Secondary metabolites from the unripe pulp of Persea americana and their antimycobacterial activities

Secondary metabolites from the unripe pulp of Persea americana and their antimycobacterial activities

Food Chemistry 135 (2012) 2904–2909 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/food...

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Food Chemistry 135 (2012) 2904–2909

Contents lists available at SciVerse ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

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 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 b

a r t i c l e

i n f o

Article history: Received 3 April 2012 Received in revised form 8 June 2012 Accepted 12 July 2012 Available online 20 July 2012 Keywords: Persea americana Avocado Lauraceae Pulp Fatty alcohol Antimycobacterial activity

a b s t r a c t The fruits of Persea americana (Avocado) are nowadays used as healthy fruits in the world. Bioassayguided fractionation of the active ethyl acetate soluble fraction has led to the isolation of five new fatty alcohol derivatives, avocadenols A–D (1–4) and avocadoin (5) from the unripe pulp of P. americana, along with 12 known compounds (6–17). These structures were elucidated by spectroscopic analysis. Among 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 tuberculosis H37RV in vitro, with MIC values of 24.0, 33.8, 24.9, and 35.7 lg/ml, respectively. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Persea americana Mill. (Lauraceae) is an evergreen tree, distributed in tropical and subtropical regions around the world. Its fruit, avocado, is edible and now wildly cultivated in tropical and subtropical 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 (Werman, 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 (Adikaram, Ewing, Karunaratne, & Wijeratne, 1992), antibacterial (Neeman, Lifshitz, & Kashman, 1970), antiviral (Miranda et al., 1997), nitric oxide and superoxide generation inhibition (Kim et al., 2000), antioxidant (Terasawa, Sakakibara, & Murata, 2006), anticardiovascular disease (Adeboye, Fajonyomi, Makinde, & Taiwo, 1999), acetyl-CoA carboxylase inhibition (Hashimura, Ueda,

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 antimycobacterial 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. tuberculosis 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 soluble 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 antimycobacterial activity, are described herein.

2. Materials and methods ⇑ 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: [email protected] (I.-S. Chen). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.07.073

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

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R3

R2

OH OH R1

R1

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

2 R1= OH, Δ6 8 R1= OH 11 R1= OAc

OH OH OH

4 Δ16 6 9 Δ6 OH OH O O

5 H3CO HO

7

7

13

O

O

H3CO

O OH

HO

O

O

14

HO

15

16 17 Δ 22

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 campus 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 obtain an EtOAc-soluble fraction (280 g) and an H2O-soluble fraction (283 g). An aliquot of the active EtOAc-soluble fraction (100 g) was

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 nhexane–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-45). 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-12m-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 chromatographed 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 A14-8-8 (11.3 mg) was subjected to a silica gel column, eluting with

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CH2Cl2–MeOH (1:1) to afford 3 (3.7 mg). Fraction A-6 (1.6 g) was subjected to a column containing silica gel, eluting with a gradient of n-hexane–acetone, to yield six fractions (A-6-1–A-6-6). Fraction A-6-2 (30.7 mg) was applied to a RP-C18 column, eluting with MeOH–H2O (5:2) to obtain 14 (1.5 mg). Fraction A-6-2-5 (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 (7:2) to produce 5 (6.7 mg). Fraction A-6-3-9 (21.6 mg) was chromatographed 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–A4-9). 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-4-51–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 ([email protected]) 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 ([email protected]) 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 ([email protected]) 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).

2.3.4. Avocadenol D (4) Colourless powder; ½a25 D 9.4 (c 0.04, CHCl3); IR (KBr) mmax 3334 (OH), 1676 ([email protected]) cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.2717 [M + Na]+ (calcd. for C19H38O3Na, 337.2718). 2.3.5. Avocadoin (5) Colourless prisms (MeOH); m.p. 85–87 °C; ½a25 D 5.1 (c 0.12, CHCl3); IR (KBr) mmax 3271 (OH), 1722 ([email protected]), 1644 ([email protected]) 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 recommended, 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 suspension with Middlebrook 7H9 broth, and were used as the initial inoculum. These inocula were prepared by diluting the initial inoculum 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 incubated at 35 °C with 10% CO2 for 2 weeks. The MIC is the lowest concentration 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

3.63 3.49 3.97 1.59

(dd, 11.2, 3.2) (dd,11.2, 6.0) (m) (m)

3.61 3.49 3.95 1.59

3.88 2.23 2.14 5.37 5.55 2.02 1.27 1.27 1.27 2.02 5.81 4.97 4.92

(m) (m) (m) (dt, 15.2, 7.0) (dt, 15.2, 7.0) (m) (br s) (br s) (br s) (m) (ddt, 17.1, 10.1, 6.8) (ddt,17.1, 2.3, 1.4) (ddt, 10.1, 2.3, 1.4)

3.87 (m) 2.18 (m)

1

2 3 4 5 6 7 8 9–12 13 14 15 16 17 18 19 OHb OHb OHb OHb a 1 b

2

2.50 (br s) 2.71 (br s) 3.77 (br s)

5.37 5.54 2.01 1.28 1.28 1.28 2.18

3 (dd, 11.2, 3.6) (dd, 11.2, 6.4) (m) (m)

(dt, 15.2, 6.6) (dt, 15.2, 6.6) (m) (br s) (br s) (br s) (m)

1.94 (t, 2.6)

2.64 (br s) 2.86 (br s) 3.84–3.98 (br s)

4

3.45 3.42 3.83 1.65 1.44 3.80 1.46

(dd, 10.8, 4.8) (dd, 10.8, 6.0) (m) (dt, 14.4, 3.0) (m) (m) (m)

1.30 1.30 1.30 1.30 1.48 1.44 4.02 5.85 5.15 5.00

(br s) (br s) (br s) (br s) (m) (m) (m) (ddd, 17.4, 10.2, 6.0) (ddd,17.4, 2.1, 1.8) (ddd,10.2, 2.1, 1.8)

3.63 3.66 4.02 4.14

(br s) (d, 4.8) (m) (br s)

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. D2O exchangeable.

3.65 3.49 3.97 1.57

(dd, 10.5, 3.0) (t, 10.5) (m) (m)

3.91 (m) 1.48 (m) 1.25 1.25 1.25 1.25 1.25 1.25 1.95 5.38 5.45

(br s) (br s) (br s) (br s) (br s) (br s) (m) (dt, 15.6, 5.7) (dt, 15.6, 5.7)

1.98 0.96 2.05 2.49 3.48

(m) (t, 7.8) (br s) (br s) (br s)

Y.-C. Lu et al. / Food Chemistry 135 (2012) 2904–2909 Table 2 13 C NMR spectroscopic data of compounds 1–4.a Position 1

2

3

4

1 2 3 4 5 6 7 8 9

66.7 72.3 38.6 71.3 41.4 124.8 135.5 32.6

66.7 72.3 38.5 71.3 41.4 124.9 135.3 32.6

68.0 73.7 41.6 72.3 38.9

66.8 72.59 39.0 72.56 38.3

10

(C-9–14) 28.9, 29.1, 29.2, 29.38, 29.40, 29.42

11 12 13 14 15 16 17 18 19

(C-6–C-11) (C-6–C-14) 29.2, 29.3, 31.0, 31.05, 29.51, 29.56, 29.60, 31.08, 31.2 29.61, 29.62, 29.69

33.8 139.2 114.1

(C-9–14) 28.4, 28.7, 29.0, 29.1, 29.2, 29.3 26.89 26.86 39.6 73.5 144.2 114.0

18.4 84.8 68.1

32.6 129.4 131.9 25.6 14.1

a 13 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.

Table 3 1 H NMR and Position

13

C NMR spectroscopic data of compound 5.a

5 dH (J in Hz)

dC

4.12 4.00 4.10 1.60 3.88 1.25 1.25

(dd, 12.2, 3.2) (dd, 12.2, 7.6) (m) (m) (m) (br s) (br s)

68.3

1.25 1.48 2.04 5.81 6.8) 4.98 4.92

(br s) (m) (q, 6.8) (ddt, 16.8, 10.4, (ddt, 16.8, 2.0, 1.2) (ddt, 10.4, 2.0, 1.2)

1 2 3 4 5–13

2.35 1.64 1.25 1.25

(t, 7.6) (m) (br s) (br s)

14 15 16 OHb OHb

1.25 1.25 0.88 2.70 3.30

(br s) (br s) (t, 7.2) (br s) (br s)

10 20 30 40 50 60 –120 130 140 150 160 170

70.9 39.1 72.5 25.3 29.3, 29.4, 29.46, 29.49, 29.58, 29.65, 29.7 28.9 38.1 33.8 139.3 114.1 174.1 34.2 24.9 29.1 29.3, 29.4, 29.46, 29.49, 29.58, 29.65, 29.7 31.9 22.7 14.1

a 1 H NMR data (d) were measured in CDCl3 at 400 MHz and were measured in CDCl3 at 100 MHz. b D2O exchangeable.

13

C NMR data (d)

3. Results and discussion 3.1. Structure elucidation of new fatty alcohol derivatives Avocadenol A (1) was isolated as an optically active colourless oil, with ½a25 D 14.6 (c 0.08, CHCl3). The molecular formula was established as C17H32O3 by ESIMS and HRESIMS analysis

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(307.2248 [M + Na]+), with two degrees of unsaturation. The IR spectrum showed hydroxy group absorption at 3364 cm1 and [email protected] stretch vibration absorption at 1644 cm1. The 1H NMR spectrum (Table 1) was similar to that of (2R,4R)-1,2,4-trihydroxyheptadec-16-ene (7) (Sugiyama, Sato, & Yamashita, 1982), also isolated in this study, and showed three hydroxy signals [d 2.50 (1H, br s, OH, D2O exchangeable), 2.71 (1H, br s, OH, D2O exchangeable), 3.77 (1H, br s, OH, D2O exchangeable)], and three vinyl protons [d 4.92 (1H, ddt, J = 10.1, 2.3, 1.4 Hz, Hb-17), 4.97 (ddt, J = 17.1, 2.3, 1.4 Hz, Ha-17), 5.81 (1H, ddt, J = 17.1, 10.1, 6.8 Hz, H-16)], except that two trans olefinic protons [d 5.37 (1H, dt, J = 15.2, 7.0 Hz, H6), 5.55 (1H, dt, J = 15.2, 7.0 Hz, H-7)] of 1 replaced two methylenes d 1.26 (4H, br s, H-6 and H-7) in C-6 and C-7 of 7. A double bond of 1, located at C-6 and C-7, was confirmed from the COSY spectrum (Fig. 2), which showed correlations between H-5 (d 2.23, 2.14) and H-4 (d 3.88), H-6 (d 5.37); H-7 (d 5.55) and H-6 (d 5.37), H-8 (d 2.02). Thus, the planar structure of 1 was proposed as (6E)-1,2,4trihydroxyheptadec-6,16-diene. Sugiyama et al. (1982) have synthesized all four stereoisomers of 1,2,4-trihydroxyheptadec16-ene. After comparison of 1 with ½a25 D 14.6 (c 0.08, CHCl3) to the synthetic (2R,4R)-1,2,4-trihydroxyheptadec-16-ene with [a]D6.4 (c 1.1, CHCl3), the absolute configuration of 1 as 2R,4R was suggested. The structure of 1 (avocadenol A) was elucidated as (2R,4R,6E)-1,2,4-trihydroxyheptadec-6,16-diene. Support for 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 used to determine the molecular formula as C17H30O3, with two fewer hydrogens than 1. The IR showed hydroxy group absorption at 3365 cm1, an acetylene group absorption at 3311 („CAH), 2118 (C„C) cm1, and [email protected] stretch vibration absorption at 1664 cm1. The 1H NMR data (Table 1) of 2 was similar to that of (2R,4R)-1,2,4-trihydroxyheptadec-16-yne (8) (Oberlies et al., 1998), also isolated in this study, except that two olefinic protons [d 5.37 (1H, dt, J = 15.2, 6.6 Hz, H-6), 5.54 (1H, dt, J = 15.2, 6.6 Hz, H-7)] with trans in 1 replaced two methylenes d 1.27 (4H, br s, H-6 and H-7) at C-6 and C-7 in 8, as in the case of 1. Thus, the planar structure of 2 was proposed as (6E)-1,2,4-trihydroxyheptadec6-en-16-yne. The absolute configuration of 2 was also proposed as 2R,4R due to its levorotatory specific rotation and comparison with the [a]D6.4 (c 1.1, CHCl3) in (2R,4R)-1,2,4-trihydroxyheptadec16-ene (Sugiyama et al., 1982). Based on the above evidence, the structure of 2, named avocadenol B, was elucidated as (2R,4R,6E)1,2,4-trihydroxyheptadec-6-en-16-yne, which was further confirmed by the COSY (Fig. 2) and HMBC (Fig. 2) techniques. Avocadenol C (3) was obtained as optically active colourless prisms, with ½a25 D +11.9 (c 0.08, CH3OH). Analysis of the ESIMS and HRESIMS of 3 revealed a molecular formula of C17H34O4, with one more oxygen than 7. The 1H (Table 1) and 13C NMR (Table 2) spectra were similar to those of 7, except that an additional oxymethine [dH 4.02 (1H, m, H-15); dC 73.5 (C-15)] was in place of the methylene [dH 2.03 (2H, q, J = 6.8 Hz, H-15); dC 33.8 (C-15)] at C-15 in 7. The presence of the oxymethine group in C-15 was confirmed by correlations in HMBC spectrum (Fig. 2) between H-17 (dH 5.15, 5.00) and C-16 (dC 144.2), C-15 (dC 73.5); H-16 (dH 5.85) and C-15 (dC 73.5); H-15 (dH 4.02) and C-14 (dC 39.6) and by the HRESIMS. Thus, the planar structure of 3 was proposed as 1,2,4,15-tetrahydroxyheptadec-16-ene. The absolute series configuration of this long chain fatty alcohol with 1,2,4-trioxygenated pattern from the unripe pulp of avocado was proposed as 2R,4R (Kashman, Neeman, & Lifshitz, 1970; Kashman et al., 1969; Sugiyama et al., 1982). The same configuration was also proposed for 3. According to the above evidence, the structure of 3 was elucidated as (2R,4R)-1,2,4,15-tetrahydroxyheptadec-16-ene. However, the absolute configuration of C-15 remains uncertain.

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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.

a

Compounds

MIC (lg/ml)

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

24.0 33.8 55.5 >200 24.9 35.7 60.4 100.5 6.25

Positive control.

(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, J = 7.6 Hz, H-2); dC 174.1 (C-1)] was in place of an acetoxy group [dH 2.11 (3H, s, OCOCH3); dC 20.9 (OCOCH3), 171.2 (OCOCH3)] in 10. The ester linkage between the two long chain moieties was confirmed from HMBC spectrum (Fig. 2), which revealed the correlations between H-10 (dH 4.12, 4.00) and C-1 (dC 174.1); H-2 (dH 2.35) and C-1 (dC 174.1), C-3 (dC 24.9), C-4 (dC 29.1) and by HRESIMS. The absolute configuration of 5 was proposed as 20 R,40 R after comparing the 1H, 13C NMR data and the ½a25 D 47.3 (c 0.02, CHCl3) 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

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 C6 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 double bond of 4 locating at C-16 and C-17, was confirmed from the HMBC spectrum (Fig. 2), which showed correlations between H18 (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-trihydroxynonadec-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)-1,2,4trihydroxyheptadec-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 ½a25 D 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, [email protected] group absorption at 1722 cm1, and [email protected] 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)

The known compounds (2R,4R)-1,2,4-trihydroxynonadecane (6) (Oberlies et al., 1998), (2R,4R)-1,2,4-trihydroxyheptadec16-ene (7) (Oberlies et al., 1998), (2R,4R)-1,2,4-trihydroxyheptadec-16-yne (8) (Oberlies et al., 1998), (2R,4R,6E)-1,2, 4-trihydroxynonadec-6-ene (9) (Abe et al., 2005), (2R,4R)-1-acetoxy-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), scopoletin (14) (Chen, Chen, Chang, Teng, & Lin, 1999), cedrelopsin (15) (Simonsen et al., 2004), and a mixture of b-sitosterol (16) and stigmasterol (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 evaluated for their in vitro antimycobacterial activities against M. tuberculosis H37RV in vitro. The results were shown as in Table 4. Compounds 1–2, 6, and 7 showed minimal inhibitory concentration (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 double 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 antimycobacterial 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 (calculated 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.

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