Isolation and Structure Elucidation of Avocado Seed (Persea

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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

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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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†

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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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‡

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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])

ACS Paragon Plus Environment


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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

Isolation and Structure Elucidation of Avocado Seed (Persea

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