Isolation and Structure Elucidation of Avocado Seed (Persea

Jul 5, 2013 - (5) However, nitrites can produce N-nitroso compounds, most of which are known potent carcinogens.(5) Substitutes to nitrites as antimic...
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Isolation and structure elucidation of avocado seed (Persea americana) lipid derivatives that inhibit Clostridium sporogenes endospore germination Dariana Graciela Rodríguez-Sánchez, Adriana Pacheco, María Isabel GarcíaCruz, Janet A. Gutierrez-Uribe, Jorge Benavides, and Carmen Hernandez-Brenes J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf401407s • Publication Date (Web): 05 Jul 2013 Downloaded from http://pubs.acs.org on July 19, 2013

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Isolation and Structure Elucidation of Avocado Seed (Persea americana)

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Lipid Derivatives that Inhibit Clostridium sporogenes Endospore

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Germination

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Dariana Graciela Rodríguez-Sánchez,†, ‡ Adriana Pacheco,§ María Isabel García-Cruz,# Janet

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Alejandra Gutiérrez-Uribe,† Jorge Alejandro Benavides-Lozano,† and Carmen Hernández-Brenes†,‡*

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Tecnológico de Monterrey-Campus Monterrey. E. Garza Sada 2501 Sur, C.P. 64849, Monterrey,

Department of Biotechnology and Food Engineering, School of Biotechnology and Food.

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N.L., México

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Tecnológico de Monterrey. Batallón San Patricio 112, Col. Real de San Agustin, C.P. 66278, San

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Pedro Garza García, N.L. México

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§

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Tecnológico de Monterrey-Campus Monterrey. E. Garza Sada 2501 Sur, C.P. 64849, Monterrey,

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N.L., México

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#

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64849, Monterrey, N.L., México

Institute of Cardiology and Vascular Medicine, Zambrano-Hellion Hospital. Tec Salud del Sistema

Department of Agrobiotechnology and Agribusiness, School of Biotechnology and Food.

School of Medicine. Tecnológico de Monterrey-Campus Monterrey. E. Garza Sada 2501 Sur, C.P.

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* Corresponding autor (Tel.: +52-818-358-1400 Ext. 4817; Fax: +52-818-328-4136; E-mail:

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[email protected])

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ABSTRACT

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Avocado fruit extracts are known to exhibit antimicrobial properties. However, effects on bacterial

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endospores and identity of antimicrobial compounds have not been fully elucidated. In this study,

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avocado seed extracts were tested against Clostridium sporogenes vegetative cells and active

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endospores. Bioassay-guided purification of a crude extract based on inhibitory properties, linked

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antimicrobial action to six lipid derivatives from the family of acetogenins compounds. Two new

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structures and four compounds known to exist in nature were identified as responsible for the

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activity. Structurally, most potent molecules shared features of an acetyl moiety and trans-enone

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group. All extracts produced inhibition zones on vegetative cells and active endospores. Minimum

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inhibitory concentrations (MIC) of isolated molecules ranged from 7.8-15.6 µg/mL, and bactericidal

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effects were observed for an enriched fraction at 19.5 µg/mL. Identified molecules showed potential

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as natural alternatives to additives and antibiotics used by food and pharmaceutical industries to

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inhibit gram-positive spore-forming bacteria.

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Keywords: acetogenins; avocado; Clostridium sporogenes; endospores; antimicrobial

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INTRODUCTION

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The genus Clostridium represents a group of ubiquitous anaerobe spore-forming microorganisms of

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great concern in the medical and food industries. C. difficile is known to be related to post-antibiotic

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and hospital-acquired infections.1,2 C. botulinum and C. perfringens3 are important food-poisoning

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agents, whereas C. sporogenes causes food spoilage.4 In this regard, nitrites are the most widely used

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food additives to control spore-forming bacteria in processed meats.5 However, nitrites can produce

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N-nitroso compounds, most of which are known potent carcinogens.5 Substitutes to nitrites as

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antimicrobial additives, which do not present the same functionality on meat flavour and colour,

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include nisin and ethyl lauroyl arginate, each compound with its own limitations such as high costs of

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production,6 limited solubility and bitter taste.7 Therefore, there is a growing consumer and industrial

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demand for new antimicrobials from natural sources.

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Avocado (Persea americana; Lauraceae) is a tropical fruit rich in biologically active

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phytochemicals.8 Industrially, avocados are processed into oil and paste, which leaves 21-30% of the

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fruit weight to be discarded.9,10 However, avocado seeds and peels have comparable or even greater

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contents of bioactive compounds than the pulp,9,10 therefore, these waste products represent a

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potential source of molecules with applications in the food, pharmaceutical and cosmetic industries.

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Previous reports indicated that crude extracts from different avocado tissues exhibited antimicrobial

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activity against yeast,11,12 fungal spores,10,14 fungal vegetative cells13 and bacteria vegetative

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cells.10,11,15-20 It has been hypothesized that the antimicrobial activity may be attributed to β-

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sitosterol,12 phenolics and fatty acids.10 Antimicrobial properties of isolated fatty alcohol

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derivatives11,19,20 and acetogenins11,14,20 have been characterized. However, there are no reports

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regarding effects of crude avocado extracts or pure compounds on bacterial endospores with known

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chemical identity.

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Our group reported in a preliminary antimicrobial screening that an acetone extract from avocado

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seed exhibited significant inhibitory properties against vegetative cell growth and endospore

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germination of Clostridium sporogenes.21 This microorganism was used as a surrogate of proteolytic

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C. botulinum22 and as a representative of anaerobic spore-forming bacteria. Therefore, in this work,

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we described the bioassay-guided purification of a crude avocado seed extract against C. sporogenes

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vegetative cell growth and endospore germination by the disc diffusion assay, which led to the

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discovery of the chemical nature of the bioactive molecules. Inhibition properties of an enriched

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extract and purified compounds were further characterized against microbial growth from activated

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C. sporogenes endospores by determination of the minimum inhibitory and minimum bactericidal

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concentrations.

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MATERIALS AND METHODS

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Plant material

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Avocados (Persea americana Mill, cv. var. Hass) were obtained by Avomex International, S.A. de

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C.V. (Sabinas, CL, México) from Uruapan, Michoacán, México. Avocados were collected at full

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fruit expansion stage (~20% dry weight), stored and transported unripe under controlled temperature

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conditions (10°C) in containers with fresh air supply. Following commercial practices, avocados

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were treated with 80-100 ppm ethylene for 3 h and considered ripe after 7 d when fruit color and

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softness were adequate (black fruit with a firmness value of < 40 N). Seeds were manually separated

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from the pulp, washed with water, vacuum packed and stored at -80 °C prior to use.

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Chemicals

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Analytical grade methanol, heptane, ethyl acetate, hexane and acetone were purchased from DEQ

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(San Nicolás de los Garza, NL, México). HPLC grade water and methanol were purchased from

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Fisher Scientific Int. (Winnipeg, MB, Canada) and isopropyl alcohol was acquired from Omnisolv

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(Gibbstown, NJ, USA).

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Crude extract preparation and partition

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Unfrozen avocado seeds were ground in a colloidal mill. Ground seed compounds (2 kg) were

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extracted with acetone (1:2 w/v) for 24 h at 25 °C and concentrated at reduced pressure to obtain

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extract F001. Dry extract F001 was then partitioned in biphasic system 1 (heptane:methanol, 1:1 v/v),

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at an extract-to-each-phase of the solvent system of 1:1 w/v. Phases were separated and concentrated

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to yield extracts: F002 (heptane-soluble compounds) and F003 (methanol-soluble compounds).

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Extract F003 was further partitioned in biphasic solvent system 2 (ethyl acetate:water, 1:1 v/v), using

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the previously described procedure to yield extracts: F004 (ethyl acetate-soluble compounds) and

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F005 (water-soluble compounds). Extracts F001 to F005 were adjusted with methanol to 2.5 mg/mL

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of solids and subjected to antimicrobial screening as described below.

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Centrifugal partition chromatography (CPC) fractionation of extract F002

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After assessment of bioactivity, extract F002 was further fractionated in a 1 L CPC system

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(Kromaton Technologies, Angers, ML, France), using heptane:methanol (1:1 v/v) as the solvent

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system. CPC column was, entirely filled with the upper phase of the solvent system to serve as

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stationary phase, and then lower phase was pumped at a 10 mL/min flow rate to establish

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hydrodynamic equilibrium. Extract F002, dissolved in 65 mL of lower phase, was then injected.

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Following the elution-extrusion approach,23 lower phase was used to elute fractions during 170 min,

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and then upper phase was pumped during 100 more min. A total of 240 fractions were collected (10

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mL/fraction), and their corresponding partition coefficients (KD) were calculated accordingly to

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Berthod et al.23 Fractions were individually concentrated to dryness and 70 groups of fractions were

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composed by mixing equal weights of consecutive fractions so that each group had a final

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concentration of 2.5 mg/mL of solids. Bioactivity of the fractions was evaluated by the disc diffusion

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method as described below.

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Purification and further characterization of active compounds

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Chromatographic profile analysis

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Antimicrobial screening identified the most potent CPC fractions against C. sporogenes. Bioactive

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fractions were then mixed together to obtain an enriched active fraction (EAF) extract. The extract

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was then adjusted to 1 mg/mL of solids and injected (2 µL) into two independent analytical Agilent

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1100 HPLC systems (Agilent Technologies, Santa Clara, CA, USA), one of the instruments coupled

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to a photodiode (PDA) detector G1315D and the other to a MS-TOF detector G1969A, the latter

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equipped with an electrospray ionization interface (ESI). Separation was performed in a 100 x 3 mm

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i.d., 3.5 µm Zorbax Extend-C18 column (Agilent Technologies, Santa Clara, CA, USA), with mobile

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phases that consisted of water 100% (A) and methanol 100% (B) with 0.1% formic acid when used

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for LC-MS. Solvent was pumped at 0.38 mL/min using a gradient of 0-2 min, 70-85% B linear; 4-22

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min, 85-90% B linear; 22-24 min, 90-100% B linear; 24-26, 100% B isocratic, followed by 6 min of

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re-equilibration. ESI conditions included analysis in positive-ion mode; N2 as nebulizing gas at 45

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psi; drying gas flow rate and temperature of 8 L/min and 300 °C, respectively; voltage at the capillary

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entrance set at 2500 V; fragmentation, skimmer and Oct RFV voltages at 100, 60 and 250 V,

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respectively. PDA detection at 220 nm produced a chromatograph with seven peaks (eluting at 7.1,

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10.0, 12.1, 12.5, 13.5, 15.0 and 15.8 min, respectively) labeled as peaks A, B, C, D, E, F and G,

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respectively. Peaks B-F were selected for further purification.

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Preparative isolation of chromatographic peaks

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Initial isolation of peaks contained in EAF extract (at 50 mg/mL of solids) was achieved in a

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preparative Agilent 1100 HPLC system, equipped with a PDA detector G1315B and fraction

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collector G1364B. The column used was a 250 x 20 mm i.d., 5 µm, Phenomenex Prodigy C18

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(Torrance, CA, USA) and the mobile phases were water 100% (A) and methanol 100% (B) at a flow

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rate of 20 mL/min. Solvent gradient was: 0-4 min, 70-85% B linear; 4-22 min, 85-90% B linear; 22-

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24 min, 90-100% B linear; 32-35 min, 100% B isocratic; followed by 10 min of re-equilibration.

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Detection at 220 nm produced a similar chromatograph to the one obtained in the previous

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chromatographic profile analysis, from which peaks previously labeled as B, C, D, E and F were

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collected from each run. Peaks were independently pooled, concentrated in a stream of nitrogen and

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stored at -20 °C until use. HPLC-MS analysis of the isolated peaks showed that in most cases, further

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purification was needed to obtain pure compounds.

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Final purification of active compounds

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Peak B contained a single component (compound 1); therefore, additional purification was not

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necessary. Purification of all the other peaks was conducted in a Phenomenex Synergi Hydro-RP

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column (250 x 4.6 mm i.d., 4 µm), using water 100% and methanol 100% as mobile phases (A and

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B, respectively) at a flow rate of 1 mL/min. Isocratic methods were optimized for each peak.

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Individually, peaks C-F were dissolved in methanol HPLC grade, and then injected into a semi-

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preparative Agilent 1100 HPLC system coupled to a PDA detector G1314A and fraction collector

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G1364C. Compounds 2, 3 and 4 were recovered from peaks C, D and E, respectively, whereas peak

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F, was resolved in two major components, designated as compounds 5 and 6, which were co-eluting

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under the method described in the chromatographic profile analysis section. Each isolated compound

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was collected separately, concentrated and immediately stored at -20 °C in amber vials until use. The

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antimicrobial activity of the EAF extract and pure compounds 1-6 at 0.5 mg/mL of solids was

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evaluated as by the disc diffusion method as described subsequently. Antimicrobial activity of the

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EAF extract and pure compounds 1-6 at 0.5 mg/mL of solids was evaluated as described below.

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Nuclear magnetic resonance (NMR) spectroscopy

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The 1H-NMR and

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compounds were performed on a 500 MHz Avance III NMR spectrometer from Bruker

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(Rheinstetten, Germany). CDCl3 was used as solvent, and chemical shifts were referenced to the

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solvent signal.

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+ Persediene (2): colourless oil; α  +1.10° (c 0.22, CHCl3); LC-MS (ESI ) m/z (rel. int): 727

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[2M+Na]+ (15), 375 [M+Na]+ (100), 353 [M+H]+ (40), 335 [M+H-H2O]+ (15), 293 [M+H-HOAc]+

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(23), 275 [M+H-HOAc-H2O]+ (8); LC-MS-TOF: m/z 353.2706 [M+H]+ (calcd. for C21H37O4,

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353.2692); 1H and 13C NMR spectroscopic data is presented in Table 1.

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+ Persenone-C (3): colourless oil; α  +9.50° (c 0.22, CHCl3); LC-MS (ESI ) m/z (rel. int): 727

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[2M+Na]+ (28), 375 [M+Na] +(67), 353 [M+H]+ (100), 335 [M+H-H2O]+ (22), 275 [M-HOAc-

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H2O+H]+ (11); LC-MS -TOF: m/z 353.2708 [M+H]+ (calcd. for C21H37O4, 353.2692); for 1H and 13C

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NMR spectroscopic data see Table 1.

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Quantitation of compounds in the enriched active fraction (EAF) extract

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The compounds in the EAF extract were quantitated by external calibration in triplicate using the

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analytical HPLC-PDA method described in the chromatographic profile analysis section. Standard

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curves were constructed using compounds isolated and purified in our laboratory from avocado seeds

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(>96% purity). Data was expressed as mg of individual compound per mg of total solids, on a dry

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weight basis.

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Determination of antimicrobial activity

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C-NMR, COSY, HSQC and HMBC spectroscopic experiments for the isolated

Preparation of vegetative cells and endospore suspensions

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Putrefactive anaerobe Clostridium sporogenes PA 3679 (ATCC 7955) was obtained from the

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American Type Culture Collection (Manassas, VA, USA). Vegetative cell cultures were grown in

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trypticase-peptone-glucose-yeast extract (TPGY) medium at 37 °C for 24 h in a Difco Laboratories

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GasPak anaerobic jar (Sparks, MD, USA). TPGY medium consisted of (per liter of distilled water) 50

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g tryptone, 5 g peptone, 4 g dextrose, 20 g yeast extract and 1 g sodium thioglycolate.24 Endospore

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cultures were prepared by growing C. sporogenes in reinforced clostridial medium (RCM) at 37 °C

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for 14 d in an anaerobic jar. After, endospores were harvested by centrifugation at 2862 X g for 20

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min at 22 ºC and resuspended in sterile 1X phosphate buffered saline (PBS). The suspension received

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ultrasonic treatment for 15 min to remove endospores from mother cells and, by washing several

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times with 1X PBS, a suspension primarily consisting of endospores was obtained, which was

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confirmed by phase-contrast microscopy.25 All microbiology reagents were purchased from Difco

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Laboratories (Sparks, MD, USA).

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Disc diffusion method

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For antimicrobial screening of avocado seed extracts at different stages of purification, C. sporogenes

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stocks of vegetative cells or endospore suspensions were adjusted to an initial optical density (OD) at

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600 nm of 0.1, which corresponded to an initial average cell concentration of 8 X 106 CFU/mL. In

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addition, the endospore suspension received a heat-shock treatment (80 ºC for 15 min) to stimulate

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spore germination and inactivate any remaining vegetative cells.24 After treatment, the suspension

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was placed in ice before inoculating the plates. Aliquots (100 µL) of vegetative cells or endospore

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suspensions were evenly spread with a sterile plastic rod in the surface of TPGY agar plates. Sterile

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filter paper discs (6 mm diameter) were impregnated with aliquots (5 µL) of the extracts and air-dried

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for approximately 2 h in a biological safety cabinet before placing the discs on the inoculated plates.

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A disc impregnated with ethanol or methanol (5 µL) was used as a solvent control for the EAF extract

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and purified compounds, respectively. Nisaplin (Danisco, Beaminster, DO, UK), which contains

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2.5% w/w of nisin, served as a positive control. A stock solution of 50 mg/mL Nisaplin in sterile

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water was prepared to obtain in 5 uL a residual concentration of 6.25 µg of nisin per disc. All samples

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were assayed in triplicates. Each plate contained six discs evenly spaced; four tested samples and the

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positive and solvent controls. Plates were incubated for 36 h at 37 °C in an anaerobic jar.

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Subsequently, diameters of inhibition zones were measured to the nearest millimeter.

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Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of

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activated endospore suspensions

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Further characterization of C. sporogenes microbial growth from activated endospores by the EAF

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extract and isolated compounds 3-5 was performed in liquid medium by determining the MIC and

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MBC. Serial two-fold dilutions of the samples were prepared in test tubes with TPGY broth to a final

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volume of 1 mL. Each tube was inoculated (100 uL) with an endospore suspension adjusted to an

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initial OD at 600 nm of 0.1, as previously described. The EAF extract in ethanol was tested at

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concentrations of 5 to 2500 µg/mL and purified compounds 3-5 in methanol from 0.5 to 15.6 µg/mL.

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All concentrations were compared to the corresponding concentration of solvent. Neither alcohol

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alone was inhibitory unless the concentration exceeded 3.12%; therefore, results are presented for

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samples with solvent concentrations below this limit. A growth control (no antimicrobial added), a

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non-inoculated tube (aseptic technique control) and a positive control (nisin) were also contemplated.

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After 18-24 h of anaerobic incubation at 37 ºC, the MIC was defined as the lowest concentration at

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which no endospore outgrowth was assessed by OD at 600 nm.25,28 To evaluate the effect of killing

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of activated endospores suspensions (bactericidal effect), aliquots (100 uL) of cultures at and above

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the MIC were subcultured in antimicrobial-free medium (1 mL TPGY broth) and inoculated for 18-

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24 h at 37 ºC in anaerobic conditions. The MBC was defined as the lowest concentration not showing

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endospore outgrowth determined by OD.25,28 Turbidity was assessed against an appropriate blank

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because samples showed some light absorbance. Concentrations limiting OD to