Plant Foods Hum Nutr (2012) 67:10–16

DOI 10.1007/s11130-012-0280-6

ORIGINAL PAPER

Hypolipidemic Effect of Avocado (Persea americana Mill)

Seed in a Hypercholesterolemic Mouse Model
María Elena Pahua-Ramos & Alicia Ortiz-Moreno &
Germán Chamorro-Cevallos &
María Dolores Hernández-Navarro &
Leticia Garduño-Siciliano &
Hugo Necoechea-Mondragón &
Marcela Hernández-Ortega

Published online: 2 March 2012

# Springer Science+Business Media, Inc. 2012

Abstract Avocado seed contains elevated levels of pheno- content of dietary fiber was found (34.8%). The oral LD50
lic compounds and exhibits antioxidant properties. We in- for ASF was 1767 mg/kg body weight, and treatment with
vestigated the effect of Avocado Seed Flour (ASF) on the ASF significantly reduced the levels of total cholesterol,
lipid levels in mice on a hyperlipidemic diet. The concen- LDL-C, and prediction of the atherogenic index. Therefore,
tration of phenols was determined by high-performance the antioxidant activity of phenolic compounds and dietary
liquid chromatography, antioxidant activity was evaluated fiber in ASF may be responsible for the hypocholesterole-
using the Trolox equivalent antioxidant capacity method, mic activity of ASF in a hyperlipidemic model of mice.
and dietary fiber was measured using the Association of
Official Analytical Chemists (AOAC) method. The LD50 Keywords Avocado . Dietary . Fiber . Hypolipidemic .
of ASF was determined using Lorke’s method and hypolipi- Phenolic compounds . Seed
demic activity was evaluated in a hypercholesterolemic
model in mice. Protocatechuic acid was the main phenolic Abbreviations
compound found in ASF, followed by kaempferide and ABTS 2, 2′-azino-bis (3-ethylbenzthiazoline-6-
vanillic acid. The total phenolic content in the methanolic sulphonic acid)
extract of ASF was 292.00±9.81 mg gallic acid equivalents/ AI Atherogenic Index
g seed dry weight and the antioxidant activity resulted in ASF Avocado Seed Flour
173.3 μmol Trolox equivalents/g DW. In addition, a high BW Body Weight

M. E. Pahua-Ramos : A. Ortiz-Moreno (*) : M. D. Hernández-Navarro

M. Hernández-Ortega Departamento de Farmacia, Facultad de Química,
Departamento de Ingeniería Bioquímica, Universidad Autónoma del Estado de México,
Escuela Nacional de Ciencias Biológicas, Paseo Colón esq. Paseo Tollocan, Col. Residencial Colón,
Instituto Politécnico Nacional, C.P. 50120 Toluca, Estado de México, Mexico
Plan de Ayala y Prol. Carpio s/n, Col. Casco de Santo Tomás,
C.P. 11340 México, D.F., Mexico
e-mail: [email protected]

G. Chamorro-Cevallos : L. Garduño-Siciliano
Departamento de Farmacia,
Escuela Nacional de Ciencias Biológicas, H. Necoechea-Mondragón
Instituto Politécnico Nacional, Coordinación de Operación de Redes de Investigación,
Av. Wilfrido Massieu s/n, esq. Manuel L. Stampa, Instituto Politécnico Nacional,
Col. Unidad Profesional Adolfo López Mateos, Edificio Secretaría Académica. Av. Miguel Othón
Del. Gustavo A. Madero, de Mendizábal s/n, Col. La Escalera,
C.P. 07738 México, D.F., Mexico C.P. 07738 México, D.F., Mexico
Plant Foods Hum Nutr (2012) 67:10–16 11

GAE Gallic Acid Equivalents Materials and Methods

HDL-C High-Density Lipoprotein Cholesterol
LDL-C Low-Density Lipoprotein Cholesterol Seed Flour Preparation
LD50 Median Lethal Dose
TC Total Cholesterol Fresh seeds of Persea americana Mill were obtained from an
TG Triglycerides orchard in Uruapan, Michoacán, Mexico. The seeds were
Trolox 6-hydroxy-2, 5, 7, 8 tetramethylchroman- identified and authenticated with the number 15658 at the
2-carboxylic acid herbarium of the Centro Médico Nacional of the Instituto
Mexicano del Seguro Social. The seeds were washed, cut into
small pieces, and dried in an oven (Fisher Scientific, Isotemp
Introduction model 718F, USA) at 40 °C for 48 h until achieving a moisture
level of 4%. The small pieces were then crushed into powder
The treatment of hypercholesterolemia and related car- with a hammer (Wiley Mill standard model No. 3, Arthur
diovascular diseases with medicinal plants has increased Thomas Co., USA) until the flour passed through US 20 mesh
in recent years [1]. Reasons for the increased popularity (0.844 mm).
of these herbal medicines may include their relatively
low cost compared to orthodox medicines, availability Phenol Extraction
(since they are almost always derived from available
plants in the local region), and efficacy. Although poisonous Phenol extraction was performed according to the method
plants are ubiquitous throughout the world, herbal medicine is described by Asaolu et al. [1]. One hundred grams of seed
still used by up to 80% of the total population in developing flour were sloped into a beaker and then extracted with 75%
countries. Despite widespread use, few scientific studies have methanol overnight in a soxhlet extractor (Electro thermal
explored the safety and efficacy of traditional herbal remedies model ME466, England). The methanolic extract was con-
[2] Substances such as chlortalidone and propranolol, which centrated and allowed to evaporate to dryness at 50 °C using
are used in the treatment of hypercholesterolemia, can have a rotary evaporator (Büchi 461, Brinkmann Instruments,
adverse effects that affect therapy compliance and total quality Switzerland). The extract was then dissolved in water at a
of life of the patient [3]. concentration of 4 g/100 ml.
Cardiovascular disease is a growing health problem
throughout the world and represents a leading cause of Total Phenolic Content
mortality and morbidity in humans [4]. Several factors,
such as a high caloric diet, age, lack of exercise, smok- HPLC-PDA Analysis of Phenolic Compounds
ing, alcohol consumption, and genetic predisposition
have been linked with cardiovascular disease [1]. Elevat- Twenty microlitres of the methanolic extract were analyzed
ed cholesterol levels predispose patients to a condition using an HPLC-PDA system (Varian 920-LC) and a C18
known as hypercholesterolemia [5], which increases the column (Onmisher 5; 150×4.6 mm i.d.). The solvent mixture
risk of fatal and nonfatal coronary heart disease in people system contained a mixture of 5% formic acid in water (A) and
over the age of 50 [6]. 100% methanol (B), with a flow rate of 0.9 ml/min, and the
Several beneficial medicinal properties of compounds gradient flow was as follows: 0 min—5% B; 3 min—15% B;
present in the avocado seed and peel have been reported, 13 min—25% B; 25 min—30% B; 35 min—35% B; 39 min—
which are related to the elevated levels of phenolic com- 45% B; 42 min—45% B; 44 min—50% B; 45 min—70% B;
pounds (64% in seed, 23% in peel, and 13% in pulp). In 50 min—70% B; 56 min—75% B; and 61 min—80% B.
addition, the seeds and peels of avocado also contribute Detection was achieved with a photo diode array detector.
57% and 38% of the antioxidant capacities of the entire Spectrophotometric data from all peaks were monitored at
fruit, respectively [7]. 220–280 nm and chromatograms were recorded at 340 nm.
Several studies on the hypolipidemic effects of the The data were processed with Varian Galaxie™ Chromatogra-
avocado seed have been focused on methanolic extracts phy Data Software version 1.9.302.952 (Agilent Technologies
[1] and aqueous extracts [8]; however, the study of the USA). Phenolic compound quantification was determined
hypolipidemic effects of the entire avocado seed provides using the retention times and absorbance recorded in the
an interesting alternative, since the seed represents 13-18% chromatograms relative to external standards [10].
[9] of the avocado fruit and is discarded during avocado Total phenol content was quantified using the Folin
pulp processing. Therefore, the aim of this study was to Ciocalteu [11]. Gallic acid was used as a standard for the
investigate the effect of ASF on the lipid levels of mice calibration curve. The results were expressed as gallic
on a hyperlipidemic diet. acid equivalents (GAE) mg/g seed dry weight (DW).
12 Plant Foods Hum Nutr (2012) 67:10–16

Antioxidant Activity was supplied to the animals ad libitum for six days in
order to induce hypercholesterolemia [16, 17]. The diet
Total antioxidant capacity was evaluated using the method formula used is shown in Table 1. Group 2–5 received
reported by Re et al. [12] with Trolox as the standard. A the hypercholesterolemic diet, which was administered
stock solution was prepared by reacting 7 mM ABTS with with distilled water (group 2) or different doses of ASF
2.45 mM potassium persulfate to generate the ABTS cation (groups 3, 4 and 5 received 125, 250, and 500 mg ASF/kg
chromophore. This solution was diluted with absolute BW, respectively, once a day) by gavage. The doses were
ethanol until reaching an absorbance of 0.7 ± 0.02 at chosen according to the acute toxicological study divided by a
734 nm. A sample of 10 μl of ASF extract was added security factor of 10.
to 990 μL of ABTS solution and the reaction was followed At the end of six days, the TC, HDL-C, LDL-C and TG
over a 7 min time course. Total antioxidant activity or capacity concentrations were determined according to the methodology
was calculated relative to the reactivity of Trolox as the described by Argüelles et al. [17].
standard under the same conditions. The results were
expressed as μmol Trolox equivalent/g of seed DW. Statistical Analysis

Proximate Analysis All data for acute toxicity were statistically analyzed by the
Student’s t-test using Sigma-Stat version 3.5 (Jandel San
Moisture, protein, ether extract, ash, and crude fiber Raphael, CA) and P<0.001 was considered statistically
content in ASF were determined in triplicate following significant. Hypolipidemic activity data were statistically
the standard methods from the Association of Official analyzed by one-way analysis of variance (ANOVA) and
Analytical Chemists International (AOAC) [13], and total Tukey’s test using Sigma-Stat version 3.5 (Jandel San
carbohydrate content was calculated as the difference to a Raphael, CA). The data were reported as mean ± standard
total of 100%. Dietary fiber was also determined in deviation (SD). A P<0.05 was considered statistically
triplicate using AOAC methods 997.08 and 999.03 [13]. significant for hypolipidemic activity data.

Animals
Results and Discussion
For the hypolipidemic activity assay, 40 8-10-week-old
adult male CD-1 mice with an average weight of 28±2 g Identification of Phenolic Compounds
were obtained from the Centro de Investigación y Estudios
Avanzados (CINVESTAV) at the Instituto Politécnico Nacional Analysis of methanolic extract from avocado seed by
(IPN-México). Each mouse was weighed and randomly HPLC-PDA identified eleven major peaks. Seven phenolic
assigned to groups by body weight (BW). Each group of mice compounds were identified using external standards, spectra
was housed in cages with wooden chip bedding and maintained characteristics, and retention time. Protocatechuic acid
under a 12 h light/dark cycle. Mice were fed with standard (128.18±0.01 μg/g DW) was the main phenolic compound
laboratory chow (5001 Lab Rodent Diet, PMI Nutrition identified, followed by kaempferide (107.42± 0.04 μg/g
International, Inc., Bienwood, MO) and provided water DW) and vanillic acid (28.67±0.001 μg/g DW). In addition,
ad libitum. The animal experiments and study design clorogenic acid, syringic acid, rutin, and kaempferol were
were approved by the Laboratory Animal Care Commit- present in small amounts (Table 2). Recently, Rodríguez-
tee of IPN and were conducted in compliance with the Carpena et al. [18] analyzed and classified phenolic
Official Mexican standard NOM-062-ZOO-1999 regarding compounds from two avocado varieties as catechins,
technical specifications for production, care, and use of labo- (sum of catechin and epicatechin), hydroxybenzoic acids
ratory animals [14].

Oral Acute Toxicity Table 1 Formulation

for the hypercholesterol- Ingredients % of total weight
emic diet
Acute toxicity tests were conducted according to Lorke’s Lab rodent diet 53.5
methodology [15]. 5001
Sucrose 30
Hypolipidemic Activity of ASF Casein 10
Butter 5
Five groups of CD-1 mice (8 mice per group) were formed, Cholesterol 1
with group 1 serving as the control. A diet rich in cholesterol Sodium cholate 0.5
Plant Foods Hum Nutr (2012) 67:10–16 13

Table 2 Phenolic compounds in methanolic extract from Persea rather than just discarded as waste [21]. Importantly, our
americana Mill
results are in agreement with the findings by Wang et al.
Peak number Rt (min) UV (nm) μg/g [17] and Soong & Barlow [21] showing that the antioxidant
activity of fruit seeds components may be responsible for
1 Protocatechuic acid 6.12 243, 322 128.18±0.01 the hypocholesterolemic activity observed.
2 Clorogenic acid 7.37 242,278,439 0.516±0.02
3 Syringic acid 8.98 242,314, 443 2.51±0.002
Proximate Analysis and Dietary Fiber
4 Vanillic acid 9.87 242,380,436 28.67±0.001
5 NI 11.00 242,307,446 –
We found that the ASF preparation contained 4.0±0.8
6 Rutin 11.67 242,277,319,386 9.63±0.008
moisture, 2.2 ± 0.14 ash, 4.75 ± 0.01 protein, 6.39 ± 0.5
7 NI 13.28 242,315,363 –
crude fiber, 4.38±0.8 ether extract, and 79.10±0.8 car-
8 NI 14.48 242,318,446 –
bohydrates (data expressed as mean ± standard deviation
9 Kaempferol 16.30 242,311,386,429 2.19±0.002
g/100 g sample fresh weight; n03). The low oil content
10 Kaempferide 23.81 216,242 107.42±0.04
of the ASF suggested that oil and its fatty acids could
11 NI 51.71 241,334,380 –
have a minimal effect on cholesterol and LDL-C reduc-
Data expressed as mean ± standard deviation; n03. NI0not identified tion, since it only represented a small portion of the total
daily oil intake of the treated mice. Nijjar et al. [22]
found that nuts are a good source of mono and poly-
(p-coumaric, caffeic, ferulic, and sinapic), hydroxycin- unsaturated fatty acids and also contain dietary fiber,
namic acids (p-hydroxybenzoic, protocatechuic, vanillic, phytosterols, and polyphenols. These components likely
syringic, and gallic), flavonols, and procyanidins (sum of combine to a reduction in LDL-C levels beyond the
dimers, oligomers, and polymers). These authors reported effects predicted by equations based solely on fatty acid
that the seed and peel contained the highest amount of profiles. Nevertheless, the high crude fiber content
phenols in the entire fruit. Moreover, Terpinc et al. [19] (6.39 g/100 g DW sample) of ASF could have a beneficial
reported that flavonoids, rutin, catechin, and quercetin effect on total cholesterol and LDL-C reduction in the
are widespread in nature and may act as powerful plasma of the groups of mice treated [23].
antioxidants. The ASF preparation was found to contain 34.8±3.4 g
These findings and our results provide evidence for the dietary fiber/100 g DW sample, which is relevant in this
importance of phenols present in avocado seed, since study, since the natural gel-forming or viscous fibers
phenolic compounds have been shown to reduce plasma (pectin, gums, mucilage, algal polysaccharides, some
lipid levels in human body through the upregulation of storage polysaccharides, and some hemicelluloses) are
LDL receptor expression, inhibition of hepatic lipid synthesis water-soluble and resistant to digestion by human gastro-
and lipoprotein secretion, and increase in cholesterol intestinal enzymes that are part of the dietary fiber.
elimination through bile acids [20]. Moreover, this content has been shown to be associated
with a cholesterol-lowering effect [24]. The dietary fiber
Total Phenolic Content and Antioxidant Activity of ASF content of the avocado seed is similar to another Mexican
seed called chia [25]. Reyes-Caudillo et al. [25] reported that
The total phenolic content and antioxidant activity of the chia seeds from Jalisco and Sinaloa States contain a total
methanolic extract of ASF was determined to be 292.00± dietary fiber content of 39.9% and 36.9%, respectively.
9.81 mg GAE/g seed DW and 173.3 μmol Trolox equiv- Therefore, the dietary fiber content in chia seeds is of
alents/g seed DW, respectively. Rodríguez-Carpena et al. sufficient level to promote beneficial health effects,
[18] previously reported a total phenolic content of 351.1± including a reduction of cholesterolaemia, modification of
9.88 mg GAE/g seed for the Hass variety and 416.4 ± the glycemic and insulinaemic responses, changes in intestinal
10.48 mg GAE/g seed dry matter for the Fuerte variety. function, and antioxidant activity. The high content of
According to Wang et al. [7], seeds contain the strongest dietary fiber in ASF found in the present study
antioxidant properties and highest phenol and procyanidin suggests that dietary fiber could play an important role in
content compared to the pulp. Soong & Barlow [21] the hypocholesterolemic activity in mice.
reported a significantly higher total antioxidant capacity
and phenolic content of fruit seeds than the edible portions. Oral Acute Toxicity
In most fruits, the contribution of the fruit seed fraction
compared to the total antioxidant activity and phenolic In the first stage of the oral acute toxicity study, the
content was more than 95%, and therefore these authors animals did not exhibit any toxicological signs, including
suggested that the fruit seeds should be further utilized depression, writhing, diarrhea, hypermotility, or aggression
14 Plant Foods Hum Nutr (2012) 67:10–16

Table 3 Daily food and water consumption and relative weight of liver and kidney in CD-1 mice treated with different doses of avocado seed flour

Groups Dose (mg/kg) Daily food consumption (g) Daily fluid consumption (ml) LBW (%) KBW (%)

Control 1st stepa – 38.3±10.2 40.8±12.8 6.80±0.15 1.58±0.09

a
ASF 10 28.4±10.4 34.4±14.3 6.75±0.37 2.67±0.02 a
100 31.9±8.3 47.1±11.2 5.48±0.11ª,b 1.83±0.05a,b
1000 33.7±7.3 50.2±10.4a 5.04±0.22ª,b 1.75±0.09a,b
Control 2nd stepc – 35.5±6.2 53.4±9.7 5.50±0.28 1.54±0.16
ASF 1250 40.8±8.0 46.9±7.6 5.14±0.14 1.44±0.10
2500 21.3±14.5c 28.1±15.8c ND ND

Data expressed as mean ± standard deviation; n08. ASF, Avocado Seed Flour; LBW, liver-to-body weight ratio; KBW, kidney-to-body
weight ratio. a P<0.001, significant difference with respect to control 1st step,. b P<0.001; significant difference with respect to 10 mg/kg of ASF;
c
P<0.001, significant difference with respect to control 2nd step. ND: Not determined.

compared to the control group. No signs of toxicity or death results of this study suggest that the compounds present
were observed in any of the animals, and all animals survived in avocado leaves and seeds are different and have an
to the end of the 14 day study period. Weight gain in the opposite influence on the liver.
control animals was minimal (< 4%), while the treated In the second stage of the acute toxicity study, no significant
animals exhibited a slight increase in weight, although differences (P>0.001) were found in daily food and water
there was no significant difference in the percent weight intake between mice treated with 1250 mg ASF/kg BW and
change between the groups (P<0.05). In the second stage control mice; however, significant differences (P≤0.001)
of the study, we observed 100% mortality by day six in were found in daily food and water intake between mice
the group fed with 2500 mg ASF/kg BW. Table 3 lists treated with 2500 mg ASF/kg BW and control mice.
the effects of different doses of ASF on daily food and Based on these results, we determined the oral LD50 for
water intake and on the weight of the main organs, ASF to be 1767 mg/kg BW by using the geometric mean of
which was expressed as a ratio of relative weight (RW) the dose that caused 100% mortality and the dose that
to total body weight. Mice administered 100 and caused no mortality, as suggested by Lorke [15]. The oral
1000 mg ASF/kg BW exhibited significant differences LD50 of ASF in mice indicated that it exhibited a low
(P ≤ 0.001) compared with the control, whereby liver toxicity [17]. It has previously been shown that ether
weight was lower and kidney weight was higher than and aqueous extracts of Persea americana Mill seed
control group. An increase in the RW of the kidney has administered by intraperitoneal injection in rats also had
also been reported by Ozolua et al. [8] in adult rats fed low toxicity, with LD50 values of 751.6 mg/kg BW and
aqueous seed extract from avocado. In addition, Brai et al. 10 g/kg BW, respectively [8, 27].
[26] found that liver weights were significantly increased in
albino rats fed avocado aqueous leaf extract after induction of Hypolipemic Activity of ASF
a hyperlipidemic diet compared to normal control rats,
which was accompanied with a significant increase in To determine the hypolipemic activity of ASF, mice were
liver cholesterol level. These findings together with the dosed according to the LD50 of ASF found in this study. The

Table 4 Effect of avocado seed flour on lipid profile of mice

Treatment ASF Dosis (mg/kg) TC (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L) AI

Normocholesterolemic – 31.9±7.16 12.50±3.02 18.92±4.25 1.05±0.13 1.6±0.05

Hypercholesterolemic – 106.7±9.70 92.1±10.26 15.00±2.1 0.852±0.09 7.9±1.86
ASF 125 70.9±4.49a 55.8±5.35 a
15.6±1.79 0.872±0.09 3.9±0.59a
250 69.0±5.15a 56.1±5.28a 13.5±0.78 0.914±0.07 4.3±0.58a
500 67.6±4.92a 54.4±5.37a 13.8±1.21 0.930±0.12 4.3±0.72a

Data expressed as mean ± standard deviation; n08, analyzed by ANOVA and Tukey-Kramer test.a P≤0.0001; significant difference with
respect to the hypercholesterolemic control. ASF, Avocado Seed Flour. TC, Total Cholesterol; LDL-C, Low-density Lipoprotein
Cholesterol; HDL-C, High-density Lipoprotein Cholesterol; TG, Triglycerides; AI, Atherogenic Index.
Plant Foods Hum Nutr (2012) 67:10–16 15

hypolipidemic effect of ASF was evaluated at doses at 125, and crude fiber content of the seed. Further research is
250, and 500 mg/kg BW. Acute supplementation of required in order to identify the components of ASF that
cholesterol produced a significant (P≤0.05) elevation in are responsible for the observed hypocholesterolemic
plasma cholesterol levels in the hypercholesterolemic effects.
control compared to the normocholesterolemic control.
In addition, the TC increased from 31.9±7.16 to 106.7± Acknowledgments This research was partly funded by the Consejo
9.70 mmol/L, LDL-C increased from 12.5±3.02 to 92.1± Nacional de Ciencia y Tecnología (CONACyT) scholarship, Secretaria
10.26 mmol/L, and the calculated AI increase from 1.6± de Investigación y Posgrado-IPN Proyect Number. 20100788,
Comisión de Operación y Fomento de Actividades Académicas del
0.05 to 7.9±1.86 between the two groups, respectively IPN (COFAA-IPN), and Universidad Autónoma del Estado de México.
(Table 4). No significant (P>0.05) changes were found
in the plasma HDL-C (18.92±4.25 vs 15.0±2.1 mmol/L,
respectively) and TG (1.05±0.13 vs. 0.85±0.09 mmol/L,
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