fractions (A-12-m-1–A-12-m-7).
Fraction A-12-m-4 (1.0 g from 7.3 g) was chromatographed on a
RP-C18 column, eluting with acetone–H2O (1:1) to obtain 2
(25.2 mg), 8 (113 mg) and five fractions (A-12-m-4-1–A-12-m-4-
5). Fraction A-12-m-4-5 (855 mg) was applied to a RP-C18 column,
eluting with acetone–H2O (3:2) to afford 1 (8.1 mg), 7 (522 mg)
and 11 fractions (A-12-m-4-5-1–A-12-m-4-5-11). Fraction A-12-
m-4-5-11 (221 mg) was subjected to a RP-C18 column, eluting with
acetone–H2O (3:1) to gain 9 (1.2 mg) and 6 (13.3 mg). Fraction
A-14 (3.81 g) was subjected to a column containing silica gel,
eluting with a gradient of n-hexane–acetone, to yield 13 fractions
(A-14-1–A-14-13). Fraction A-14-3 (42.3 mg) was chromato-
graphed on a RP-C18 column, eluting with MeOH–H2O (2:1), to
obtain eight fractions (A-14-3-1–A-14-3-8). Fraction A-14-3-7
(34.3 mg) was chromatographed on a RP-C18 column, eluting with
MeOH–H2O (5:2) to obtain 11 (0.8 mg). Fraction A-14-4 (46.0 mg)
was submitted to a RP-C18 column, eluting with MeOH–H2O (3:1),
to obtain 13 fractions (A-14-4-1–A-14-4-13). Fraction A-14-4-12
(34.5 mg) was subjected to an RP-C18 column, eluting with
MeOH–H2O (4:1) to afford 10 (1.3 mg). Fraction A-14-8 (286 mg)
was submitted to a RP-C18 column, eluting with acetone–H2O
(1:1), to obtain 15 fractions (A-14-8-1–A-14-8-15). Fraction A-
14-8-8 (11.3 mg) was subjected to a silica gel column, eluting with
2906 Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909

CH2Cl2–MeOH (1:1) to afford 3 (3.7 mg). Fraction A-6 (1.6 g) was 2.3.4. Avocadenol D (4)
subjected to a column containing silica gel, eluting with a gradient Colourless powder; ½a25
D 9.4 (c 0.04, CHCl3); IR (KBr) mmax 3334
of n-hexane–acetone, to yield six fractions (A-6-1–A-6-6). Fraction (OH), 1676 (C@C) cm1; for 1H and 13C NMR spectroscopic data, see
A-6-2 (30.7 mg) was applied to a RP-C18 column, eluting with Tables 1 and 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.2717
MeOH–H2O (5:2) to obtain 14 (1.5 mg). Fraction A-6-2-5 [M + Na]+ (calcd. for C19H38O3Na, 337.2718).
(18.4 mg) was chromatographed on a RP-C18 column, eluting with
MeOH–H2O (7:2) to afford 15 (1.3 mg). Fraction A-6-3 (125 mg)
was submitted to a silica gel column, eluting with n-hexane–EtOAc 2.3.5. Avocadoin (5)
(7:2) to produce 5 (6.7 mg). Fraction A-6-3-9 (21.6 mg) was chro- Colourless prisms (MeOH); m.p. 85–87 °C; ½a25
D 5.1 (c 0.12,

matographed on a silica gel, eluting with CH2Cl2–acetone (30:1)
to afford 12 (4.6 mg). Fraction A-4 (9.2 g) was recrystallized from
MeOH to give a mixture of 16 and 17 (157 mg) and the mother
liquor was applied to a column containing silica gel, eluting with
a gradient of n-hexane–acetone, to yield nine fractions (A-4-1–A-
4-9). Fraction A-4-5 (1.16 g) was subjected to a RP-C18 column,
eluting with MeOH–H2O (4:1), to obtain seven fractions (A-4-5-
1–A-4-5-7). Fraction A-4-5-7 (1.0 g) was subjected to a RP-C18
column, eluting with MeOH–H2O (5:1) to gain 13 (352 mg).
2.3.1. Avocadenol A (1)
Colourless oil; ½a25
D  14:6ðc 0:08; CHCl3 Þ; IR (neat) mmax 3364
(OH), 1644 (C@C) cm1; for 1H and 13C NMR spectroscopic data,
see Tables 1 and 2; ESIMS m/z 307 [M + Na]+; HRESIMS m/z
307.2248 [M + Na]+ (calcd. for C17H32O3Na, 307.2249).
2.3.2. Avocadenol B (2)
Colourless oil; ½a25
D 5.1 (c 0.15, CHCl3); IR (neat) mmax 3365
(OH), 3311 („CAH), 2118 (C„C), 1664 (C@C) cm1; for 1H and
13
C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 305
[M + Na]+; HRESIMS m/z 305.209 [M + Na]+ (calcd. for C17H30O3Na,
305.2093).
2.3.3. Avocadenol C (3)
Colourless prisms (MeOH); m.p. 82–85 °C; ½a25
D +11.9 (c 0.08,
CH3OH); IR (KBr) mmax 3435 (OH), 1640 (C@C) cm1; for 1H and
13
C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 325
[M + Na]+; HRESIMS m/z 325.2352 [M + Na]+ (calcd. for
C17H34O4Na, 325.2354).
CHCl3); IR (KBr) mmax 3271 (OH), 1722 (C@O), 1644 (C@C) cm1;
for 1H and 13C NMR spectroscopic data, see Table 3; ESIMS m/z
547 [M + Na]+; HRESIMS m/z 547.4699 [M + Na]+ (calcd. for
C33H64O4Na, 547.4702).
2.4. Antimycobacterial activity assay
The in vitro antimycobacterial activity of each compound was
evaluated using M. tuberculosis H37Rv. Middlebrook 7H10, and
their corresponding MIC values were determined, as recom-
mended, by the proportion method (Inderlied & Nash, 1996).
Briefly, each test compound was added to Middlebrook 7H10 agar
and supplemented with oleic acid–albumin–dextrose–catalase
(OADC) at 50–56 °C by serial dilution, to yield a final concentration
of 100–0.8 lg/ml. Then, 10 ml samples of each test compound
were dispensed into plastic quadrant Petri dishes. Several colonies
of the test isolate of M. tuberculosis were selected to make a sus-
pension with Middlebrook 7H9 broth, and were used as the initial
inoculum. These inocula were prepared by diluting the initial inoc-
ulum in Middlebrook 7H9 broth until turbidity was reduced to
reach an equivalent to that of the McFarland No. 1 standard. Final
suspensions were prepared by adding Middlebrook 7H9 broth and
preparing 102 dilutions of the standardized bacterial suspensions.
After solidification of the Middlebrook 7H10 medium, 33 ll of the
102 standardized bacterial suspension dilutions were placed on
each quadrant of the agar plates. The agar plates were then incu-
bated at 35 °C with 10% CO2 for 2 weeks. The MIC is the lowest con-
centration of test compound that completely inhibited the growth
of the test isolate of M. tuberculosis, as detected visually.

Table 1
1
H NMR spectroscopic data of compounds 1–4.a

Position dH (J in Hz)
1 2 3 4
1 3.63 (dd, 11.2, 3.2) 3.61 (dd, 11.2, 3.6) 3.45 (dd, 10.8, 4.8) 3.65 (dd, 10.5, 3.0)
3.49 (dd,11.2, 6.0) 3.49 (dd, 11.2, 6.4) 3.42 (dd, 10.8, 6.0) 3.49 (t, 10.5)
2 3.97 (m) 3.95 (m) 3.83 (m) 3.97 (m)
3 1.59 (m) 1.59 (m) 1.65 (dt, 14.4, 3.0) 1.57 (m)
1.44 (m)
4 3.88 (m) 3.87 (m) 3.80 (m) 3.91 (m)
5 2.23 (m) 2.18 (m) 1.46 (m) 1.48 (m)
2.14 (m)
6 5.37 (dt, 15.2, 7.0) 5.37 (dt, 15.2, 6.6) 1.30 (br s) 1.25 (br s)
7 5.55 (dt, 15.2, 7.0) 5.54 (dt, 15.2, 6.6) 1.30 (br s) 1.25 (br s)
8 2.02 (m) 2.01 (m) 1.30 (br s) 1.25 (br s)
9–12 1.27 (br s) 1.28 (br s) 1.30 (br s) 1.25 (br s)
13 1.27 (br s) 1.28 (br s) 1.48 (m) 1.25 (br s)
14 1.27 (br s) 1.28 (br s) 1.44 (m) 1.25 (br s)
15 2.02 (m) 2.18 (m) 4.02 (m) 1.95 (m)
16 5.81 (ddt, 17.1, 10.1, 6.8) 5.85 (ddd, 17.4, 10.2, 6.0) 5.38 (dt, 15.6, 5.7)
17 4.97 (ddt,17.1, 2.3, 1.4) 1.94 (t, 2.6) 5.15 (ddd,17.4, 2.1, 1.8) 5.45 (dt, 15.6, 5.7)
4.92 (ddt, 10.1, 2.3, 1.4) 5.00 (ddd,10.2, 2.1, 1.8)
18 1.98 (m)
19 0.96 (t, 7.8)
OHb 2.50 (br s) 2.64 (br s) 3.63 (br s) 2.05 (br s)
OHb 2.71 (br s) 2.86 (br s) 3.66 (d, 4.8) 2.49 (br s)
OHb 3.77 (br s) 3.84–3.98 (br s) 4.02 (m) 3.48 (br s)
OHb 4.14 (br s)
a 1
H NMR data (d) were measured in CDCl3 at 400 MHz for 1 and 2, in acetone-d6 at 600 MHz for 3, in CDCl3 at 600 MHz for 4.
b
D2O exchangeable.
 Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909 2907

Table 2 (307.2248 [M + Na]+), with two degrees of unsaturation. The IR

13
C NMR spectroscopic data of compounds 1–4.a spectrum showed hydroxy group absorption at 3364 cm1 and
Position 1 2 3 4 C@C stretch vibration absorption at 1644 cm1. The 1H NMR spec-
1 66.7 66.7 68.0 66.8 trum (Table 1) was similar to that of (2R,4R)-1,2,4-trihydroxyhep-
2 72.3 72.3 73.7 72.59 tadec-16-ene (7) (Sugiyama, Sato, & Yamashita, 1982), also isolated
3 38.6 38.5 41.6 39.0 in this study, and showed three hydroxy signals [d 2.50 (1H, br s,
4 71.3 71.3 72.3 72.56 OH, D2O exchangeable), 2.71 (1H, br s, OH, D2O exchangeable),
5 41.4 41.4 38.9 38.3
6 124.8 124.9
3.77 (1H, br s, OH, D2O exchangeable)], and three vinyl protons [d
7 135.5 135.3 4.92 (1H, ddt, J = 10.1, 2.3, 1.4 Hz, Hb-17), 4.97 (ddt, J = 17.1, 2.3,
8 32.6 32.6 1.4 Hz, Ha-17), 5.81 (1H, ddt, J = 17.1, 10.1, 6.8 Hz, H-16)], except
9 (C-6–C-11) (C-6–C-14) 29.2, 29.3, that two trans olefinic protons [d 5.37 (1H, dt, J = 15.2, 7.0 Hz, H-
31.0, 31.05, 29.51, 29.56, 29.60,
6), 5.55 (1H, dt, J = 15.2, 7.0 Hz, H-7)] of 1 replaced two methylenes
31.08, 31.2 29.61, 29.62, 29.69
10 (C-9–14) 28.9, (C-9–14) 28.4, d 1.26 (4H, br s, H-6 and H-7) in C-6 and C-7 of 7. A double bond of
29.1, 29.2, 28.7, 29.0, 1, located at C-6 and C-7, was confirmed from the COSY spectrum
29.38, 29.40, 29.1, 29.2, (Fig. 2), which showed correlations between H-5 (d 2.23, 2.14) and
29.42 29.3 H-4 (d 3.88), H-6 (d 5.37); H-7 (d 5.55) and H-6 (d 5.37), H-8 (d
11
2.02). Thus, the planar structure of 1 was proposed as (6E)-1,2,4-
12 26.89
13 26.86 trihydroxyheptadec-6,16-diene. Sugiyama et al. (1982) have
14 39.6 synthesized all four stereoisomers of 1,2,4-trihydroxyheptadec-
15 33.8 18.4 73.5 32.6 16-ene. After comparison of 1 with ½a25 D 14.6 (c 0.08, CHCl3) to
16 139.2 84.8 144.2 129.4
the synthetic (2R,4R)-1,2,4-trihydroxyheptadec-16-ene with
17 114.1 68.1 114.0 131.9
18 25.6 [a]D6.4 (c 1.1, CHCl3), the absolute configuration of 1 as 2R,4R
19 14.1 was suggested. The structure of 1 (avocadenol A) was elucidated
a 13
as (2R,4R,6E)-1,2,4-trihydroxyheptadec-6,16-diene. Support for
C NMR data (d) were measured in CDCl3 at 100 MHz for 1 and 2, in acetone-d6
at 150 MHz for 3, in CDCl3 at 150 MHz for 4.
this structural assignment obtained from 13C NMR (Table 2), DEPT,
HSQC, NOESY and HMBC (Fig. 2) experiments.
Avocadenol B (2) was obtained as an optically active colourless
oil, with ½a25
D 5.1 (c 0.15, CHCl3). ESIMS and HRESIMS data were

Table 3
used to determine the molecular formula as C17H30O3, with two
1
H NMR and 13
C NMR spectroscopic data of compound 5.a fewer hydrogens than 1. The IR showed hydroxy group absorption
at 3365 cm1, an acetylene group absorption at 3311 („CAH),
Position 5
2118 (C„C) cm1, and C@C stretch vibration absorption at
dH (J in Hz) dC 1664 cm1. The 1H NMR data (Table 1) of 2 was similar to that of
10 4.12 (dd, 12.2, 3.2) 68.3 (2R,4R)-1,2,4-trihydroxyheptadec-16-yne (8) (Oberlies et al.,
4.00 (dd, 12.2, 7.6) 1998), also isolated in this study, except that two olefinic protons
20 4.10 (m) 70.9
[d 5.37 (1H, dt, J = 15.2, 6.6 Hz, H-6), 5.54 (1H, dt, J = 15.2, 6.6 Hz,
30 1.60 (m) 39.1
40 3.88 (m) 72.5 H-7)] with trans in 1 replaced two methylenes d 1.27 (4H, br s,
50 1.25 (br s) 25.3 H-6 and H-7) at C-6 and C-7 in 8, as in the case of 1. Thus, the pla-
60 –120 1.25 (br s) 29.3, 29.4, 29.46, 29.49, 29.58, 29.65, nar structure of 2 was proposed as (6E)-1,2,4-trihydroxyheptadec-
29.7
6-en-16-yne. The absolute configuration of 2 was also proposed as
130 1.25 (br s) 28.9
140 1.48 (m) 38.1 2R,4R due to its levorotatory specific rotation and comparison with
150 2.04 (q, 6.8) 33.8 the [a]D6.4 (c 1.1, CHCl3) in (2R,4R)-1,2,4-trihydroxyheptadec-
160 5.81 (ddt, 16.8, 10.4, 139.3 16-ene (Sugiyama et al., 1982). Based on the above evidence, the
6.8) structure of 2, named avocadenol B, was elucidated as (2R,4R,6E)-
170 4.98 (ddt, 16.8, 2.0, 1.2) 114.1
1,2,4-trihydroxyheptadec-6-en-16-yne, which was further con-
4.92 (ddt, 10.4, 2.0, 1.2)
1 174.1 firmed by the COSY (Fig. 2) and HMBC (Fig. 2) techniques.
2 2.35 (t, 7.6) 34.2 Avocadenol C (3) was obtained as optically active colourless
3 1.64 (m) 24.9 prisms, with ½a25 D +11.9 (c 0.08, CH3OH). Analysis of the ESIMS
4 1.25 (br s) 29.1
and HRESIMS of 3 revealed a molecular formula of C17H34O4, with
5–13 1.25 (br s) 29.3, 29.4, 29.46, 29.49, 29.58, 29.65,
29.7
one more oxygen than 7. The 1H (Table 1) and 13C NMR (Table 2)
14 1.25 (br s) 31.9 spectra were similar to those of 7, except that an additional oxy-
15 1.25 (br s) 22.7 methine [dH 4.02 (1H, m, H-15); dC 73.5 (C-15)] was in place of
16 0.88 (t, 7.2) 14.1 the methylene [dH 2.03 (2H, q, J = 6.8 Hz, H-15); dC 33.8 (C-15)] at
OHb 2.70 (br s)
C-15 in 7. The presence of the oxymethine group in C-15 was con-
OHb 3.30 (br s)
firmed by correlations in HMBC spectrum (Fig. 2) between H-17
a 1 13
H NMR data (d) were measured in CDCl3 at 400 MHz and C NMR data (d) (dH 5.15, 5.00) and C-16 (dC 144.2), C-15 (dC 73.5); H-16 (dH 5.85)
were measured in CDCl3 at 100 MHz.
b and C-15 (dC 73.5); H-15 (dH 4.02) and C-14 (dC 39.6) and by the
D2O exchangeable.
HRESIMS. Thus, the planar structure of 3 was proposed as
1,2,4,15-tetrahydroxyheptadec-16-ene. The absolute series config-
3. Results and discussion uration of this long chain fatty alcohol with 1,2,4-trioxygenated
pattern from the unripe pulp of avocado was proposed as 2R,4R
3.1. Structure elucidation of new fatty alcohol derivatives (Kashman, Neeman, & Lifshitz, 1970; Kashman et al., 1969; Sugiy-
ama et al., 1982). The same configuration was also proposed for 3.
Avocadenol A (1) was isolated as an optically active colourless According to the above evidence, the structure of 3 was elucidated
oil, with ½a25
D 14.6 (c 0.08, CHCl3). The molecular formula was as (2R,4R)-1,2,4,15-tetrahydroxyheptadec-16-ene. However, the
established as C17H32O3 by ESIMS and HRESIMS analysis absolute configuration of C-15 remains uncertain.
2908 Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909
Fig. 2. Key COSY and HMBC correlations for compounds 1–5.
Table 4
Anti-tubercular activities of isolates from the unripe pulp of Persea americana against
M. tuberculosis H37RV.
Compounds
Avocadenol A (1)
Avocadenol B (2)
Avocadenol C (3)
Avocadoin (5)
(2R,4R)-1,2,4-trihydroxynonadecane (6)
(2R,4R)-1,2,4-trihydroxyheptadec-16-ene (7)
(2R,4R)-1,2,4-trihydroxyheptadec-16-yne (8)
1,4-Diacetoxy-2-hydroxyheptadec-16-ene (12)
Ethambutola
a
Positive control.
Avocadenol D (4) was isolated as optically active colourless
powder, with ½a25
D 9.4 (c 0.04, CHCl3). The ESIMS and HRESIMS
of 4 was established as C19H38O3 with one degree of unsaturation,
same as (2R,4R,6E)-1,2,4-trihydroxynonadec-6-ene (9) (Abe et al.,
2005), also isolated in this study. The 1H (Table 1) and 13C NMR
(Table 2) spectra of 4 were similar to those of 9, but the double
bond locating at C-16 and C-17 [dH 5.38 (1H, dt, J = 15.6, 5.7 Hz,
H-16), dC 129.4 (C-16); dH 5.45 (1H, dt, J = 15.6, 5.7 Hz, H-17), dC
131.9 (C-17)] in 4 was in place of the double bond locating at C-
6 and C-7 [dH 5.38 (1H, dt, J = 15.6, 7.2 Hz, H-6), dC 124.7 (C-16);
dH 5.56 (1H, dt, J = 15.6, 7.2 Hz, H-7), dC 135.7 (C-17)] in 9. The dou-
ble bond of 4 locating at C-16 and C-17, was confirmed from the
HMBC spectrum (Fig. 2), which showed correlations between H-
18 (dH 1.98) and C-19 (dC 14.1), C-17 (dC 131.9), C-16 (dC 129.4).
Thus, the planar structure of 4 was proposed as (16E)-1,2,4-tri-
hydroxynonadec-16-ene. The absolute configuration of 4 was also
proposed as 2R,4R due to its levorotatory specific rotation and
comparison with the [a]D6.4 (c 1.1, CHCl3) of (2R,4R)-1,2,4-
trihydroxyheptadec-16-ene (Sugiyama et al., 1982). Based on the
above evidence, the structure of 4 was elucidated as (2R,4R,16E)-
1,2,4-trihydroxynonadec-16-ene, namely avocadenol D, which
was further confirmed by COSY (Fig. 2), DEPT, HSQC, NOESY, and
HMBC (Fig. 2) experiments.
Avocadoin (5) was obtained as optically active colourless
prisms, with ½a25D 5.1 (c 0.12, CHCl3). Analysis of the ESIMS and
HRESIMS of 5 represented a molecular formula of C33H64O4, with
two degrees of unsaturation. The IR spectrum showed hydroxy
group absorption at 3271 cm1, C@O group absorption at
1722 cm1, and C@C stretch vibration absorption at 1644 cm1.
The 1H and 13C NMR data (Table 3) spectra were similar to
those of (2R,4R)-1-acetoxy-2,4-dihydroxyheptadec-16-ene (10)
(Domergue, Helms, & Prusky, 2000), also isolated in this study,
except that the hexadecanoyloxyl group [dH 0.88 (3H, t, J = 7.2 Hz,
H-16), 1.25 (24H, br s, H-4–H-15), 1.64 (2H, m, H-3), 2.35 (2H, t,
MIC (lg/ml) J = 7.6 Hz, H-2); dC 174.1 (C-1)] was in place of an acetoxy group
24.0
[dH 2.11 (3H, s, OCOCH3); dC 20.9 (OCOCH3), 171.2 (OCOCH3)] in
33.8 10. The ester linkage between the two long chain moieties was
55.5 confirmed from HMBC spectrum (Fig. 2), which revealed the corre-
>200 lations between H-10 (dH 4.12, 4.00) and C-1 (dC 174.1); H-2 (dH
24.9
2.35) and C-1 (dC 174.1), C-3 (dC 24.9), C-4 (dC 29.1) and by HRE-
35.7
60.4 SIMS. The absolute configuration of 5 was proposed as 20 R,40 R after
100.5 comparing the 1H, 13C NMR data and the ½a25 D 47.3 (c 0.02, CHCl3)
6.25 in 10. Thus, the structure of 5 was elucidated as (20 R,40 R)-20 ,
40 -dihydroxyheptadec-160 -enyl palmitate, namely avocadoin.
3.2. Structure identification of the known isolates
The known compounds (2R,4R)-1,2,4-trihydroxynonadecane (6)
(Oberlies et al., 1998), (2R,4R)-1,2,4-trihydroxyheptadec-
16-ene (7) (Oberlies et al., 1998), (2R,4R)-1,2,4-trihydroxyhep-
tadec-16-yne (8) (Oberlies et al., 1998), (2R,4R,6E)-1,2,
4-trihydroxynonadec-6-ene (9) (Abe et al., 2005), (2R,4R)-1-acet-
oxy-2,4-dihydroxyheptadec-16-ene (10) (Domergue et al., 2000),
(2R,4R)-1-acetoxy-2,4-dihydroxyheptadec-16-yne (11) (Domergue
et al., 2000), 1,4-diacetoxy-2-hydroxyheptadec-16-ene (12)
(Huang, 2004), oleic acid (13) (Pouchert & Behnke, 1993), scopole-
tin (14) (Chen, Chen, Chang, Teng, & Lin, 1999), cedrelopsin (15)
(Simonsen et al., 2004), and a mixture of b-sitosterol (16) and stig-
masterol (17) (De-Eknamkul & Potduang, 2003) were identified by
comparison of their physical and spectroscopic data ([a]D, IR, 1H,
13
C NMR, and MS) with values reported in the literature.
3.3. Biological studies
The isolates obtained from the pulp of P. americana were evalu-
ated for their in vitro antimycobacterial activities against M. tuber-
culosis H37RV in vitro. The results were shown as in Table 4.
Compounds 1–2, 6, and 7 showed minimal inhibitory concentra-
tion (MIC) values of 24.0–50.0 lg/ml, respectively (Table 4). The
clinically used anti-tubercular agent ethambutol was used as the
positive control. Based on the antimycobacterial activities results,
the following conclusion can be drawn: the compounds with a triol
moiety exhibited ascending degrees of antimycobacterial activities
in the following order: with a terminal methylene and a trans dou-
ble bond at C-6 and C-7 > with an alkyl group > with a terminal
acetylene and a trans double bond at C-6 and C-7 > with a terminal
methylene > with a terminal acetylene, hence avocadenol A (1),
 Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909
with a terminal methylene, showed the strongest activity, with an
MIC of 24.0 lg/ml. The recent findings that most saturated fatty
acids are inactive towards mycobacteria, whereas unsaturated
fatty acids show activity depends on the degree of unsaturation,
chain length, and the bacterial species tested (Carballeria, 2008).
The isolates in this study were of a different type compared to
the previous antimycobacterial compounds. These findings against
tuberculosis could provide more ideas of the fatty acids structural
characteristics.
4. Conclusion
The antimycobacterial activities of P. americana have never been
conducted. This is the first report about avocado’s antimycobacteri-
al activity. In this study, five long chain fatty alcohol analogues as
new compounds were additionally found from the unripe pulp of
P. americana. In our preliminary test, both unripe and ripe avocado
showed antimycobacterial activities against M. tuberculosis H37RV
in vitro. The MeOH extract of the unripe pulp of avocado showed
the MIC value of 15 lg/ml, however, no constituent with MIC value
less than 15 lg/ml was isolated. The major constituent 7 (calcu-
lated yield over 5.9 g) with MIC value of 35.7 lg/ml might play a
synergistic effect for the antimycobacterial activity in the unripe
pulp of avocado. Avocado is now popularly used as a healthy fruit
throughout the world (Hirasawa et al., 2008; Ledesma et al.,
1996; Plaza, Sanchez-Moreno, De Pascual-Teresa, De Ancos, & Cano,
2009), and its antimycobacterial activity in unripe pulp was first
found in this study. Further evidence is needed to support whether
avocado fruits are helpful to tuberculosis patients.
Acknowledgement
This work was supported by the National Science Council of the
Republic of China and the Kaohsiung Medical University Reasearch
Foundation (KMUER013).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.
2012.07.073.
References
Abe, F., Nagafuji, S., Okawa, M., Kinjo, J., Akahane, H., Ogura, T., et al. (2005).
Trypanocidal constituents in plants 5. Evaluation of some Mexican plants for
their trypanocidal activity and active constituents in the seeds of Persea
americana. Biological and Pharmaceutical Bulletin, 28, 1314–1317.
Adeboye, J., Fajonyomi, M., Makinde, J., & Taiwo, O. (1999). A preliminary study on
the hypotensive activity of Persea americana leaf extracts in anaesthetized
normotensive rats. Fitoterapia, 70, 15–20.
Adikaram, N., Ewing, D., Karunaratne, A., & Wijeratne, E. (1992). Antifungal
compounds from immature avocado fruit peel. Phytochemistry, 31, 93–96.
Bailey, L. H. (1970). Manual of cultivated plants: Most commonly grown in the
continental United States and Canada (rev. ed.). New York: MacMillan Publishing
Company.
Carballeria, N. M. (2008). New advances in fatty acids as antimalarial,
antimycobacterial and antifungal agents. Progress in Lipid Research, 47, 50–61.
Chen, I. S., Chen, T. L., Chang, Y. L., Teng, C. M., & Lin, W. Y. (1999). Chemical
constituents and biological activities of the fruit of Zanthoxylum integrifoliolum.
Journal of Natural Products, 62, 833–837.
2909
De Almeida, A., Miranda, M., Simoni, I., Wigg, M., Lagrota, M., & Costa, S. (1998).
Flavonol monoglycosides isolated from the antiviral fractions of Persea
americana (Lauraceae) leaf infusion. Phytotherapy Research, 12, 562–567.
De-Eknamkul, W., & Potduang, B. (2003). Biosynthesis of b-sitosterol and
stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene
units. Phytochemistry, 62, 389–398.
Domergue, F., Helms, G. L., & Prusky, D. (2000). Antifungal compounds from
idioblast cells isolated from avocado fruits. Phytochemistry, 54, 183–189.
Gross, J., Gabai, M., Lifshitz, A., & Sklarz, B. (1974). Structures of some carotenoids
from the pulp of Persea americana. Phytochemistry, 13, 1917–1921.
Hashimura, H., Ueda, C., Kawabata, J., & Kasai, T. (2001). Acetyl-CoA carboxylase
inhibitors from avocado (Persea americana Mill.) fruits. Bioscience, Biotechnology,
and Biochemistry, 65, 1656–1658.
Hirasawa, M., Shimura, K., Shimizu, A., Mura, K., Tokue, C., & Arai, S. (2008).
Quantification and functional analysis of dietary fiber and polyphenols in
avocado. Nippon Shokuhin Kagaku Kogaku Kaishi, 55, 95–101.
Huang, T. T. (2004). Studies on the constituents of the leaves of Persea americana
var. americana. Master Thesis, Graduate Instituent of Pharmacognosy Science.
Taipei Medical University, Taipei.
Inderlied, C. B., & Nash, K. A. (1996). Antibiotics in Laboratory Medicine (4th ed.).
Philadelphia: Lippincott Williams & Wilkins.
Kashman, Y., Neeman, I., & Lifshitz, A. (1969). New compounds from avocado pear.
Tetrahedron, 25, 4617–4631.
Kashman, Y., Neeman, I., & Lifshitz, A. (1970). New compounds from avocado pear–
II. Tetrahedron, 26, 1943–1951.
Kawagishi, H., Fukumoto, Y., Hatakeyama, M., He, P., Arimoto, H., Matsuzawa, T.,
et al. (2001). Liver injury suppressing compounds from avocado (Persea
americana). Journal of Agricultural and Food Chemistry, 49, 2215–2221.
Kim, O. K., Murakami, A., Nakamura, Y., Takeda, N., Yoshizumi, H., & Ohigashi, H.
(2000). Novel nitric oxide and superoxide generation inhibitors, persenones A
and B, from avocado fruit. Journal of Agricultural and Food Chemistry, 48,
1557–1563.
Ledesma, R. L., Munari, A. C. F., Dominguez, B. C. H., Montalvo, S. C., Luna, H. H.,
Juarez, C., et al. (1996). Monounsaturated fatty acid (avocado) rich diet for mild
hypercholesterolemia. Archives of Medical Research, 27, 519–523.
Miranda, M., Almeida, A., Costa, S., Santos, M., Lagrota, M., & Wigg, M. (1997). In
vitro activity of extracts of Persea americana leaves on acyclovir-resistant and
phosphonoacetic resistant herpes simplex virus. Phytomedicine, 4, 347–352.
Nagaraj, M., Sandhya, V., Supriya, G., Manju, R., Kumari, P., Bole, S., et al. (2010).
Antioxidant and antibacterial activity of avocado (Persea gratissima Gaertner.)
seed extract. World Applied Sciences Journal, 9, 695–698.
Neeman, I., Lifshitz, A., & Kashman, Y. (1970). New antibacterial agent isolated from
the avocado pear. Applied Microbiology, 19, 470–473.
Oberlies, N. H., Rogers, L. L., Martin, J. M., & McLaughlin, J. L. (1998). Cytotoxic and
insecticidal constituents of the unripe fruit of Persea americana. Journal of
Natural Products, 61, 781–785.
Pino, J., Rosado, A., & Aguero, J. (2000). Volatile components of avocado (Persea
americana Mill.) fruits. The Journal of Essential Oil Research, 12, 377–378.
Plaza, L., Sanchez-Moreno, C., De Pascual-Teresa, S., De Ancos, B., & Cano, M. P.
(2009). Fatty acids, sterols, and antioxidant activity in minimally processed
avocados during refrigerated storage. Journal of Agricultural and Food Chemistry,
57, 3204–3209.
Pouchert, C. J., & Behnke, J. (1993). The Aldrich library of 13C and 1H FT NMR spectra.
In Non-aromatic carboxylic acids (1st ed., vol. I, p. 782) Winsconsin: Aldrich
Chemical Co. Inc.
Rodriguez-Saona, C., Millar, J. G., & Trumble, J. T. (1997). Growth inhibitory,
insecticidal, and feeding deterrent effects of (12Z,15Z)-1-acetoxy-2-hydroxy-4-
oxo-heneicosa-12,15-diene, a compound from avocado fruit, to Spodoptera
exigua. Journal of Chemical Ecology, 23, 1819–1831.
Sciancalepore, V., & Dorbessan, W. (1982). Sterol composition of oil of avocado
(Persea americana Mill.). Grasas y Aceites, 33, 273–275.
Scora, R., & Scora, P. (1998). Leaf oils of two new avocado varieties endemic to Costa
Rica. The Journal of Essential Oil Research, 10, 705–707.
Simonsen, H. T., Adsersen, A., Bremner, P., Heinrich, M., Wagner Smitt, U., &
Jaroszewski, J. W. (2004). Antifungal constituents of Melicope borbonica.
Phytotherapy Research, 18, 542–545.
Sugiyama, T., Sato, A., & Yamashita, K. (1982). Synthesis of all four stereoisomers of
antibacterial component of avocado. Agricultural and Biological Chemistry, 46,
481–485.
Terasawa, N., Sakakibara, M., & Murata, M. (2006). Antioxidative activity of avocado
epicarp hot water extract. Food Science and Technology Research, 12, 55–58.
Werman, M., Mokady, S., & Neeman, I. (1990). Partial isolation and characterization
of a new natural inhibitor of lysyl oxidase from avocado seed oil. Journal of
Agricultural and Food Chemistry, 38, 2164–2168.