Antioxidant capacities, procyanidins and pigments in avocados of different strains and cultivars

Food Chemistry 122 (2010) 1193–1198

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Antioxidant capacities, procyanidins and pigments in avocados of different strains and cultivars Wei Wang, Terrell R. Bostic, Liwei Gu * Department of Food Science and Human Nutrition, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, United States

a r t i c l e

i n f o

Article history: Received 6 July 2009 Received in revised form 17 March 2010 Accepted 26 March 2010

Keywords: Avocado Antioxidants Flavanols Pigments Procyanidins

a b s t r a c t Avocado (Persea americana) is an important tropical fruit, but little is known about their antioxidant capacities and phytochemical composition. The objective of this research was to determine antioxidant capacities, total phenolic content and identify and quantify major antioxidant compounds in avocados of different strains and cultivars. Ripe Florida avocados from seven cultivars (Slimcado, Booth 7, Booth 8, Choquette, Loretta, Simmonds, and Tonnage) of West Indian or Guatemalan strains were separated into seeds, peels and pulp, and freeze dried. Hass avocado of Mexican strain was chosen for comparison. Samples were extracted with acetone/water/acetic acid solvent and analysed using the Folin–Ciocalteu assay for total phenolic content. Antioxidant capacities were determined by oxygen radical absorbance capacity (ORAC) and DPPH assays. Procyanidins were identified and quantified using HPLC-MSn. Antioxidant pigments (chlorophylls and carotenoids) were estimated spectrophotometrically. For all varieties, seeds contained the highest antioxidant capacities, phenolic content, and procyanidins, whereas the pulp had the lowest. Procyanidins, including catechin, epicatechin, A- and B-type dimers, A- and B-type trimers, tetramers, pentamers and hexamers, were identified in peels and seeds using normal-phase HPLC–ESI-MSn. Antioxidant capacities, phenolic contents and procyanidins in avocados were highly correlated, suggesting that procyanidins were the major phenolic compounds that contributed to antioxidant capacities. Carotenoids and chlorophylls were found to be concentrated in avocado peels but did not correlate with antioxidant capacities. This study suggested that avocado seeds and peels, industrial wastes of avocado processing, can be exploited as source of antioxidants. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Avocado (Persea americana) is an important commercial tropical fruit. Botanically, avocados can be classified into four strains: West Indian, Guatemalan, West Indian–Guatemalan hybrid, and Mexican (Edward Evans, 2006; Morton, 1987). The well-known Hass avocado is a Mexican strain and grows primarily in Mexico and California. Avocados of the other three strains grow in Florida, Puerto Rico, the Dominican Republic and other Caribbean countries, where the high humidity is not suitable for Hass. The West Indian and Guatemalan avocados have less fat content than the Hass avocado (Gomez-Lopez, 1999), and are often marketed under trademark name ‘‘Slimcado” or simply referred as ‘‘Florida avocado” in the United States. Florida avocados are easily distinguishable from Hass by having much larger fruit sizes and smooth green skins. About 60 varieties of avocados are grown in Florida and the

* Corresponding author. Address: Food Science and Human Nutrition, P.O. Box 110370, Newell Drive, University of Florida, Gainesville, FL 32611, United States. Tel.: +1 352 392 1991x210; fax: +1 352 392 9467. E-mail address: LGu@ufl.edu (L. Gu). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.114

fruits are harvested from May to February. Each year, Florida produces approximately 10%, about 200,000 tonnes, of the avocados for the total avocados produced in the United States (Crane, Evans, & Balerdi, 2007). A unique feature of avocados is that the fruits mature on the tree and ripen after harvest. The ripening process takes 5–7 days under room temperature. The fruits are ripe when they yield to gentle pressure (Ozdemir & Topuz, 2004). Avocados are rich in unsaturated fatty acids, fibre, vitamins B and E, and other nutrients (GomezLopez, 1998). Studies on avocados showed that they contained potentially anti-carcinogenic lipophilic components such as carotenoids (Ding, Chin, Kinghorn, & D’Ambrosio, 2007). The lipophilic extract of avocado inhibited prostate cancer cell growth (Lu et al., 2005), induced apoptosis in human breast cancer cells (Butt et al., 2006), and suppressed liver injury (Kawagishi et al., 2001). A few studies have focused on the phytochemical compositions of avocados. There is no knowledge about the total phenolic content and antioxidant capacities among avocados from different strains and cultivars. Industrial processing of avocados generates a large amount of peels and seeds as wastes. Exploiting the phytochemicals content of these wastes may lead to new products and add value to the avocado industry. Therefore,

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the objective of this research was to determine the antioxidant capacities, phenolic content, and quantify the major antioxidant phytochemicals in the seeds, pulp, and peels of different avocados.

the antioxidant capacity of fruit extracts were expressed as lmol Trolox equivalents (TE) per gram of samples on the basis of fresh weight (lmol TE/g).

2. Materials and methods

2.6. DPPH assay

2.1. Chemicals

The DPPH scavenging activities of avocado samples were measured using a published method (Brand-Williams, Cuvelier, & Berset, 1995). DPPH stock solution was prepared by dissolving 20 mg of DPPH in 100 ml methanol and stored at 20 °C prior to use. DPPH working solution was freshly prepared by mixing 3.5 ml DPPH stock solution and 6.5 ml methanol. Absorbance at 515 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA). The initial absorbance of DPPH working solution was between 0.9 and 1.0. Diluted avocado extracts (50 ll) were added to 950 ll DPPH working solution and incubated in darkness for 60 min. Trolox solutions from 100 to 1000 lM were added to DPPH working solution as standards. The results of the DPPH scavenging activity of fruit extracts were expressed as lmol Trolox equivalents per gram of samples on the basis of fresh weight (lmol TE/g).

AAPH (2,20 -azotis(2-amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich (St. Louis, MO). Folin–Ciocalteu reagent and others chemicals were products of Fisher Scientific (Pittsburg, PA). Purified cocoa procyanidins that contained monomer through decamers were a gift from Mars Inc. (McLean, VA). A polymeric procyanidin fraction with an average degree of polymerisation (DP) of 36.1 was used as a polymer standard. This polymer fraction did not contain procyanidins with DP 6 10. Characterisation of this polymer fraction has been described previously (Gu et al., 2002). 2.2. Sample preparation

2.7. HPLC–ESI-MSn analyses of procyanidins Ripe fruits of six cultivars of Florida avocados were obtained from local growers in south Florida. One variety of Florida avocado with trademark ‘‘Slimcado” was purchased from a local grocery store. Hass avocado was a product of Mexico and was purchased from a local grocery store. All seven varieties of Florida avocado were harvested from July through October of 2008. The harvesting time of the Hass avocado was not known. All fruits were kept in room temperature until ripe. Fruits were manually separated into peel, seed and pulp, and then freeze dried. The moisture losses were recorded. Freeze dried fruit portions were ground into powder (90% passed a standard 40-mesh sieve) for solvent extraction. 2.3. Polyphenol extraction One gram of pulp samples or 0.5 g of seeds or peels were extracted in 10 ml of acetone/water/acetic acid (70:29.7:0.3, v/v/v) solvent. The extraction tubes were vortexed for 30 s and sonicated for 5 min, and kept at room temperature for 20 min and sonicated for another 5 min. The tubes were centrifuged at 1277g for 10 min. Supernatants were kept at 20 °C for phenolic content, antioxidant capacity and procyanidin analyses.

An Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Avocado extracts were filtered through 0.45 lm filter units and injected without further purification. Procyanidins were separated on a 250  4.6 mm Phenomenex Luna Silica (2) column (Phenomenex, Torrance, CA). The binary mobile phase consisted of (A) methylene chloride/methanol/acetic acid/ water (82:14:2:2, v/v/v/v) and (B) methanol/acetic acid/ water (96:2:2, v/v/v). The 70 min gradient was as follows: 020 min, 0.011.7% B linear; 2050 min, 11.725.6% B linear; 5055 min, 25.687.8% B linear; 5565 min, 87.8% B isocratic; 6570 min, 87.80.0% B linear; followed by 5 min of re-equilibration of the column before the next run. Electrospray ionisation at negative mode was performed using nebuliser 50 psi, drying gas 10 l/min, and drying temperature 350 °C, capillary 4000 V (Gu, Kelm, Hammerstone, Zhang, et al., 2003). Excitation and emission of the fluorescent detector were set at 231 and 320 nm, respectively (Robbins et al., 2009). Purified cocoa or blueberry procyanidins were used to generate standard curves for oligomers and polymers. Data were collected and calculated using the Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA).

2.4. Folin–Ciocalteu assay 2.8. Pigment extraction and estimation Total phenolic content of each sample was determined by the Folin–Ciocalteu assay. Fruit extracts were mixed with diluted Folin–Ciocalteu reagent and 15% sodium carbonate. Absorbance at 765 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA) after 30 min of incubation at room temperature. Gallic acid was used to generate a standard curve. Results of total phenolics for fruit extracts were expressed as milligram gallic acid equivalents (GAE) per gram of samples on the basis of fresh weight (mg GAE/g). 2.5. Oxygen radical absorbance capacity (ORAC) assay Fruit extracts were incubated with fluorescein as a free radical probe and AAPH as a free radical generator (Prior et al., 2003). The kinetics of fluorescein degradation was read on a Spectra XMS Gemini microplate reader (Molecular Devices, Sunnyvale, CA). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used to generate a standard curve. The results of

A method reported by Lichtenthaler et al. was adopted with minor modification (Lichtenthaler & Wellburn, 1983). Freeze dried samples (0.5 g) were extracted in 10 ml of chloroform/methanol solvent (2:1, v/v). The extraction tubes were vortexed for 30 s and sonicated for 2 min, and kept on ice for 20 min. After filtration, 4 ml of extract was dried under vacuum in a Speedvac (ISS110, Fisher Scientific, Pittsburg, PA). The dried extracts were dissolved in 2 ml of 80% of acetone and sonicated for 30 s. The extracts were centrifuged at 2095g for 5 min. Absorbance of supernatants was measured at 663, 646, and 470 nm on a UV/Vis spectrophotometer (DU 370, Beckman Coulter, Fullerton CA) using 80% of acetone as a blank. Chlorophyll a and b concentration (lg/g) were calculated using 12.21  A663  2.81  A646 and 20.13  A646  5.03  A663, respectively. Total chlorophyll concentration (lg/g) was the sum of chlorophylls a and b. Total carotenoid content (lg/g) was calculated using A1% 1cm ¼ 2290 at 470 nm reported by Lichtenthaler and Wellburn (1983).

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2.9. Statistical and multivariate analyses

Table 2 Total phenolic content and antioxidant capacities in avocados of different cultivars.

Samples were analysed in triplicates and data were expressed as mean ± standard deviation unless noted otherwise. One-way analyses of variance with Tukey–Kramer pair-wise comparison of the means were performed using JMP software (Version 7.0, SAS Institute Inc., Cary, NC). A difference of p 6 0.05 was considered as significant. Hierarchical clustering of avocado cultivars were done with the same software using phenolic content, antioxidant capacity (average of ORAC and DPPH assay), procyanidin content, chlorophyll and carotenoid content in seeds and peels as variables. 3. Results

Portions

Cultivars

Total phenolic content (mg GAE/ g)

ORAC (lmol TE/g)

DPPH (lmol TE/g)

Seeds

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

19.2 ± 3.3d 40.2 ± 1.9b 31.5 ± 2.2c 33.4 ± 1.5bc 33.4 ± 5.0bc 35.7 ± 0.8bc 33.1 ± 0.9bc 51.6 ± 1.6a

229.0 ± 16.2d 459.3 ± 56.6a 299.0 ± 17.6cd 348.9 ± 43.4bc 319.8 ± 42.2cd 368.7 ± 23.8abc 464.4 ± 42.1a 428.8 ± 12.0ab

128.3 ± 17.4c 240.2 ± 36.6a 159.7 ± 18.5bc 157.8 ± 13.4bc 188.1 ± 15.0ab 207.3 ± 30.6ab 162.9 ± 8.1bc 164.6 ± 5.1bc

Peels

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

4.6 ± 0.3c 7.4 ± 0.2b 7.6 ± 0.8b 13.9 ± 1.1a 13.2 ± 0.2a 8.1 ± 0.6b 4.3 ± 0.1c 12.6 ± 0.3a

58.2 ± 1.3e 226.8 ± 12.2b 92.6 ± 9.5de 174.8 ± 11.9c 164.9 ± 2.9c 110.5 ± 6.9d 187.6 ± 39.8bc 631.4 ± 4.2a

39.7 ± 5.3c 84.9 ± 5.0b 38.0 ± 2.3c 90.8 ± 5.4b 80.0 ± 6.5b 52.6 ± 1.7c 51.9 ± 4.0c 189.8 ± 10.8a

Pulp

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

1.0 ± 0.1b 0.6 ± 0.1b 1.0 ± 0.1b 0.6 ± 0.0b 1.3 ± 0.0b 1.2 ± 0.1b 0.8 ± 0.0b 4.9 ± 0.7a

4.7 ± 0.2bc 4.1 ± 0.4c 3.9 ± 0.5c 2.6 ± 0.2d 4.4 ± 0.5bc 5.1 ± 0.4bc 5.6 ± 0.7b 11.6 ± 0.4a

3.1. Fruit characterisation Five cultivars of West Indian–Guatemalan strain, one cultivar of West Indian, and a Mexican avocado were studied. The avocado with trademark name ‘‘Slimcado” is a Florida avocado of unknown cultivar (Table 1). Fruit sizes of non-Hass avocado ranged from 283 to 860 g and were much larger than the Hass (174 g). On average, 18% and 13% of a fresh avocado are peels and seeds, respectively. The edible pulp portion was 69% of whole fruit. The pulp, peels, and seeds lost 85%, 78%, and 68% of weight on average as moisture after freeze drying. 3.2. Total phenolic content and antioxidant capacities For all cultivars, seeds contained the highest total phenolic content and antioxidant capacities, whereas the pulp had the lowest (Table 2). Total phenolic content in the seeds ranged from 19.2 to 51.6 GAE mg/g. Hass avocado peel and pulp contained higher phenolic content and antioxidant capacities than all the non-Hass cultivars. Among the Florida avocados tested, Simmonds had the highest phenolic content and antioxidant capacities in the seeds and peels. The antioxidant capacity determined by DPPH assay was about half of the values determined by ORAC. This is likely caused by the differences in the chemistry of these two methods. ORAC assay is based on a hydrogen atom transfer reaction, whereas DPPH assay uses an electron transfer mechanism (Huang, Ou, & Prior, 2005). 3.3. Procyanidin identification and quantitation The chromatograms of procyanidins in Choquette avocado are shown in Fig. 1. B-type procyanidin oligomers (monomers to octamers) and polymers were identified from seeds and peels accord-

Table 1 Fruit, seed, and peel weight of fresh avocados of different strains and cultivars. Cultivar

Slimcadoa Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass a b

Strain

West Indian West Indian– Guatemalan West Indian– Guatemalan West Indian– Guatemalan West Indian– Guatemalan West Indian– Guatemalan Mexican

Fruit weight (g)b

Peel weight (% fruit weight)b

Seed weight (% fruit weight)b

769 587 860

15.2 14.2 15.7

14.4 7.0 6.2

640

18.3

13.9

378

20.9

18.3

283

24.4

16.4

433

20.4

16.6

174

17.1

13.8

Trademark name for any Florida avocado. Data were averages of 2–4 fruits for each cultivar.

1.3 ± 0.2a 0.7 ± 0.0c 0.4 ± 0.1de 0.4 ± 0.0e 1.1 ± 0.1ab 0.6 ± 0.1cd 1.0 ± 0.0b 1.3 ± 0.1a

Data are mean ± standard deviation on fresh weight basis. Data for the same type of sample in the same column with different superscript differed significantly (p 6 0.05).

ing to mass spectra and comparing to the retention time of the external standards. A B-type trimer eluted at 22.4 min and had a deprotonated molecular ion of 865.3m/z. The product ion spectrum of this trimer is shown as insert A in Fig. 1. An A-type trimer eluted at 21.9 min and had a deprotonated molecular ion of 863.3m/z. Its product ion spectrum is depicted as insert B in Fig. 1. The A-type linkage in an A-type procyanin trimer can be present between top flavanols unit and middle unit or between middle unit and terminal unit. According to the fragmentation patterns of procyanidin oligomers reported in our previous publication (Gu, Kelm, Hammerstone, Zhang, et al., 2003), the position of A-type linkage in A-type trimers in avocado was determined to be between the top flavanol unit and the middle unit. A peak eluted at 13 min in the chromatogram of seed extract was not procyanidin on the basis of mass spectrum. Chromatograms of avocado pulp contained many peaks that were not procyanidins. However, compounds in these peaks cannot be identified. B-type procyanidin monomers to tetramers were identified in Choquette avocado pulp (Fig. 1). Table 3 shows procyanidin contents in avocados of different cultivars. The content was higher in seeds than in peels. About 40% of procyanidins in the seeds or peels were polymers with a degree of polymerisation above 10. Procyanidins were not quantifiable in pulp of other avocado cultivars except for Choquette. 3.4. Chlorophyll and carotenoid content The content of chlorophyll a, b, total chlorophylls and total carotenoids are shown in Table 4. Chlorophylls in non-Hass avocados were highly concentrated in peels. Seeds of non-Hass avocados contained less amounts of chlorophyll. By contrast, chlorophyll in Hass avocado appeared to be evenly distributed in the seeds, peels, and pulp. Concentrations of chlorophyll in peels of non-Hass avocado were consistent with their green coloured skins. Tonnage avocado had darker green skin and the chlorophyll content in the peels was significantly higher than other cultivars. Carotenoid contents were the highest in peels and lowest in seeds.

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Fig. 1. Chromatograms of procyanidins in seed, peel, and pulp of Choquette avocado using fluorescent detection (Ex = 231 nm, Em = 320 nm). Numbers 1, 2, 3, to 8 on the peak denote procyanidin monomers, dimmers, trimers, to octomers. 2A and 3A indicate dimmers and trimers with one A-type linkage. Peak with label u was an unidentified peak. Product ion spectra of a B-type procyanidin trimer and an A-type procyanidin trimer are shown in inserts A and B, respectively.

3.5. Correlation among phenolic content, antioxidant capacities, procyanidin and pigment contents The phenolic content, ORAC, DPPH, and procyanidin content in pulp, peels, and seeds were significantly correlated to each other (correlation coefficients r P 0.79). The phenolic content and antioxidant capacities did not correlate with the chlorophyll or carotenoid contents (r < 0.1). The chlorophyll content and carotenoid content were correlated (r = 0.92). When ORAC values and DPPH assay from seeds were correlated, the coefficient (r = 0.57) was lower than that from peels (0.96) or all three portions of avocados (0.94). 3.6. Hierarchical clustering of cultivars Overall similarity of the eight avocado cultivars is depicted in a dendrogram in Fig. 2. Hass avocado of Mexican strain had the least similarity to others. Among the non-Hass avocados, Slimcado stood out from the rest. Choquette, Simmonds, Booth 7, and Booth 8 had similar compositions, and were different from Loretta and Tonnage.

4. Discussion Avocados are eaten raw or processed into products such as Guacamole. Because 18% and 13% of a fresh avocado are peel and seed, respectively, large amounts of industrial waste are produced during avocado processing. Data from this study indicate that avocado peel and seed contained high amounts of phenolic compounds and antioxidant capacities. The phenolic content and antioxidant capacity of avocado seeds and peels are several folds of those reported for raw blueberry (5.3 GAE mg/g and 65 lmol TE/g ORAC), a fruit well known for its higher antioxidant capacity (Wu et al., 2004). The amount of phenolic components in seeds, peels and pulp were 64%, 23%, and 13% of those in a whole fruit. Seeds and peels also contributed 57% and 38% of the antioxidant capacities of a whole fruit. The high phenolic content in avocado seeds had also been reported in a previous study (Soong & Barlow, 2004). Worth noting were the differences in antioxidant capacities measured by two assays of different chemistry and mechanism. ORAC assay applies a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. DPPH assay

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W. Wang et al. / Food Chemistry 122 (2010) 1193–1198 Table 3 Procyanidins in avocados of different cultivars. Portions

Cultivars

Monomer

Dimers

Trimers

4–6mers (mg/g)

7–10mers

Polymers

Total

Seeds

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

1.8 ± 0.1 2.7 ± 0.2 2.3 ± 0.7 2.0 ± 0.1 2.6 ± 0.1 2.7 ± 0.1 1.9 ± 0.1 2.1 ± 0.0

1.7 ± 0.1 2.6 ± 0.2 2.2 ± 0.7 4.3 ± 0.2 4.2 ± 0.2 5.8 ± 0.2 3.0 ± 0.2 4.5 ± 0.0

1.9 ± 0.2 3.9 ± 0.1 2.6 ± 0.1 2.6 ± 0.2 2.9 ± 0.1 3.4 ± 0.1 4.7 ± 0.2 4.1 ± 0.0

5.6 ± 1.6 11.2 ± 0.4 7.6 ± 0.4 7.7 ± 0.2 8.1 ± 0.1 8.5 ± 0.3 12.5 ± 0.3 10.6 ± 0.3

3.6 ± 0.1 9.2 ± 0.6 6.4 ± 0.4 6.3 ± 0.3 7.2 ± 1.1 7.5 ± 0.6 9.9 ± 0.6 8.5 ± 0.4

9.5 ± 1.0 26.6 ± 0.6 16.6 ± 0.6 16.8 ± 0.5 17.8 ± 0.8 18.3 ± 0.3 15.0 ± 0.1 18.6 ± 0.2

23.7 ± 1.0f 55.6 ± 1.8a 37.2 ± 1.1e 39.1 ± 1.4de 42.2 ± 2.1cd 45.3 ± 1.2bc 46.4 ± 1.5b 47.7 ± 0.6b

Peels

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

0.4 ± 0.0 1.5 ± 0.0 0.4 ± 0.0 0.8 ± 0.0 0.9 ± 0.0 0.6 ± 0.0 0.8 ± 0.0 3.4 ± 0.0

0.3 ± 0.0 1.1 ± 0.1 0.7 ± 0.0 1.5 ± 0.0 1.3 ± 0.0 0.8 ± 0.0 1.1 ± 0.0 5.5 ± 0.2

0.2 ± 0.0 0.9 ± 0.1 0.5 ± 0.0 1.1 ± 0.0 1.0 ± 0.0 0.6 ± 0.0 0.8 ± 0.0 4.4 ± 0.3

0.8 ± 0.1 3.2 ± 0.3 1.7 ± 0.1 3.6 ± 0.0 3.3 ± 0.0 1.9 ± 0.0 2.3 ± 0.0 11.9 ± 1.1

0.4 ± 0.1 1.8 ± 0.5 1.0 ± 0.0 2.2 ± 0.1 2.1 ± 0.1 1.2 ± 0.0 1.5 ± 0.2 6.7 ± 1.5

2.7 ± 0.2 8.3 ± 0.7 4.3 ± 0.2 7.0 ± 0.2 6.6 ± 0.2 4.9 ± 0.2 7.1 ± 0.5 7.0 ± 0.7

4.9 ± 0.5d 16.8 ± 0.5a 8.6 ± 0.5c 16.2 ± 0.5a 15.0 ± 0.5a 10.0 ± 0.5c 15.0 ± 0.5c 38.9 ± 0.5a

Pulp

Choquettea

0.3 ± 0.0

0.3 ± 0.1

0.3 ± 0.1

0.2 ± 0.0

ND

ND

1.1 ± 0.2

Data are mean ± standard deviation on fresh weight basis. Data for the same type of sample in the same column with different superscript differed significantly (p 6 0.05). ND, not detected. a Sample was analysed in duplicates; Procyanidins were not quantifiable in pulp of other cultivars.

Table 4 Pigment content in avocados of different cultivars. Portions

Cultivars

Chlorophyll a (lg/g)

Chlorophyll b (lg/g)

Total chlorophyll (lg/g)

Seeds

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnagea Hassa

0.1 ± 0.0b 0.5 ± 0.1b 0.4 ± 0.1b 1.1 ± 0.1b 0.9 ± 0.1b 0.9 ± 0.2b 1.1 ± 0.9b 21.0 ± 4.0a

0.1 ± 0.1b 0.8 ± 0.2b 0.7 ± 0.2b 2.0 ± 0.1b 1.3 ± 0.2b 1.7 ± 0.2b 1.9 ± 1.5b 20.2 ± 1.8a

0.2 ± 0.1b 1.3 ± 0.2b 1.1 ± 0.4b 3.0 ± 0.2b 2.1 ± 0.2b 2.6 ± 0.4b 3.0 ± 2.5b 41.2 ± 5.7a

Total carotenoids (lg/g)

Peel

Slimcado Simmonds Loretta Choquette Booth 7 Booth 8 Tonnage Hass

28.7 ± 0.9 bcd 22.7 ± 4.6cde 33.4 ± 0.9b 30.9 ± 2.9bc 18.6 ± 2.1e 21.9 ± 2.3cde 47.5 ± 6.4a 19.2 ± 3.6de

6.2 ± 0.9c 14.0 ± 2.3ab 13.9 ± 1.9ab 11.2 ± 0.6bc 6.5 ± 1.0c 9.6 ± 2.3bc 19.4 ± 2.4a 9.5 ± 2.9bc

34.8 ± 1.7bcd 36.7 ± 6.8bcd 47.4 ± 2.9b 42.1 ± 3.6bc 25.1 ± 2.6d 31.5 ± 4.6cd 66.9 ± 8.9a 28.8 ± 6.2cd

9.3 ± 1.1c 12.8 ± 2.9abc 17.3 ± 1.3a 11.5 ± 1.1bc 8.9 ± 0.7c 10.4 ± 1.8bc 17.7 ± 1.6a 15.2 ± 2.7ab

Pulp

Slimcado Simmonds Lorettaa Choquette Booth 7 Booth 8 Tonnage Hass

1.8 ± 0.1bc 2.9 ± 0.5bc 1.4 ± 0.3bc 0.5 ± 0.3c 2.1 ± 0.5bc 2.7 ± 0.7bc 3.9 ± 0.4b 14.8 ± 1.9a

1.9 ± 0.1b 3.2 ± 0.6b 1.4 ± 0.5b 1.8 ± 0.5b 1.7 ± 0.4b 3.0 ± 1.1b 4.3 ± 0.8b 13.8 ± 4.9a

3.7 ± 0.2bc 6.1 ± 1.0bc 2.7 ± 0.7c 3.3 ± 0.8c 3.9 ± 0.8bc 5.8 ± 1.8bc 8.2 ± 1.2b 28.7 ± 3.3a

1.5 ± 0.2d 4.2 ± 0.4b 2.4 ± 0.4cd 1.6 ± 0.2d 2.2 ± 0.2cd 3.4 ± 0.7bc 4.7 ± 0.6b 7.1 ± 0.6a

0.7 ± 0.1c 1.5 ± 0.1bc 1.2 ± 0.2bc 2.1 ± 0.1b 2.1 ± 0.3b 1.8 ± 0.2bc 1.8 ± 0.7bc 6.3 ± 0.9a

Data are mean ± standard deviation on fresh weight basis. Data for the same type of sample in the same column with different superscript differed significantly (p 6 0.05). a Samples were analysed in duplicates.

Fig. 2. Dendrogram of avocado cultivars using hierarchical cluster analyses.

measures the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced (Huang et al., 2005). Nevertheless, ORAC values were highly correlated with DPPH assays, and they all correlated with total phenolic content in the pulp, peels and seeds. The correlation of antioxidant capacities and total phenolic content in foods were widely observed (Shan, Cai, Sun, & Corke, 2005; Velioglu, Mazza, Gao, & Oomah, 1998). A comparative study correlated the ORAC values with antioxidant capacities measured by ferric reducing antioxidant power (FRAP, another electron-transfer assay) and found a wide range of correlation coefficients (r = 0.00–0.96) in different vegetables (Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002). The degrees of correlation were associated with phytochemical compositions and appeared to be higher in vegetables with a wider span of antioxidant capacities. These are consistent with our observations in avocados.

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Procyanidins are the oligomers and polymers of flavanols and are one of the major polyphenol compounds in foods. Procyanidins from grape seeds or cocoa have been reported to offer many health benefits, such as preventing cancers and other chronic diseases (Prior & Gu, 2005). Avocado seeds had been used to extract A- or B-type procyanidins three decades ago (Acques & Aslam, 1973), yet their procyanidin content and profiles had not been reported. Our data suggested that a small percentage of procyanidins in avocado seeds and peels contained A-type linkage. This was consistent with our previous observations (Gu, Kelm, Hammerstone, Beecher, et al., 2003). B- and A-type procyanidin trimers have molecular weight of 866 and 864, respectively. Thus A-type procyanidin trimers can be distinguished from B-type by their deprotonated ion 863m/z (insert B in Fig. 1). Based on the characteristic product ions 573.1m/z and 289.0m/z and the fragmentation patterns (Gu, Kelm, Hammerstone, Zhang, et al., 2003), the position of the A-type bond was determined to be between the top flavanol unit and the middle unit. The procyanidin content in avocado seeds and peels were comparable to those found in natural cocoa powder and dark chocolates which have been known for their high procyanidin content (Gu, House, Wu, Ou, & Prior, 2006). The existence of A-type procyanidins in avocado may cause additional health benefits because A-type procyanidins found in cranberries have been suggested to prevent urinary tract infections (Howell et al., 2005). High correlation of procyanidin content with phenolic content and antioxidant capacities suggested that procyanidins were the major polyphenols in avocado that contributed to their antioxidant capacities. Chlorophylls are the most ubiquitous pigments in the plant and possess potent antioxidant activities (Lanfer-Marquez, Barros, & Sinnecker, 2005). Carotenoids are among the best known antioxidants and precursors of vitamin A. Pigments in avocados are concentrated in the skins and the colours decreased in pulp and seeds. The green colour of non-Hass avocados were due to high chlorophyll content in the peel, whereas the dark brown colour of the Hass avocado was caused in part by anthocyanins (Ashton et al., 2006). Carotenoids in avocado have been reported to consist of zeaxanthin, lutein, a- and b-carotene. Total carotenoid content in peels and pulp observed in this research was consistent with a previous study (Ashton et al., 2006). Similar content of chlorophyll in the pulp of Hass avocado was also reported (Ashton et al., 2006). The chlorophyll and carotenoid contents in peels and seeds were much lower than those of procyanidins. The low correlation between pigment contents and antioxidant capacity also indicated that pigments were not the major antioxidant compounds in avocado. Avocados from different strains and cultivars differed in antioxidant capacities and phytochemical content. Hierarchical cluster analysis was useful in revealing the overall likeness of different cultivars. The dendrogram may help to illustrate taxonomical differences or phytochemical genealogy of these cultivars. 5. Conclusions Avocado seeds and peels of different cultivars were found to contain high levels of procyanidins as major phenolic compounds and antioxidants. Procyanidins in avocados were predominantly B-type with A-type as minor components. Chlorophyll and carotenoids were concentrated in the peels but were not the major antioxidant compounds. Avocado seeds and peels generated from avocado processing may be used as sources of phenolic nutraceuticals. Overall cultivar differences were illustrated by Hierarchical cluster analyses. References Acques, D. J., & Aslam, E. H. (1973). Structure of the dirneric proanthocyanidin-A2 and its derivatives. Journal of the Chemical Society, Chemical Communications, 1, 518–520.

Ashton, O. B. O., Wong, M., McGhie, T. K., Vather, R., Wang, Y., Requejo-Jackman, C., et al. (2006). Pigments in avocado tissue and oil. Journal of Agricultural and Food Chemistry, 54(26), 10151–10158. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT – Food Science and Technology, 28(1), 25–30. Butt, A. J., Roberts, C. G., Seawright, A. A., Oelrichs, P. B., Macleod, J. K., Liaw, T. Y., et al. (2006). A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells. Molecular Cancer Therapeutics, 5(9), 2300–2309. Crane, J., Evans, E., & Balerdi, C. (2007). A review of the Florida avocado industry. In Proceedings VI world avocado congress (Vols. 12–16). Chile. Ding, H., Chin, Y. W., Kinghorn, A. D., & D’Ambrosio, S. M. (2007). Chemopreventive characteristics of avocado fruit. Seminars in Cancer Biology, 17(5), 386–394. Edward Evans, S. N. (2006). World, US and Florida avocado situation and outlook. Electronic data information source (Vol. FE639). Food and Resource Economics Department. GomezLopez, V. M. (1998). Characterization of avocado (Persea americana Mill.) varieties of very low oil content. Journal of Agricultural and Food Chemistry, 46(9), 3643–3647. Gomez-Lopez, V. M. (1999). Characterization of avocado (Persea americana Mill.) varieties of low oil content. Journal of Agricultural and Food Chemistry, 47(7), 2707–2710. Gu, L., House, S. E., Wu, X., Ou, B., & Prior, R. L. (2006). Procyanidin and catechin contents and antioxidant capacity of cocoa and chocolate products. Journal of Agricultural and Food Chemistry, 54(11), 4057–4061. Gu, L., Kelm, M., Hammerstone, J. F., Beecher, G., Cunningham, D., Vannozzi, S., et al. (2002). Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normalphase HPLC-MS fluorescent detection method. Journal of Agricultural and Food Chemistry, 50(17), 4852–4860. Gu, L., Kelm, M. A., Hammerstone, J. F., Beecher, G., Holden, J., Haytowitz, D., et al. (2003). Screening of foods containing proanthocyanidins and their structural characterization using LC–MS/MS and thiolytic degradation. Journal of Agricultural and Food Chemistry, 51(25), 7513–7521. Gu, L., Kelm, M. A., Hammerstone, J. F., Zhang, Z., Beecher, G., Holden, J., et al. (2003). Liquid chromatographic/electrospray ionization mass spectrometric studies of proanthocyanidins in foods. Journal of Mass Spectrometry, 38(12), 1272–1280. Howell, A. B., Reed, J. D., Krueger, C. G., Winterbottom, R., Cunningham, D. G., & Leahy, M. (2005). A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry, 66(18), 2281–2291. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841–1856. 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(5), 2215–2221. Lanfer-Marquez, U. M., Barros, R. M. C., & Sinnecker, P. (2005). Antioxidant activity of chlorophylls and their derivatives. Food Research International, 38(8–9), 885–891. Lichtenthaler, H. K., & Wellburn, A. R. (1983). Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions, 11, 591–592. Lu, Q. Y., Arteaga, J. R., Zhang, Q., Huerta, S., Go, V. L., & Heber, D. (2005). Inhibition of prostate cancer cell growth by an avocado extract: Role of lipid-soluble bioactive substances. The Journal of Nutritional Biochemistry, 16(1), 23–30. Morton, J. F. (1987). Avocado. In Fruits of warm climates (pp. 91–102). Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002). Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) Assays: A comparative study. Journal of Agricultural and Food Chemistry, 50(11), 3122–3128. Ozdemir, F., & Topuz, A. (2004). Changes in dry matter, oil content and fatty acids composition of avocado during harvesting time and post-harvesting ripening period. Food Chemistry, 86(1), 79–83. Prior, R. L., & Gu, L. W. (2005). Occurrence and biological significance of proanthocyanidins in the American diet (pp. 2264–2280). Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., et al. (2003). Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry, 51(11), 3273–3279. Robbins, R. J., Leonczak, J., Johnson, J. C., Li, J., Kwik-Uribe, C., Prior, R. L., et al. (2009). Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography–fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. Journal of Chromatography A, 1216(24), 4831–4840. Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. Journal of Agricultural and Food Chemistry, 53(20), 7749–7759. Soong, Y. Y., & Barlow, P. J. (2004). Antioxidant activity and phenolic content of selected fruit seeds. Food Chemistry, 88, 411–418. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food Chemistry, 46(10), 4113–4117. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52(12), 4026–4037.