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IlokiAssanga et al.

BMC Res Notes (2015) 8:396

DOI 10.1186/s13104-015-1388-1


Solvent effects onphytochemical

constituent profiles andantioxidant
activities, using four different extraction
formulations foranalysis ofBucida buceras L.
andPhoradendron californicum
SimonB.IlokiAssanga1*, LidianysM.LewisLujn1, ClaudiaL.LaraEspinoza1, ArmidaA.GilSalido1,
DanielaFernandezAngulo1, JoseL.RubioPino1 andDavidD.Haines2

Background: The present investigation evaluated 4 different solvent compositions for their relative capacity to
extract total phenolic and total flavonoid (TF) components of the leaves, trunks, and stems of Bucida buceras L. (Com
bretaceae), and the stems of Phoradendron californicum (Viscaceae), plus mesquite and oak species endemic to the
Southwestern United States, northern Mexico, and tropical regions of Central and South America, as well as to profile
the composition of these plant materials and to measure their antioxidant capacity.
Methods: The total phenolic content of plant material used in the present investigation was measured using the
FolinCiocalteau assay. Total flavonoids were assayed by AlCl3 and 2,4-dinitrophenylhydrazin colorimetry. Nitroblue
tetrazolium was utilized for scavenging of superoxide anion, and invitro antioxidant activity was evaluated using the
2, 2-diphenyl-1-picrylhydrazyl and Ferric Reducing/Antioxidant Power assays.
Results: Phytochemical screening of each plant extract evaluated revealed the following major results: (1) No
evidence of alkaloids for each of the extraction phases tested was detected in the hexanic, ethanolic, or aqueous
phases of Bucida buceras and Phoradendron californicum (oak and mesquite); (2) Analysis of the hexane phase of B.
buceras and P. californicum (mesquite) extracts revealed the presence of carotenes, triterpenes/steroids, and lactonic
groups; (3) Analysis of the ethanol and aqueous extraction phases for both plants revealed the presence of a diverse
range of compounds, including tripterpenes/steroids, lactonics groups, saponins, phenols/tannins, amines and/or
amino acids, and flavonoids/anthocyanins; and (4) The highest total phenolic and flavonoid content were observed
in P. californicum (oak): 523.88651.457g GAE/mg extract and 409.65123.091g/mg of extract for methanol
and aqueous fractions, respectively. The highest flavonoid content was 237.27321.250g PNE/mg extract in the
acetone extract of Bucida buceras stems; while the flavonol content (260.68523.031g CE/mg extract) was higher
in the ethanol extract of P. californicum (oak). The acetone extract of B. buceras trunk extract showed the highest levels
of DPPH radical-scavenging activity (IC50=4.1360.446g/mL) and reducing power (4928.392281.427M AAE/
mg extract). The highest superoxide radical scavenging activity (IC50) was 55.2499.829g/mL, observed in acetone
extracts of B. buceras leaves.

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2015 Iloki-Assanga etal. This article is distributed under the terms of the Creative Commons Attribution 4.0 International
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IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 2 of 14

Conclusions: The results of the present investigation demonstrated the effects of extraction solvent on phenolic
and flavonoid content yieldand antioxidant activities by Bucida buceras and Phoradendron californicum. The present
investigation further revealed that Bucida buceras exhibited optimal antioxidant capacity when acetone was used as
extraction solvent; and the highest yield of phenols and flavonoids were obtained from the P. californicum oak, using
methanol and aqueous solvents, respectively.
Keywords: DPPH, FRAP, Solvents, Phenols, Flavonoids, Phytochemical screening, Bucida buceras L., Phoradendron

Background are a major focus of investigation. Current isolation and

Free radicals contribute to more than one hundred dis- chemical purification methods used include solvent
orders in humans, including atherosclerosis, arthritis, extraction processes that utilize solvent polarity as a
ischemia and repercussion injury of many tissues, cen- major separation technique. These methods frequently
tral nervous system injury, gastritis, and cancer [1]. Envi- include the use of ethyl acetate, phenol/chloroform,
ronmental pollutants, including radiation, chemicals, aqueous, and several other approaches [1, 9, 1115].
and dietary toxicants, along with physical trauma, cause This study examines the effects of type solvents on
dysregulation of immune activity, and may alter gene extraction of bioactive compounds from Bucida buceras
expression to induce expression of abnormal proteins. L., a timber and shade evergreen tree that belongs to the
Oxidative processes, which trigger the production of free family Combretaceae. It is endemic to the tropical regions
radicals, resulting in tissue damage, are a major contribu- of Northern South America, and is commonly referred
tor to diminished health, and manifested in a wide spec- to as the nonedible black olive tree [16]. The plants in
trum of disease [2]. this family are used for the treatment of various diseases
Plant-derived antioxidants, especially polyphenolic in humans, including abdominal pains, chest coughs,
compounds, have gained considerable importance colds, conjunctivitis, diarrhea, earache, fever, infertility in
due to their potential health benefits. Antioxidants are women, leprosy, pneumonia, heart diseases, sore throat,
important compounds, which protect organisms from and nose bleeds [17, 18]. Diverse authors have found that
damage caused by free radical-induced oxidative stress Bucida buceras has high antifungal activity [1921].
[3]. The antioxidant activity of phenolic compounds is This investigation will also evaluate effects of the
mainly due to their redox properties, which allow them extraction of the solvent on Phoradendron californi-
to act as reducing agents, hydrogen donors, singlet oxy- cum (known in United States as dessert mistletoe and
gen quenchers and metal chelators [35]. Many plants toji in Mexico), which is an autotrophic hemiparasite,
respond to environmental stressors by producing anti- surviving at the expense of its higher vascular plant host
oxidants such as polyphenols. These absorb and neutral- [2224]. The leaves and bark of this plant are used to
ize free radicals, quenching singlet and triplet oxygen, or treat stomachache and digestive disorders. It is also used
inducing expression of peroxides and other toxic metab- with other plants in mixed herbal teas against venereal
olites [6, 7]. The medicinal value of plants is related to diseases. In a recent study, toji was found to have multi-
their phytochemical component content and secondary ple biological activities, including anti-inflammatory and
metabolites, including: phenolic compounds, flavonoids, anti-proliferative effects [25]. Bucida buceras and Pho-
alkaloids, tannins, and other stress gene response prod- radendron californicum (mesquite and oak) are medici-
ucts [8, 9]. nal plants and both have been attributed with beneficial
Several major groups of plant products with antioxi- health effects, but there are only a few studies related to
dant and anti-inflammatory capacity have been identi- this topic. The aim of this work has been to investigate
fied. A particularly valuable class of health-enhancing the effect of this solvent type on the yield and profile of
plant compounds is flavonoids. These are polyphenolic component phenolics and flavonoids and antioxidant
molecules with properties that include free radical scav- activity.
enging, inhibition of hydrolytic and oxidative enzymes,
anti-inflammatory action, reduction of blood-lipids and Methods
glucose, and the enhancement of human immunity [10]. Sample sources andcollection
Anti-inflammatory plant metabolites are currently the The leaves, trunks and stems of Bucida buceras L. were
focus of intensive research in the development of novel collected in Hermosillo, Sonora, Mexico, during the sum-
preventive and therapeutic strategies. Accordingly, meth- mer of 2012. This genus grows in tropical areas and the
ods of extracting these compounds from source material tree blooms and fructifies irregularly throughout the
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 3 of 14

year. The stems of Phoradendron californicum of Pros- sodium carbonate, sodium acetate were obtained from
opis species (mesquite) and Quercus (oak) were collected J. T. Baker (Deventer, Holland). Absolute methanol and
from Carbo and Moctezuma towns, Sonora, Mexico, ethanol, acetic acid, sudan III reactive, glycerin, sodium
at the same year. Members of this genus grow in warm chloride, ferric chloride, ninhydrin, chloroform, sulfuric
temperate and desert regions. Fruit ripening occurs in acid, magnesium metal, amyl alcohol, iron chloride, hex-
late winter to early spring. The plants were taxonomi- ane and ethylenediaminetetraacetic acid (EDTA) from
cally identified by Dr. Jose Cosme Guerrero Ruiz at Fagalab (Edo. de Mexico, Mexico), hydrochloric acid,
the Agronomy Department of Sonora University and picric acid from Fermont (Monterrey, Mexico), anti-
assigned voucher specimen designations as follows: For mony chloride (Carr Price reactive) and Dragendorff rea-
Bucida buceras: RP2014-10, for Phoradendron californi- gent from Fluka (St. Gallen, Switzerland), fehling A and
cum (mesquite): RP2014-11; and for Phoradendron cali- fehling B reactives from Meyer Reagents (Tlahuac, Mex-
fornicum (oak): RP2014-12 deposited at the herbarium of ico) and distilled water with mili-Q system (Millipore,
Sonora University. All the plants were washed, dried and Bedford, MA, USA) was used.
stored in polyethylene bags protected from light at 25C
until analysis. Phytochemical screening
A preliminary screenings of each extract was performed
Preparation ofplant extracts following the standard phytochemical analysis protocol
The different parts of Bucida buceras and Phoraden- described by Chhabra etal. [26], with some modifications.
dron californicum were dried at room temperature for Briefly, 10 g of the collected materials were extracted
7 days, finely ground and used for extraction. The pow- successively with hexane, ethanol and water in a Soxhlet
der obtained (500 g) was mixed with 1.5 L of organic extractor for 1820 h. The extracts were concentrated
solvents, including acetone, methanol and ethanol for using a rotary evaporator (Buchi R-210, Switzerland) at
leaves, trunks and stems of B. buceras; and for the stems 37C and preserved at 4C. All extracts were subjected to
of Phoradendron californicum (mesquite and oak). All qualitative chemical tests for the identification of selected
the extracts were shaken mechanically for 3 days at phytoconstituents such as alkaloids, using Mayers and
room temperature in an orbital shaker. The supernatant Draggendorffs test, carotenes, using the Carr-Price test,
was next filtered through Whatman No. 41 filter paper triterpenes/steroids using the Liebermann-Burchard test,
by vacuum and fresh solvent was added to the samples, quinines, using the Borntrager test, lactonic groups, using
which were extracted for another 3 days. For aqueous the Baljet test, lipids and essential oils, using the Sudan III
extracts of B. buceras and Phoradendron californicum test, reducing compounds, using the Fehling test, sapo-
(mesquite and oak), 100g of dried plants were extracted nins, using the Frothing test, phenols/tanines, using the
with 1L of water at 100C for 30min. The residues were ferric chloride test, flavonoids, using the Shinona test,
subsequently extracted twice with fresh aqueous solvent amines/amino acids, using the Ninhidrin test and cardiot-
(water) and the extracts were combined. onic glycosides, using the Kedde test.
Finally, the two supernatants of the different extracts of
B. buceras and P. californicum were concentrated sepa- Determination oftotal phenols andflavonoids
rately in a rotary evaporator (Buchi R-210, Switzerland) Total phenols
at 37C, then frozen at 77C and finally lyophilized at The total phenolic content of plant extracts was deter-
46 C. At last, the extracts were powdered in a coffee mined using the Folin-Ciocalteu reagent described by
grinder and dissolved in dimethyl sulfoxide to a final con- Singleton and Rossi [27] with some modification, as
centration of 200mg of dry matter/mL. All extracts were reported by Iloki etal. [28].
stored at 4C until use. Briefly, 500 L of plant extract samples in a range of
concentrations (25500g/mL) were mixed with 500L
Solvents andreagents of Folin-Ciocalteu reagent (1:4) and 500 L of a 10 %
Catechin, rutin, pinocembrin, gallic acid, 2,2 difenil- sodium carbonate solution (w/v) were added. The sample
1-picrylhydrazyl (DPPH), Folin-Ciocalteu phenol rea- was left standing at room temperature (~25 C) for 2 h
gent, Dimetilsulfoxide (DMSO), 3,5-dinitrobenzoic and the absorbance was measured at 760nm in a micro-
acid, sodium nitrite, aluminum chloride, ascorbic acid, plate spectrophotometer reader (Thermo Scientific).
2,4,6-tripyridyl-s-triazine, riboflavin, nitroblue tetrazo- Based in the calibration curve established as preparation
lium and phosphate saline buffer were purchased from for the experiments, (0.006250.1 mg/mL), the results
Sigma Chemical Co. (St. Louis, MO, USA). Sodium and were expressed as micrograms of gallic acid equivalent
pottasium hydroxide reactive grade, acetic anhydride, per milligram of extract (g GAE/mg).
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 4 of 14

Flavonoids reader (Thermo Scientific) at 517 nm. The assay vehicle

The total flavonoid content of each batch of plant mate- without extract, served as an absolute control (A blank).
rial was measured by two complementary colorimetric The free radical scavenging of DPPH was calculated
methods, one by the method aluminum chloride (AlCl3) according to the following formula:
for the quantification of flavones and flavonols that react  
% inhibition = Ablank Asample /Ablank 100
better with AlCl3 and other by the method of 2,4-dini-
trophenylhydrazin (DNP) for flavanones and flavanonols where Ablank is the absorbance of the control reaction
[29, 30]. (containing DPPH solution adequately diluted with
ethanol) and Asample is the absorbance of the test com-
Aluminum chloride (AlCl3) method pound. Extract concentrations mediating 50% inhibition
The colorimetric aluminum chloride method was used (IC50) was calculated from a graphic plot of inhibition
to determine flavonoid content according to the method percentage versus extract concentration. The lower the
proposed by Zou et al. [31]. The method is based on IC50, the higher the antioxidant activity of the examined
quantification of the yellow-orange color produced by compound.
the interaction of flavonoid with AlCl3. A 50L aliquot
of plant extract appropriately diluted, was mixed with Ferric reducing antioxidant power assay (FRAP assay)
1250 L of deionized water and 75 L of 5 % sodium The FRAP reagent was prepared in acetate buffer (pH
nitrite, after 6 min, 150 L of 10 % AlCl3 solution was 3.6), 10 mmol 2,4,6-tripyridyl-s-triazine (TPTZ) solu-
added and the mixture was allowed to stand for 5 min; tion in 40 mmol hydrochlorin acid and 20 mmol iron
followed by addition of 500L of 1M sodium hydroxide. (III) chloride solution in proportions of 10:1:1 (v/v),
After 30min of reaction, absorbance was read at 510nm. respectively. The FRAP reagent was prepared daily. 5L
The flavonoid content was assessed by reference to a cali- of samples at 0.5 to 2mg/mL diluted with 20L of dis-
bration curve of catechin (0.21.0mg/mL) and expressed tilled water were added to 150L of FRAP reagent [34].
as g of catechin equivalent per mg of extract (g CE/ The absorbance of the mixture was measured using
mg). microplate spectrophotometer reader Thermo Scientific
at 593nm after 8min. The standard curve was prepared
2,4Dinitrophenylhidrazin (DNP) method with ascorbic acid (AA) and the results were expressed as
Quantification of flavanones and flavanonols was accom- mol AA Equivalent/mg polyphenol-rich extract.
plished using a spectrophotometric method proposed by
Nagy and Grancai [32], which was based on the interac- Superoxide radical scavenging
tion of these compounds with DNP in acid medium, to Superoxide anion radical scavenging activity was deter-
form phenylhydrazones. 40L of sample were dissolved mined by the method proposed by Beauchamp and Fri-
in 80 L of DNP solution (For 5 mL: 50 mg of DNP in dovich [35] with little modifications. The assay is based
100 L of 96 % sulfuric acid and 4850 L of methanol), on the capacity of the sample to inhibit formazan blue
mixed and incubated at 50C for 50min in a water bath, formation by scavenging the superoxide radicals gener-
cooled at room temperature and 280 L of 10 % potas- ated in the riboflavin-light-nitroblue tetrazolium (NBT)
sium hydroxide (KOH) in water were added. Finally, the system. The reaction medium contains 625 L phos-
absorbance was measured at 486 nm. The total content phate buffer (pH 7.6), 25 L riboflavin (0.2 mg/mL),
of flavanones was determined using a calibration curve 50L EDTA (12mM), 25L NBT (1mg/mL) and 25L
based on pinocembrin (0.55.0 mg/mL) and expressed of different concentration of sample. Illuminating the
as g of pinocembrin equivalent per milligram of extract reaction mixture for 10min started the reaction. Super-
(g PNE/mg). oxide radical content of each sample was measured spec-
trophotometrically by the increase in the amount of the
Antioxidant activity absorbance at 590 nm. Blank standardization was per-
Freeradical scavenging activity (DPPH assay) formed in the same way with 25 L of ethanol in the place
The free-radical scavenging activity of each plant mate- of samples being of tested. The concentration of test sam-
rial was measured as described previously with some ple required to inhibit NBT reduction by 50% (IC50) was
modifications [33]. Briefly, 500L of (0.2mM) 1,1-diphe- calculated from dose-inhibition curves.
nyl-2-picrylhydrazyl (DPPH) in ethanol was added to
500 L of selected concentrations (12100g/mL) of the Statistical analysis
extracts and the mixture was incubated for 30min in the All data were expressed as mean standard deviation.
dark, at room temperature. Absorbance of the mixture Statistical analysis was performed by analysis of variance
was measured using a microplate spectrophotometer (ANOVA). A post hoc test (Turkey) was carried out when
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 5 of 14

the differences shown by data were significant (p<0.05). Major outcomes of the present investigation revealed
NCSS (version 2007) statistical program was used for all that the samples tested contained high concentrations
analysis included the Pearsons correlation coefficient. of health-enhancing phytochemical constituents, includ-
ing flavonoids, phenols and/or tannins, saponins, amines
Results anddiscussion and/or amino acids [25]. Plants endemic to Northwest
Phytochemical screening Mexico such as Phoradendron californicum contained
Preliminary phytochemical screening of Bucida buceras compounds with significant antioxidant and antiprolif-
L. and Phoradendron californicum extract show the pres- erative activities. Hayashi et al. [36, 37] identified pres-
ence of various bioactive components which are shown ence of potent cytotoxic agents including Bucidarasins
in Table1. The results provide evidence of the presence of and Buceracidins in Bucida buceras. Moreover, many
carotenes, triterpenes/steroids, lactonic groups, phenols/ flavonoids and terpenoids are potent antioxidants, with
tannins, amines or amino acids, flavonoids/anthocyanins, anti-inflammatory, antibacterial, antiviral and anticancer
saponins, reducing compounds and lipids/essential oils in properties [38, 39]. Other compounds previously identi-
selected extracts of B. buceras and P. californicum (mesquite fied in these plant materials include phenolics, which
and oak), except the last group for B. buceras. The results possess antioxidative, antidiabetic, anticarcinogenic,
also revealed the absence of alkaloids and quinines with antimicrobial, antiallergic, anti-inflammatory and anti-
the exception of P. californicum (mesquite) on the ethanolic mutagenic activities [40, 41]. These plants also contain
phase in which the presence of quinines was detected. steroids, which are known to mediate cardiotonic activi-
Some authors have found the presence of diterpenes ties and possess insecticidal and antimicrobial properties,
and flavanones (flavonoids) in Bucida buceras [36, 37], while tannins, which are also found in these plants, are
this result agree with some of the results obtained in this known to possess general antimicrobial and antioxidant
investigation. Meanwhile, Iloki et al. [33] found that P. activities [42]. The saponins fraction of extracts described
californicum is a plant rich in flavonoids, saponins, phe- here, are typically used in treatment of hypercholes-
nols and/or tannins mainly. terolemia, hyperglycemia, as antioxidants, anticancer,

Table1 Results ofphytochemical screening forthe different plant extracts

Phase Secondary metabolite Plant extracts
B. buceras P. californicum mesquite P. californicum Oak

Hexanic Alkaloids
Carotenes +++ +++ +
Tripterpenes/steroids +++ ++ +
Lactonic groups +++ +++ +
Lipids and/or essential oils + +
Ethanolic Alkaloids
Tripterpenes/steroids ++ ++ +++
Quinines ++
Lactonic groups +++ ++ +
Reducing compounds ++ ++
Saponins +++ ++ +
Phenols/tannins +++ +++ +++
Amines and/or amino acids + +++ +++
Flavonoids/anthocyanins ++ ++ +
Cardiac glycosides +
Aqueous Alkaloids
Saponins ++ ++ +++
Phenols/tannins +++ +++ +++
Reducing compounds + ++ ++
Flavonoids/anthocyanins + +++ +++
, absent; +, low in abundance; ++, moderate in abundance; +++, high in abundance
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 6 of 14

antifungal, antibacterial, anti-inflammatory and in weight >aqueous (321.05 g GAE/mg) ethanol (304.34 g
loss [40, 43]. GAE/mg) acetone (283.49 g GAE/mg). In general,
extractability of a particular component appeared to
Total phenolic andflavonoid content determination depend on extraction medium polarity and the ratio of
Total phenolic content (TPC) solute to solvent. Moreover, recovery of phenolic com-
Sample content of phenolic compounds (g GAE/mg of pounds appeared dependent on the type of solvent used,
extract) in different solvent extracts of the leaves, stems its polarity index (PI) and the solubility of phenolic com-
and trunks of B. buceras L. and stems of P. californicum pounds in the extraction solvents.
of mesquite and oak are summarized in Fig.1. The solubility of polyphenols was observed to depend
Results of the assays for phenolics described in the mainly on the presence and position of hydroxyl groups
present report, indicated a wide variation in the total and the molecular size and the length of constituent
phenolic content in the different extracts, ranging from hydrocarbon chains. Phenolic compounds are often
82.818 to 523.886 g GAE/mg of extract, showing the extracted in higher amounts in more polar solvents. In
higher phenolic content the methanol, ethanol Oak such cases aqueous extract (PI = 9) was not more effi-
toji extracts and acetone trunk and leaves of B. buceras cient in extracting polyphenolic compounds than metha-
extract. nol (PI = 6.6), a phenomenon that may be due at least
Results of these assays, demonstrated significant vari- in part, to the thermal decomposition of some phenolic
ability in total yield of phenolic compounds (p<0.05). In antioxidants at the higher temperatures which were
the present study, methanol proved to be the most effec- used for this extraction. This effect was evident, despite
tive solvent for isolation of phenolic compounds (327.91 the observation that increased heating temperature may
523.89g GAE/mg of extract) from samples of B. buceras increase the yield of phenols in an extraction. Results
and P. californicum, whereas much lower yields were shown here demonstrate that solvents with similar polar-
obtained from samples extracted with acetone (82.82 ity ethanol (PI = 5.2) and acetone (PI = 5.4) have sig-
480.11g GAE/mg). The order of effectiveness in extrac- nificantly different effects on polyphenol content, mainly
tion of phenolics was methanol (388.04 g GAE/mg) for trunk and stem of B. buceras and Toji Oak. These

Fig.1 Total phenols content from B. buceras and P. californicum of oak and mesquite in different solvent type. Values as meanstandard deviation
(n=3)triplicate. Different lowercase letter indicate significant differences (p<0.05) between different solvent type in the same plant part and
different capital letter indicate significant differences (p<0.05) between the same solvent type in different plant part for B. buceras and same solvent
type in same part (stems) for P. californicum (oak and mesquite)
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 7 of 14

significant variations indicated that change in phenolic for extraction of phenolic compounds [14]. Conversely,
solubility may be altered dependent on particular extrac- Jayaprakasha etal. [49] demonstrated that acetone, meth-
tion conditions. anol and water were relatively ineffective for the extrac-
Whereas certain compounds are common to most tion of total phenols from grapes seeds.
parts of a particular plant, none are ubiquitously abun- The recovery of phenolic contents in different samples
dant and most are restricted in distribution to a defined is influenced by the polarity of extracting solvents and the
location of plant anatomy (e.g. seeds, stems, leaves, bark, solubility of each compound in the solvent used for the
etc.). The present study revealed that the distribution of extraction process [50, 51]. Therefore, it is difficult to select
polyphenols differed significantly between different of B. an optimally appropriate solvent for extraction of phenolics
buceras parts for all solvents. For example, leaf content from multiple plant material samples [52]. The profile and
of these compounds was 417.41 g GAE/mg of extract; yield of polyphenol content and antioxidant activity appears
and stems contained 355.53g GAE/mg of extract which higher in more polar solvents [16], hence these seem good
were approximately 15 and 30% more than the phenolic choices for screening of multi-sample substances.
content of the trunks (314.24 g GAE/mg). This differ- In the context of these observations, it should be noted
ence was particularly marked in ethanol and aqueous that the phenolic compounds are often associated with
solvents. Significant differences were found between other biomolecules (polysaccharides, proteins, terpenes,
extraction yields of phenolics, dependent on types of chlorophyll, inorganic compounds) and a solvent suitable
solvent used; and origin of a sample on plant anatomy. It for the extraction of particular classes of compound must
was observed that the highest extraction yield of phenolic be used based on the structural features and related level
compounds was achieved in acetone for B. buceras leaves of aqueous solubility of a particular target molecule [9].
and trunk. Using this general strategy, the phenolic extraction effi-
Significant solvent-dependent differences in content ciencies of the different parts of Bucida and Toji may be
of phenolic compounds were observed when samples optimized, based on the relative polarities of target com-
from P. californicum (oak and mesquite) were evaluated. pounds. Thus different classes of solvent may therefore
Contributing factors may be related to environments in be required depending on the known distribution of tar-
which the plants lived. Oaks from highland forests are get compounds in various plant anatomical locations.
adapted to cold and acid soils. Environmental stressors to
which the plants are subjected favor expression of stress- Total flavonoid content (TFC)
response compounds, including polyphenols. In the Total flavonoid content was evaluated in the present
present study, methanolic extraction protocols were the study using two colorimetric methods. The flavonol/fla-
most efficient for recovery of phenols. vones content in the different extracts of plant materials
Different parts of the same plant may synthesize and was evaluated using catechin equivalent as a reference;
accumulate different compounds or different amounts and the flavanones/flavanonols content was evaluated
of a particular compound due to their differential gene in pinocembrin equivalent as a reference. Some insight
expression, which in turn, affects antioxidant activities into the molecular mechanisms contributing to sol-
and other biological properties of the plant extracts [44, vent extraction efficiency may be gained by considering
45]. Many studies have confirmed that the amount and major features of target compounds. For example, the
composition of phenolic and flavonoid compounds is aluminum chloride method involves formation of stable
diversified at the sub-cellular level and within plant tis- acid complexes between the AlCl3 reagent and the C-4
sues as well [46, 47]. keto group; and either the C-3 or C-5 hydroxyl group of
In general, the results of the study revealed that meth- flavones and flavonols. In addition, aluminum chloride
anol extracted higher levels of phenolics from Toji Oak forms labile acid complexes with the ortho-dihydroxyl
than the other solvent systems tested. These results were groups in the A- or B-ring of flavonoids.
similar to those reported for Toji Oak by Iloki etal. [33]. The 2, 4-dinitrophenyl-hydrazine reagent, (DNP) reacts
In these investigations, methanolic extracts of Toji Oak with ketones and aldehydes to form 2,4 dinitrophenyl-
and Mesquite contained the highest total amounts of hydrazones, although flavones, flavonols and isoflavones
phenolics, flavonoids and condensed tannin compounds, with the C2-C3 double bond fail to react with 2,4-dini-
when compared to extraction yields of the other solvent trophenyl-hydrazine and only flavanones strongly react.
systems: (acetone, methanol and water). Further, results Of the plant materials studied here, the highest yields
shown here are similar to those reported by Sun and of flavanones and flavanonols were found in Bucida
Ho [48] where methanol solvent was most effective in buceras. In trunk and stem samples, flavanones yields
extracting phenolic components from oat bran. Metha- were 23 times greater than those of flavones/flavonols
nol and ethanol have been proven as effective solvents (Table2). The flavanone content in the trunk was greater
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 8 of 14

Table2 Flavonoids content bythe two colorimetric methods onB. buceras andP. californicum indifferent solvent type
Plant Part Solvent Flavonols Flavanones Total flavonoids
(g ofCE/mg ofextract) (g ofPNE/mg ofextract) (g/mg ofextract)
Bucida Leaf AC 72.0758.791A b
69.8974.481C a
b A a A ab
ET 37.2199.716 95.8376.389 133.05612.590A
a A c B ab
ME 71.18311.033 54.4756.355 90.06733.427AB
b A b B b
A 51.7049.873 67.92210.195 120.7857.593A
a B a B a
Bucida Trunk AC 51.1806.242 119.53811.551 169.03416.231B
d B a A c
ET 19.1192.018 93.9998.614 112.7109.607A
c B a A b
ME 33.9765.117 104.26915.674 138.24615.296A
b A a A ab
A 40.3062.892 107.88614.794 148.94716.008A
a B a A a
Bucida Stem AC 49.9806.171 237.27321.254 287.25420.597A
c A b B c
ET 24.6432.957 49.0133.959 73.6565.214B
b B b B b
ME 34.8753.671 64.1225.262 98.9976.601B
b A b B b
A 41.4302.510 62.9795.292 104.4094.293A
d B a B d
Toji mesquite Stem AC 25.8113.523 42.9774.286 68.7886.022B
c B a A c
ET 68.0947.528 42.9874.897 114.86710.811B
a A b B a
ME 153.67311.553 34.5423.395 190.1258.410B
b B a B b
A 90.5167.289 49.456714.190 138.1008.310B
d A b A d
Toji oak Stem AC 87.6326.974 63.4143.651 151.0979.766A
b A b A b
ET 260.68523.031 54.7195.671 330.29515.120A
c A b A c
ME 173.37714.982 59.1704.895 233.38414.325A
a A a A a
A 316.85813.580 83.02311.668 409.65123.091A
The results are expressed as meanstandard deviation (n=3) of triplicate samples. Values in the same column followed by a different lowercase letter are
significantly different (p<0.05) in the same plant and part with different solvent. Different capital letter indicate significant differences (p<0.05) between different
part of the plant in the same solvent for B. buceras and same part and solvent for P. californicum (oak and mesquite)
ET ethanol, A aqueous, AC acetone, ME methanol, CE Catechin, PNE Pinocembrine

than the leaves and stem, except in acetone extracts of amount of total flavonoids observed in the aqueous
the stem. Non-significant differences between solvents extract of oak, whereas the lowest yield was obtained in
were observed in analysis of phenolic content of trunks, the acetone extract of mesquite.
whereas for leaves and stems, ethanol and acetone con- Results of the present study demonstrated higher total
tained the highest flavanone content. These findings sug- flavonoid content in Phoradendron californicum (mes-
gest that acetone extraction appears superior to other quite) than was previously reported by Jimnez et al.
solvents for recovery of flavonols/flavones and flavanones [25]. It has been established that phenolics are the major
(total flavonoids) from Bucida buceras. plant compounds with antioxidant activity, a property
Assays conducted on Phoradendron californicum derived from their redox abilities. Phenolic compounds
revealed that the content of flavonols/flavones was are a class of antioxidant agents, which can quench and
approximately 3-fold higher than that of flavanones. neutralize the free radicals [53]. Flavonoids and flavonols
Analysis of plant materials showed that Toji (Oak) con- are two other major classes of health-enhancing plant
tained the highest flavonol/flavone content compared to compounds and are among the most widely distributed
toji (Mesquite). In contrast to Bucida buceras, aqueous groups of compound found in the plants with known
and methanol extracts exhibited the highest content of benefit to vertebrate viability. Both flavonoids and fla-
phenolic compounds; while the acetone solvent was less vonols possess antioxidant activity as a result of a native
effective in extracting flavonols/flavones components scavenging or chelating attribute [54].
from Toji (oak and mesquite). These outcomes indicate
that polyphenols from Phoradendron californicum were Evaluation ofthe antioxidant activity
extracted most efficiently in more polar solvents. DPPH radical scavenging assay
The amount of total flavonoids extracted from Bucida DPPH radical scavenging activity of the various Bucida
buceras and Phoradendron californicum using dif- buceras and Phoradendron californicum (mesquite and
ferent solvents type ranged from 68.788 6.022 to oak) extracts evaluated in the present investigation are
409.651 23.091 g/mg of extract, with the highest shown in Fig. 2. The extracts of each plant examined in
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 9 of 14

Fig.2 DPPH radical scavenging activity of the different extracts from B. buceras and P. californicum of mesquite and oak. Values as meanstandard
deviation (n=3)triplicate. Different lowercase letter indicate significant differences (p<0.05) between different solvent type in the same plant
part and different capital letter indicate significant differences (p<0.05) between the same solvent type in different plant part for B. buceras and
same solvent type in same part (stems) for P. californicum (oak and mesquite)

the present study, exhibited free radical scavenging prop- attributed to their high phenolic contents. These results
erties and low IC50 values, which are indicative of robust were much better than those obtained in an acetone
antioxidant capacities. The IC50 values for free radical extract of trunks of Delonix regia (44.64 1.8 g/mL)
scavenging activity were observed to be in a range of conducted by Shabir etal. [55]. Meanwhile, Masoko and
4.136 0.44690.265 6.032 g/mL. Maximum scav- Eloff [56] found a strong antioxidant activity in different
enging activity in the samples studied, was observed in by plants of the family Combretaceae in acetone solvent.
Bucida buceras (4.1312.79 g/mL), followed by Phora- Also, this value was higher than the values of standards
dendron californicum-oak (16.0333.73g/mL) and Pho- like ascorbic acid and rutin (10.0 and 29.0g/mL respec-
radendron californicum-mesquite (28.4590.265g/mL). tively) [57] showing high scavenging of DPPH radical.
For the purpose of the present investigation, acetone Significant influence of the polarity solvent on antioxi-
extracts of leaves and trunk of Bucida buceras exhib- dant activity was observed for different parts of Bucida
ited potent scavenging capacity against the free radical buceras plant anatomy. While the DPPH scavenging
DPPH. Moreover, with this same solvent (acetone), the activity was not significantly different when comparisons
highest content of polyphenols was observed. Significant were made between leaves, trunk and stem in high polar-
differences (p < 0.05) in free radical scavenging capac- ity solvents (water and methanol), significant differences
ity between extracts using the different solvent were emerged when moderately polar solvents (acetone and
observed. In decreasing order of scavenging effectiveness, ethanol) were used to extract Bucida buceras stem sam-
this included: acetone > methanol aqueous > ethanol ples. Sultana et al. [15] found that the ethanolic extract
(Fig. 2). These outcomes demonstrated that high-polar- of Terminalia arjuna showed the highest scavenging
ity solvents (aqueous and methanol) were less effective activity. Contrary, Jegadeesware etal. [58] found the low-
for extraction of antioxidants with efficient free radical est IC50 value in ethyl acetate, a solvent much less polar
scavenging properties versus an intermediate-polarity (PI=4.4).
solvent (acetone) but not ethanol. Acetone extracts of Conversely, methanol, ethanol and aqueous extracts
Bucida buceras trunk showed the highest DPPH radical- from Phoradendron californicum-oak exhibited higher
scavenging activity (4.1360.446g/mL), which can be total phenolic and flavonol/flavone content than Bucida
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 10 of 14

buceras extracts, but not antioxidant activity. It is pos- activities vary with high levels of total phenols and total
sible in this context phenolic compounds (flavanones) flavonoids and exhibit high free radical scavenging activ-
produced by Bucida buceras possess an ideal structure ity [59, 60].
for the scavenging of free radicals since structure of Previous investigations have demonstrated that
these molecules includes a number of hydroxyls acting alteration in solvent polarity may be used to differen-
as hydrogen donators which makes them very powerful tially precipitate selected antioxidant compounds [1,
antioxidant agents. The higher efficiency of free radical 61]. In the present study, significant differences in free
inhibition of the Bucida buceras extracts can be related radical scavenging ability for extracts of Phoradendron
to its higher flavanone content, rather than to flavonols/ californicum-mesquite were observed dependent on the
flavones, which are predominant in the extract. In addi- kind of extraction solvent used, while in Phoradendron
tion, a moderate and significant correlation (r = 0.52, californicum-oak the effect of solvent was minimized.
p < 0.001) between total phenolic and free radical scav- Aqueous and methanol extracts showed the strong-
enging was observed for this plant. Also the high abun- est scavenging ability, followed by acetone and ethanol
dance of carotene detected by phytochemical screening extract.
of Bucida buceras may account for this activity.
Among the variants of Phoradendron californicum, Ferric reducing antioxidant power assay (FRAP assay)
toji-Oak exhibited 24 times greater antioxidant activity The reducing power of the plant extracts ranged from
than toji-Mesquite. The observed ratio of oak-mesquite 283.899 3.912 to 4928 281.427 M AAE/mg of
activity was more variable: approximately 2:1 in the aque- extract. Figure 3 shows the ferric reducing antioxidant
ous and methanol extracts, 3:1 in the acetone and 4.5:1 power of the different extracts of B. buceras and P. califor-
in the ethanol extract. Results of the present study reveal nicum (oak and mesquite). The highest reducing power
high and significant correlations between the content of was exhibited by the acetone trunk Bucida buceras wish
total phenolic (r = 0.78, p < 0.001) and flavonols/fla- is also high in phenolic content (480.113 26.044 g
vones (r=0.73, p<0.001) with the antiradical activity GAE/mg of extract) and the acetone P. californicum
of Phoradendron californicum-oak. Similar results were (mesquite) showed lowest activity. Diverse studies have
reported on Zingiber officinale Roscoe (ginger). DPPH shown that acetone or its combinations with water show

Fig.3 Reducing power of different extracts from B. buceras and P. californicum of mesquite and oak. Values as meanstandard deviation
(n=3)triplicate. Different lowercase letter indicate significant differences (p<0.05) between different solvent type in the same plant part and
different capital letter indicate significant differences (p<0.05) between the same solvent type in different plant part for B. buceras and same solvent
type in same part (stems) for P. californicum (oak and mesquite)
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 11 of 14

better reducing antioxidant power and partially accord californicum), the correlation between FRAP with phe-
with the results obtained in this investigation [52, 62, 63]. nolic and flavonoid contents was 0.806 (p < 0.001) and
In this assay, the presence of antioxidants in the extracts 0.590 (p < 0.001) respectively. In B. buceras among dif-
reduced Fe+3/TPTZ complex to the ferrous form. The ferent parts, trunk and leaves exhibited the highest ferric
intensity of the blue color formation is proportional to reducing antioxidant power with respect to stem sam-
the concentration of the ferrous form and the antioxi- plesan effect that might be due to the greater polyphe-
dant capacity of the extract. Antioxidant compounds that nol content in these parts. P. californicum-oak exhibited
exhibit antioxidant capacity in FRAP assay are usually higher reducing power than P. californicum-mesquite
electron donors. FRAP assay had a similar trend to DPPH mainly in aqueous and ethanolic extracts.
radical assay. A significant correlation was found between
IC50 values of DPPH and FRAP (r = 0.69, p < 0.001). Superoxide radical scavenging activity (O2)
The reducing capacity of the extracts may serve as an Superoxide radical is a potent reactive oxygen species
indicator of potential antioxidant activities through the and is highly toxic at fairly low tissue concentrations [66].
action of breaking the free radical chain by donating Moreover, although this anion is a weak oxidant, it gives
hydrogen atom [64]. reacts with other molecules in tissues to form highly
Results shown here indicate that methanol solvent toxic hydroxyl radicals as well as single oxygen, both of
exhibits comparatively high reducing power for differ- which are significant contributors to oxidative stress [67].
ent parts of B. buceras and type of P. californicum due Results of O2- activity assays conducted in this study,
to the high polarity of the solvent system (Fig.3). These yielded, values ranging between 55.249 9.829 and
results agree with the results obtained by Barchan et al. 1062.17116.172g/mL. Figure4 shows the results of
[65] were the methanolic extract of Mentha tree showed assays for superoxide radical scavenging activity of the
high reducing power. The reducing potential of antioxi- different extracts of Bucida buceras and Phoradendron
dant components is closely associated with their total californicum (mesquite and oak). The acetone leaves and
phenolic content. The extracts with higher levels of total trunk Bucida buceras showed the best superoxide radical
phenolics, also exhibit greater ferric reducing power. In scavenging activity and the aqueous P. californicum gave
the aforementioned examples (B. buceras and type of P. the lowest.

Fig.4 Superoxide radical scavenging activity of the different extracts from B. buceras and of mesquite and oak. Values as meanstandard devia
tion (n=3)triplicate. Different lowercase letter indicate significant differences (p<0.05) between different solvent type in the same plant part and
different capital letter indicate significant differences (p<0.05) between the same solvent type in different plant part for B. buceras and same solvent
type in same part (stems) for P. californicum (oak and mesquite)
IlokiAssanga et al. BMC Res Notes (2015) 8:396 Page 12 of 14

The low IC50 values in Bucida buceras followed by validation of scientific content and refining the text so as to improve its clarity
for a wide spectrum of scientific audiences, as well as scientifically literate non-
Phoradendron californicum oak show a high capacity scientists. All the authors read and approved the final manuscript.
for superoxide scavenging of these extracts while Pho-
radendron californicum-mesquite exhibited a fivefold Author details
Rubio Pharma y Asociados S.A. de C.V., Blvd. Garca Morales, Km. 6.5 # 330. El
less activity. Similar results were observed in assays for Llano, 83210Hermosillo, Sonora, Mexico. 2Summative Synergy Pharmaceuti
the ability to scavenge the DPPH radical and significant cals Group (SSPG) LLC, 2040 S. Alma School Road, Suite 1, No. 255, Chandler,
association was observed between these antioxidant AZ 85286, USA.
activities (r = 0.7444, p < 0.001). Methanolic extracts Acknowledgements
exhibited the most robust superoxide scavenging abil- The authors would like to thank the National Council of Science and Technol
ity for phenolic compounds (r = 0.760, p < 0.001). ogy of Mexico (CONACYT) for financing this project and to the botanist Dr.
Jose Cosme Guerrero Ruiz for the botanical identification of the plant speci
Extracts of trunk and leaves from Bucida buceras were mens. The authors are sincerely grateful to Stephanie C. Fox, J.D. of Queen
found to possess a strong and potent superoxide scaveng- BeeEdit in Bloomfield Connecticut, U.S.A. ( for her
ing capacity with respect to analyses for these parameters hard work in organizing, formatting, and editing this article.
in stems. The scavenging activities of leaves and trunk of Compliance with ethical guidelines
Bucida buceras were similar to that of standard reference
compounds such as scopoletin (54.5 g/mL), quercetin Competing interests
The authors declare that they have no competing interests.
(70.3g/mL) and catechin (73.3g/mL).
Received: 3 December 2014 Accepted: 24 August 2015
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