Stability of the main Aloe fractions and Aloe-based commercial products under different storage conditions

Abstract

Aloe-based household preparations and commercial products are used worldwide as a source of nutraceutical compounds, among which anthraquinones and acemannan are the most known. However, it is reported that improper storage conditions could lead to significant degradation processes, resulting in a low content of active substances. The aim of this work was to study the stability of aloin and β-polysaccharides in aloe-based products during storage, as a function of temperature and chemical agents added.
Generally, aloin degradation in whole leaf homogenate was fast, and neither ascorbate nor the antimicrobial agents could increase stability; a lower temperature slightly improved stability, (DT50 up to 26 days at +4 °C). Regarding the stability of β-polysaccharides, a strong influence of the storage temperature was observed, while antimicrobial agents did not show significant reduction of degradation. For both compounds, household and commercial products were significantly more stable.

Full text

Stability of the main Aloe fractions and Aloe-based commercial
products under different storage conditions
M. PELLIZZONI, G.P. MOLINARI, L. LUCINI*
Institute of Environmental and Agricultural Chemistry, Università Cattolica del Sacro Cuore, via Emilia
Parmense, 84, 29122 Piacenza, Italy
Keywords: Anthraquinones, antimicrobial agents, antioxidant agents, degrada-
tion, nutraceuticals, β-polysaccharides
I. – Aloe barbadensis Miller (Aloe vera L.) and Aloe
arborescens Miller are members of the Liliaceae family which are
widely used as source of functional foods, for the application of health
drinks and for pharmaceutical and cosmetic purposes (G and
C-V, 2006; R and S R, 2008).
Many studied have reported that biological activity of Aloe includes pro-
motion of wound healing, antidiabetic, antimicrobial, immunomodulatory,
antioxidant and gastroprotective properties (L and C,
2001; H 2008). Some characteristic polysaccharides seem to pos-
sess the most of pharmacological and physiological properties besides a
synergic action between different components is reported by other authors
(R and S R, 2008; L, 1978). Among these
further active compounds, hydroxyanthraquinones are the most important.
Among polysaccharides, some acetylated mannans, namely aceman-
nan, are the main active compounds (H, 2008; MA,
1993). Acemannan has a backbone of β-1,4-D-mannosyl and β-1,4-D-
glucosyl residues, acetylated at the C2 and C3 positions on the mannose
residues, and some side chains of D-galactose attached to its C6.
Anthraquinones are secondary phenolic metabolites including
C-glucosyl derivatives such as barbaloin (10-glucopyranosyl-1,8-di-
hydroxy-3-hydroxymethyl-9-10H-anthracenone), a mixture of the two
diastereoisomers aloin A and B, as well as glucose-free compounds such
as aloeresin, aloenin and aloe-emodin (F et al. 2010). They were
found in the yellowish bitter exudate seeping out from freshly cut leaves
Agrochimica, Vol. LV - N. 5 September-October 2011
* Corresponding author: luigi.lucini@unicatt.it
Received XX Xx 2011 – Received in revised form XX Xx 2011 – Accepted XX Xx 2011

M. PELLIZZONI ET AL.
204
and were reported to have cathartic effects, anti-inflammatory effects in
vivo, to increase the peristaltic movements of the intestinal musculature
and also prevent the colon from reabsorbing water (G and
C-V, 2006; MA, 1993).
The acemannans are stored in the inner mucilaginous parenchyma of
the leaves, while anthraquinones are mainly localized in the outer rind
of leaves.
Processing of Aloe leaves, and in particular leaf pulp of A. bar-
badensis, has become a big industry worldwide due to the application in
the food industry (R and S R, 2008). Besides
a rapid degradation of the active compounds in different conditions
was described, the real content of aloin and acemannan in commercial
products is not usually declared (G and C-V,
2006; R and S R, 2008).
Industrial processes often involve heating, dehydration and grind-
ing, which may cause irreversible modifications to the active substances,
promoting important changes in the physiological and pharmaceutical
properties (T et al. 1992). Because of these improper process-
ing procedures, many Aloe products contain very little active ingredients
R and S R (2008). Improper storage conditions
can lead to degradation processes as well, as a consequence of tempera-
ture, light and humidity (G and C-V (2006).
In fact, leaves loose their biological activity at room temperature starting
from 6 hours from harvest (R and S R, 2008).
At present, few and discrepancy information is available on aloin
and polysaccharides content in Aloe-based commercial products and on
the stability of bioactives during storage. On this basis, our aim was to
investigate the stability of anthraquinones and acemannans during stor-
age at different conditions. With this purpose, whole leaf homogenate,
leaf gel homogenate, a very common household preparation from folk
medicine (father Zago’s recipe) and two commercial products (one
based on the whole leaf and another based on the sole leaf gel), were
considered. The stability of β-polysaccharides and aloin were investi-
gated in all the matrices considered as a function of temperature, oxygen
and addition of antimicrobial and antioxidant agents.
M  . – Leaves of A. barbadensis and A. arborescens were
taken randomly from three years old plants supplied by Dester Garden, Brescia, Italy.
Plant materials were immediately stored in freezer at -30°C until the beginning of the
experimental phase.

STABILITY OF DIFFERENT ALOE FRACTIONS
205
Preparation of test items. – Gel and whole leaf homogenates. – Leaf gel was gained
from A. barbadensis plants according to industrial applications for gel-based products.
Three fresh leaves from a basal, central and apical position were taken from 3 different
plants, and after manual removal of the peel, the gel fractions were pooled and homog-
enized by a blender.
As far as concerns A. arborescens, however, three fresh leaves from the central
and three fresh leaves from the lateral stem were taken from 3 plants. The leaves were
pooled and homogenized by a blender to prepare a representative sample of whole leaf
homogenate.
Samples with antimicrobial or antioxidant agents. – To inhibit microbial growth,
a preservative of either 0.1% sodium azide or 0.1% of benzoate were added to different
samples of A. arborescens whole leaf homogenate and A. barbadensis gel.
In order to test the effect of antioxidants, samples of A. arborescens whole leaf
homogenate were prepared by adding 0.05% and 0.005% of ascorbate.
In both cases, an aqueous solution of each agent was prepared and then a volume
added to an accurate weight of matrix.
Commercial and household prepared products. – The most common worldwide
household preparation (father Zago’s recipe) was prepared by mixing in a blender 350 g
of A. arborescens whole leaf homogenate, 40 mL of grape brandy and 500 g of honey.
Furthermore, an A. barbadensis stabilized gel and a whole leaf homogenate (again
prepared according to father Zago’s recipe) commercial products were bought in a local
supermarket.
Test conditions. – All samples were stored for 65±5 days in sealed glass tubes and
in dark. The quantitative analysis of aloin and β-polysaccharides was carried out at 5 dif-
ferent time points as minimum and the concentration values were compared with those
at the beginning of the test.
The tests with A. barbadensis gel and A. arborescens whole leaf homogenate with
the addition of an antioxidant agent were done at two different temperature conditions:
in refrigerator (+4°C) and at room temperature (+22°C). Those with the addition of anti-
microbial agents were instead carried out at room temperature (+22°C) only, in order to
enhance the effect of benzoate or sodium azide in a shorter time. As far as the oxygen
conditions are concerned, a further test with A. arborescens whole leaf homogenate to
which the antimicrobial agents were added, was done after an initial removal of air by
degassing with nitrogen.
Regarding the commercial products, the stability tests were carried out according to
the recommended or actual storage conditions. Tests with the preparation done accord-
ing to father Zago’s recipe was carried out stored at refrigerator temperature (+4°C)
while those with the stabilized gel was done at both refrigerator (+4°C) and room tem-
perature (+22°C). These different temperature conditions were chosen in according to
information in commercial labels.
Determination of anthraquinones and acemannan content. – Prior to be analysed,
each sample was visually examined in order to evaluate changes in appearance: color,
consistency, moulds development were checked at each sampling point. Anthraquinones
were then determined in each sample by liquid chromatography followed by tandem
mass spectrometry with electrospray ionisation source (LC-ESI/MS/MS) in the negative
mode, and quantified by the external standard method. A 1200 series liquid chromato-
graph system was used, equipped with quaternary pump, electrospray ionization system

M. PELLIZZONI ET AL.
206
and coupled to a G6410A triple quadrupole mass spectrometer detector (all from Agilent
technologies, Santa Clara, CA, USA). The plant material (2 g) was extracted by Ultra-
Turrax in 8 + 4 ml of ethyl acetate / methanol mixture (9:1 by volume) after adding 4 ml
of a 20% NaCl aqueous solution. After centrifugation (2000 g for 15 min) the extract
was diluted with methanol, filtered through a 0.45 m membrane and then analysed
by reversed phase LC-MS/MS using a Zorbax Eclipse plus C18 column (300 mm, 3.5
m) from Agilent. The solvent system used was water (solvent A) and acetonitrile (sol-
vent B) at a flow rate of 0.3 ml min-1; the gradient was designed to decrease solvent A
from 35% at 0 min to 20% at 2.5 min. The injection volume was 10 l and the drying gas
was nitrogen at 5 l min-1. Data handling was performed by ChemStation software under
Multiple Reaction Monitoring (MRM) acquisition: aloin transition was from m z-1 417
(M-H)- to 297 (collision energy 15 V).
Polysaccharides were instead determined colorimetrically at 540 nm, after binding
with the Congo red dye, on the basis of the work described by Eberendu et al. (2005).
Colorimetric measurements were done using a Perkin Elmer UV/VIS spectrometer
lambda 12. Aloe pulp (4 g) was extracted in 10 ml of double distilled water, on a hori-
zontal shaker for 2 h; 500 l of 1.3% KOH was added to the solution and then 2 ml
of Congo red solution (obtained diluting a saturated aqueous solution by a factor 50)
was also added. The solution was left for 1 h in dark and then analysed at λ = 540 nm.
Semi-quantitative determination of the solutions was carried out using a pure β-glucan
standard. Three measurements on three different extracts were performed for each leaf
pulp sample.
Raw data elaboration. – Single first order kinetic models were applied to describe
the degradation curves of bioactive substances in different Aloe products. Logarithmic
transformation of the measured concentrations was done and then the different log
concentrations were included in graphics in connection of the time, and modeled using
linear functions. Sufficient and adequate time points were chosen to ensure a robust esti-
mation of parameters; five points were used in all cases as minimum. Regression linear
coefficients (R2) were calculated to each linear regression curves.
The time for decrease in the concentration of β-polysaccharides and aloin from
100% to 50% (DT50) and to 10% (DT90) of the initial amount corresponding for each
analyte were numerically obtained through mathematical elaboration of different linear
regression curves slopes (k). The DT50 and DT90 were calculated as ln2/k and ln10/k
respectively.
R  . – The regression linear coefficients
showed a good correlation, ranging between 0.766-0.999 for the stability
of aloin and between 0.798-0.999 for β-polysaccharides. A worst corre-
lation (R2 = 0.502) was observed regarding the stability of the gel-based
commercial preparation only.
As far as visual assessments are concerned, the colour of Aloe gel
after preparation changed slightly from whitish to brownish after only
2 days of storage, and red mildews growth was observed after 7 days of
storage at room temperature. A similar behaviour was observed in Aloe
gel stored at +4°C, although the brown colouring occurred after 7 days of

STABILITY OF DIFFERENT ALOE FRACTIONS
207
storage and red mildews growth was observed after 14 days. Stabilised
gel commercial preparation stored at room temperature showed colour
alteration after 22 days of storage and any mildew grown for the whole
test period. Analogously, mildew growth was not observed in samples
with antimicrobial agents.
Regarding the whole leaf homogenate, a colour change from natural
dark green to light green was observed in all preparations, including
those with antioxidant agents. White mildews growth was observed in
all the preparations without antimicrobial agents, starting from 12 and
21 days of storage for room and refrigerate conditions respectively. The
colour of whole leaf based commercial product and that of the household
preparation (Father Zago’s recipe) stored at refrigerator temperature
remained stable and any microbial growth was observed.
The whole data concerning the stability of preparations, expressed
as DT50 and DT90, together with the corresponding correlation coef-
ficients, are presented in tables 1 and 2 for aloin and β-polysaccharides
respectively. The aloin content in the household preparation stored at
+4°C resulted more stable (DT50 = 108 days) than in all the whole leaf
homogenates tested, including those with antimicrobial or antioxidant
agents. Additionally, the samples at +4°C were more stable than those
at room temperature.
The aloin content in whole leaf based commercial product was
stable for the whole 70 days of testing, and therefore DT50 and DT90
T 1. – Stability of aloin in whole leaf homogenate and in the Father Zago’s household preparation.
Test item Storage conditions Additives Correlation
coefficient
DT50
(days)
DT90
(days)
T (°C) Atmosphere
Whole leaf homogenate +22 air* - 0.924 20 66
Whole leaf homogenate +22 Air 0.1% Na benzoate 0.766 11 37
Whole leaf homogenate +22 Air 0.1% Na azide 0.760 11 38
Whole leaf homogenate +4 Air 0.005% ascorbate 0.960 14 45
Whole leaf homogenate +22 air 0.005% ascorbate 0.944 14 47
Whole leaf homogenate +4 air 0.05% ascorbate 0.999 26 86
Whole leaf homogenate +22 air 0.05% ascorbate 0.930 12 41
father Zago’s preparation +4 air - 0.889 108 360
* degassed with N2 immediately after homogenization

M. PELLIZZONI ET AL.
208
were not calculated. Regarding aloin stability in whole leaf homogenate
stored under vacuum conditions, the concentration was stable for 20
and 40 days at +22 and +4°C respectively. Hence, DT values were not
calculated because a linear disappearance trend could not be accurately
defined.
The stability of aloin in homogenates with antimicrobial agents
(DT50 = 11 days) was not significantly different from those without these
additives. Furthermore, there was no difference in stability between the
use of sodium azide and sodium benzoate.
In all cases, aloe emodin was detected in traces and sporadically,
proving that it is not the direct oxidative or microbial degradation prod-
uct of aloin.
As far as the stability of β-polysaccharides is regarded, the whole
gel samples stored at +4°C was more stable (DT50 = 11 days) than those
conserved at room temperature (DT50 = 2-3 days). The addition of either
sodium azide or sodium benzoate did not affect significantly the stability
of β-polysaccharides in the preparations, without significant differences.
However, the stabilised gel commercial preparation DT50 ranged from
12 days at room temperature to 44 days at +4°C, proving a higher stabil-
ity. Hence, the stability of β-polysaccharides is strongly affected by the
temperature of storage, while antimicrobial agents did not show signifi-
cant reduction of degradation. Similar results were achieved by Yaron
(1993), who evidenced changes in rheological properties at 40°C even
with antimicrobial agents, and by Chang et al. (1993) who observed a
relationship between degradation and temperature.
Generally, aloin stability in whole leaf homogenate was poor, and
neither ascorbate nor the antimicrobial agents could increase disappear-
T 2. – Stability of β-polysaccharides in aloe leaf gel and stabilised gel commercial.
Test item Storage conditions Additives Correlation
coefficient
DT50
(days)
DT90
(days)
T (°C) Atmosphere
Leaf gel +4 air - 0.818 11 36
Leaf gel +22 air - 0.999 3 9
Leaf gel +22 air 0.1% Na benzoate 0.798 3 10
Leaf gel +22 air 0.1% Na azide 0.831 2 7
Commercial preparation +4 air - 0.502 44 148
Commercial preparation +22 air - 0.847 12 41

STABILITY OF DIFFERENT ALOE FRACTIONS
209
ance time. Temperature seemed to be quite more effective in reducing
aloin degradation, although storage at +4°C alone could not provide
acceptable stability as well. The results are in contrast with the findings
from Gutterman and Chauser-Volfson (2006) who observed a stability
of aloin in leaf exudate aqueous suspension at +4°C. The difference
may account in the different matrix from our study. On the other hand,
Chang et al. (2006) reported that Aloe polysaccharides stability was
higher at 70°C, do decrease at both lower and higher temperature; they
also described that aloin degrades quickly in these conditions. A further
evidence on aloin degradation when temperature increases has been
given by direct observations from Gulia et al. (2010) and by indirect
evaluations from Miranda et al. (2009).
F. 1. – Representative regression lines through which degradation times of aloin (upper) and
β-polysaccharides (lower) were calculated.

M. PELLIZZONI ET AL.
210
Generally, the poor stability observed for both aloin and
β-polysaccharides in most of the conditions tested, helps to explain the
findings of Ramachandra and Srinivasa Rao (2008) who found a very
little amount of active ingredients in several aloe products.
Concluding, the preparation done according to Father Zago’s
recipe and the commercial one (prepared in a very similar recipe)
were stable at +4°C; all the other preparations showed a poor stability.
The improved stability might arise from the components of the recipe
different from Aloe components; the commercial product contained
citrate, ascorbate and sorbate which could affect the stability of the
preparation. Generally, the degradation of aloin does not seem to be
related to antioxidant or antimicrobial agents, albeit its degradation
mechanism is no clear yet.
The stability of β-polysaccharides is strongly affected by the tem-
perature of storage, while additives such as antimicrobial agents did not
lead to significant reduction of degradation. The stabilized gel commer-
cial preparation, is instead stable; although benzoate is reported among
ingredients, this might be due to the patented stabilization process.
Therefore, albeit temperature has been proved to play a major role
in aloin and β-polysaccharides stability, further attempts still need to be
done to clarify the key factors affecting the stability of Aloe fractions.
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S. – Aloe-based household preparations and commercial products are used
worldwide as a source of nutraceutical compounds, among which anthraquinones and
acemannan are the most known. However, it is reported that improper storage conditions
could lead to significant degradation processes, resulting in a low content of active sub-
stances. The aim of this work was to study the stability of aloin and β-polysaccharides
in aloe-based products during storage, as a function of temperature and chemical agents
added.
Generally, aloin degradation in whole leaf homogenate was fast, and neither
ascorbate nor the antimicrobial agents could increase stability; a lower temperature
slightly improved stability, (DT50 up to 26 days at +4°C). Regarding the stability of
β-polysaccharides, a strong influence of the storage temperature was observed, while
antimicrobial agents did not show significant reduction of degradation. For both com-
pounds, household and commercial products were significantly more stable.