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April 30, 2021
Archives • 2021 • vol.1 • 426-446
http://pharmacologyonline.silae.it
ISSN: 1827-8620
BIOASSAY-GUIDED FRACTIONATION IN Anacardium excelsum (Bert. &
Balb. Ex Kunth) SKEELS (ANACARDIACEAE)
Luis G. Sequeda-Castañeda 1*, Crispín A. Celis-Zambrano 1*,
Rubén D. Torrenegra-Guerrero 2
1Departament of Chemistry, Sciences Faculty, Pontificia Universidad Javeriana, Bogotá,
Colombia. 2Sciences Faculty, Universidad de Ciencias Aplicadas y Ambientales, Bogotá,
Colombia
* crispin.celis@javeriana.edu.co, lsequeda@javeriana.edu.co
Abstract
Anacardium excelsum is a tree between 15 and 50 meters high, with a thick trunk and is
present in some departments of Colombia. Its nut-shaped fruits were used in the old Upar
Valley by natives (Valledupar) to make Caracolí bread. Most research has been done at the
forest level, but there are few phytochemical and biological activity studies. This bioguided
work shows the application of antioxidant methods (ABTS, DPPH and DMPD
) in the search
for active compounds present in the fraction and subfractions of greater activity. The
antioxidant capacity was measured to the crude extract in ethanol and the antioxidant activity
of the fractions in petroleum ether, dichloromethane, and ethanol-butanol of testa, tegument,
flower, fruit, seed, bark, and leaf. Column Chromatography, Thin Layer Chromatography and
Gas Chromatography coupled to Mass Spectrometry were performed on the petroleum ether
fraction of testa with greater antioxidant capacity, finding the following compounds: 3-
pentadec(en)il-phenol, 3-pentadecyl-phenol, 3-heptadeca(dien)il-phenol, 3-heptadec(en)il-
phenol, 3-heptadecyl-phenol, 3-nonadec(en)il-phenol, ethyl ester of hexadecanoic acid, ethyl
ester of heptadecanoic acid , ethyl ester of linolenic acid, hexadecanoic acid butyl ester, ethyl
ether of octadecanoic acid, ethyl ester of (Z)-9-octadecenoic acid, ethyl ester of 9,12-
octadecadienoic acid and ethyl ester of eicosanoic acid. The results indicate that Anacardium
excelsum is a potential source of bioactive compounds.
Keywords: Antioxidant, Anacardium excelsum, phenolic lipids, 3-pentadecyl-phenol, 3-
heptadecyl-phenol
PhOL Sequeda-Castañeda, et al. 427 (pag 426-446)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Introduction
The Anacardiaceae family includes 73 genera and
850 species of trees, shrubs and lianas distributed
worldwide grouped into five tribes: Anacardieae,
Spondiadeae, Semecarpeae, Rhoeae and Dobineae,
approximately 41% of its genera are native to
America [1, 2]. The Anacardiaceae family includes
trees and shrubs with alternate pinnate-compound
or simple leaves. The flowers are regular and can be
bisexual or unisexual, composed of 5 joined sepals, 5
free petals and 5 to 10 stamens inserted into a fleshy
disc [3]. The fruit can be a drupe, a walnut, or a
samara, also, some species exude irritating resins
for the skin [4, 5]. This family is distributed mainly in
tropical and subtropical zones, some of them
present in temperate zones, with economic
importance to produce edible fruits, gums, resins,
tannins, dyes, and woods of commercial importance
[5-9]. Among the most valuable species of fruit trees
are Anacardium occidentale L. [10, 11], Mangifera
indica L. [12, 13] and Pistacia vera L. [14, 15], and
among those for timber use are Anacardium
excelsum (Bert. & Balb. ex Kunth) Skeels [16-19],
Astronium graveolens Jacq. [20, 21] and Spondias
mombin L. [22, 23]. Anacardium occidentale L. shows
antioxidant activity due to the content of phenolic
compounds [24, 25], and the antitumor activity to
long-chain derivatives of salicylic acid commonly
called anacardic acids, such as, 6-[8(Z)-
pentadecenyl] salicylic acid [26, 27]. Lannea
coromandelica (Houtt.) Merr. shows sporicidal
activity against zoospores of Aphanomyces
cochlioides attributed to the content of
poliflavonoid tannins and antihelminth activity
against Caenorhabditis elegans [28, 29]. From
Pistacia lentiscus L., known as lentiscus, essential oils
are extracted for different use; antioxidant [30],
antitumoral [31] and antibacterial against Escherichia
coli, Staphylococcus aureus and Bacillus subtilis
whose action derives in the terpenes content such
as -pineno (40%), -pineno (1.5%), -myrcene (9.0%),
limonene (1.0%) and caryophyllene (5%) [32].
Pistacia vera L. commonly known as pistachio is
highly appreciated for its drupes that are used to
make sweets and sausages [33, 34], shows
antibacterial activity against Corynebacterium
xerosis, Bacillus brevis, Bacillus megaterium, Bacillus
cereus, Mycobacterium smegmatis, Pseudomonas
aeruginosa, Staphylococcus aureus, Klebsiella
pneumoniae, Klebsiella oxytocica A, Enterococcus
faecalis, Micrococcus luteus, Escherichia coli, Yersinia
enterocolitica, and also against pathogenic yeasts
like Kluvyeromyces fragilis, Rhodotorula rubra and
Candida albicans, all this spectrum of bactericidal
action is due to the content of pinene (75.6%), -
pinene (9.5%), trans-verbenol (3.0%), camphene
(1.4%), trans-pinocarveol (1.2%) and limonene (1.0%)
[35, 36]. Magnifera indica L. commonly known as
mango has antioxidant activity attributed to the
metabolites; mangiferin, isomangiferin and
quercetin, all quercetin 3-O-rhac derivatives [37, 38].
The mango seed is associated with functional
properties when used as a prebiotic in feed of Labeo
rohita fingerlings, increasing the immune response
and resistance to diseases caused by Aeromonas
hydrophila infections [37, 39]. Rhus coriaria L.
recognized as Sumac is grown to obtain tannins for
industrial usage and presents antioxidant activity,
finding the gallic and protocatechuic acids as
responsibles for this activity [40, 41]. Rhus
toxicodendron L. is characterized by producing skin
dermatitis on contact with urushiol resin, this type
of pathology (dermatitis) is detected on contact
with poison ivy [42], however, homeopathic
medicine used this plant for the treatment against
herpes and in recent years studies were conducted
to evaluate anti-inflammatory activity of different
extracts (tincture in alcohol) [43-45]. Schinus mole L.
demonstrates antidepressant activity when testing
for tail immobilization and forced swimming in mice,
two predictive models of depression [46], and
repellent activity of ovocidal and adulticidal type on
Triatoma infestans and Blattella germanica related to
the content of tannins, alkaloids, terpenes, and
flavonoids, among others [47, 48]. Sclerocarya birrea
(A. Rich.) Hochst. shows antiproliferative and
apoptotic activity against breast cancer cells (MCF-
7) and has antioxidant activity due to its content of
glucosidated derivatives of quercetin and
kaempferol such as quercetin 3-O- -D-(6"-alloyl)
glucopyranoside and kaempferol 3-O- -D-(6"-galloyl)
glucopiranoside [49, 50]. Spondias mombin L. grows
in the rainforest and coastal area of Africa, is used in
traditional medicine as a diuretic, against diarrhea,
dysentery, fevers, hemorrhoids, gonorrhea and
leucorrhoea, as an expectorant, stomach pain and
to expel the tapeworm. Faced with a spectrum of
PhOL Sequeda-Castañeda, et al. 428 (pag 426-446)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
broad action in medicine, researchers like Ayoka
conducted studies for the treatment of psychiatric
disorders evaluating anxiolytic, antiepileptic and
antisychotic activity with excellent results in rats
ns,
anthraquinones, flavonoids and cardatonic
glycosides were found [51, 52]. Likewise,
anthelminthic activity was evidenced when extracts
were evaluated on earthworms of the genus
Eudrilus eugeniae, this activity is attributed to the
content of phenolic acids and ellagitannins [53].
In Colombia we find species as Anacardium
excelsum (Bert. & Balb. ex Kunth) Skeels, a tree
between 15 and 50 m high, with a thick trunk,
sympodial ramification, kidney-shaped fruits and
leaves simple, alternate, obovate with entire margin
and a size between 10-60 cm long 6-12 cm wide.
Inflorescences are clusters (panicles) 23-45 cm long
with white-yellow, pink-creamy, white, or yellow
flowers, see Figure 1 [54, 55]. The fruits are about 3
cm in diameter, reniform nuts of floury consistency
called noses due their pear shape. This nuts where
used in the old Upar Valley to make a kind of edible
bread called snail bread and consumed cooked in
syrup or cakes [56]. The pendulums of Anarcardium
excelsum (Bert. & Balb. ex Kunth) Skeels are curved
and fleshy and serve as food for monkeys and bats.
The exudate serves as food for Sanguinus oedipus,
the white-headed titi monkey. Nutritional analysis
reveals that the exudate is a rich source of calcium,
proteins, carbohydrates, and water. It is currently
cultivated as a shade tree and its edible fruit has not
been economically exploited [57]. Its wood was
formerly used to build canoes and some kitchen
tools such as rafts, bongos, trays, and plates [17].
Also has been used for veneres, floors of modern
constructions and some expensive furniture [16, 55].
Anacardium excelsum (Bert. & Balb. ex Kunth) Skeels
has few studies in the field of Pharmacognosy &
Phytochemistry [18, 58-61], however, it is widely
studied in the field of reforestation as an alternative
for the exchange of carbon dioxide-oxygen and for
the acquisition and fixation of phosphorus and
nitrogen in soils (biomass) [19, 62]. Therefore, this
bioguided research focused on the evaluation of the
antioxidant capacity of the total extracts and
fractions of the Anacardium excelsum (Bert. & Balb.
ex Kunth) Skeels plant using ABTS
, DPPH,
DMPD methods and subsequent identifying the
compounds present in the most active fraction
using Gas Chromatography coupled to Mass
Spectrometry (GC-MS)
Methods
Collecting botanical material
Sample of plant material for leaves, flowers,
fruits, seed coat, seed, integument, and bark, were
collected during the flowering season in
municipality of Bucaramanga (department of
Santander, Colombia), under the following
elevation of 986m. The National Herbarium of
Colombia identified the sample as Anacardium
excelsum (Bert. & Balb. ex Kunth) Skeels, under
classification number COL 520397.
Extraction and fractionation
The material was dried to the environment for 15
days, until humidity less than 8%. Between 50800 g
was soxhlet extracted with 4 liters of ethanol. After
filtration, the extracts were concentrated using
vacuum at 40°C. Partial liquid-liquid extractions was
made in continuously using solvents in different
polarities obtaining fractions in petroleum ether
(PE), dichloromethane (CH2Cl2) and ethanol-butanol
(EtOH-BuOH), as shown in Table 1. The extract
bioactive was fractionated by Column
Chromatography (4 x 60 cm) using SiliaFlash
Irregular Silica Gel G60, (60 - 200 µm, 60 Å, SiliCycle)
eluted with mixtures of chloroform (CHCl3) and
methanol (MeOH) in a relationship [95:5], [70:30]
and [5:95]. The fractionation of the sub-fractions
was performed using a discontinuous gradient with
n-Hexane, ethyl acetate (EtOAc) and acetic acid
(AcOH) in the proportion of [90:10:1], [80:20:1] and
[50:50:1]. The fractions and subfractions were
monitored and regroupded by Thin Layer
Chromatography (TLC) using SiliaPlate F254 (20 x 20
cm, SiliCycle) plates, and mixtures of n-hexane,
EtOAc, AcOH and MeOH as mobile phase (Plates
and Solvents of Merck). Vainillin was used as
developer. The extracts, fractions and sub-fractions
using antioxidant capacity and activity, were
bioassay-guided study [63].
PhOL Sequeda-Castañeda, et al. 429 (pag 426-446)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Methods of antioxidant capacity and activity
The measurement of the antioxidant capacity of
the crude ethanol extract from each part of the
plant was preparaded using several methodologies
(ABTS
, DPPH and DMPD), while the antioxidant
activity of the fraction and group of subfractions
was carried out using the most sensitive method
[64]. All crude extracts were prepared in the range
CH2Cl2, and EtOH-BuOH between 10 and 1,000
/mL or fractions capable of
inhibiting 50% radical absorbance (IC50). The pooled
fractions and subfractions from the PE fraction of
testa were prepared at one single concentration
determined as percentage of radical inhibition.
ABTS method of antioxidant activity
The antioxidant capacity of whole extracts,
fractions and subfractions was determined by the
method described by Re et al. [65, 66]. The stock
solution of ABTS
-Azinobis-(ethyl-
benzothiazoline-6-sulfonic acid) was prepared per
solubilizing 20mg of ABTS in 10mL of deionized
water and 2.45 mg of potassium persulfate
(K2S2O8). The solution could react at room
temperature for 16 hours in the dark. After working
solutions were prepared in MeOH, obtaining an
absorbance at 0.75 ± 0.02 to 744nm in a
spectrophotometer Sanyo SP50 and quartz cuvettes
using a 10x10x45 mm. The solution of ABTS loses
its color blue-green reaching colorless when added
an antioxidant compound, reducing the absorbance
of the solution, which is measured at 744 nm, which
is expressed as percentage inhibition. The
percentage of inhibition (turning blue-green to
transparent color) was determined by using the I =
[(Ao - Ae) / Ao] x 100 equation, where Ao is
absorbance without extract, and Ae is the
absorbance with extracts. To get IC50 of extracts
turning to transparent color that indicate
antioxidant capacity, was calculated by plotting % of
inhibition vs. extract concentration; then linear
regression equation was calculated (y = mx + b), and
50% inhibitory concentration by the equation IC50 =
(50 - b) / m. As antioxidant standards, 6-Hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid
(trolox), butylhydroxytoluene (BHT), L-ascorbic acid
-tocopherol (vitamin E) were
used [54, 66, 67].
DPPH method of antioxidant activity
We used the methodology proposed by Brand-
Williams et al (1995) [68, 69], where 10 mg of DPPH
-diphenyl--picrylhydrazyl radical) were
solubilized in 10 mL of MeOH (4.34 mM), then
working solutions were prepared to obtain an
absorbance of 0.75 ± 0.05 for all cases to a
wavelength of 514 nm and read in a
spectrophotometer Sanyo SP50, quartz cells using
1.0 cm. When an antioxidant compound is added to
the solution of DPPH radical, it loses its violet,
causing the decrease of the initial absorbance
causing an inhibition. The conditions for assessing
the antioxidant capacity of the extracts were the
same of ABTS assay [54, 68, 70, 71].
DMPD method of antioxidant activity
The method developed by Fogliano et al. (1999)
was implemented to determine the antioxidant
activity [72]. 41.8 mg of DMPD (N,N-dimethyl-p-
phenylenediamine dihydrochloride) were solubilized
in 2 mL of distilled water, a 0.25 mL aliquot was
diluted to 25 mL with acetate buffer solution pH
5.25. The cation radical DMPD was formed by
adding 50 mL of 0.5 M FeCl3 solution and monitored
at 505 nm [54, 72]. The conditions for assessing the
antioxidant capacity of the extracts were the same
of ABTS assay.
Antioxidant method selection and statistical
analysis
The antioxidant method that delivers greater
sensitivity and moderate cost will be selected, to
perform the bioguided study to find the fraction or
subfractions with high antioxidant activity, then, a
tentative identification of the compounds
responsible for the activity will be made using Gas
Chromatography coupled to Mass Spectrometry
(GC-MS). All experiments were done by triplicate (n
= 3) and results were expressed as means ±
standard deviations [73].
PhOL Sequeda-Castañeda, et al. 430 (pag 426-446)
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ISSN: 1827-8620
Gas Chromatography-Mass Spectrometry (GC-
MS)
Analyses were performed in a gas chromatograph
Agilent 6850 Series II Network System equipped
with mass selective detector Agilent 5975B VL
(Electron impact ionization, EI, 70 eV), a
split/splitless injector (1:100 split ratio) and
Enhanced ChemStation MSD D.03.00.52 data
system, that included the spectral libraries Wiley and
Nist. A fused-silica capillary column is a HP-1MS (30
Chromatographic conditions were: The GC oven
temperature was programmed from 100 °C (2 min)
to 285 °C (10 min) at 25 °C/min slope and post run to
320 °C (3 min). The temperatures the injection port,
ionization chamber and the transfer line were set at
300, 185 and 285 °C, respectively. Helium (99.999%,
AGA-Fano) was used as carrier gas, with 85 kPa
column head pressure and linear velocity at
constant flow (1 mL/min). Mass spectra and
reconstructed (total) ion chromatograms were
obtained by scanning in the mass range m/z 30-500
Da at 2.2 scan/s. Chromatographic peaks were
checked for homogeneity with the aid of the mass
chromatograms for the characteristic fragment ions
and with the help of the peak purity program.
Tentative identification criteria of compounds were
obtained compounds compared to Wiley 7n.1 and
Nist 05a.L mass spectra libraries [71].
Results
Obtaining extracts and fractions
Extraction and fraction yields for each plant organ
are found in Table 1. Extract values between 19.8
and 45.6% were observed for flowers and tegument,
respectively. Fractions highest yield corresponds to
petroleum ether and EtOH-BuOH of fruits and
tegument, 57.7 and 54.0%, respectively. The lower
efficiency corresponds to leaves and testa (0.4 and
0.9%, respectively) where mixture of EtOH-BuOH
solvents was used. The highest accumulated
fraction (PE, CH2Cl2, EtOH-BuOH) corresponds to
tegument (86.2%).
Antioxidant method selection and the best
antioxidant fraction
The antioxidant capacity (AOC) for crude extracts
(Figure 2A) and fractions (Figure 2B) was measured
by different methods. Comparison of all methods
using 50% inhibition (IC50) as criteria, indicates that
all EtOH extract from testa exhibit high values of
IC50, 164.3 ± 7.0, 239.5 ± 54.8 and 2929.0 ± 100.4
, DPPH and DMPD
, respectively.
Besides, the highest sensitivity method corresponds
to ABTS
statistically comparable with the values of standart
Table 2A). The
comparison of antioxidant methods is shown in
Table 2B. It was observed that DMPD method is
the least suitable to perform the AOC
measurements because it is not stable under
working conditions, adittionally, presents
interferences and it is necessary to adjust the pH of
the working solution precisely (pH 5.25). Moreover,
between the DPPH and ABTS methods were
observed differences in the price per milligram, U$
0.3 for DPPH against U$ 0.02 for ABTS
. In the
other hand, some antioxidants such as carotenoids
present UV-Vis spectra like DPPH, which can lead to
erroneous interpretations of AOA measurement, so
this method could present interferences. Another
difference between methods is solubility, DPPH is
soluble in polar medium and ABTS presents
solubility in polar and non-polar medium, offering a
great advantage to evaluate hydrophilic and
liposoluble antioxidants [74].
ABTS method was selected as the one with the
highest sensitivity, the shortest work time, the
lowest cost, the best solubility, and the highest
stability to measure the antioxidant activity in the
fractions and subfractions. Testa extract was
selected with the purpose of looking for fractions
and subfractions with potential antioxidant activity.
The subfractions with the highest activity were
analyzed using GC-MS.
Fractionation
The PE testa fraction with the highest antioxidant
) was
subjected to fractionation and sub-fractionation,
results that are recorded in Table 3. The
fractionation of 2.49 g of the active fraction was
performed on silica gel (146 g) using as eluent a
PhOL Sequeda-Castañeda, et al. 431 (pag 426-446)
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ISSN: 1827-8620
discontinuous gradient of 1,440 mL CHCl3-MeOH
[100: 0], followed by 2,880 mL [95:5], then 1,440 mL
[70:30], and ending with 1,320 mL [5:95]. In total 59
fractions were collected (120 mL each) and
monitored by TLC (normal phase) using CHCl3-PE
[80:20] and CHCl3-MeOH [95: 5] as the mobile
phase. According to the results of TLC, the fractions
were regrouped into 10 groups registered as G1
(Fr.1-3), G2 (Fr.4-11), G3 (Fr.12-20), G4 (Fr.21-24), G5
(Fr.25-35), G6 (Fr.36-41), G7 (Fr.42-45), G8 (Fr.46-51),
G9 (Fr.52-54) and G10 (Fr. Fr.55-59), which were
evaluated by the ABTS. The bioguided work found
the following groups of fractions with greater
antioxidant activity: G2 (Fr.4-11), G3 (Fr.12-20) and G8
(Fr.46-51) with inhibition values of 49.3 ± 2.0, 45.9 ±
1.5 and 41.6 ± 1.2%, respectively (Table 3). For
subfractionation of the groups with the highest
activity fractions (G1, G2 and G8), a solvent gradient
of Hexane-OEtAc-AcOH in the ratio of [90:10:1],
[80:20:1] and [50:50:1] was used. The subfractions
groups with higher antioxidant activity were: G1S1,
G2S2 and G3S2 with 44.2 ± 3.1, 38.1 ± 3.9 and 41.6 ±
4.0% percentage of inhibition values of, respectively.
GC-MS analyzed these sub-fractions to perform the
tentative identification of the compounds
responsible for the antioxidant activity.
Tentative identification of compounds by Gas
Chromatography-Mass Spectrometry (GC-MS)
The tentative identification of compounds, with
percentages of coincidence (> 85%) and area (1.0%)
in the active subfractions is shown in Table 4.
Discussion
Table 4 shows the compounds identified in each
of the active subfractions (G1S1, G2S2 and G3S2)
analyzed by GC-MS. Identified compounds in
subfractions G1S1 and G2S2 correspond to the family
of saturated and unsaturated fatty acids in their
ethyl ester form, such as ethyl oleate and ethyl ester
of 9,12-octadecadienoic acid, among others. 3-
pentadecylphenol compound was identified in G3S2
subfraction, with an 8.01 minutes tR and molecular
peak [M]+ in m/z 304, corresponding to the phenolic
lipids family also called long chain phenols
(alkylphenols). These compounds are characteristic
of the Anacardiaceae family [75-80]. However, only
one compound (3-pentadecylphenol) from this
fraction was identified by comparison with Wiley
and Nist database spectra, the other five
compounds are not found in the GC-MS equipment
data library. However, the relative intensities of
fragments in the mass spectra were collected to
identify base and diagnostic ions of each family of
compounds and with it the most probable structure
for the 5 unidentified compounds in G3S2 fractions,
see Table 5.
Analysis of fragmentation patterns of
unidentified compounds
Compounds 1, 3, 4, 5 and 6 of G3S2 subfraction
with retention times 7.96, 8.55, 8.64, 8.66 and 9.25
minutes, presented molecular ions [M]+ in m/z 302,
328, 446, 332 and 358, respectively; a base ion in m/z
108 appears in all mass spectra which is typical for
cardanols (Table 5). This fragment (m/z 108) is
formed by a McLafferty type rearrangement in the
phenolic ring and the fragment in m/z 107 of lower
intensity corresponding to the hydroxypropyl ion
formed by a direct beta break of the aromatic ring.
The fragments in m/z 91 and 77 indicate the
presence of a monosubstituted aromatic ring to an
aliphatic chain [76, 81], see Figure 3A. Tyman,
identified by Gas-Liquid Chromatography (GLC) and
thin-layer chromatography (TLC), both coupled to
mass spectrometry, homologs corresponding to
lipid phenols of the family of cardanols. Length of
the saturated alkyl chain was found in the order of
C13, C15 and C17, whose molecular masses were 276,
304 and 332 uma's, for 3-tridecyl-phenol, 3-
pentadecyl-phenol, and 3-heptadecyl-phenol,
respectively [77]. Liu and Abreu (2006) identified 41
compounds belonging to the alkyl- and alkenyl-
phenols (cardanols) family, characterized by GC-MS,
NMR-13C and NMR-1H techniques, by alpha, beta-
thiomethylated derivatives. Researchers determined
that, 3-pentadec(en)il-phenol and 3-nonadec(en)il-
phenol compounds have 302 and 358 uma's,
respectively [76].
Given the above and based on the pattern of
fragmentation in the mass spectrum by electron
impact (Table 5), it is possible to assign a structure
to the unidentified compounds of the active fraction
G3S2.
Compound 1, with tR 7.96 minutes, [M] 302,
base peak at m/z 108 for cardanols, mono
substituted aromatic group evidenced by ions in m/z
PhOL Sequeda-Castañeda, et al. 432 (pag 426-446)
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ISSN: 1827-8620
91, 77 and 65, unsaturated aliphatic chain due to
ions CnH2n+1, CnH2n-1, and CnH2n-3 series in m/z 53,
55, 57, 67, 69, 79, 81 and 83, correlate for compound
3-pentadec(en)il-phenol [76, 82, 83]. Compound 3,
with tR 8.55 minutes, [M] 328, base peak at m/z
108 for cardanols, mono substituted aromatic group
evidenced by ions in m/z 91, 77 and 65, unsaturated
C17 aliphatic chain due to ions CnH2n+1, CnH2n-1 and
CnH2n-3 series in m/z 53, 55, 57, 67, 69, 79, 81 and
83, correlate for the compound 3-heptadeca(dien)il-
phenol [76, 77, 82, 83]. Compound 4, with tR 8.64
minutes, [M] 330, base peak at m/z 108 for
cardanols, mono substituted aromatic group
evidenced by ions in m/z 91, 77 and 65, unsaturated
aliphatic chain due to ions series CnH2n+1, CnH2n-1
and CnH2n-3 in m/z 53, 55, 57, 67, 69, 79, 81 and 83,
correlate for the compound 3-heptadec(en)il-phenol
[76, 77, 82, 83]. Compound 5, with tR 8.66 minutes,
[M] 332, base peak at m/z 108 for cardanols, mono
substituted aromatic group evidenced by ions in m/z
91, 77 and 65, unsaturated aliphatic chain due to ions
series CnH2n+1, CnH2n-1, and CnH2n-3 in m/z 53, 55,
57, 67, 69, 79, 81 and 83, correlate for the compound
3-heptadecyl-phenol (69, 71, 73, 74). Compound 6,
with tR 9.25 minutes, [M] 358, base peak at m / z
108 for cardanols, mono substituted aromatic group
evidenced by the ions in m/z 91, 77 and 65,
unsaturated aliphatic chain due to the series of ions
CnH2n+1, CnH2n-1 and CnH2n-3 in m/z 53, 55, 57, 67,
69, 79, 81 and 83, correlate for the compound 3-
nonadec(en)il-phenol [76, 77, 82, 83]. The active
compounds present in the subfraction G3S2
tentatively identified by comparison with the
spectra of the databases (Nist and Wiley), by
fragmentation patterns and by bibliographic review,
are part of the family of phenolic lipids
corresponding to the alkyl phenols (3-pentadecyl-
phenol, 3-heptadecyl-phenol) and alkenyl-phenols
(3-pentadec(en)yl-phenol, 3-heptadeca(dien)il-
phenol, 3-heptadec(en)yl-phenol, and 3-
nonadec(en)il-phenol) [78-80], however for the
latter it was not possible to assign the position of
the double bonds .
Analysis of fragmentation patterns of identified
compounds
In the subfractions G2S1 and G2S2 mass spectra
(Table 5), typical fragmentation patterns are
observed for ethyl esters of fatty acids (FAEE's),
characterized by a base peak in m/z 88 that
corresponds to an McLafferty arrangement, also the
ions in m/z 269, 239, 73, 29 and 15 are from an alpha
rupture [81, 84-87]. In Figure 3B the fragmentation
pattern for hexadecanoic acid as ethyl ester is
shown.
Acknowledgments
Authors thank to Fisicoquímica Integral SAS
(Commercial Director) and COLCIENCIAS
(Administrative Department of Science, Technology,
and Innovation). Academic Vice-Rectory and Vice-
Rectory for Research of the Pontificia Universidad
Javeriana funded this work (Projects 0027 and
4033).
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Table 1. Mass [g] of total extracts and fractions from Anacardium excelsum.
Part Plant
Dry weight
Extract (*)
Fraction (*)
PE
CH2Cl2
EtOH-BuOH
Integument
68.2
31.1 (45.6)
4.12 (20.6)
2.32 (11.6)
10.81 (54.0)
Seed
785.8
183.4 (23.3)
2.02 (4.1)
1.31 (2.6)
13.65 (27.3)
Fruit
50.2
19.4 (38.6)
8.65 (57.7)
1.01 (6.7)
2.01 (13.4)
Flower
336.4
66.7 (19.8)
6.15 (12.3)
5.45 (10.9)
2.31 (4.6)
Seed coat
309.1
65.4 (21.2)
8.52 (17.0)
8.95 (17.9)
0.45 (0.9)
Bark
349.4
95.6 (27.4)
1.61 (3.2)
1.21 (2.4)
12.35 (24.7)
Leave
416.8
97.5 (23.4)
2.71 (5.4)
6.62 (13.2)
0.21 (0.4)
* Percent yield (%Y). Amount to start the fractionation: 15 g (fruit), 20 g (integument), and 50 g (seed, flower,
seed coat, bark, and leave). Bold number corresponds the higher and minor percent yield. PE: Petroleum ether.
CH2Cl2: Dichloromethane. EtOH-BuOH: Ethanol-Butanol.
PhOL Sequeda-Castañeda, et al. 439 (pag 426-446)
http://pharmacologyonline.silae.it
ISSN: 1827-8620
Table 2A. Antioxidant methods comparation: IC50 ( *
Part plant
ABTS+•
DPPH•
DMPD+•
Integument
221.5 ± 8.8
271.6 ± 20.8
5728.6 ± 176.2
Seed
179.7 ± 6.1
257.3 ± 17.7
4082.9 ± 144.7
Fruit
228.0 ± 9.0
1288.9 ± 79.3
3324.7 ± 69.0
Flower
1599.0 ± 51.8
1497.1 ± 78.8
7995.1 ± 182.3
Seed coat
164.3 ± 7.0
239.5 ± 54.8
2929.0 ± 100.4
Bark
852.3 ± 22.8
1137.1 ± 74.3
3223.7 ± 114.2
Leave
927.1 ± 33.1
1114.1 ± 84.8
5241.4 ± 135.6
Trolox
146.0 ± 4.2
158.3 ± 2.4
319.8 ± 4.1
Vitamin E
153.3 ± 9.3
164.5 ± 4.0
347.3 ± 12.0
Vitamin C
135.6 ± 6.4
152.4 ± 5.3
337.5 ± 24.6
BHT
250.2 ± 13.9
270.1 ± 5.9
429.4 ± 25.1
*Average of four determinations in crude extracts. Bold number corresponds the best capacity
antioxidant.
Table 2B. Antioxidant methods comparation: Variables
Variable
ABTS+•
DPPH•
DMPD+•
Stable
Yes
Yes
No
Radical
No
Yes
No
Time Ao [min]
6
20
10
Interference
No
Yes
Si
Solubility
Polar/Apolar
Apolar
Polar
Wavelength [nm]
744
517
505
pH adjustment
No
No
5.25
U$/mg
0.02
0.3
0.003
PhOL Sequeda-Castañeda, et al. 440 (pag 426-446)
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ISSN: 1827-8620
Table 3. % Inhibition for fractions and subfractions groups from the fraction in petroleum ether of Seed coad
Groups (Fractions)
Mass [mg] 1
% Inhibition 2
G1 (Fr.01 to Fr.03)
16.9
18.6 ± 1.4
G2 (Fr. 04 to Fr.11)
120.5
49.3 ± 2.0 *
G3 (Fr. 12 to Fr.20)
78.5
45.9 ± 1.5 *
G4 (Fr.21 to Fr.24)
1307.1
15.3 ± 1.1
G5 (Fr.25 to Fr.35)
115.2
20.4 ± 1.1
G6 (Fr.36 to Fr.41)
51.4
24.4 ± 1.2
G7 (Fr.42 to fr.45)
106.5
16.1 ± 1.5
G8 (Fr.46 to Fr.51)
75.9
41.6 ± 1.2
G9 (Fr.52 to Fr.54)
50.0
21.1 ± 1.5
G10 (Fr.55 to Fr.59)
21.9
7.1 ± 1.2
Group G2 (Subfractions)
Mass [mg] 1
% Inhibition 2
G2S1 (Subfr. 01)
53.1
44.2 ± 3.1 *
G2S2 (Subfr. 02)
9.4
38.1 ± 3.9 *
G2S3 (Subfr. 03 to Subfr.12)
5.6
17.6 ± 2.3
Group G3 (Subfractions)
Mass [mg] 1
% Inhibition 2
G3S1 (Subfr. 01)
21
4.9 ± 0.7
G3S2 (Subfr. 02)
38.7
41.6 ± 4.0 *
G3S3 (Subfr. 03)
14.7
19.0 ± 1.6
G3S4 (Subfr. 04 to Subfr.12)
4.1
3.7 ± 0.4
Group G8 (Subfractions)
Mass [mg] 1
% Inhibition 2
G8S1 (Subfr. 01 to subfr.09)
2.1
4.4 ± 1.0
G8S2 (Subfr. 10 to Subfr.16)
4.3
8.7 ± 1.6
G8S3 (Subfr. 17 to Subfr.24)
12.4
17.5 ± 1.6
G8S4 (Subfr. 25 to Subfr.30)
52.5
11.9 ± 1.6
1 Mass obtained by grouping of fractions or subfractions.
2 Average for four determinations (100 g/mL).
* In bold number corresponds the best capacity antioxidant.
PhOL Sequeda-Castañeda, et al. 441 (pag 426-446)
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ISSN: 1827-8620
Table 4. Identification tentative of compounds in active subfractions 1 from Anacardium excelsum by GC-MS (Databases Wiley 7n.1 and Nist
05a.L.) 2
Subfraction G2S1
Subfraction G2S2
No.
tR [min]
[M+•
]
Compount
No.
tR [min]
[M+•]
Compount
1
6.31
284
Hexadecanoic acid, ethyl ester
1
6.31
284
Hexadecanoic acid, ethyl ester
2
6.68
298
Heptadecanoic acid, ethyl ester
2
6.91
308
9,12-Octadecadienoic acid, ethyl ester
3
6.91
308
Linoleic acid ethyl ester
3
6.95
310
Ethyl Oleate
4
6.96
310
Ethyl Oleate
4
7.05
312
Octadecanoic acid, ethyl ester
5
7.02
312
Hexadecanoic acid, butyl ester
Subfraction G3S2
6
7.05
312
Octadecanoic acid, ethyl ester
1
7.96
302
3 Phenol, 3-pentadec(en)yl-
7
7.61
338
(Z)-9-Octadecenoic acid butyl ester
2
8.01
304
Phenol, 3-pentadecyl-
8
7.70
340
Octadecanoic acid, 2-methylpropyl ester
3
8.55
328
3 Phenol, 3-heptadeca(dien)yl-
9
7.73
340
Eicosanoic acid, ethyl ester
4
8.64
446
3 Phenol, 3-heptadec(en)yl-
10
8.38
368
Docosanoic acid, ethyl ester
5
8.66
332
3 Phenol, 3-heptadecyl-
11
8.98
396
Ethyl tetracosanoate
6
9.25
358
3 Phenol, 3-nonadec(en)yl-
1 Active subfractions of petroleum ether fraction of Seed Coat.
2 Matching percentage >85%. Percentage of area >1.0%.
3 Assignment given by the fragmentation pattern (see Table 5, Figure 3).
PhOL Sequeda-Castañeda, et al. 442 (pag 426-446)
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Table 5. Relative intensities (%) of characteristic fragments in the mass spectra of the active subfractions*
Subfraction G2S1
No.
tR
[min]
[M+•]
Base
peak
Diagnostic ions, m/z (%)
Other ions, mz (%)
1
6.31
284
88(100)
101(63), 157(23), 55(21), 73(16)
57(16), 284(15), 70(15), 239(14), 69(13), 83(10), 143(9), 61(9), 199(8)
2
6.68
298
88(100)
101(65), 55(49), 69(33), 83(26)
97(24), 57(24), 74(21), 157(20), 70(20), 87(18), 84(18), 98(17), 67(17)
3
6.92
308
67(100)
81(94), 95(73), 79(57), 55(57)
82(42), 96(37), 68(37), 109(36), 69(32), 54(29), 80(29), 93(25), 263(22)
4
6.96
310
55(100)
69(75), 83(67), 88(67), 97(63)
96(60), 101(57), 264(56), 84(56), 265(54), 98(50), 67(47), 81(46), 222(37)
5
7.02
312
56(100)
257(77), 57(54), 239(38), 73(36)
55(36), 129(30), 60(25), 69(22), 71(20), 83(19), 61(18), 101(17), 97(15)
6
7.05
312
88(100)
101(65), 157(23), 55(23), 89(20)
57(18), 312(17), 269(16), 73(15), 70(14), 69(14), 83(11), 267(10), 213(9)
7
7.61
338
55(100)
69(80), 83(78), 97(74), 57(68)
265(66), 98(52), 56(49), 96(48), 84(48), 81(48), 67(48), 264(44), 111(40)
8
7.70
340
56(100)
55(62), 57(61), 285(41), 69(39)
73(36), 83(33), 97(30), 129(28), 101(25), 71(25), 60(25), 98(17), 61(17)
9
7.73
340
88(100)
101(66), 55(24), 89(23), 57(23)
157(21), 69(16), 340(14), 70(14), 73(14), 83(13), 97(11), 71(11), 143(8)
10
8.38
368
88(100)
101(72), 89(27), 57(27), 55(24)
69(18), 368(17), 83(14), 70(14), 73(13), 71(13), 97(12), 143(8), 85(8)
11
8.98
396
88(100)
57(32), 89(29), 55(26), 157(25)
69(20), 396(19), 83(16), 71(16), 97(14), 73(14), 70(14), 353(9), 85(9)
Subfraction G2S2
No.
tR
[min]
[M+•]
Base
peak
Diagnostic ions, m/z (%)
Other ions, mz (%)
1
6.31
284
88(100)
101(62), 157(21), 55(21), 241(16)
89(16), 73(16), 70(15), 57(15), 284(13), 69(13), 239(12), 83(10) , 61(9)
2
6.91
308
67(100)
81(94), 95(74), 55(58), 79(54)
82(43), 96(39), 68(38), 109(36), 69(33), 54(29), 80(28), 93(24), 110(22)
3
6.95
310
55(100)
69(78), 88(66), 97(64), 96(60)
101(55), 84(55), 98(49), 67(47), 264(46), 81(46), 265(45), 95(38), 70(35)
4
7.05
312
88(100)
101(62), 55(22), 157(21), 89(19)
57(18), 73(15), 70(14), 69(14), 269(13), 312 (11), 83(11), 97(9), 213 (8)
Subfraction G3S2
No.
tR
[min]
[M+•]
Base
peak
Diagnostic ions, m/z (%)
Other ions, mz (%)
1
7.96
302
108(100)
107(38), 120(18), 55(13), 121(10)
109(8), 302(6), 147(6), 77(6), 133(5), 69(5), 161(3), 149(3), 79(3)
2
8.01
304
108(100)
107(38), 304(10), 121(9), 109(8)
120(5), 149(4), 77(4), 55(4), 305(2), 150(2), 91(2), 79(2), 57(2)
3
8.55
328
108(100)
107(83), 120(47), 147(39), 67(37)
81(28), 95(23), 133(21), 79(19), 109(18), 55(18), 77(17), 328 (16), 121(16)
4
8.64
330
108(100)
107(38), 55(12), 330(10), 121(10)
109(8), 147(6), 133(5), 77(5), 69(5), 331(3), 161(3), 149(3), 67(3)
5
8.66
332
108(100)
107(33), 121(9), 109(8), 332(7)
120(7), 55(6), 149(4), 77(4), 133(3), 333(2), 330(2), 150(2), 147(2), 95(2)
6
9.25
358
108(100)
107(38), 120(16), 55(13), 121(10)
358(9), 109(8), 147(6), 69(6), 133(4), 77(4), 359(3), 149(3), 83(3)
* Active subfractions of petroleum ether fraction of Seed Coat from Anarcadium excelsum.
PhOL Sequeda-Castañeda, et al. 443 (pag 426-446)
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ISSN: 1827-8620
Figure 1. Anacardium excelsum: Tree (A). Leaves and flower-bud (B). Inflorescences (C). Flowers (D).
Infrutescences (E). Fruits and seed-wet (F). Taken from Smithsonian Tropical Research Institute (B-F, 2016)
PhOL Sequeda-Castañeda, et al. 444 (pag 426-446)
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ISSN: 1827-8620
Figure 2. Capacity antioxidant by ABTS, DPPH and DMPD methods from Anacardium excelsum
total extract (A). Activity antioxidant by method ABTS
from Anacardium excelsum fractions (B)
0
2000
4000
6000
8000
IC50 [mg/mL]
Part plant and reference compounds
(A) - Antioxidant capacity by ABTS+• , DPPH• and DMPD+• methods
from Anacardium excelsum total extract
ABTS
DPPH
DMPD
0
200
400
600
800
1000
IC50 [mg/mL]
Part plant
(B) - Antioxidant activity by method ABTS+• from Anacardium
excelsum fractions
Ep
CH2Cl2
BuOH
PhOL Sequeda-Castañeda, et al. 445 (pag 426-446)
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Figure 3 A. Fragmentation pattern for phenolic lipids [76, 81, 84, 85, 87-89].
PhOL Sequeda-Castañeda, et al. 446 (pag 426-446)
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Figure 3 B. Fragmentation pattern for ethyl esters of fatty acids (hexadecanoic acid)
[76, 81, 84, 85, 87-89].
