Characterisation of gels from different Aloe spp. as antifungal treatment: Potential crops for industrial applications

Industrial Crops and Products 42 (2013) 223–230

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Characterisation of gels from different Aloe spp. as antifungal treatment: Potential crops for industrial applications P.J. Zapata a , D. Navarro a , F. Guillén a , S. Castillo a , D. Martínez-Romero a , D. Valero a , M. Serrano b,∗ a b

Dept. Food Technology, EPSO, University Miguel Hernández, Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, Spain Dept. Applied Biology, EPSO, University Miguel Hernández, Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, Spain

a r t i c l e

i n f o

Article history: Received 18 January 2012 Received in revised form 31 May 2012 Accepted 1 June 2012 Keywords: Aloe spp. Aloe gel composition Aloin Antifungal activity Polyamines Phenolics Antioxidant activity

a b s t r a c t The leaf characteristics and gel chemical composition of eight Aloe species (Aloe arborescens Mill., Aloe aristata Haw., Aloe claviflora Strydenburg, Aloe ferox Mill., Aloe mitriformis Mill., Aloe saponaria Ait., Aloe striata Haw., and Aloe vera L.) were studied in freshly harvested leaves at three different seasons within the Mediterranean climate: winter, spring and summer. Results revealed that differences existed in leaf properties and chemical composition of the gels of the several Aloe spp. and harvest seasons. The highest gel percentage was obtained from A. vera and A. claviflora (≈62–65%) followed by A. ferox and A. mitriformis (≈50–58%). Harvest season greatly affected gel properties with increases in lipids, proteins, aloin, total phenolics, total antioxidant activity (hydrophilic and lipopihilic fractions) and polyamines (putrescine and spermidine) were obtained in the summer season, whilst they were no differences in leaf characteristics. In addition, the growth potential of fruit pathogenic fungi (Botrytis cinerea, Penicillium digitatum, Penicillium expansum and Penicillium italicum), artificially inoculated on the whole leaves, was evaluated. The highest antifungal activity, measured as absence of or low percentage of infected wounds, was obtained for A. ferox, A. mitriformis and A. saponaria, this antifungal activity being positively correlated with gel aloin content. A. ferox, A. mitriformis and A. saponaria could be good alternatives to A. vera for commercial gels and other practical applications. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The genus Aloe comprises over 500 species, ranging from diminutive shrubs to large tree-like forms and it is represented in several biodiversity hotspots. However, information on the ethnobotanical significance and chemical composition of different Aloe spp. has remained largely inaccessible, despite this knowledge being of potential importance in biodiversity conservation and ecotourism (Grace, 2011). Aloe vera L. is undoubtedly one of the most important medicinal plants in the world, providing the raw materials for different uses (medicinal, cosmetic, tonic drinks, and numerous other uses in the food industry). Statistics given by the International Aloe Science Council (2012) indicate that about 24,000 ha of A. vera are cultivated globally, with an economic value of US$300 billion. However, in view of the large number of Aloe species it is surprising that little research has been done to investigate the commercial potential of other species. In fact, Aloe arborescens Mill. was used to treat nuclear irradiation burns from the atomic bomb explosion over Hiroshima during the Second World War, and is currently under commercial development

∗ Corresponding author. Tel.: +34 96 674 9616; fax: +34 96 674 9678. E-mail address: [email protected] (M. Serrano). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.06.002

in Japan and Italy as a possible alternative source of gel products (Van Wyk and Gericke, 2000; Van Wyk et al., 2009). Similarly, for Aloe ferox Mill. there is almost no commercially relevant data available, with the exception of South Africa, where it is a commercial Aloe plant, with a gel yield ca. 50%, being strongly influenced by the water content of the leaves at time of harvest (Chen et al., in press; O’Brien et al., in press). The most studied gel from any Aloe spp. has been on A. vera, for which the gel is found in a clear internal zone located between the abaxial and adaxial mesophyll. This central zone is called different names including pulp, mucilaginous tissue, mucilaginous gel, and parenchyma tissue and is composed of cell degenerate organelles, and the viscous liquid contained in the cells (Li et al., 2003). The chemical composition of the gel is very complex, composed mainly of polysaccharides and soluble sugars, followed by proteins, many of which are enzymes, aminoacids, vitamins and anthraquinones (Liu et al., 2007). The gel of this species has been used for pre-harvest or postharvest treatments to maintain fruit quality attributes and reduction in fungal decay of sweet cherry (Martínez-Romero et al., 2006), table grape (Valverde et al., 2005; Serrano et al., 2006; Castillo et al., 2010), and nectarine (Navarro et al., 2011). In these papers the antifungal activity of A. vera gel in vitro on (potato-dextrose-agar) PDA cultures has been also shown for Botritys cinerea, Rhizopus stolonifer and Penicillium digitatum.

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In view of the large number of Aloe species (more than 400 have been described) it is surprising that only the above commented three species have been commercialised, and so little, or no data, have been published describing the properties of other Aloe species. In this sense, we have investigated the leaf characteristics and gel chemical composition of eight Aloe species (A. arborescens Mill., Aloe aristata Haw., Aloe claviflora Strydenburg, A. ferox Mill., Aloe mitriformis Mill., Aloe saponaria Ait., Aloe striata Haw., and A. vera L.) harvested at three different seasons. In addition, the antifungal activity of the leaves from the eight species against the most typical fruit pathogenic fungi (B. cinerea, P. digitatum, Penicillium expansum and Penicillium italicum) was also evaluated. With this information there is the possibility of using other Aloe species, apart from A. vera, to obtain gels usable as either pre-harvest or postharvest treatments.

2. Materials and methods 2.1. Plant material and experimental design In this work eight Aloe species were used: A. arborescens Mill., A. aristata Haw., A. claviflora Strydenburg, A. ferox Mill., A. mitriformis Mill., A. saponaria Ait., A. striata Haw., and A. vera L. plants were grown under standard organic farming practices in Orihuela, Alicante, Spain, and the external leaves from 3-year-old plants were used in the experiments. Soil characteristics were: Clayey-Icam, pH 8.52 and electrical conductivity 0.16 dS m−1 . The aloe plants were cultivated according to the National Regulation for Ecological Agriculture (Comité de Agricultura Ecológica de la Comunidad Valenciana http://www.caecv.com/), without any addition of synthetic fertilizers, herbicides or pesticides. In Experiment 1 (Year 2010), for each Aloe spp., six mature leaves were taken from six different plants at 3-time intervals: end of February (Winter), end of May (Spring) and end of August (Summer). Climatic data were recorded from the local meteorology station (Estación MU21, http://siam.imida.es, GPS Coordinates: N38◦ 2 4.33 and W0◦ 59 58.72 ). Media of maximum and minimum temperatures were 15.37 and 5.41, respectively in February, 26.37 and 14.12, respectively in May and 30.42 and 23.36, respectively in August. Rainfall was 29.8, 23.2 and 47.4 mm, for, winter, spring and summer, respectively. Radiation was 125.7, 290.86 and 248.32 W m−2 and sun hours were 202, 369 and 335 in February, May and August, respectively. The experiment was carried out at the three seasons within the same year by using the same experimental plot for the eight Aloe species (same location and culture practises) and so the differences along the year are attributed to the typical climatic conditions for each particular season and the differences amongst species to genetic characteristics of each one. The leaves (harvested 3 h after sunrise) were transferred to the laboratory where weight, length, thickness and width were determined, and then the parenchymatous tissue was manually removed to obtain the gel from each leave. The analytical determinations in the gel were: total soluble solids (TSS), pH, total acidity, total proteins, total lipids, free polyamine content (putrescine, spermidine and spermine), and aloin A (barbaloin) concentration by HPLC. In Experiment 2 (Year 2011), leaves were picked from the same Aloe species at the end of August (Summer), and inoculated with several plant pathogenic fungi (B. cinerea, P. digitatum, P. expansum and P. italicum). The fungi were purchased from the Spanish collection of type culture, and routinely cultured in potato dextrose agar (PDA). For the Aloe species with small leaves (A. mitriformis, A. clavoiflora and A. aristata) eight leaves were used to inoculate each fungal species. In each of these leaves, 2 injuries (2 mm × 2 mm in length and width and 2 mm in depth) were performed along the leaf surface with a sterile lancet for fungal inoculation. For the

remaining Aloe species four leaves were used to be inoculated with each fungal species. In these leaves, four injuries were done as above. For each injury, 20 ␮L containing 100 spores of the corresponding fungi stock were deposited, and after 7-days the presence or absence of fungus growth in the injury was determined, and results expressed as percentage of infected wounds.

2.2. Leaf dimensions and gel yield The leaves from the selected plants were removed at the point of attachment with a sharp knife, washed with tap water to remove the adhering soil particles and deposited inside a container for 1 h to permit the efflux of yellow latex. Then, the leaves were weighed and results expressed as grams. Leaf length (cm), the width at midpoint and base of the leaves and thickness (cm) at the mid-point of the leaf were measured by a measuring stick and Vernier calliper. For each leaf, the spikes placed along their margins were removed before longitudinally slicing to separate the epidermis from the parenchyma. The parenchyma fillets were crushed to yield a mucilaginous gel which was filtered to discard the fibrous fraction. The gel yield was expressed as percentage of the obtained gel with respect to the whole leaf weight. In the fresh gel pH (pH metre Crison, Spain) and total soluble solids as ◦ Brix (Atago refractometer, Japan) were analysed. Then, the gel was frozen using liquid N2 , stored at −40 ◦ C until performing the following analytical determinations.

2.3. Aloin concentration Gel (5 g) was homogenised in 25 mL of methanol (50%, v/v) containing 2 mL HCL 0.1 N and NaF 5 mM by using a PolytronTM at 9500 rpm for 2 min. After homogenisation, samples were sonicated at 10 ◦ C for 60 min and then centrifuged at 20,000 × g for 15 min. The supernatant was filtered through 0.45 ␮m Millipore filter and then 1 mL injected into HPLC-DAD (Hewlett-Packard HPLC-1100 Series), system equipped with a C18-column (Supelcogel C-610H, 30 cm × 7.8 mm, Supelco Park, Bellefonte, USA). Aloin A was eluted isocratically by methanol-water as mobile phase (64:36, v/v containing 0.5% formic acid) at flow rate of 1 mL min−1 and detected at 254 nm wavelength. A calibration curve was performed by using aloin A (barbaloin) standard (Sigma, Madrid, Spain) at concentrations ranging 0–100 mg L−1 (y = 4.71x + 4.25, R2 = 0.9979). Results were expressed as mg 100 g−1 fresh weight).

2.4. Polyamine extraction and quantification For each gel, 1 g fresh tissue was extracted with 10 mL of 5% cold perchloric acid. 1,6 hexanediamine (100 nmol g−1 of tissue) was added as an internal standard. The homogenate was then centrifuged for 30 min at 20,000 × g. A 2 mL aliquot of the supernatant was used to determine free polyamines by benzoylation, and derivatives analysed by HPLC according to previous work (Serrano et al., 2003). The elution system consisted of MeOH/H2 O (64:36) solvent, running isocratically with a flow rate of 0.8 mL min−1 . The benzoyl-polyamines were eluted through a reversed-phase column (LiChroCart 250–4,5 ␮m) and detected by absorbance at 254 nm. A relative calibration procedure was used to determine the polyamines in the samples, using 1,6 hexanediamine as the internal standard and standard curves covered the range 1–320 nM. The calibration curves were y = 10.66x + 170.00, R2 = 0.94 for Putrescine, y = 10.19x − 39.96, R2 = 0.96 for Spermidine, and y = 11.52x − 4.32, R2 = 0.90 for Spermine. Results were expressed as nmol g−1 fresh weight).

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Table 1 Leaf dimensions and weight of the Aloe species.a Leaf dimensions (cm) Aloe spp

Length

Width at base

Width at half

Thickness

Weight (g)

A. arborescens A. aristata A. claviflora A. ferox A. mitriformis A. saponaria A. striata A. vera

42.43 ± 1.08 A 21.40 ± 0.52 B 26.20 ± 1.60 C 66.00 ± 3.42 D 14.36 ± 0.28 E 26.86 ± 1.03 C 36.86 ± 0.90 F 68.29 ± 5.14 D

4.29 ± 0.14 A 4.91 ± 0.23 B 6.78 ± 0.63 C 11.21 ± 0.83 D 5.20 ± 0.27 B 9.86 ± 0.67 E 10.50 ± 0.66 DE 11.79 ± 0.48 D

2.97 ± 0.11 A 3.92 ± 0.15 B 6.16 ± 0.52 C 12.79 ± 0.83 D 4.64 ± 0.28 E 7.93 ± 0.56 C 12.29 ± 0.96 D 9.57 ± 0.36 F

0.49 ± 0.02 A 0.86 ± 0.02 B 2.56 ± 0.21 C 2.87 ± 0.17 C 0.66 ± 0.03 D 1.10 ± 0.10 E 1.99 ± 0.14 F 2.14 ± 0.17 F

50.6 ± 4.2 A 32.21 ± 1.6 B 77.74 ± 3.5 C 725.3 ± 90.7 D 33.8 ± 1.0 B 170.2 ± 10.3 E 343.3 ± 24.6 F 559.9 ± 38.9 G

a

Data are the mean ± SE of determinations made at the 3 seasons. For each column, different capital letters show significant differences (p < 0.05) amongst Aloe spp.

2.5. Total proteins The gels (5 g) were centrifuged at 15,000 × g for 10 min at 4 ◦ C. The supernatant was separated and used for soluble protein measurement. The protein content was determined in the supernatant according to Bradford’s dye binding method, using bovine serum albumin (BSA) as standard (Bradford, 1976). 2.6. Total lipids Total lipids were extracted according to Valero et al. (1990). Briefly, 2 g of Aloe gel were homogenised in 10 mL chloroform:methanol:0.1 N HCl (200:100:1) and then 10 mL of 0.1 N HCl were added before centrifugation at 4000 × g for 10 min. The lower organic phase containing the total lipids was collected and taken to dryness in a water bath at 45 ◦ C under continuous flushing of N2 gas. The residue was weighed and total lipids were expressed as g 100 g−1 . 2.7. Statistical analysis Data from analytical determinations were subjected to analysis of variance (ANOVA). Sources of variation were Aloe spp. and harvest season. Mean comparisons were performed using the high significant difference (HSD) Tukey’s test to examine if differences were significant at p < 0.05 and are shown in tables and figures with significant letters. All analyses were performed with statistical products and service solutions (SPSS) software package v. 11.0 for Windows. Linear regression between aloin concentration and fungal growth on inoculated wounds was performed by using SigmaPlot 11 for Windows. 3. Results and discussion 3.1. Leaf characteristics and composition During the experiment, no significant differences were obtained in leaf dimensions and weight amongst the 3 harvest seasons since only the external leaves from the plants were picked. Then, data are expressed as the mean of the three measurements (winter, spring and summer). Accordingly, Saks and Ish-shalom-Gordon (1995) did not find differences in leaf size from A. vera plants grown in different climactic and soil conditions. However, the leaves of the several Aloe spp. differed significantly in leaf dimension and weight (Table 1), in which A. mitriformis and A. aristata showed the smallest leaves (≈14–21 cm in length and ≈32–34 g of weight) and the largest leaves were found in A. ferox (≈66 cm in length and ≈725 g of weight). In addition, the leaves of A. ferox also had the greatest thickness and width, both at the basal and mid-points. This variability in the leaf dimensions of the several Aloe spp. probably reflects a combination of both genetic and environmental effects. In a recent paper on 24 plants of A. ferox from eight populations,

O’Brien et al. (in press) reported mean leaf weight of 650 g, mean length of 48.5 cm and width of 10.8 cm, which are quite in agreement with our results. The Aloe gel yield generally decreased from winter to summer (Table 2) for all Aloe spp., although the decrease depended on the specific species, with 22% being found for A. saponaria and non-significant decreases in A. claviflora, A. striata, and A. vera. In addition, the percentage of obtained gel was also dependent on the species, with the highest gel yield being obtained from the leaves of A. vera followed by A. claviflora, and the lowest gel yield from A. arborescens. The pH also diminished from winter to summer and the values differed amongst Aloe spp., especially during the winter season. These results would indicate that during winter the photosynthetic capacity and acid accumulation during the night was lower than in summer. Since leaves were harvested 3 h after sunrise, a lower pH value would indicate that higher acid accumulation in the vacuole occurred during the night period (Walker and Leegood, 1996). The pH values found in the gel of the different species for the samples taken on spring and summer are within the range reported previously for A. vera gel (Eshun and He, 2004; Vega-Gálvez et al., 2011). With respect to the content of total soluble solids, differences were also found amongst Aloe spp. (Table 2) although generally no differences could be attributed to harvest season, with the exception of A. claviflora, A. ferox and A. striata for which significant increases were found from winter to summer. The analysis of total lipids and total proteins revealed that the gels obtained during the summer season showed the highest concentration (Table 3), although differences existed amongst the Aloe spp. Thus, A. arborescens had the highest concentration of both lipids and proteins followed by A. mitriformis and A. striata. Beppu et al. (2004) studied the chemical components of A. arborescens but using the whole leaf (epidermis and parenchyma) and found that total proteins were also higher in the warm than in the cold season, whilst no significant differences were found for total saccharides (◦ Brix). In the case of A. vera, the soluble solids in the gel was found at 0.56% with some seasonal fluctuations, whilst levels of protein and lipids were 7 and 4% (Ahlawat and Khatkar, in press), or 3.7 and 4.5% (Vega-Gálvez et al., 2011), respectively, on dry matter basis.

3.2. Aloin concentration One of the main biologically active constituents of Aloe extracts is aloin or barbaloin (10-glucopyranosyl-1.8-dihydroxy-3(hydroxymethyl)-9 (10H)-anthracenone), which is found in nature as a mixture of two diastereosiomers, aloin A (10R) and aloin B (10S). These two compounds are generally used as key components for the quality control of this plant and its derivatives. Aloin is generally contained in the bitter, smelly exudate seeping out from freshly cut leaves, whilst very low amounts of aloin exist in Aloe gel obtained from the internal mass of Aloe leaf (Fanalli et al., 2010). In

38.10 ± 1.75 Ab 40.57 ± 2.50 Bb 62.57 ± 4.45 Ca 51.72 ± 3.54 Db 54.79 ± 2.95 Da 52.38 ± 1.05 Db 56.28 ± 1.05 Da 65.76 ± 1.13 Ca

B Winter Spring Summer

C

8 B -1

Aloin (mg 100 g )

Data are the mean ± SE. For each column, different capital letters show significant differences (p < 0.05) amongst Aloe spp. For each row, different small letters show significant differences (p < 0.05) amongst harvest seasons. a

1.91 ± 0.04 Aa 1.15 ± 0.02 Ba 1.50 ± 0.06 Cb 1.06 ± 0.06 Bc 1.62 ± 0.06 Da 1.51 ± 0.06 Ca 1.69 ± 0.05 Db 1.15 ± 0.03 Ba 2.10 ± 0.07 Aa 1.15 ± 0.02 Ba 1.45 ± 0.05 Cb 0.86 ± 0.07 Db 1.71 ± 0.06 Ea 1.45 ± 0.05 Ca 1.60 ± 0.04 Eb 1.11 ± 0.03 Ba

Spring Winter

2.15 ± 0.02 Aa 1.10 ± 0.10 Ba 1.25 ± 0.02 Ba 0.63 ± 0.08 Ca 1.75 ± 0.03 Da 1.41 ± 0.03 Ea 1.30 ± 0.09 Ba 1.12 ± 0.02 Ba 4.66 ± 0.06 Ab 4.71 ± 0.03 Ac 5.01 ± 0.07 Bb 4.67 ± 0.02 Ab 4.69 ± 0.03 Ab 4.76 ± 0.05 Ab 4.98 ± 0.03 Bb 4.58 ± 0.01 Ab

Summer Spring

4.78 ± 0.05 Ab 4.90 ± 0.02 Ab 4.84 ± 0.06 Ab 4.63 ± 0.05 Ab 4.64 ± 0.02 Ab 4.86 ± 0.02 Ab 4.96 ± 0.04 Ab 4.61 ± 0.03 Ab 7.09 ± 0.07 Aa 7.48 ± 0.25 Ba 7.52 ± 0.13 Ba 6.82 ± 0.02 Ca 7.79 ± 0.09 Da 5.52 ± 0.03 Ea 5.73 ± 0.07 Fa 5.30 ± 0.28 Ea

Winter Spring Winter Aloe spp

43.78 ± 1.03 Aa 50.28 ± 2.12 Ba 65.80 ± 1.36 Ca 58.98 ± 1.88 Da 58.89 ± 1.56 Da 62.10 ± 0.83 Da 57.24 ± 2.13 Da 66.47 ± 1.59 Ca

Summer

pH Aloe gel yield (%)

Table 2 Values of gel yield (%), pH and total soluble solids of the Aloe species.a

A. arborescens A. aristata A. claviflora A. ferox A. mitriformis A. saponaria A. striata A. vera

Total soluble solids (g 100 g−1 )

Summer

P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

32.27 ± 1.34 Ac 43.89 ± 0.78 Bb 61.72 ± 3.80 Ca 51.34 ± 3.59 Db 50.67 ± 2.25 Db 48.86 ± 2.35 Db 56.90 ± 3.05 Ca 63.38 ± 1.04 Ca

226

6

B

C A

4

A

A

B

C

B A

C

C

A

B

A

B

B 2

A A A

s ore arb A.

ns ce

A

s a a ta ox ora mi iat ari sta fer vifl str for on ari A. itri ap A. cla s . A. m . A A A.

A

ra ve A.

Fig. 1. Aloin concentration in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

fact aloin concentration ranged in between 1 and 9 mg 100 g−1 fw depending on Aloe spp. and harvest season. Most of the literature about aloin content has been reported for the most studied species, that is A. vera, for which concentrations between 0.3 and 15 mg 100 g−1 fw were found (Bozzi et al., 2007; Miranda et al., 2009; Vega-Gálvez et al., in press). However, in other leaf parts such as in the inner epidermal layer, higher aloin concentrations have been reported. Thus, in this tissue aloin concentrations of 0.7, 0.5 and 0.07 mg 100 g−1 were found for A. arborescens, A. vera and A. striata, respectively (Okamura et al., 1996). Apart from aloin, an oxanthrone (10-hydroxy B 6 -OAcetate) was identified as a member of the anthrones in A. claviflora (Dagne et al., 1998), whilst in the case of A. ferox, aloeresin A and aloesin are also abundant, and 8-O-methyl-7-hydroxyaloin A and B are characteristics of A. vera (Fanalli et al., 2010). These compounds could serve as fingerprint phytochemicals for these Aloe species. In a recent paper, 5-hydroxyaloin A, and the aloinosides A and B have been described as anthrone-C-glucosides typical of Cape aloe (A. ferox), which can be metabolised to aloin A, aloin B and a hydroxyl metabolite after ingestion (Chen et al., in press). It is interesting to point out that harvest season has a strong influence on aloin content, since aloin significantly increased from winter to summer for all Aloe spp., the highest increase (≈10-fold) being found in A. mitriformis and A. striata (Fig. 1). However, A. ferox and A. saponaria showed less change from winter to summer. In a study with A. arborescens seasonal variations were also found, the aloin levels being higher during the warm season than in the cold ones (Beppu et al., 2004). For A. hereroensis, a type of phenolics named homonataloin was the highest during the summer season and the lowest at beginning of the winter (Chauser-Volfson and Gutterman, 1997). The increase of aloin concentration in the gels obtained during the summer season could be a consequence of the high radiation level, which was ≈300 W m−2 day−1 according to Regional Meteorological Institute for 2010. 3.3. Phenolic compounds and antioxidant activity Total phenolic concentration was dependent on the Aloe spp., with A. arborescens having the highest phenolics and the lowest being found for A. aristata, A. saponaria and A. vera (Fig. 2). In addition, harvest season also influenced the content of total phenolics, for which those samples picked at either winter or summer had

P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

227

Table 3 Total lipids and proteins of the Aloe species.a Total lipids (g 100 g−1 )

Total proteins (g 100 g−1 )

Aloe spp

Winter

Spring

Summer

Winter

Spring

Summer

A. arborescens A. aristata A. claviflora A. ferox A. mitriformis A. saponaria A. striata A. vera

0.13 ± 0.03 Aa 0.10 ± 0.01 Aa 0.14 ± 0.02 Aa 0.07 ± 0.03 Aa 0.11 ± 0.01 Aa 0.13 ± 0.03 Aa 0.12 ± 0.02 Aa 0.11 ± .0.02 Aa

0.15 ± 0.01 Aa 0.06 ± 0.01 Bb 0.07 ± .0.01 Bb 0.07 ± 0.01 Ba 0.09 ± 0.01 Ba 0.09 ± .0.01 Ba 0.09 ± 0.01 Ba 0.08 ± 0.01 Ba

0.42 ± 0.02 Ab 0.18 ± 0.03 Bc 0.29 ± 0.03 Cc 0.17 ± 0.03 Bc 0.36 ± 0.02 Ab 0.19 ± 0.03 Bb 0.36 ± 0.04 Ab 0.19 ± 0.02 Bb

0.26 ± 0.01 Aa 0.14 ± 0.01 Ba 0.26 ± 0.05 Aa 0.05 ± 0.01 Ca 0.28 ± 0.01 Aa 0.11 ± 0.02 Ba 0.14 ± 0.01 Ba 0.20 ± 0.03 Aa

0.31 ± 0.02 Aa 0.06 ± 0.02 Bb 0.24 ± 0.05 Aa 0.02 ± 0.01 Ba 0.11 ± 0.02 Cb 0.09 ± 0.01 Ca 0.06 ± 0.02 Bb 0.16 ± 0.05 Ca

0.54 ± 0.05 Ab 0.12 ± 0.01 Ba 0.24 ± 0.01 Ca 0.15 ± 0.03 Bb 0.39 ± 0.01 Dc 0.17 ± 0.01 Bb 0.25 ± 0.01 Cc 0.22 ± 0.01Ca

-1

30

20

A A A

A

B

C

C B

C

A

B

A 10 B

B

C

A

A

B

A A B

C B

ra ve A.

Fig. 3. Hydrophilic total antioxidant activity (H-TAA) in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

contribution of ascorbic acid (vitamin C, a well-known hydrophilic antioxidant moiety) to H-TAA should not be discharged, since it is present in A. vera gel (Vega-Gálvez et al., 2011) at high concentration (160 mg 100 g−1 , dw). With respect to L-TAA, an increase was generally found from winter to summer, although

-1

C

Winter Spring Summer

40 -1

Total Phenolics (mg 100 g )

Winter Spring Summer

C

s s a ta ra ox ria mi iat en iflo sta na fer sc str for ari po A. lav itri A. ore sa .c A. m b . r . A a A A A.

Lipophilic Total Antioxidant Activity (mg 100 g )

higher phenolic concentrations than those taken during the spring. These results would indicate that environmental low and high temperatures could induce an accumulation of total phenolics as a stress response. In fact, in 2010 the winter samples were harvested after 3 months with temperatures below 5 ◦ C (average minimum temperature), whilst the summer samples after 3 months with temperature over 30 ◦ C (average maximum temperature) according to Regional Meteorological Institute. Total antioxidant activity (TAA) of the Aloe gels was measured separately in two fractions: hydrophilic (H-TAA) and lipophilic (L-TAA). For all Aloe spp., H-TAA (Fig. 3) was higher than L-TAA (Fig. 4). H-TAA showed a similar behaviour to that of total phenolic concentrations, that is higher activities during the winter and summer seasons than during spring, the highest H-TAA levels being found for A. arborescens. In fact high correlation could be observed between H-TAA and total phenolics (y = 0.69x + 3.62; R2 = 0.808), showing that phenolics are the main hydrophilic compounds contributing to H-TAA, according to previous reports in a wide range of fruit and vegetables (Valero and Serrano, 2010). Amongst phenolic compounds identified in the latex from Aloe harlana, the highest antioxidant activity corresponded to 7-O-methylaloeresin A followed by aloin (Asamenew et al., 2011). In A. ferox, TAA expressed as ORAC (in both hydrophilic and lipophilic fractions) was also reported, although the concentration of individual polyphenols antioxidants are not the only factors influencing antioxidant capacity, since other compounds such as indoles, alkaloids are also known to possess antioxidant activities (Loots et al., 2007). Although it was not determined in this work, the

Hydrophilic Total Antioxidant Activity (mg 100 g )

a Data are the mean ± SE. For each column, different capital letters show significant differences (p < 0.05) amongst Aloe spp. For each row, different small letters show significant differences (p < 0.05) amongst harvest seasons.

30 A 20

A

A

A

A

B B

10

B

A B

A A B

C

B AA

AA

B

s s a ta ra ox ria mi iat en iflo sta na fer sc str for ari po A. lav itri A. ore sa .c A. m b . r . A a A A A.

A A B ra ve A.

Fig. 2. Total phenolic concentration in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

12

Winter Spring Summer

C A

10

B

C

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Fig. 4. Lipophilic total antioxidant activity (L-TAA) in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

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Fig. 5. Free putrescine concentration in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

Fig. 6. Free spermidine concentration in the gel of different Aloe species from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital letters amongst seasons show significant differences at p < 0.05.

the activity was different amongst the Aloe spp., with A. mitriformis having the highest L-TAA and A. saponaria and A. vera the lowest. In A. vera occurrence of vitamin E has been reported (Vega-Gálvez et al., 2011), which might contribute to L-TAA due to its lipophilic nature and its role in protecting the fatty acids of the membranes against damage caused by free radicals. Very recently it has been reported that both methanolic and acetone extracts from A. vera gel exhibited the highest antioxidant activity compared with those from ethanolic, hexane and chloroform extracts (Saritha et al., 2010), which would indicate that hydrophilic compounds contribute more than lipophilic ones to antioxidant activity of Aloe gels according to our results. Although the Aloe gels show antioxidant capacity lower than tea extract or ascorbic acid, they have more activity than the synthetic BHT or ␣-tocopherol (Khaing, 2011). It has been suggested that growth stage of Aloe plants play a vital role in the composition and antioxidant activity of the gel (Hu et al., 2003). However, in our experiment the leaves were harvested from plants aged 3-year-old and then the observed differences should be attributed to the Aloe species and harvest season within the Mediterranean climate.

postharvest treatment with putrescine or spermidine led to a delay of the ripening process of plums (Serrano et al., 2003), alleviated mechanical damage in lemon (Martínez-Romero et al., 1999) and reduced chilling injury in pomegranate (Mirdehghan et al., 2007), as a consequence of increased endogenous polyamine concentration. In addition, pre-harvest application of putrescine increased the functional ovules of apricot flowers (Alburquerque et al., 2006). The pre-harvest application of A. vera gel on table grape led to reduction on fruit metabolism during postharvest storage (Castillo et al., 2010), which could be induced by the polyamine content of the gel. However, more studies are necessary to correlate the relationship between the effects of Aloe gels used as pre- and postharvest treatments and polyamines.

3.4. Polyamines The levels of free polyamines (putrescine, spermidine and spermine) were determined by HPLC-DAD in the gels of the several Aloe species, and results revealed that concentration of putrescine were higher than spermidine and spermine. Generally, levels of putrescine and spermidine increased along the season, and especially from spring to summer, although increases were different depending on the Aloe species. Thus, putrescine increased ≈10–20fold for A. vera, A. saponaria and A. ferox (Fig. 5), whilst for spermidine the highest increases were found for A. vera and A. saponaria (Fig. 6). However, this behaviour was not found for spermine which increased from spring to summer just in A. vera (data not shown). In a study with the whole leaf of A. arborescens (Beppu et al., 2004), putrescine and spermidine levels were high in summer and low in either spring or winter, which is in agreement with our results. This is the first time in which the content of free polyamines has been carried out in a wide range of Aloe spp. Since polyamines have been recognised as anti-senescence plant hormones (Valero et al., 2002), the occurrence of free polyamines in the Aloe gels could be considered as a goof source of antiageing agents for fruit pre- and postharvest treatments. In fact,

3.5. Fungal growth in Aloe species leaves Given that the highest aloin, total phenolics, total antioxidant activity, total lipid and protein concentrations were found in the gels obtained from the leaves collected in summer, the following year leaves were picked at this season for determining the Aloe leaves sensitivity to be infected by several fruit pathogenic fungi. The assayed fungi were B. cinerea, P. digitatum, P. expansum and P. italicum. The results revealed that after 7 days of inoculation 3 distinctive types of injuries were found: (a) injuries with the same initial size and necrosis (cured); (b) injuries with increased injury size due to an initial fungus development but with further necrosis and (c) injuries with active growth of the fungus. Accordingly, the results of the latter were shown in Fig. 7. It is clear that B. cinerea was the most invasive fungus since it was able to grow on all Aloe spp., although differentially depending of the species, and the most effective Aloe species on inhibiting the growth of this fungus was A. ferox, whilst B. cinerea grew up in ≈70–80% of the injuries for the remaining Aloe species. Overall, the most effective Aloe spp., on inhibiting fungal growth were A. mitriformis and A. saponaria, in which absence of Penicillium spp. growth was obtained, followed by A. arborescens and A. ferox, in which P. italicum and P. digitatum grew up just in 10% of the injuries, respectively. In previous reports, the efficacy of A. vera gel on reducing fungal growth on PDA plates was higher for P. digitatum than for B. cinerea, although for both fungi the inhibition of mycelium growth increased as did the Aloe gel concentration (Castillo et al., 2010). In addition, A. vera pulp showed an inhibitory effect on the mycellium development of Rhizoctonia solani, Fusarium oxysporum, and Colletotrichum

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Acknowledgements Botrytis cinerea P. digitatum P. expansum P. italicum

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This work has been co-funded by the Spanish Ministry of Science and Innovation (MICINN) and FEDER Funds through Project AGL2009-10857 (ALI).

Infected wounds (%)

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References

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Aloe spp Fig. 7. Percentage of infected wounds on the leaves of several Aloe species harvested at summer season and artificially inoculated with several fungi spp.

coccodes (Jasso de Rodríguez et al., 2005). Moreover, in a recent paper, the application of A. vera gel on two nectarine cultivars significantly reduced the growth of Rhizopus stolonifer, B. cinerea and P. digitatum, which were inoculated after the Aloe application (Navarro et al., 2011). Interestingly, taking into account data of aloin content and the percentage of injuries in which the fungus occurred, a negative correlation was found between these parameters (y = −0.11x + 9.17; R2 = 0.743), showing that aloin could be responsible for the antifungal effects of these Aloe species. The mechanism of action by which Aloe extracts act is not fully understood although it has been proposed that aloin or barbaloin affect dramatically phospholipid membranes, leading to remarkable changes in membrane physical properties. These alterations consist in changes of the lipid/water interface in negatively charged phospholipids and perturbations of the core of the bilayer, which are related to the antimicrobial activity of aloin (Alves et al., 2004). Accordingly, in a wide range of Gram-negative and Gram-positive bacteria and fungi strains, aloin and 7-O-methylaloeresin A showed strong antimicrobial activity (Asamenew et al., 2011). 4. Conclusions This is the first time in which a comprehensive and comparative study has been carried out on eight Aloe species, and results showed that significant differences existed on leaf properties and chemical composition of the gels depending on the Aloe spp. Harvest season also influences gel composition since increases in lipids, proteins, aloin, total phenolics, total antioxidant activity (hydrophilic and lipopihilic fractions) and polyamines (putrescine and spermidine) were obtained in the summer season. The Aloe leaves affected differentially fungal growth, with A. ferox, A. mitriformis and A. saponaria being the most effective in controlling the growth of the artificially inoculated fungi on the leaves, which was related to increased concentration of aloin, one of the most important Aloe components related to antifungal activity. It is clear that active ingredients and biological function of Aloe has attracted the interest of more and more researchers, although most of the work done has been carried out in A. vera. However, A. ferox, A. mitriformis and A. saponaria could be good alternatives to A. vera for commercial gels with putative practical applications, since gel yield percentage is similar (50–65%) and contains high aloin concentration.

Ahlawat, K.S., Khatkar, B.S. Processing, food applications and safety of aloe vera products: a review. Journal of Food Science and Technology, in press. Alburquerque, N., Egea, J., Burgos, L., Martínez-Romero, D., Valero, D., Serrano, M., 2006. The influence of polyamines on apricot ovary development and fruit set. Annals of Applied Biology 149, 27–33. Alves, D.S., Pérez-Fons, L., Estepa, A., Micol, V., 2004. Membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin. Biochemical Pharmacology 66, 549–561. Asamenew, G., Bisrat, D., Mazumder, A., Asres, K., 2011. In vitro antimicrobial and antioxidant activities of anthrone and chromone from the latex of Aloe harlana Reynolds. Phytotherapy Research 25, 1756–1760. Beppu, H., Kawai, K., Shimpo, K., Chihara, T., Tamai, I., Ida, C., Ueda, M., Kuzuya, H., 2004. Studies on the components of Aloe arborescens from Japan-monthly variation and differences due top art and position of the leaf. Biochemical Systematics and Ecology 32, 783–795. Bozzi, A., Perrin, C., Austin, S., Arce Vera, F., 2007. Quality and authenticity of commercial Aloe vera gel powders. Food Chemistry 103, 22–30. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Castillo, S., Navarro, D., Zapata, P.J., Guillén, F., Valero, D., Serrano, M., MartínezRomero, D., 2010. Antifungal efficacy of Aloe vera in vitro and its use as a preharvest treatment to maintain postharvest table grape quality. Postharvest Biology and Technology 57, 183–188. Chauser-Volfson, E., Gutterman, Y., 1997. Content and distribution of secondary phenolic compound homonataloin in Aloe hereroensis leaves according to leaf part, position and monthly changes. Journal of Arid Environments 37, 115–122. Chen, W., Van Wyk, B.E., Vermaak, I, Viljoen, A.M. Cape aloes – A review of the phytochemistry, pharmacology and commercialization of Aloe ferox. Phytochemistry Letters, in press. de Agricultura Ecológica de la Comunidad Valenciana Comité http://www.caecv.com/ 2012 (accessed on May, 2012).  Dagne, E., Bisrat, D., Wyk, B.E., Viljoen, A., 1998. 10-Hydroxyaloin B 6 -O-Acetate, an oxanthrone from Aloe claviflora. Journal of Natural Products 61, 256–257. Eshun, K., He, Q., 2004. Aloe vera: a valuable ingredient for the food pharmaceutical and cosmetic industries – a review. Critical Reviews in Food Science and Nutrition 44, 91–96. Estación MU21, http://siam.imida.es 2012 (accessed on May, 2012). Fanalli, S., Aturki, Z., D’Orazio, G., Rocco, A., Ferranti, A., Mercolini, L., Raggi, M.A., 2010. Analysis of Aloe-based phytotherapeutic products by using nano-LC–MS. Journal of Separation Science 33, 2663–2670. Grace, O.M., 2011. Current perspectives on the economic botany of the genus Aloe L. (Xanthorrhoeaceae). South African Journal of Botany 77, 980–987. Hu, Y., Xu, J., Hu, Q., 2003. Evaluation of antioxidant potential of aloe vera (Aloe barbadensis Miller) extracts. Journal of Agricultural and Food Chemistry 51, 7788–7791. International Aloe Science Council. www.iasc.org. 2011 (accessed 8.01.12). Jasso de Rodríguez, D., Hernández-Castillo, D., Rodríguez-García, R., Angulo-Sánchez, J.L., 2005. Antifungal activity in vitro of Aloe vera pulp and liquid fraction against plant pathogenic fungi. Industrial Crops and Products 21, 81–87. Khaing, T.A., 2011. Evaluation of the antifungal and antioxidant activities of the leaf extract of Aloe vera (Aloe barbadensis Miller). World Academy of Science, Engineering and Technology 75, 610–612. Li, J.Y., Wang, T.X., Shem, Z.G., Hu, Z.H., 2003. Relationship between leaf structure and aloin content in six species of Aloe L. Acta Botanica Sinica 45, 549–600. Liu, C.H., Wang, C.H., Xu, Z.Y., Wang, Y., 2007. Isolation, chemical characterization and antioxidant activities of two polysaccharides from the gel and the skin of Aloe barbadensis Miller irrigated with sea water. Process Biochemistry 42, 961–970. Loots, D.T., van der Westhuizen, F.H., Botes, L., 2007. Aloe ferox leaf gel phytochemical content, antioxidant capacity, and possible health benefits. Journal of Agricultural and Food Chemistry 55, 6891–6896. Martínez-Romero, D., Alburquerque, N., Valverde, J.M., Guillén, F., Castillo, S., Valero, D., Serrano, M., 2006. Postharvest sweet cherry quality and safety maintenance by Aloe vera treatment: a new edible coating. Postharvest Biology and Technology 39, 93–100. Martínez-Romero, D., Valero, D., Serrano, M., Martínez-Sánchez, F., Riquelme, F., 1999. Effects of postharvest putrescine and calcium treatments on reducing mechanical damage and polyamines and abscisic acid levels during lemon storage. Journal of the Science of Food and Agriculture 79, 1589–1595. Miranda, M., Maureira, H., Rodríguez, K., Vega-Gálvez, A., 2009. Influence of temperature on the drying kinetics, physicochemical properties, and antioxidant capacity of Aloe vera (Aloe barbadensis Miller) gel. Journal of Food Engineering 91, 297–304. Mirdehghan, S.H., Rahemi, M., Castillo, S., Martínez-Romero, D., Serrano, M., Valero, D., 2007. Pre-storage application of polyamines by pressure or immersion

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P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

improves shelf life of pomegranate stored at chilling temperature by increasing endogenous polyamine levels. Postharvest Biology and Technology 44, 26–33. Navarro, D., Díaz-Mula, H.M., Guillén, F., Zapata, P.J., Castillo, S., Serrano, M., Valero, D., Martínez-Romero, D., 2011. Reduction of nectarine decay caused by Rhizopus stolonifer, Botritys cinérea and Penicillium digitatum with Aloe vera gel alone or with the addition of thymol. International Journal of Food Microbiology 151, 241–246. O’Brien, C., Van Wyk, V., Van Heerden, F.R. Physical and chemical characteristics of Aloe ferox leaf gel. South African Journal of Botany, in press. Okamura, N., Asai, M., Hine, N., Yagi, A., 1996. High-performance liquid chromatographic determination of phenolic compounds in Aloe species. Journal of Chromatography A 746, 225–231. Saks, Y., Ish-shalom-Gordon, N., 1995. Aloe vera L., a potential crop for cultivation under conditions of low-temperature winter and basalt soils. Industrial Crops and Products 4, 85–90. Saritha, V., Anilakumar, K.R., Farhath, K., 2010. Antioxidant and antibacterial activity of Aloe vera gel extracts. International Journal of Pharmaceutical and Biological Archive 1, 376–384. Serrano, M., Martínez-Romero, D., Guillén, F., Valero, D., 2003. Effects of exogenous putrescine on improving shelf life of four plum cultivars. Postharvest Biology and Technology 30, 259–271. Serrano, M., Valverde, J.M., Guillén, F., Castillo, S., Martínez-Romero, D., Valero, D., 2006. Use of Aloe vera gel coating preserves the functional properties of table grapes. Journal of Agricultural and Food Chemistry 54, 3882–3886. Valero, D., López-Frías, M., Llopis, J., López-Jurado, M., 1990. Influence of dietary fat on the lipid composition of perirenal adipose tissue in rats. Annals of Nutrition and Metabolism 34, 327–332.

Valero, D., Martínez-Romero, D., Serrano, M., 2002. The role of polyamines in the improvement of the shelf life of fruit. Trends in Food Science & Technology 13, 228–234. Valero, D., Serrano, M., 2010. Postharvest Biology and Technology for Preserving Fruit Quality. CRC Press-Taylor & Francis, Boca Raton, Florida, USA. Valverde, J.M., Valero, D., Martínez-Romero, D., Guillén, F., Castillo, S., Serrano, M., 2005. Novel edible coating based on of Aloe vera gel to maintain table grape quality and safety. Journal of Agricultural and Food Chemistry 53, 7807–7813. Van Wyk, B.E., Gericke, N., 2000. People Plants: A Guide to Useful Plants of Southern Africa. BRIZA Publications, Pretoria. Van Wyk, B.E., Van Oudtshoorn, B., Gericke, N., 2009. Medicinal plants of South Africa, 2nd ed. Briza Publications, Pretoria. Vega-Gálvez, A., Giovagnoli, A., Pérez-Won, M., Reyes, J.E., Vergara, J., Miranda, M., Uribe, E., Di Scala, K. Application of high hydrostatic pressure to aloe vera (Aloe barbadensis Miller) gel: microbial inactivation and evaluation of quality parameters. Innovative Food Science & Emerging Technologies, in press. Vega-Gálvez, A., Miranda, M., Aranda, M., Henriquez, K., Vergara, J., Tabilo-Munizaga, G., Pérez-Won, M., 2011. Effect of high hydrostatic pressure on functional properties and quality characteristics of Aloe vera gel (Aloe barbadensis Miller). Food Chemistry 129, 1060–1065. Walker, R.P., Leegood, R.C., 1996. Phosphorylation of phosphoenolpyruvate carboxykinase in plants: Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds. Biochemical Journal 317, 653–658.