Industrial Crops and Products 42 (2013) 223–230

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Industrial Crops and Products

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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
Dept. Food Technology, EPSO, University Miguel Hernández, Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, Spain
b
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 a b s t r a c t

Article history: The leaf characteristics and gel chemical composition of eight Aloe species (Aloe arborescens Mill., Aloe
Received 18 January 2012 aristata Haw., Aloe claviflora Strydenburg, Aloe ferox Mill., Aloe mitriformis Mill., Aloe saponaria Ait., Aloe
Received in revised form 31 May 2012 striata Haw., and Aloe vera L.) were studied in freshly harvested leaves at three different seasons within
Accepted 1 June 2012
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 high-
Keywords:
est gel percentage was obtained from A. vera and A. claviflora (≈62–65%) followed by A. ferox and A.
Aloe spp.
mitriformis (≈50–58%). Harvest season greatly affected gel properties with increases in lipids, proteins,
Aloe gel composition
Aloin
aloin, total phenolics, total antioxidant activity (hydrophilic and lipopihilic fractions) and polyamines
Antifungal activity (putrescine and spermidine) were obtained in the summer season, whilst they were no differences in
Polyamines leaf characteristics. In addition, the growth potential of fruit pathogenic fungi (Botrytis cinerea, Penicillium
Phenolics digitatum, Penicillium expansum and Penicillium italicum), artificially inoculated on the whole leaves, was
Antioxidant activity 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 corre-
lated 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 in Japan and Italy as a possible alternative source of gel products

(Van Wyk and Gericke, 2000; Van Wyk et al., 2009). Similarly, for
The genus Aloe comprises over 500 species, ranging from Aloe ferox Mill. there is almost no commercially relevant data avail-
diminutive shrubs to large tree-like forms and it is represented able, with the exception of South Africa, where it is a commercial
in several biodiversity hotspots. However, information on the eth- Aloe plant, with a gel yield ca. 50%, being strongly influenced by the
nobotanical significance and chemical composition of different water content of the leaves at time of harvest (Chen et al., in press;
Aloe spp. has remained largely inaccessible, despite this knowl- O’Brien et al., in press).
edge being of potential importance in biodiversity conservation The most studied gel from any Aloe spp. has been on A. vera,
and ecotourism (Grace, 2011). Aloe vera L. is undoubtedly one for which the gel is found in a clear internal zone located between
of the most important medicinal plants in the world, providing the abaxial and adaxial mesophyll. This central zone is called dif-
the raw materials for different uses (medicinal, cosmetic, tonic ferent names including pulp, mucilaginous tissue, mucilaginous
drinks, and numerous other uses in the food industry). Statistics gel, and parenchyma tissue and is composed of cell degenerate
given by the International Aloe Science Council (2012) indicate that organelles, and the viscous liquid contained in the cells (Li et al.,
about 24,000 ha of A. vera are cultivated globally, with an economic 2003). The chemical composition of the gel is very complex, com-
value of US$300 billion. However, in view of the large number posed mainly of polysaccharides and soluble sugars, followed by
of Aloe species it is surprising that little research has been done proteins, many of which are enzymes, aminoacids, vitamins and
to investigate the commercial potential of other species. In fact, anthraquinones (Liu et al., 2007). The gel of this species has been
Aloe arborescens Mill. was used to treat nuclear irradiation burns used for pre-harvest or postharvest treatments to maintain fruit
from the atomic bomb explosion over Hiroshima during the Sec- quality attributes and reduction in fungal decay of sweet cherry
ond World War, and is currently under commercial development (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
∗ Corresponding author. Tel.: +34 96 674 9616; fax: +34 96 674 9678. shown for Botritys cinerea, Rhizopus stolonifer and Penicillium digi-
E-mail address: [email protected] (M. Serrano). tatum.

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.06.002
224 P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

In view of the large number of Aloe species (more than 400 have remaining Aloe species four leaves were used to be inoculated with
been described) it is surprising that only the above commented each fungal species. In these leaves, four injuries were done as
three species have been commercialised, and so little, or no data, above. For each injury, 20 ␮L containing 100 spores of the corre-
have been published describing the properties of other Aloe species. sponding fungi stock were deposited, and after 7-days the presence
In this sense, we have investigated the leaf characteristics and gel or absence of fungus growth in the injury was determined, and
chemical composition of eight Aloe species (A. arborescens Mill., results expressed as percentage of infected wounds.
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 2.2. Leaf dimensions and gel yield
activity of the leaves from the eight species against the most typical
fruit pathogenic fungi (B. cinerea, P. digitatum, Penicillium expansum The leaves from the selected plants were removed at the point
and Penicillium italicum) was also evaluated. With this informa- of attachment with a sharp knife, washed with tap water to remove
tion there is the possibility of using other Aloe species, apart from the adhering soil particles and deposited inside a container for 1 h
A. vera, to obtain gels usable as either pre-harvest or postharvest to permit the efflux of yellow latex. Then, the leaves were weighed
treatments. and results expressed as grams. Leaf length (cm), the width at mid-
point and base of the leaves and thickness (cm) at the mid-point
of the leaf were measured by a measuring stick and Vernier cal-
2. Materials and methods liper. For each leaf, the spikes placed along their margins were
removed before longitudinally slicing to separate the epidermis
2.1. Plant material and experimental design from the parenchyma. The parenchyma fillets were crushed to yield
a mucilaginous gel which was filtered to discard the fibrous frac-
In this work eight Aloe species were used: A. arborescens Mill., A. tion. The gel yield was expressed as percentage of the obtained gel
aristata Haw., A. claviflora Strydenburg, A. ferox Mill., A. mitriformis with respect to the whole leaf weight. In the fresh gel pH (pH metre
Mill., A. saponaria Ait., A. striata Haw., and A. vera L. plants were Crison, Spain) and total soluble solids as ◦ Brix (Atago refractome-
grown under standard organic farming practices in Orihuela, Ali- ter, Japan) were analysed. Then, the gel was frozen using liquid N2 ,
cante, Spain, and the external leaves from 3-year-old plants were stored at −40 ◦ C until performing the following analytical determi-
used in the experiments. Soil characteristics were: Clayey-Icam, nations.
pH 8.52 and electrical conductivity 0.16 dS m−1 . The aloe plants
were cultivated according to the National Regulation for Ecolog-
ical Agriculture (Comité de Agricultura Ecológica de la Comunidad 2.3. Aloin concentration
Valenciana http://www.caecv.com/), without any addition of syn-
thetic fertilizers, herbicides or pesticides. Gel (5 g) was homogenised in 25 mL of methanol (50%, v/v) con-
In Experiment 1 (Year 2010), for each Aloe spp., six mature taining 2 mL HCL 0.1 N and NaF 5 mM by using a PolytronTM at
leaves were taken from six different plants at 3-time intervals: 9500 rpm for 2 min. After homogenisation, samples were sonicated
end of February (Winter), end of May (Spring) and end of August at 10 ◦ C for 60 min and then centrifuged at 20,000 × g for 15 min.
(Summer). Climatic data were recorded from the local meteorol- The supernatant was filtered through 0.45 ␮m Millipore filter and
ogy station (Estación MU21, http://siam.imida.es, GPS Coordinates: then 1 mL injected into HPLC-DAD (Hewlett-Packard HPLC-1100
N38◦ 2 4.33 and W0◦ 59 58.72 ). Media of maximum and mini- Series), system equipped with a C18-column (Supelcogel C-610H,
mum temperatures were 15.37 and 5.41, respectively in February, 30 cm × 7.8 mm, Supelco Park, Bellefonte, USA). Aloin A was eluted
26.37 and 14.12, respectively in May and 30.42 and 23.36, respec- isocratically by methanol-water as mobile phase (64:36, v/v con-
tively in August. Rainfall was 29.8, 23.2 and 47.4 mm, for, winter, taining 0.5% formic acid) at flow rate of 1 mL min−1 and detected
spring and summer, respectively. Radiation was 125.7, 290.86 and at 254 nm wavelength. A calibration curve was performed by using
248.32 W m−2 and sun hours were 202, 369 and 335 in February, aloin A (barbaloin) standard (Sigma, Madrid, Spain) at concentra-
May and August, respectively. The experiment was carried out at tions ranging 0–100 mg L−1 (y = 4.71x + 4.25, R2 = 0.9979). Results
the three seasons within the same year by using the same exper- were expressed as mg 100 g−1 fresh weight).
imental 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 2.4. Polyamine extraction and quantification
differences amongst species to genetic characteristics of each one.
The leaves (harvested 3 h after sunrise) were transferred to the For each gel, 1 g fresh tissue was extracted with 10 mL of 5%
laboratory where weight, length, thickness and width were deter- cold perchloric acid. 1,6 hexanediamine (100 nmol g−1 of tissue)
mined, and then the parenchymatous tissue was manually removed was added as an internal standard. The homogenate was then
to obtain the gel from each leave. The analytical determinations in centrifuged for 30 min at 20,000 × g. A 2 mL aliquot of the super-
the gel were: total soluble solids (TSS), pH, total acidity, total pro- natant was used to determine free polyamines by benzoylation, and
teins, total lipids, free polyamine content (putrescine, spermidine derivatives analysed by HPLC according to previous work (Serrano
and spermine), and aloin A (barbaloin) concentration by HPLC. et al., 2003). The elution system consisted of MeOH/H2 O (64:36)
In Experiment 2 (Year 2011), leaves were picked from the same solvent, running isocratically with a flow rate of 0.8 mL min−1 .
Aloe species at the end of August (Summer), and inoculated with The benzoyl-polyamines were eluted through a reversed-phase
several plant pathogenic fungi (B. cinerea, P. digitatum, P. expansum column (LiChroCart 250–4,5 ␮m) and detected by absorbance at
and P. italicum). The fungi were purchased from the Spanish col- 254 nm. A relative calibration procedure was used to determine the
lection of type culture, and routinely cultured in potato dextrose polyamines in the samples, using 1,6 hexanediamine as the inter-
agar (PDA). For the Aloe species with small leaves (A. mitriformis, A. nal standard and standard curves covered the range 1–320 nM. The
clavoiflora and A. aristata) eight leaves were used to inoculate each calibration curves were y = 10.66x + 170.00, R2 = 0.94 for Putrescine,
fungal species. In each of these leaves, 2 injuries (2 mm × 2 mm y = 10.19x − 39.96, R2 = 0.96 for Spermidine, and y = 11.52x − 4.32,
in length and width and 2 mm in depth) were performed along R2 = 0.90 for Spermine. Results were expressed as nmol g−1 fresh
the leaf surface with a sterile lancet for fungal inoculation. For the weight).
 P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230 225

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 42.43 ± 1.08 A 4.29 ± 0.14 A 2.97 ± 0.11 A 0.49 ± 0.02 A 50.6 ± 4.2 A
A. aristata 21.40 ± 0.52 B 4.91 ± 0.23 B 3.92 ± 0.15 B 0.86 ± 0.02 B 32.21 ± 1.6 B
A. claviflora 26.20 ± 1.60 C 6.78 ± 0.63 C 6.16 ± 0.52 C 2.56 ± 0.21 C 77.74 ± 3.5 C
A. ferox 66.00 ± 3.42 D 11.21 ± 0.83 D 12.79 ± 0.83 D 2.87 ± 0.17 C 725.3 ± 90.7 D
A. mitriformis 14.36 ± 0.28 E 5.20 ± 0.27 B 4.64 ± 0.28 E 0.66 ± 0.03 D 33.8 ± 1.0 B
A. saponaria 26.86 ± 1.03 C 9.86 ± 0.67 E 7.93 ± 0.56 C 1.10 ± 0.10 E 170.2 ± 10.3 E
A. striata 36.86 ± 0.90 F 10.50 ± 0.66 DE 12.29 ± 0.96 D 1.99 ± 0.14 F 343.3 ± 24.6 F
A. vera 68.29 ± 5.14 D 11.79 ± 0.48 D 9.57 ± 0.36 F 2.14 ± 0.17 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 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 agree-
The gels (5 g) were centrifuged at 15,000 × g for 10 min at 4 ◦ C. ment with our results.
The supernatant was separated and used for soluble protein mea- The Aloe gel yield generally decreased from winter to sum-
surement. The protein content was determined in the supernatant mer (Table 2) for all Aloe spp., although the decrease depended
according to Bradford’s dye binding method, using bovine serum on the specific species, with 22% being found for A. saponaria and
albumin (BSA) as standard (Bradford, 1976). non-significant decreases in A. claviflora, A. striata, and A. vera. In
addition, the percentage of obtained gel was also dependent on the
2.6. Total lipids 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.
Total lipids were extracted according to Valero et al. (1990). arborescens. The pH also diminished from winter to summer and
Briefly, 2 g of Aloe gel were homogenised in 10 mL chloro- the values differed amongst Aloe spp., especially during the win-
form:methanol:0.1 N HCl (200:100:1) and then 10 mL of 0.1 N HCl ter season. These results would indicate that during winter the
were added before centrifugation at 4000 × g for 10 min. The lower photosynthetic capacity and acid accumulation during the night
organic phase containing the total lipids was collected and taken was lower than in summer. Since leaves were harvested 3 h after
to dryness in a water bath at 45 ◦ C under continuous flushing of N2 sunrise, a lower pH value would indicate that higher acid accu-
gas. The residue was weighed and total lipids were expressed as g mulation in the vacuole occurred during the night period (Walker
100 g−1 . and Leegood, 1996). The pH values found in the gel of the differ-
ent species for the samples taken on spring and summer are within
2.7. Statistical analysis the range reported previously for A. vera gel (Eshun and He, 2004;
Vega-Gálvez et al., 2011).
Data from analytical determinations were subjected to analy- With respect to the content of total soluble solids, differences
sis of variance (ANOVA). Sources of variation were Aloe spp. and were also found amongst Aloe spp. (Table 2) although generally
harvest season. Mean comparisons were performed using the high no differences could be attributed to harvest season, with the
significant difference (HSD) Tukey’s test to examine if differences exception of A. claviflora, A. ferox and A. striata for which signifi-
were significant at p < 0.05 and are shown in tables and figures cant increases were found from winter to summer. The analysis
with significant letters. All analyses were performed with statis- of total lipids and total proteins revealed that the gels obtained
tical products and service solutions (SPSS) software package v. during the summer season showed the highest concentration
11.0 for Windows. Linear regression between aloin concentration (Table 3), although differences existed amongst the Aloe spp. Thus,
and fungal growth on inoculated wounds was performed by using A. arborescens had the highest concentration of both lipids and pro-
SigmaPlot 11 for Windows. teins followed by A. mitriformis and A. striata. Beppu et al. (2004)
studied the chemical components of A. arborescens but using the
3. Results and discussion whole leaf (epidermis and parenchyma) and found that total pro-
teins were also higher in the warm than in the cold season, whilst
3.1. Leaf characteristics and composition no significant differences were found for total saccharides (◦ Brix).
In the case of A. vera, the soluble solids in the gel was found at
During the experiment, no significant differences were obtained 0.56% with some seasonal fluctuations, whilst levels of protein and
in leaf dimensions and weight amongst the 3 harvest seasons since lipids were 7 and 4% (Ahlawat and Khatkar, in press), or 3.7 and
only the external leaves from the plants were picked. Then, data are 4.5% (Vega-Gálvez et al., 2011), respectively, on dry matter basis.
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 dif- 3.2. Aloin concentration
ferent climactic and soil conditions. However, the leaves of the
several Aloe spp. differed significantly in leaf dimension and weight One of the main biologically active constituents of Aloe
(Table 1), in which A. mitriformis and A. aristata showed the small- extracts is aloin or barbaloin (10-glucopyranosyl-1.8-dihydroxy-3-
est leaves (≈14–21 cm in length and ≈32–34 g of weight) and the (hydroxymethyl)-9 (10H)-anthracenone), which is found in nature
largest leaves were found in A. ferox (≈66 cm in length and ≈725 g as a mixture of two diastereosiomers, aloin A (10R) and aloin B
of weight). In addition, the leaves of A. ferox also had the great- (10S). These two compounds are generally used as key compo-
est thickness and width, both at the basal and mid-points. This nents for the quality control of this plant and its derivatives. Aloin is
variability in the leaf dimensions of the several Aloe spp. probably generally contained in the bitter, smelly exudate seeping out from
reflects a combination of both genetic and environmental effects. freshly cut leaves, whilst very low amounts of aloin exist in Aloe gel
In a recent paper on 24 plants of A. ferox from eight populations, obtained from the internal mass of Aloe leaf (Fanalli et al., 2010). In
226 P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

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.
1.69 ± 0.05 Db
1.62 ± 0.06 Da
B

1.91 ± 0.04 Aa

1.50 ± 0.06 Cb
1.15 ± 0.02 Ba

1.15 ± 0.03 Ba
1.51 ± 0.06 Ca
1.06 ± 0.06 Bc
Winter
Spring

Summer
8 C Summer

B
B C

Aloin (mg 100 g )

6

-1
C A B
A

0.86 ± 0.07 Db
1.45 ± 0.05 Cb
2.10 ± 0.07 Aa

1.60 ± 0.04 Eb
1.15 ± 0.02 Ba

1.11 ± 0.03 Ba
1.45 ± 0.05 Ca
1.71 ± 0.06 Ea
A
A C
B
Total soluble solids (g 100 g−1 )

4 C B
Spring

A
A
B
B
A
2
A
A
1.75 ± 0.03 Da

A
2.15 ± 0.02 Aa

1.25 ± 0.02 Ba

1.12 ± 0.02 Ba
1.10 ± 0.10 Ba

1.30 ± 0.09 Ba
0.63 ± 0.08 Ca

1.41 ± 0.03 Ea

A
Winter

ns ta ora ox mi
s
ari
a
iat
a ra
ce sta vifl fer for on str ve
ore
s ari cla A. itri ap A. A.
A. . m s
arb A A. A .
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
4.66 ± 0.06 Ab

4.67 ± 0.02 Ab
4.69 ± 0.03 Ab
4.76 ± 0.05 Ab

4.58 ± 0.01 Ab
4.98 ± 0.03 Bb
5.01 ± 0.07 Bb
4.71 ± 0.03 Ac

amongst seasons show significant differences at p < 0.05.

Summer

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 concen-
4.78 ± 0.05 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
4.90 ± 0.02 Ab

trations 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).
Spring

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 -O-
7.79 ± 0.09 Da
7.09 ± 0.07 Aa
7.48 ± 0.25 Ba
7.52 ± 0.13 Ba
6.82 ± 0.02 Ca

5.52 ± 0.03 Ea

5.30 ± 0.28 Ea
5.73 ± 0.07 Fa

Acetate) was identified as a member of the anthrones in A. claviflora

Winter

(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
pH

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
51.34 ± 3.59 Db

48.86 ± 2.35 Db
50.67 ± 2.25 Db
43.89 ± 0.78 Bb
32.27 ± 1.34 Ac

61.72 ± 3.80 Ca

63.38 ± 1.04 Ca
56.90 ± 3.05 Ca

been described as anthrone-C-glucosides typical of Cape aloe (A.

ferox), which can be metabolised to aloin A, aloin B and a hydroxyl
Summer

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

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
51.72 ± 3.54 Db

52.38 ± 1.05 Db
38.10 ± 1.75 Ab

54.79 ± 2.95 Da

56.28 ± 1.05 Da
40.57 ± 2.50 Bb
62.57 ± 4.45 Ca

65.76 ± 1.13 Ca

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
Spring

cold ones (Beppu et al., 2004). For A. hereroensis, a type of phe-

nolics 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
Aloe gel yield (%)

58.98 ± 1.88 Da
58.89 ± 1.56 Da

57.24 ± 2.13 Da
62.10 ± 0.83 Da
43.78 ± 1.03 Aa
50.28 ± 2.12 Ba

66.47 ± 1.59 Ca
65.80 ± 1.36 Ca

gels obtained during the summer season could be a consequence

of the high radiation level, which was ≈300 W m−2 day−1 according
Winter

to Regional Meteorological Institute for 2010.

3.3. Phenolic compounds and antioxidant activity

Total phenolic concentration was dependent on the Aloe spp.,

A. arborescens

A. mitriformis
A. saponaria
A. claviflora

with A. arborescens having the highest phenolics and the lowest

A. aristata

A. striata
Aloe spp

A. ferox

being found for A. aristata, A. saponaria and A. vera (Fig. 2). In addi-
A. vera
Table 2

tion, harvest season also influenced the content of total phenolics,

a

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 0.13 ± 0.03 Aa 0.15 ± 0.01 Aa 0.42 ± 0.02 Ab 0.26 ± 0.01 Aa 0.31 ± 0.02 Aa 0.54 ± 0.05 Ab
A. aristata 0.10 ± 0.01 Aa 0.06 ± 0.01 Bb 0.18 ± 0.03 Bc 0.14 ± 0.01 Ba 0.06 ± 0.02 Bb 0.12 ± 0.01 Ba
A. claviflora 0.14 ± 0.02 Aa 0.07 ± .0.01 Bb 0.29 ± 0.03 Cc 0.26 ± 0.05 Aa 0.24 ± 0.05 Aa 0.24 ± 0.01 Ca
A. ferox 0.07 ± 0.03 Aa 0.07 ± 0.01 Ba 0.17 ± 0.03 Bc 0.05 ± 0.01 Ca 0.02 ± 0.01 Ba 0.15 ± 0.03 Bb
A. mitriformis 0.11 ± 0.01 Aa 0.09 ± 0.01 Ba 0.36 ± 0.02 Ab 0.28 ± 0.01 Aa 0.11 ± 0.02 Cb 0.39 ± 0.01 Dc
A. saponaria 0.13 ± 0.03 Aa 0.09 ± .0.01 Ba 0.19 ± 0.03 Bb 0.11 ± 0.02 Ba 0.09 ± 0.01 Ca 0.17 ± 0.01 Bb
A. striata 0.12 ± 0.02 Aa 0.09 ± 0.01 Ba 0.36 ± 0.04 Ab 0.14 ± 0.01 Ba 0.06 ± 0.02 Bb 0.25 ± 0.01 Cc
A. vera 0.11 ± .0.02 Aa 0.08 ± 0.01 Ba 0.19 ± 0.02 Bb 0.20 ± 0.03 Aa 0.16 ± 0.05 Ca 0.22 ± 0.01Ca
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.

higher phenolic concentrations than those taken during the spring.

Hydrophilic Total Antioxidant Activity (mg 100 g )

-1
These results would indicate that environmental low and high tem-
C Winter
peratures could induce an accumulation of total phenolics as a Spring
stress response. In fact, in 2010 the winter samples were harvested Summer
30
after 3 months with temperatures below 5 ◦ C (average minimum
temperature), whilst the summer samples after 3 months with tem-
perature over 30 ◦ C (average maximum temperature) according to
Regional Meteorological Institute. A
20 A A
Total antioxidant activity (TAA) of the Aloe gels was measured B
A
C C
separately in two fractions: hydrophilic (H-TAA) and lipophilic B A
C
(L-TAA). For all Aloe spp., H-TAA (Fig. 3) was higher than L-TAA A B
(Fig. 4). H-TAA showed a similar behaviour to that of total phenolic 10
B B
concentrations, that is higher activities during the winter and sum- C C A A
mer seasons than during spring, the highest H-TAA levels being A A
B B
B
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 en
s ta ra ox mi
s ria iat
a ra
sc sta iflo fer for na str ve
ore ari lav A. itri po A. A.
contributing to H-TAA, according to previous reports in a wide r b A. A .c . m . sa
a A A
A.
range of fruit and vegetables (Valero and Serrano, 2010). Amongst
phenolic compounds identified in the latex from Aloe harlana, the Fig. 3. Hydrophilic total antioxidant activity (H-TAA) in the gel of different Aloe
highest antioxidant activity corresponded to 7-O-methylaloeresin species from the leaves harvested at 3 different seasons. Data are the mean ± SE.
A followed by aloin (Asamenew et al., 2011). In A. ferox, TAA Different capital letters amongst seasons show significant differences at p < 0.05.
expressed as ORAC (in both hydrophilic and lipophilic fractions)
was also reported, although the concentration of individual contribution of ascorbic acid (vitamin C, a well-known hydrophilic
polyphenols antioxidants are not the only factors influencing antioxidant moiety) to H-TAA should not be discharged, since
antioxidant capacity, since other compounds such as indoles, it is present in A. vera gel (Vega-Gálvez et al., 2011) at high
alkaloids are also known to possess antioxidant activities (Loots concentration (160 mg 100 g−1 , dw). With respect to L-TAA, an
et al., 2007). Although it was not determined in this work, the increase was generally found from winter to summer, although
Lipophilic Total Antioxidant Activity (mg 100 g )
-1

C
Winter 12 Winter
C
Spring Spring
40
Summer Summer
A
Total Phenolics (mg 100 g )
-1

10
B
C C
30
8
A B
B
6
20 A
A A A A
A AA
B A A 4 C BB
A A
C B B
10 B B
B A
B 2 B B
B A A A B AA
AA AA B
A

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

Fig. 2. Total phenolic concentration in the gel of different Aloe species from the Fig. 4. Lipophilic total antioxidant activity (L-TAA) in the gel of different Aloe species
leaves harvested at 3 different seasons. Data are the mean ± SE. Different capital from the leaves harvested at 3 different seasons. Data are the mean ± SE. Different
letters amongst seasons show significant differences at p < 0.05. capital letters amongst seasons show significant differences at p < 0.05.
228 P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230

C
Winter
Winter
Spring C Spring
Summer 80 Summer
150 C

Sperrmidine (nmol g )
-1
Putrescine (nmol g )
-1

60

100

40 C
C
B B
B AC
C B B AA
50 A C
B B 20
B
C A BB A A AA B
B A C B A AA
A C A AA A AA B A A
AB A

en
s ta ra ox mi
s ria iat
a era ns ta ora ox s a a ra
sc sta iflo fer for na str v ce sta fer mi ari iat ve
ore ari lav A. itri po A. A. s ari vifl A. for on str A.
r b A. A .c . m . sa ore A. . cla mitri s ap A.
A.
a A A arb A A. A.
A.

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

the activity was different amongst the Aloe spp., with A. mitriformis postharvest treatment with putrescine or spermidine led to a delay
having the highest L-TAA and A. saponaria and A. vera the lowest. of the ripening process of plums (Serrano et al., 2003), alleviated
In A. vera occurrence of vitamin E has been reported (Vega-Gálvez mechanical damage in lemon (Martínez-Romero et al., 1999) and
et al., 2011), which might contribute to L-TAA due to its lipophilic reduced chilling injury in pomegranate (Mirdehghan et al., 2007), as
nature and its role in protecting the fatty acids of the membranes a consequence of increased endogenous polyamine concentration.
against damage caused by free radicals. Very recently it has been In addition, pre-harvest application of putrescine increased the
reported that both methanolic and acetone extracts from A. vera functional ovules of apricot flowers (Alburquerque et al., 2006). The
gel exhibited the highest antioxidant activity compared with pre-harvest application of A. vera gel on table grape led to reduc-
those from ethanolic, hexane and chloroform extracts (Saritha tion on fruit metabolism during postharvest storage (Castillo et al.,
et al., 2010), which would indicate that hydrophilic compounds 2010), which could be induced by the polyamine content of the
contribute more than lipophilic ones to antioxidant activity of Aloe gel. However, more studies are necessary to correlate the relation-
gels according to our results. ship between the effects of Aloe gels used as pre- and postharvest
Although the Aloe gels show antioxidant capacity lower than treatments and polyamines.
tea extract or ascorbic acid, they have more activity than the syn-
thetic BHT or ␣-tocopherol (Khaing, 2011). It has been suggested 3.5. Fungal growth in Aloe species leaves
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 Given that the highest aloin, total phenolics, total antioxidant
experiment the leaves were harvested from plants aged 3-year-old activity, total lipid and protein concentrations were found in the
and then the observed differences should be attributed to the Aloe gels obtained from the leaves collected in summer, the following
species and harvest season within the Mediterranean climate. year leaves were picked at this season for determining the Aloe
leaves sensitivity to be infected by several fruit pathogenic fungi.
3.4. Polyamines The assayed fungi were B. cinerea, P. digitatum, P. expansum and
P. italicum. The results revealed that after 7 days of inoculation 3
The levels of free polyamines (putrescine, spermidine and sper- distinctive types of injuries were found: (a) injuries with the same
mine) were determined by HPLC-DAD in the gels of the several initial size and necrosis (cured); (b) injuries with increased injury
Aloe species, and results revealed that concentration of putrescine size due to an initial fungus development but with further necrosis
were higher than spermidine and spermine. Generally, levels of and (c) injuries with active growth of the fungus. Accordingly, the
putrescine and spermidine increased along the season, and espe- results of the latter were shown in Fig. 7. It is clear that B. cinerea
cially from spring to summer, although increases were different was the most invasive fungus since it was able to grow on all Aloe
depending on the Aloe species. Thus, putrescine increased ≈10–20- spp., although differentially depending of the species, and the most
fold for A. vera, A. saponaria and A. ferox (Fig. 5), whilst for effective Aloe species on inhibiting the growth of this fungus was
spermidine the highest increases were found for A. vera and A. A. ferox, whilst B. cinerea grew up in ≈70–80% of the injuries for
saponaria (Fig. 6). However, this behaviour was not found for sper- the remaining Aloe species. Overall, the most effective Aloe spp.,
mine which increased from spring to summer just in A. vera (data on inhibiting fungal growth were A. mitriformis and A. saponaria, in
not shown). In a study with the whole leaf of A. arborescens (Beppu which absence of Penicillium spp. growth was obtained, followed
et al., 2004), putrescine and spermidine levels were high in sum- by A. arborescens and A. ferox, in which P. italicum and P. digita-
mer and low in either spring or winter, which is in agreement tum grew up just in 10% of the injuries, respectively. In previous
with our results. This is the first time in which the content of reports, the efficacy of A. vera gel on reducing fungal growth on
free polyamines has been carried out in a wide range of Aloe spp. PDA plates was higher for P. digitatum than for B. cinerea, although
Since polyamines have been recognised as anti-senescence plant for both fungi the inhibition of mycelium growth increased as did
hormones (Valero et al., 2002), the occurrence of free polyamines the Aloe gel concentration (Castillo et al., 2010). In addition, A.
in the Aloe gels could be considered as a goof source of anti- vera pulp showed an inhibitory effect on the mycellium develop-
ageing agents for fruit pre- and postharvest treatments. In fact, ment of Rhizoctonia solani, Fusarium oxysporum, and Colletotrichum
 P.J. Zapata et al. / Industrial Crops and Products 42 (2013) 223–230 229

Acknowledgements
100 Botrytis cinerea
P. digitatum
P. expansum This work has been co-funded by the Spanish Ministry of Sci-
P. italicum ence and Innovation (MICINN) and FEDER Funds through Project
80 AGL2009-10857 (ALI).
Infected wounds (%)

60
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