Avocado Seeds: Extraction Optimization and Possible Use as

Antioxidant in Food
Francisco Segovia Gómez,1,2 Sara Peiró Sánchez,1 Maria Gabriela Gallego Iradi,1 Nurul Aini Mohd
Azman,1 and María Pilar Almajano1,*

Author information ► Article notes ► Copyright and License information ►

Abstract
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1. Introduction
Vegetables and fruits are essential foods in our diet and also have many compounds that are
beneficial for health due to minor components. These minor components include phenolic
substances [1]. These are secondary metabolites of plants. They have an aromatic ring with
one or more hydroxyl groups. Their complexity may be high, as for example quercetin, which
is one flavone with several aromatic rings. The properties depend on the arrangement and/or
structure of the molecule [2].
In recent times, many plants have been studied in order to characterize them depending on the
amount of polyphenols they have and on their potential use [3].
The polyphenols are associated with the potential prevention of diseases which are due to the
presence of free radicals, such as cardiovascular insufficiency, hypertension, inflammatory
conditions, asthma, diabetes and Alzheimer’s [4], thanks to their antiradical power. For this
reason, they are very useful in food products, since they prevent lipid peroxidation due to the
attack of free radicals [5]. They also protect against oxidation, direct or indirect, caused by
metal cations [6]. These cations stimulate the creation of reactive oxygen species (ROS),
which are harmful to the health. In some cases, polyphenols have been used as preservatives,
protecting against microorganisms [7].
The process of food, especially for IV and V gamma products, produces many byproducts
and waste. This type of waste has a significant environmental impact due to the organic
charge. It also has associated handling, transport and storage costs, among others. Therefore,
more and more alternative uses for these residues are sought, as for instance animal feed and
fertilizers, among others. In the present case, it is interesting to obtain, through an optimized
extraction process, harmless substances with high antioxidant power. Thus, what was a waste
becomes a “high value-added” product [8,9]. Previous examples already studied [10,11,12]
are the orange juice industry, where a large amount of skin and seeds are produced with a
high content of polyphenols and the industry of processed apple, pear and peach, with a
significant amount of skin byproduct. There is evidence that the skin may even have a greater
amount of polyphenols than the flesh [13]. Also, the waste from wine and beer production
includes phenolic compounds [9]. Other studies have focused on the shells of nuts, rice and
wheat in which large amounts of polyphenols are found [14].
In the avocado industry the pulp is used, while the skin and the seeds are discarded as waste.
These residues are rich in polyphenols with antioxidant and antimicrobial power [15]. Among
the polyphenols the (+)-catechin and (−)-epicatechin [16] and chlorogenic and protocatechuic
acid, are included [14]. Previous studies on this residue have been applied to pork burgers
and have been shown to be effective in preventing oxidation and microbial growth [15].
Given the above, it can be concluded that polyphenols obtained from these industrial wastes
can be potent antioxidants and, in some cases, they are better than synthetic antioxidants such
as BHA or BHT which in high doses can become toxic [17].
In order to optimize the extraction process, response surface methodology (RSM) has been
used. Phenolic compounds extraction optimization from strawberries [18], apple pulp [9] and
residues of chestnuts [19], are examples of this. This method establishes a multivariable
mathematic model to obtain the relationship between responses and independent variables
[20,21] with the use of a minimal number of experiments.
This paper consists of two main objectives. First, a mathematical model was obtained to
predict the best conditions of extraction of polyphenols from dried avocado seed. Second, an
extract using these conditions was obtained and the effect of lyophilized powder in the delay
oxidation in oil-in-water (O/W) emulsions and beef meat burgers analyzed.
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2. Experimental Section

2.1. Materials
2,2′-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), was used as peroxyl radical
source. Trolox (6-hydroxy-2,5,8-tetramethylchroman-2-carboxylic acid), ethanol, fluorescein,
AAPH, BHA, egg albumin, p-anisidine (4-amino-anisole; 4-methoxy-aniline), isooctane,
potassium persulfate, acetic acid (glacial) and 2-thiobarbituric acid were purchased from
Sigma-Aldrich Company Ltd. (Gillingham, UK). Folin-Ciocalteu reagent, sodium carbonate
and 1,6-diaminohexane were supplied by Merck (Darmstadt, Germany). Trichloroacetic acid,
hydrochloric acid and Tween® 20 (Panreac Química S.L.U, Barcelona, Spain) were acquired
from Panreac Química S.L.U. (Barcelona, Spain). Refined sunflower oil, with no added
antioxidants, was purchased from a local retail outlet. All compounds were of reagent grade.

2.2. Avocado Preparation

The avocado (Persea americana) was obtained in the local market; the seeds were separated
from other edible parts. This waste was homogenized and frozen at −80 °C for lyophilization.
Then the seeds were ground into a powder by using a Moulinex mill (A5052HF, Moulinex,
Lyon, France). The particle size was standardized with a number 40 mesh sieve. Finally, the
powder was stored in a dark bottle in a desiccator until use.

2.3. Extraction Procedure

Extraction was carried out in dark bottles: lyophilized sample powder (0.25 g) was blended
with 15 mL of solvent of concentration specified by the experimental design (Table 1). This
mixture was placed in a bath by stirring at the required temperature and time specified by the
experimental design (Table 1). At the end, it was cooled in a refrigerator at 5 °C, centrifuged
(Orto Alresa, Madrid, Spain) at 2500 rpm for 10 min, vacuum filtered and the lost solvent
was replaced. The extract was stored at −20 °C until used for analysis.
Table 1
Experimental design and responses for extraction.

2.4. Total Phenolic Content (TPC)

TPC was determined spectrophotometrically following the Folin-Ciocalteu colorimetric
method [22]. A sample diluted 1:4 with milli-Q water was stirred in triplicate. The final
concentration in the well (96 wells plate was used) was: 7.7% v/v sample, 4% v/v Folin-
Ciocalteu’s reagent, 4% saturated sodium carbonate solution and 84.3% of milli-Q water, all
mixed. The solution was allowed to react for 1 h in the dark and the absorbance was
measured at 765 nm using a Fluostar Omega (BMG Labtech, Ortenberg, Germany). The total
phenolic content was expressed as mg Gallic Acid Equivalents (GAE)/g dry weight.

2.5. ORAC Assay

Antioxidant activities of avocado seeds extracts were determined by the ORAC assay, as
reported by Casettari et al. [23]. The assay was carried out using a Fluostar Omega equipped
with a temperature-controlled incubation chamber. The incubator temperature was set to 37
°C. The extract samples were diluted 1:20 with milli-Q water. The assay was performed as
follows: 20% of sample was mixed with Fluorescein 0.01 mM, and an initial reading was
taken with excitation wavelength, 485 nm and emission wavelength, 520 nm. Then, AAPH
(0.3 M) was added, measurements were continued for 2 h every 5 min. This method includes
the time and decrease of fluorescence. The area under the curve (AUC) was calculated. A
calibration curve was made each time with the standard Trolox (500, 400, 250, 200, 100, 50
mM). The blank was 0.01 M phosphate buffered saline (pH 7.4). ORAC values were
expressed as mg Trolox Equivalents (TE)/g of dry weight.

2.6. Statistical Analysis

RSM was used to determine the optimal conditions of polyphenol extraction. A central
composite design (CCD) was used to investigate the effects of three independent variables
with two levels (solvent concentration, extraction temperature, and extraction time) with the
dependent variables (TPC, ORAC activity). CCD uses the method of least-squares regression
to fit the data to a quadratic model.
The adequacy of the model was determined by evaluating the lack of fit, coefficient of
determination (R2) obtained from the analysis of variance (ANOVA) that was generated by
the software. Statistical significance of the model and model variables were determined at the
5% probability level (α = 0.05). The software uses the quadratic model equation shown above
to build response surfaces. Three-dimensional response surface plots and contour plots were
generated by keeping one response variable at its optimal level and plotting that against two
factors (independent variables). Response surface plots were determined for each response
variable. The coded values of the experimental factors and factor levels used in the response
surface analysis are shown in Table 1. The graphics and the RSM analysis were made by
software Matlab version R2013b (The MathWorks Inc., Natick, MA, USA, 2013). All
responses were determined in triplicate and are expressed as average ± standard deviation.
The answers have a percentage deviation less than 10%.

2.7. Water-Oil Emulsions

Oil-in-water emulsions (20.2 g) were prepared by dissolving Tween-20 (1%) in acetate buffer
(0.1 M, pH 5.4), either with or without protein, namely egg albumin (0.2% w/w) and avocado
seeds extracts (0.45% w/w, 0.225% w/w, 0.1125% w/w). The emulsion was prepared by the
dropwise addition of oil (sunflower oil) to the water phase, cooling it in an ice bath with
continuous sonication with a Vibracell sonicator (Sonics and Materials, Newtown, CT, USA)
for 4 min. All emulsions were stored in triplicate in 60 mL glass beakers in the dark (inside
an oven) at 30 °C in an incubator. Two aliquots of each emulsion (0.005–0.1 g, depending on
the extent of oxidation) were removed periodically for determination of peroxide value (PV).

2.8. Peroxide Value (PV)

PV was determined by the ferric thiocyanate method [24] (after calibrating the procedure
with a series of oxidized oil samples analyzed using the AOCS Official Method Cd 8-53).
Data from the PV measurements were plotted against time.

2.9. Meat Preparation

Fresh beef meat was purchased from a local processor 96 h postmortem. All subcutaneous
and inter-muscular fat and visible connective tissue were removed from the fresh beef
muscle. Lean meat was ground through Ø-4 mm plate using a meat grinder (PM-70, Mainca,
Barcelona, Spain). The ground meat was divided into six portions for each experiment prior
to the addition of the sodium chloride or different concentration of powder (freeze-dried
extract of powder of avocado). The lyophilized avocado and the powder of direct avocado
were mixed with the salt final concentration of 1.5% (w/w). Each portion of beef meat was
mixed manually with each solid. Each mixed sample was divided into nine smaller portions
(about 10 g each) and allocated onto trays. The meat was packed under MAP (20% CO2 and
80% O2) in polystyrene/EVOH/polyethylene trays, heat sealed with laminated barrier film
and stored at 4 ± 1 °C for 8 days. Patties were evaluated for lipid oxidation.

2.10. Thiobarbituric Reactive Substances

Fat meat oxidation was determined by the concentration of thiobarbituric acid-reactive
substances (TBARS) using the method described by Domenech Asensi (2013) [25] with
some modifications. In the dark, 1 g of burger patty was dispensed in tubes and 1 mL of
EDTA was added. The samples were homogenized for 5 min sin an Ultra-Turrax (Ika®-
Werke, Staufen, Germany) with 5 mL of TBARS reactive (Trichloroacetic acid, 9.2%;
Hydrochloric acid, 2%; Thiobarbituric acid, 0.22%, all w/w final). During homogenization,
the tubes were placed in an ice bath to minimize the development of oxidative reactions. The
sample tubes were heated at 90 °C in a boiling water bath for 20 min and then left to cool.
Two milliliters of slurry was centrifuged (10,000 rpm for 10 min). The absorbance was
measured at 531 nm in a Spectrophotometer Zuzi model 4201/20 (AUXILAB, SL, Navarra,
Spain). The result is expressed in mg of MDA/kg sample.
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3. Results and Discussion

3.1. Extraction Optimization

Experimental design was carried out to see the effects of temperature, solvent concentration
(ethanol) and time in both TPC and radical scavenging (measured by ORAC). Several authors
used ethanol/water as solvent to extract different raw material polyphenols, such as seeds,
grape marc, fruits, among others [26,27,28,29,30]. Ethanol concentration with the highest
polyphenols yield is in the range of 10%–60%. Ethanol, instead of methanol, is used when it
is necessary to reduce the toxicity of extracts [18]. The time effect was measured between 5
and 45 min, because some research reported that it is enough to achieve the maximum
amount of polyphenols [31,32]. Temperature bounds were taken between 40 and 80 °C, to
achieve the maximum temperature that does not have a negative effect on the polyphenols
stability [33]. All these parameters are collected in Table 1 which shows the experimental
design for the variables temperature (T), ethanol concentration (% EtOH) and time (t), with
responses of TPC and antiradical activity measured by ORAC.
Figure 1 shows the relationship between the variables T, % EtOH and t in polyphenol
extraction. The process is favored by high temperatures and low concentrations of ethanol (in
the studied range). This behavior can be attributed to the nature of the polyphenols present in
the sample, mainly chlorogenic acid and protocatechuic acid [34] both highly soluble in
water. The solvent plays an important role in mass transfer of the compounds; not all
polyphenols show identical behavior in the extraction process, and the less polar polyphenols
are favored by the highest concentration of ethanol [9].

Figure 1
Response surface model plot: the variable is the total phenolic content (TPC) of the extract.
% EtOH with temperature; temperature with time; % EtOH with time.
The effect of temperature on the extraction is associated with the solubility of the components
present in the avocado pit. This variable, T, has a marked influence on the diffusivity of the
substances [30]. Solubility increases with temperature. Time has no influence in the
extraction process. This means that from the beginning, the extraction is governed by the
solubility and diffusion, and both are almost complete after 5 min.
Figure 2 shows the effect of the parameters on the antioxidant power measured by ORAC.
The ORAC increases with temperature. In the investigated range, the ORAC is increased
about 44% (Table 1). Furthermore, as stated above, it is in accordance with the higher
polyphenols solubility at high temperature. This means that these kinds of polyphenols are
thermo-resistant [20].
Figure 2
Response surface model plot: the variable is the Oxygen Radical Antioxidant Capacity
(ORAC) of the extract. Temperature with time; % EtOH with temperature; % EtOH with
time.
The effect observed for the percentage of ethanol is similar to that described in the TPC. An
increase in the ethanol concentration causes a decrease in antioxidant activity. It is not a new
fact, because similar results were described in other studies and were justified by the polarity
of the compounds of the extract [18].
On TPC the variable t has no influence, while on ORAC small changes were observed, but all
of them with similar final values. One possible explanation is that there are antioxidant
compounds with slow solubilization and, therefore, the time promotes an increase in total
extraction [35].
Table 2 shows the “p values” of the mathematical model for the coefficients, with the
decoded variables. It starts with the complete model, taking the variables that have less
influence, i.e., with p > 0.05. For TPC all those that are with % EtOH and t are involved. This
means that the more important variable is T. However, on the ORAC, the variables that have
more influence are % EtOH, t, and these quadratic terms. From the data, different iterations
were made and less influential terms were eliminated; after which the values were
recalculated. With these data the reduced model was obtained and provided a better fit. In
ORAC the predicted R2 becomes 77.88 which is within the range of a good set [36].

Table 2
p-Values for each of the constants in the equation of the mathematical model.
Therefore, with the exception of T × % EtOH for the TPC, all of the crossed terms disappear
in the reduced model (which is used to adjust and to determine the optimal extraction
conditions).
Additionally, the quadratic variables % EtOH × % EtOH and t × t, as well as the linear
variable t are eliminated for the TPC. The quadratic term T × T is eliminated from the model
which determines the scavenging activity. This is summarized in Table 2.
Table 3 lists the completed model and the reduced model equations. The reduced model has a
higher R2predicted which means that it is more reliable in estimating a response.
Table 3
Mathematical equations from Response Surface Method (RSM) for each of the responses,
with their respective value of R2 and R2-predicted.
When the fitting was considered good enough, the experiment was performed in the
laboratory to obtain the real value. Table 4 contains these values for the TPC and for the
ORAC. The TPC is fitted with less than a 4% error (the predicted value is 43.6 mg GAE/g
dw, compared to an experimental value of 45.01 mg GAE/g dw). This indicates that the
initial hypothesis was correct, and demonstrates that T is the variable with the greatest
influence on the maximum TPC extraction.

Table 4
Optimal conditions for the extractions for TPC and ORAC, given by RSM.
However, the values which maximize scavenging activity (ORAC) have a greater deviation.
The value predicted by the reduced model was 200.66 mg TE/g dw, compared to an
experimental value of 154.3 mg TE/g dw, which represents a deviation of 23.1%.
The best-fitting experimental conditions were then applied, i.e., 23 min extraction with 56%
EtOH and 63 °C. This extract was lyophilized and used in subsequent experiments.

3.2. Extract Optimized Effect in Oil-in-Water Emulsions (O/W)

Figure 3 shows the evolution of peroxide value over time. In this case, the possible synergy
between the extract (with different concentrations) and egg albumin was determined. Firstly,
it should be noted that both albumin and various concentrations of the extract of avocado
produce significant protection against oxidation. For example, within the 400 h of the
experiment the amount of hydroperoxides produced is 90% higher in the control than in any
of the samples (20 mg hydroperoxides/kg of emulsion hydroperoxides vs.38 mg/kg for the
emulsion control). Notably, there were no significant differences (p < 0.05) for the three
tested avocado concentrations (0.1125%, 0.225% and 0.45% w/w), as well as egg albumin
(0.2% w/w). This fact could be explained by the solubility of the lyophilized extract in water
and the ability to coat the oil drop generated in the emulsion and prevent oxidation thereof.
The necessary concentration that allows this protection is already achieved with 0.1125% and
the results do not improve if increased. Similar behavior has been published elsewhere
[37,38,39].
Figure 3
Peroxide values vs. time in the emulsions.
In fact, putting together two different compounds (avocado pit extract and egg protein) allows
greater protection against oxidation and further differentiates the two concentrations of the
tested extract. For example, the time required to reach 15 mg hydroperoxides/kg emulsion
goes from 180 h of the control group up to 480 h for the sample containing 0.45% extract +
0.2 egg protein. This is an increase of 260% superior durability. In the intermediate areas
three avocado extract concentrations were tested, as well as the protein (an increase in the
durability between 150% and 180%) and one that contains 0.225% of avocado and 0.2%
protein, with an improvement of the durability of 220%. Almajano and Bonilo-Carbognin
already published similar results of synergy [40,41]. As a summary, it can be said that
increasing the concentration of the extract does not improve the durability. However, the
incorporation of small amounts of protein allows significant differences to be found between
the samples containing protein and those that do not contain it.
Avocado pits contain polyphenolic compounds (such as protocatechuic acid, chlorogenic
acid, syringic acid and rutin), which are very strong antioxidants [34]. In 2010, Sasaki [42]
studied the antioxidant power of chlorogenic acid in oil-water emulsions. The effects
discovered are remarkable. The authors analyzed the presence of other compounds, which in
that case were also polyphenolic compounds. Additionally, they demonstrated that the
presence of several different compounds provided better results than the added individual
effects.
As it was stated before, 1% of surfactant (Tween-20) was added to the emulsion prepared in
the present work. This eases the dissolution of the polyphenolic compounds, thus increasing
the antioxidant activity in the emulsion.

3.3. Effect of the Extract in Burger Meat

The TBARS method is widely used to determine the oxidation of fats and oils in foods
[43,44,45]. In Figure 4, the evolution of TBARs vs. time for each of the studied beef burger
meat patties is collected. Samples containing 0.1% lyophilized extract and 0.5% direct seed
powder have no significant differences compared with the BHA (0.05%), but show a big
difference compared with the control. The lower concentration (0.01% and 0.05% lyophilized
extract powder direct seed) presented intermediate behavior, as expected. The duration of the
experiment was 8 days and it was observed that the burger meat with 0.5% seed powder and
0.1% of lyophilized extract had no significant oxidation, or the protection is higher than 90%.
These results are similar to those reported by Weiss et al. [46] for pork burgers. That study
examined protecting fat oxidation also with excellent results [46]. Additional results along
the same lines have avocado oil added directly to the pork burgers. This shows a positive
effect on the conservation of the burger [47].
Figure 4
The TBARS (Thiobarbituric acid reactive substances) values for the meat emulsions.
It is not the first time avocado pits have been used in meat products. Rodríguez-Carpena et
al. (2011) [15], prepared pork meat pies and inserted the grinded avocado pits to protect the
meat against lipid oxidation. The authors indicated that one of the factors might be the
formation of chelates with the copper and iron cations. These cations, in their free ionic state,
could cause the creation of free radicals.
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4. Conclusions
RSM was used to identify the best conditions for the extraction of compounds with an
antioxidant activity from an organic residue: the avocado pit. The reduced model obtained
provides parameters that fit with those of the TPC (with a 3.13% error when compared to the
experimental value).
The lyophilized extract was used as protection from the oxidation of oils (oil-in-water
emulsions) and fat (beef burgers) with excellent results, especially in meat, in which the
durability of the burger meat is significantly increased relative to oxidation.
These studies should encourage further exploration in this area of study in order to obtain a
byproduct of the natural antioxidants that currently as waste are worthless.
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Acknowledgments
This research was conducted at the Department Chemical Engineering, University
Polytechnic of Catalonia, Spain. The authors thank Universidad Experimental Politécnica
“Antonio José de Sucre” and Carlos Barreiro, for their support in this research. The authors
want to thank Mariona Vila for the support.
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Author Contributions
Francisco Segovia and Sara Peiró have done the experimental section and the design, data
acquisition, analysis and data interpretation; Maria Gabriela Gallego and Nurul Aini Mohd,
participate in revising it critically for important intellectual content. Finally, María Pilar
Almajano, as director, had the initial idea, the constant support, the English revision and the
final approval of the version to be submitted and any revised version.
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Conflicts of Interest
The authors declare no conflict of interest.
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Persea americana Mill. Seed: Fractionation, Characterization, and

Effects on Human Keratinocytes and Fibroblasts
Maria del R. Ramos-Jerz, 1 Socorro Villanueva, 2 Gerold Jerz, 1 Peter Winterhalter, 1 and Alexandra M.
Deters 3 ,*

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

 Abstract
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1. Introduction
Originally, Persea americana Mill. (Lauraceae) was solely native to humid tropical areas of
Mexico; it was later cultivated and extended to other regions of Latin America as well as to
USA and Europe. Today, the fruits are widely grown worldwide on large scale in various
subtropical countries and are generally recognized as a popular and healthy food source
supplying proteins and lipids to the human diet. Three principal cultivars (Guatemalan,
Mexican, and West Indian) exist with obvious differences in their fruit skin structure and
size. The original Mexican wild type varieties (span.: aguacate criollo) are purple or black
colored fruits in the size of a plum showing a smooth skin and are characterized by a yellow-
green aromatic flavored flesh. The earliest record of avocado used for human nutrition was an
archaeological dig in Peru that discovered avocado seeds buried with a mummy and was
dated to the 8th century BC. In ethnomedicine, the avocado kernels and pulp were used to
treat Saint Antonius fire (ergotism caused by fungal toxic alkaloid metabolites in grain),
dander, and scabies by Mexican indigenous people [1]. An ointment made from mashed
seeds had been used for women's makeup. Avocado seed oil had been applied to treat skin
eruptions. Nowadays, avocado is commonly used as a fruit or as a rich source for pulp oil
from fruit flesh. The oil and lipid fractions are part of various dermatological and cosmetic
preparations. Furthermore, they are utilized for treatment of dry skin problems in Ayurvedic
medicine [2, 3] which had been confirmed by in vivo studies [4, 5].
Avocado extracts, especially polyhydroxylated fatty alcohols (PFA) revealed a protection
against UV-B induced damage [6] in an in vitro keratinocyte model. Further, AV119, a
patented mixture of avocado sugars (perseitol and D-mannoheptulose [7]), increased the
expression of defensins [8]. Nonsaponifiable compounds influenced collagenic structures in
fibroblasts [9], and persenone A suppressed inflammation related processes [10]. Catechins,
procyanidins, and hydroxycinnamic acids (from seeds and pulp) were described as
antioxidative and antimicrobial agents [11]. Nevertheless, most of these components had
been derived and tested from avocado fruit or leaf extracts.
The countercurrent chromatography (HSCCC) method is based on liquid-liquid partitions
effect. It is successfully used for the fractionation of plants [12] as well as microorganism
grown culture medium extracts [13]. A solid support is not used in the HSCCC method;
therefore, sample absorption effects on stationary phase material could not occur, neither
could artifact generation. More details of this technique were published by Ito [14]. This
technique was used for the fractionation of avocado seeds extracts. Abscisic acid derivatives
(ABA) and small polyphenols such as salidroside, as well as A-type dimers and trimers of
procyanidins, were identified among others as principal natural products from a methanol
extract and its methanol-water and ethyl acetate partitions from avocado seeds [15, 16].
These extracts were tested in regard to their influence on human skin keratinocytes and
fibroblasts especially with respect to their ethnomedicinal background. Appropriate bioassays
to investigate cellular reactions and behavior upon treatment with natural products are in
vitro cell cultures of normal human dermal fibroblasts (NHDF), normal human epidermal
keratinocytes (NHEK), and human adult low calcium high temperature keratinocytes (HaCaT
keratinocytes). These cells represent the protecting dermal and epidermal layer of the human
skin. These cells mainly participate in the remodeling and regeneration of skin, two crucial
stages of the wound healing process as well as of skin integrity. Proliferation, differentiation,
and metabolic activity of cells are important processes during skin regeneration. In
vitro proliferation can be quantified with the 5-bromo-2′-deoxyuridine (BrdU) incorporation
test. The cell viability or more precisely the metabolic activity is analyzed with diverse
tetrazolium salts, which are reduced to formazan by active reductases, dehydrogenases, and
reductive products of cell metabolism. The reduction of tetrazolium salts a dependency to the
point of use respectively to utilized cell types [17]. Cytotoxicity was determined on basis of
activity of extracellular lactate dehydrogenase (LDH). Differentiation of skin cells is obvious
by morphological changes accompanied by a change in protein synthesis [18]. Therefore, the
differentiation process can be measured using specific antibodies against differentiation
specific proteins as involucrin [19]. To get a direction for the potential mechanism of
avocado seed extract activity, gene expression analysis was carried out. Signaling elements
involved in pro-proliferative, cell survival, and maturation respective differentiation were
taken into consideration.
Go to:

2. Methods

2.1. General
Solvents for extraction and liquid-liquid partitioning (n-hexane, methanol, and ethyl acetate)
were purchased in HPLC quality from Fischer-Scientific (Schwerte, Germany). Petrolether
Rotisolv (Fp 40–60°C) was from Carl Roth GmbH (Karlsruhe, Germany) and
dichloromethane from MWG-Biotech (Ebersberg, Germany). Solvents used for the
preparative HSCCC fractionation (TBME, acetonitrile, and n-butanol) were of analytical
grade and were purchased from Fischer-Scientific (Schwerte, Germany), acetonitrile for LC-
ESI-MS from Honeywell Speciality Chemicals (Seelze, Germany), and Nanopure water was
generated by a laboratory clean water unit (Werner Reinstwasser System, diamond analytic,
Leverkusen, Germany). Paper Filter no. 1 for extracts filtration was from Schleicher &
Schuell (Dassel, Germany).
Standards (−)-quinic acid (1) were purchased from Merck-Schuchardt (Hohenbrunn,
Germany) and chlorogenic acid (16) (3-O-(3, 4-dihydroxy-cinnamoyl)-D-quinic acid
hemihydrate) from Sigma-Aldrich (Deisenhofen, Germany).

2.2. Extraction and Fractionation of Avocado Seeds

Avocado fruits (P. americana Mill. cv. Hass) were picked in a commercial orchard close to
Tingüidín, Michoacán, México, in the year 2000. The seeds were released from the fruit
flesh, cleaned, and entirely dried at 40°C and then vacuum packaged and stored at −20°C. For
further processing the seeds were freeze-dried, powdered in a laboratory mill, and defatted
with petroleum ether in multiple steps. Then the defatted avocado seeds powder was
macerated exhaustively with methanol to yield the methanol extract (MeOH-E) (Figure 1(a)).
In another extraction process the avocado seeds were defatted and extracted with methanol
before they were extracted with distilled water and resulted in the water extract. In a second
process, the seeds were separated in the cotyledons and testa. Avocado cotyledons were
defatted with petroleum ether and macerated with methanol. For subsequent liquid-liquid
partitioning steps, the dried methanol extract was dissolved in a mixture of Nanopure water
and methanol (2 : 1, v/v) and in the first step partitioned against petroleum ether to gain the
petroleum ether partition. In the second step the residual methanol-water phase was
partitioned against dichloromethane to yield the semipolar compounds in the CH2Cl2 partition
(not tested). In the final step ethyl acetate was used to extract polar compounds such as
proanthocyanidins and other polyphenols from the methanol-water phase to yield the EtOAc
partition (mrC.3r). The final residue was named MeOH-H2O partition M (Figure 1(b)). The
obtained extracts and partitions were filtered through paper filters and concentrated under
vacuum at 35°C. The methanol-water partition M was fractionated by preparative high-speed
countercurrent chromatography (HSCCC) on a PTR model CCC-100 (Pharma-Tech Research
Corp., Baltimore, MD, USA) equipped with three preparative coils made of a
polytetrafluoroethylene tubing (2.6 mm i.d. × 165 m) connected in series. The HSCCC
separation was run at a revolution speed with the biphasic solvent system consisting of tert.-
butylmethylether-n-BuOH-ACN-H2O (1 : 3 : 1 : 5, v/v/v/v) in the head-to-tail mode using the
lower aqueous phase as mobile phase and the upper organic phase as stationary phase. The
system was operated at a spinning velocity of 1000 rpm with a flow rate of 3.0 mL min−1.
Chromatographic UV detection of fractions was done at λ = 280 nm (UV trace) to give seven
fractions (M.1 to M.7). Compound (13) was present on M, M.5 und M.6.

Figure 1
Extraction and fractionation scheme of avocado seed material. (a) Extraction of complete
avocado seeds (Persea americana Mill. cv. Hass). (b) Extraction process for avocado
cotyledons. Extractions as well as partitions used for HSCCC separation or in ...

2.3. Phytochemical Analysis of Avocado Seed Compounds by LC-ESI-MS/MS

An HPLC system (pump 1100 series, autosampler 1200 series) from Agilent Technologies
(Bӧblingen, Germany) was coupled with an ESQUIRE LC-ESI-MS/MS ist korrekt ion-trap
system from Bruker Daltonics (Bremen, Germany).
For C18 HPLC, a ProntoSil C18Aq column (5 μm, 250 × 2.0 mm, Knauer, Berlin, Germany)
was used. Solvent A: Nanopure water; solvent B: acetonitrile. HPLC gradient: t (0, 10, 40,
55, 65, 75 min), A (97, 97, 40, 0, 0, 97), and B (3, 3, 60, 100, 100, 3). Flow rate of
0.25 mL min−1. The HPLC separations were carried out at ambient temperature with the
following conditions.
ESI-MS/MS parameter settings: drying gas was nitrogen (flow 9.0 L min−1, 310°C), and
nebulizer pressure was set to 40 psi. ESI-MS/MS ionization parameters (negative mode):
capillary 3500 V, end plate off set 500 V, capillary exit −94.6 V, trap drive 35, ICC target
50000, maximum accumulation time 200 ms, charge control on, scan range m/z 50–2200,
threshold auto MS/MS 500, MS/MS experiments afforded a fragmentation amplitude value of
1.2 V.
ESI-MS (negative ionization mode) and MS/MS fragmentation data with [M-H]− signals of
the identified compounds in the crude extract, solvent partitions, and HSCCC fractions: (−)-
Quinic acid (1): ESI-MS: m/z191, MS/MS: m/z 173, 129, 111, 100, 85. Hydroxy-salidroside
(13): ESI-MS: m/z 315, MS/MS: m/z 179, 161, 153, 135, 119, 113, 89. Chlorogenic acid [syn.
3-O-(3, 4-dihydroxycinnamoyl)-D-quinic acid] (16): ESI-MS: m/z 353, MS/MS: m/z 191,
179, 135. Tyrosol-1′-β-D-O-glucoside (salidroside) (17): ESI-MS: m/z299, MS/MS: m/z 179,
161, 143, 131, 119, 113, 101, 89.  (1′R, 3′R, 5′R, 8′S)-epi-dihydrophaseic acid  β-D-glucoside
(18): ESI-MS: m/z 443, MS/MS: m/z 425, 237, 281, 161. Proanthocyanidin dimer B1 (19)
and B2 (20): ESI-MS: m/z 577, MS/MS: m/z 407, 425, 289. (1′S,  6′R)-8′-hydroxyabscisic
acid  β-D-glucoside (21): ESI-MS: m/z 441, MS/MS: m/z 397, 330, 139, 161. The trimers
proanthocyanidin A2-(+)-catechin or proanthocyanidin A2-(−)-epicatechin (24) occurred as
trace: ESI-MS: m/z 863, MS/MS: m/z 289. ESI-MS and fragmentations patterns (MS/MS)
(m/z) of not identified compounds are described in Table 3.

Table 3
Not identified metabolite profile in the methanol-water partition Mand in the respective
HSCCC fractions.

2.4. Skin Cell Culture

All chemicals used for bioassays were of analytical quality and were purchased from
Diagonal (Muenster, Germany). Cell culture media supplements were from PAA (Coelbe,
Germany) as not mentioned otherwise.
Confined cell lines were obtained after isolation of fibroblasts (NHDF) and keratinocytes
(NHEK) from human skin, obtained from skin surgery (University Hospital of Muenster,
Germany, Department of Dermatology, Department of Pediatrics) of various Caucasian
subjects. The study was approved by the local ethical committee of University of Muenster
(acceptance no. 2006-117-f-S). NHDF and NHEK were isolated and cultivated as described
earlier [20]. The continuous keratinocytes cell line of immortalized HaCaT keratinocytes,
kindly provided by Prof. Fusenig (German Cancer Research Institute, Heidelberg, Germany),
were cultivated with D-MEM high glucose (10% FCS, 1% penicillin/streptomycin, 1%
glutamine, and 1% nonessential amino acids) in an 8% CO2 humidified atmosphere. HaCaTs
were used because there were not enough NHEK at that time to do every experiment in three
replicates with normal keratinocytes. For investigations during the 2nd to 6th passage
(confined cell lines) and 56th–60th passage (HaCaT) the cells were directly adapted to
serum-free media (test medium); fibroblasts to MEM high glucose, SerEx (10%), and L-
glutamine (1%); and keratinocytes (NHEK and HaCaT) to MCDB153 complete. For gene
expression studies cells were shortly adapted to basal media supplemented only with L-
glutamine but without growth factors, BPE, and serum (minimal medium).
2.5. Influence of Test Compounds on Skin Cell Response
All tests were performed in 96-well plates (Sarstedt, Nuembrecht, Germany) at starting cell
densities of 5 × 103 keratinocytes and 3 × 103 fibroblasts/well. Extracts, cotyledon solvent
partitions, and HSCCC fractions of P. americana were solved into a stock solution of
1 mg/mL in Aqua Millipore, sterilized by filtration through a 0.2 μm regenerated cellulose
acetate membrane, and solved in the recommended serum-free media to a final concentration
of 10 μg/mL. As positive control 10% FCS was added to test medium [21]. Incubation started
24 h after seeding when the cells had reached a confluence of 50% and ended 48 h later by
adding 5-bromo-2′-deoxyuridine (BrdU), 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT), and (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,
3-benzene disulfonate (WST-1). BrdU incorporation assay, WST-1 and LDH assays were
performed according to the manufacturer's instructions (Roche, Penzberg, Germany).
Activity of intracellular reducing enzymes was measured by MTT test. Terminal
differentiation of NHEK was elucidated as described earlier [20].

2.6. Gene Expression Analysis of Keratinocytes and Fibroblasts

Gene expression of genes related to proliferation and differentiation was investigated after
incubation of skin cells with P. americana seeds extracts for 6 and 24 h in basal media.
Appropriate for investigation of, respectively, differentiation and maturation related cell
signaling were SMAD3, a member of a protein family that are homologs of both the
Drosophila protein, mothers against decapentaplegic (MAD), and the Caenorhabditis
elegans protein SMA, which participate in TGF-β signaling [24] and involucrin [25], was
appropriate for investigation of differentiation respectively maturation related cell signaling.
Procollagen 1α2 (Col1A2; [26]) and fibronectin 1 [27], both being proteins of extracellular
matrix (ECM) as well as the epidermal growth factor receptor (EGFR [28]), were chosen
because of their partition on wound healing respective proliferation related processes. 18s
RNA was used as endogenous control since GAPDH regulated itself during the keratinocyte
differentiation process [29]. Total RNA was isolated by InnuPREP RNA Mini Kit (Analytik
Jena, Jena, Germany). After qualitative and quantitative analysis of RNA at 260 nm RNA
was transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit (Life
Technologies, Darmstadt). cDNA was diluted with RNa free water to 20 ng cDNA. RT-PCR
was performed by TaqMan gene expression assays (20×; Life Technologies, Darmstadt,
Germany) described in Table 1. Experiments were performed with TaqMan Universal
MasterMix 2× without AmpErase (Life Technologies, Darmstadt, Germany) on a 7300 Real-
Time PCR system (Life Technologies, Darmstadt, Germany). Gene expression was
calculated with the comparative Ct method. In step one a normalization of target gene
occurred to the endogenous control 18s RNA (ΔCt), followed by normalization of the
normalized sample ΔCt to the normalized calibrator sample ΔCt (untreated NHEK) in step
two (ΔΔCt). The relative values were obtained using the formula  2−ΔΔCt.

Table 1
Assay IDs (Life Technologies, Darmstadt) for gene expression analysis performed with Real-
Time PCR. NHDF and NHEK were treated with 10 µg/mL avocado seed HSCCC fractions.
18 s rRNA was used for endogenous control. KGFR and involucrin ...

2.7. Statistical Analysis

Statistical evaluation was performed by Dunnett's post hoc test after ANOVA for comparison
of three to four treatment groups after variance calculation by Levene. The results were
considered significant with  P  values less than 0.05.
Go to:

3. Results

3.1. Phytochemical Analysis of Avocado Cotyledons

The methanol-water partition M yielded 5.4% of dried avocado cotyledons. 5.4 g of
methanol-water partition M were further separated by several reproducible preparative high-
speed countercurrent chromatography (HSCCC) and resulted in six fractions (M.1 to M.6)
and the additional residual solvent fraction coil fraction M.7 containing the more apolar
components which were not eluted from the HSCCC column coil system (Figure 2). Fraction
number 2 (M.2) was obtained with the highest yield followed by M.7, M.6, and M.3 (Table
2).

Figure 2
HSCCC-separation of the methanol-water partition M from avocado seed cotyledons
material. Recovered fractions M.1 to M.6 and the coil fraction M.7 by using the polar
biphasic solvent system: TBME-n-BuOH-ACN-H2O (1 : 3 : 1 : 5, ...

Table 2
Yields of whole avocado seed extracts, cotyledon solvent partitions, and HSCCC fractions.
Specific compounds were identified by LC-ESI-MS/MS and 1D/2D-NMR spectroscopy [16].
Methanol-water partition M as well as the HSCCC fractions had been shown to be active in
the in vitrotests. They were analyzed by negative LC-ESI–MS (Figure 3). Secondary plant
metabolites from avocado seeds in the mass range m/z 191 to m/z 863 were detected (Table
3).
Figure 3
LC-ESI-MS (negative mode) of the methanol-water partition M and the recovered HSCCC
fractions (M.2, M.3, M.6, and M.7). Intensity × 105 when not another is written.
The occurrence of abscisic acid derivatives such as (1′S,  6′R)-8′-hydroxyabscisic acid  β-D-
glucoside (21) and ( 1′R,   3′R,   5′R,   8′S)-epi-dihydrophaseic acid  β-D-glucoside (18) was
confirmed by LC-ESI-MS analysis with [M-H]− signals at m/z 441 and m/z 443 and indicated
by their characteristic MS/MS fragmentation data (397, 330, 161 and 425, 237). The
structural identity of these compounds was elucidated before in avocado seed extracts by
1D/2D-NMR experiments [15]. These compounds were then identified by retention time and
MS/MS data in the HSCCC fraction M.6 which was used for the bioassays. The low
molecular weight polyphenol tyrosol-1′-β-D-O-glucoside (salidroside) (17) was identified in
the methanol soluble avocado seed extract and in the cotyledons extract. The constitution
of 17 and hydroxy-salidroside (13) was also isolated and elucidated before by preparative
HSCCC isolation from other fractions of avocado seeds [16].
The proanthocyanidin B1 (19) and B2 dimers (20) and A-type linked trimer (24) were
identified in the fractions M.7 by selected [M-H]− ion traces at m/z 577 and m/z 863,
respectively, and were in accordance with published data [16] (Figure 4).

Figure 4
Structures of the identified compounds by LC-ESI-MS analysis in the avocado seeds extracts
and the tested HSCCC fractions.
With standards of (−)-quinic acid (1) (with [M-H]− at m/z 191) and chlorogenic acid (3-O-
(3,4-dihydroxycinnamoyl)-D-quinic acid; 16) with [M-H]− at m/z 353 it was confirmed that
these compounds were present in the HSCCC fractions M.2 and M.3. Additional peaks at
[M-H]− at m/z 353 were detected and indicated positional isomers of 16, such as 4- and 5-
caffeoylquinic acid which were not distinguished by MS/MS fragmentation data.

3.2. Influence on Fibroblasts

NHDF differently responded to the incubation with the avocado cotyledon methanol-water
partition M and HSCCC fractions. The HSCCC fractions M.2, M.6, and M.7 enhanced the
proliferation rate of NHDF compared to untreated cells. A significant increase of
proliferation rates compared to untreated NHDF was observed after treatment with M.7.
HSCCC fraction M.6 enhanced the average proliferation rate considerably but not
significantly, and M.2 only showed a marginal effect. An insignificant reduction of
proliferation was seen when NHDF were incubated with the methanol-water partition M and
HSCCC fraction M.3 (Figure 5). Metabolic activity was analyzed by reduction of WST-1, a
formazan that is not able to penetrate the cell membrane. WST-1 reduction through
extracellular active reductive enzymes was reduced after treatment with the methanol-water
partition M and each HSCCC fraction. In case of M and M.7 there was a significant
inhibition of extracellular enzyme activity (Figure 5).

Figure 5
Effects of 10 μg/mL avocado cotyledon methanol-water partition and HSCCC fractions on
the fibroblasts proliferation (dark columns) determined by BrdU incorporation assay and
extracellular enzyme activity calculated with WST-1 reduction ...
Cytotoxic effects were investigated on the base of LDH activity in culture supernatants as a
result of cell membrane damage. The LDH activity of cells incubated with avocado cotyledon
fractions was normalized to the naturally occurring LDH release of untreated cells. As shown
in Table 4 no cytotoxic effects were observed when fibroblasts were incubated with 10 μg/mL
of avocado cotyledon methanol-water partition M and HSCCC fractions.

Table 4
Percentage cytotoxicity of avocado cotyledon products determined by LDH activity in cell
culture supernatants normalized to the LDH release of untreated cells (=100%).
Gene expression analysis revealed that the methanol-water partition M and
fraction M.3 induced slightly the gene expression of fibronectin (FN1) and collagen 1α2
(Col1A2), respectively, but none of the other genes whose expression was analyzed after 6 h
of incubation. Fraction M.2 increased the gene expression of epidermal growth factor
receptor (EGFR), insulin receptor (InsR), signal transducer and activator 6 (STAT6), and
Col1A2 and FN1. Treatment of NHDF with HSCCC fraction M.6 induced the gene
expression of FN1 but inhibited gene expression of SMAD3. HSCCC coil
fraction M.7 treated NHDF revealed an upregulated gene expression of InsR and FN1. A
slight stimulation of EGFR, STAT6, SMAD3, and Col1A2 gene expression was also
observed, but it was not possible to do a statistically firm calculation of this effect because of
issues with adequate endogenous control (Table 5).
Table 5
Regulation of NHDF gene expression after incubation for 6 h with 10 µg/mL avocado
cotyledon methanol-water partition M and HSCCC fractions in minimal media. Gene
expression of three independent tests was normalized to the endogenous ...

3.3. Influence on Keratinocytes

The methanol-water partition of avocado cotyledons M slightly increased the proliferation
rate and intracellular enzyme activity of HaCaT keratinocytes. The HSCCC
fractions M.3 and M.6 enhanced the proliferation. In case of M.6 the promotion of
proliferation was significant. While HSCCC fraction M.2insignificantly inhibited the HaCaT
proliferation, a significant inhibition was observed when HaCaTs were treated
with M.7 (Figure 6).

Figure 6
Effects of 10 μg/mL avocado cotyledon methanol-water partition Mand HSCCC fractions on
HaCaT keratinocytes. Proliferation rates (dark columns) were investigated with the BrdU
incorporation assay; the intracellular metabolism (bright columns) ...
The cell metabolism was not affected by the methanol-water partition M but each of tested
HSCCC fractions slightly inhibited the reduction of MTT, whereas M.6 had the least
influence (Figure 6). Methanol-water partition M and HSCCC fraction M.3 did not influence
the LDH activity in culture supernatants. Incubation of HaCaT with
fractions M.2, M.6, and M.7 increased the extracellular LDH activity in a range between
18% (M.7) and 25% (M.2; Table 4).
Normal human epidermal keratinocytes (NHEK) were incubated with 10 μg/mL avocado
cotyledon methanol-water partition M and HSCCC fractions for nine days to investigate the
keratinocyte differentiation. NHEK were chosen since the used HaCaTs did not differentiate
in several tests. After extraction of total proteins the amount of the differentiation specific
protein involucrin was semiquantitatively analyzed using Dot blot technique. As shown
in Figure 7 the methanol-water partition M and the corresponding HSCCC
fractions M.2, M.3, and M.6 increased the synthesis of involucrin. Incubation of NHEK with
the HSCCC “coil fraction” M.7 in three independent tests resulted in high standard deviations
and differences between results. When data of each test were pooled it got obvious that the
average involucrin expression was marginal elevated compared to pooled data of untreated
cells (Figure 7).
Figure 7
Involucrin amounts of NHEK incubated with 10 μg/mL avocado cotyledon methanol-water
partition M and corresponding HSCCC fractions for 9 days. Values were normalized to
involucrin amounts of untreated NEHK (=100%, —). (Error bars ...
For preliminary gene expression analysis NHEK were used instead of HaCaT because NHEK
give an account of the situation in normal skin. First results revealed minor changes in gene
expression compared to the untreated cells after 6 h. Prolonging the incubation time for 18 h
mostly resulted in a stimulation of gene expression. Methanol-water partition M exhibited the
least influence but the expression of KGFR (FGFR2b) was enhanced. To minor extend genes
of EGFR and STAT6 were upregulated. HSCCC fraction M.2 treated cells showed an
increased gene expression of KGFR (FGFR2b), EGFR, and SMAD3. No statistically firm
results were obtained with M.3 and M.7 because the 18s RNA was also influenced and a
normalization was not possible. M.6 mostly increased the gene expression of STAT6 and
insignificantly enhanced the expression of EGFR (data not shown).
Go to:

4. Discussion
For the preparative fractionation of the methanol-water partition M from avocado cotyledons,
high-speed countercurrent chromatography (HSCCC) resulted in high quantities of the
fractions later used for the biological assays, further isolation, and spectroscopical elucidation
of the active compounds.
Chlorogenic acid (3-CQA 16) and maybe its 4-, 5-CQA isomers were present in the
methanol-water partition M and in recovered HSCCC fractions M.2 and M.3. Hydroxy-
salidroside (13) and tyrosol-glucoside (syn.: salidroside 17) were present in M and HSCCC
fraction M.6 as well as derivatives of abscisic acid glucosides (ABA, 18, 21). HSCCC “coil
fraction” M.7 was mostly composed of dimers and trimers of proanthocyanidins, but also
ABA derivatives 18 and 21 were regained in traces.
Biological assays revealed a relation between composition and effects of methanol-water
partition M and its corresponding HSCCC fractions and a dependency on the used cell type.
Methanol-water partition Mslightly reduced the proliferation rates of NHDF but slightly
stimulated the proliferation of HaCaT, an effect that was also observed with NHEK in one
preliminary experiment (data not shown). The enhanced proliferation resulted in a contact
inhibition that explains the differentiation of NHEK after 9 days of incubation [18]. The
results obtained with functional in vitro tests went conform with gene expression results.
Every investigated gene that is related to proliferation in NHDF was not affected, while in
NHEK the KGFR gene, responsible for stimulation of keratinocytes' proliferation, was
upregulated [30]. HSCCC fraction M.2 composed of quinic acid (1) and chlorogenic acid
(16) exerted a slight proliferation stimulating activity on NHDF but did not affect metabolic
activity and cytotoxicity of fibroblasts. Gene expression analysis revealed an upregulation of
genes that are involved in proliferation (STAT6 and EGFR), cell survival (InsR), and/or
maturation processes (SMAD3, Col1A2, and FN1). This leads to the assumption that the two
main components quinic acid and chlorogenic acid exert different activity on fibroblasts.
Quinic acid might be responsible for the pro-proliferative effect because chlorogenic acid was
shown to inhibit growth of fibroblasts [31] and because M.3, which did not contain quinic
acid, inhibited the NHDF proliferation. Furthermore, it was previously described that quinic
acid inhibited NFkB signaling and stimulated DNA repair mechanism [32]. On the other
hand, M.2 slightly inhibited the proliferation and the metabolic activity of HaCaT and
directly induced the differentiation of NHEK. The upregulation of SMAD3 went conform
with the direct induction of differentiation. SMAD3 is part of signal pathways that are related
to cellular development respective differentiation [24]. In addition to their effect on cellular
proliferation EGFR and KGFR (FGFR2b) are involved in the regulation of keratinocyte
differentiation [30, 33]. In this context the coexpression of EGFR and KGFR (FGFR2b) also
supports the obtained results of functional tests. Quinic acid and respective or chlorogenic
acid differently affected HaCaT keratinocytes compared to fibroblasts. This outcome was
more considerable when results obtained with HSCCC fraction M.3 were regarded. Due to
the absence of further information it was difficult to allocate the effect to quinic acid or
chlorogenic acid. A circumspective suggestion is that quinic acid inhibited the HaCaT
proliferation because the absence of quinic acid in M.3 results in a clear increase in HaCaT
growth. Additionally, chlorogenic acid, the main component of M.3, was already reported to
stimulate the proliferation of other epithelial cell cultures [34].
As detected by LC-ESI-MS the HSCCC fraction M.6 was principally composed of
salidroside (17), hydroxy salidroside (13), ABA derivatives (18, 21), and structurally
unknown compounds. M.6 was the most active fraction in the functional assays because it
significantly increased the proliferation rates of both keratinocytes and fibroblasts. Gene
expression analysis showed that M.6 promoted the proliferation of NHDF and NHEK via
different signal pathways. In NHDF the repression of SMAD3 might indicate an inhibition of
maturation related signal pathways and a preferred induction of signal transduction resulting
in proliferation. The increased expression of fibronectin 1 gene led assume
that M.6 influenced NHDF proliferation via the ECM and not directly via growth factor
receptors since neither the EGFR, InsR, nor STAT6 were affected. Therefore, the induction
of proliferation took place through remodeling of ECM proteins [35].
In NHEK an induction of EGFR and STAT6 gene expression was observed after 24 h. As
mentioned before, EGFR is involved in proliferation and differentiation processes [30, 33].
From this point of view the slight increase in EGFR gene expression is jointly responsible for
the increase of HaCaT and NHEK proliferation rates. The STAT6 pathway is usually affected
by IL-4 and IL-13, but there are reports that the activation occurred upon a different stimulus
[36]. Nevertheless, the pro-proliferative effect of M.6 is based on the inhibition of pathways
that prevent proliferation. The effect of M.6 on skin cells might base on the ABA
derivatives 18 and 21. As shown in Figure 3,  21 represents the component with the highest
amount, and additionally both 18 and 21 resemble autocrine abscisic acid that acts as stress
response hormone in animal cells [37]. In contrast it has been shown that ABA derivatives
had no influence on healthy fibroblasts [38] and that ABA induction of cellular response is
based on the activation of ADP-ribose and an increase in intracellular  Ca2+  concentrations
[39]. The latter would result in an induction of keratinocyte differentiation, but this was not
observed during the tests. Among ABA derivatives salidroside (17) and hydroxy-salidroside
(13) were detected in M.6. Salidroside is reported to inhibit the proliferation of cancer cells
[40], but there are as much reports that show an antiapoptotic effect [41] and pro-proliferative
effect especially on fibroblasts [42, 43]. Furthermore, salidroside attenuates the level of
intracellular calcium [44], resulting in a support of keratinocyte proliferation. Supernatants
might relate to the increased cell number particularly with regard to the unaltered reduction of
MTT and WST-1 test compared to untreated cells.
HSCCC fractionation “coil fraction” M.7 was mostly composed of proanthocyanidin B1 and
B2 as well as an A-type linked trimer among ABA derivatives and not yet identified
components. Results showed that the difference in composition was recovered in bioactivity
of M.7 on human skin cells. Such an apparent difference in susceptibility to natural products
of keratinocytes and fibroblasts has been shown with the other fractions but not in such an
apparent way. M.7 steeply increased NHDF proliferation, did not affect proliferation and
intracellular enzyme activity of normal keratinocytes (data not shown) but significantly
inhibited HaCaT proliferation, increased LDH activity in supernatant, and slightly reduced
the intracellular enzyme activity of HaCaT. Unfortunately the analysis of NHEK gene
expression failed, and no gene expression analysis was done with HaCaT because they exert
a modified gene expression compared to NHEK. Nevertheless, the increase of NHDF
proliferation was accompanied with an increase of insulin receptor and FN1 gene expression.
These results indicate that M.7 influences the cellular survival mechanisms of fibroblasts and
the signal transduction via ECM. The question for the responsible ingredient of M.7 is not
clarified yet. As with fractions M.2 and M.6 there are two different classes of bioactive
ingredients and not identified components. On the one hand, there are traces of ABA
derivatives which were also present in M.6. On the other hand, M.7 predominantly contained
the proanthocyanidins B1 and B2 as well as A-type linked trimers. Results obtained
with M.7 are not comparable with bioactivities previously described of ABA derivatives. But
HSCCC “coil fraction” M.7 acted as being selective on normal cells (NHDF and NHEK) and
on immortalized HaCaT cells as a synthesized procyanidin, which was earlier reported Kim
et al [45]. Furthermore, the inhibitory activity on fast growing cells like HaCaT was shown
for proanthocyanidin trimers [46]. Further previous studies on the bioactivity of
proanthocyanidins support the recent findings. Procyanidin B2 was found to induce the
proliferation of different normal cells [47, 48], and redox-active proanthocyanidins of grape
seeds improve the dermal wound healing [49]. Additionally, procyanidolic oligomers
interacted with the ECM proteins of fibroblasts [50], and such an interaction might base the
upregulation of fibronectin gene expression that was measured after treatment of NHDF
with M.7.
In conclusion the recent results show that the investigated avocado extracts offered a high
impact on cellular function like proliferation, enzyme activity, and differentiation of skin cell
that base on an influence on their gene expression. The effects of HSCCC fraction differed
due to their composition and the cell type they were tested on. Although further investigation
must prove the following assumption, the bioactivity of avocado seed extracts was related to
quinic acid, chlorogenic acid, salidrosides, ABA derivatives, and proanthocyanidins. In view
of traditional use and antiproliferative activity of M.7especially on immortalized HaCaT cells
the results present a sound baseline for a successful development of new medications. Also
the activities of HSCCC fractions M.2 and M.6 are worthy to be investigated in regard to
their properties to improve wound healing.
Go to:

Acknowledgments
The authors thank Dr. Lohse, Department of Paediatric Surgery, University of Muenster, for
support with skin grafts. Financial support for the study was given by Professor Hensel,
University of Muenster, Institute for Pharmaceutical Biology and Phytochemistry. The work
of M. del R. Ramos-Jerz was financially supported by the German Academic Exchange
Service (DAAD).
Go to:
Glossary

FCS: Fetal calf serum

HaCaT: Human adult low calcium high temperature keratinocyte

NHDF: Normal human dermal fibroblasts

NHEK: Normal human epidermal keratinocytes

EGFR: Epidermal growth factor receptor

InsR: Insulin receptor

KGFR
Keratinocyte growth factor receptor or fibroblast growth factor receptor 2
(FGFR2):

STAT6: Signal transducer and activator of transcription 6

Drosophila protein, mothers against decapentaplegic (MAD) and

SMAD3:
the Caenorhabditis elegans protein (SMA), homolog 3

FN1: Fibronectin 1

Col1A2: Procollagen 1α2

ECM: Extracellular matrix

LDH: Lactate dehydrogenase

BrdU: 5-Bromo-2′-deoxyuridine

MTT: 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromid

Water-soluble tetrazolium (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-

WST-1:
tetrazolio]-1, 3-benzene disulfonate.

Go to:

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oil extraction from avocado seed using N-Heptana solvent