Arch. Pharm. Res.

DOI 10.1007/s12272-015-0587-6

RESEARCH AR TIC L E

Anti-inflammatory, antioxidant and anti-acetylcholinesterase

activities of Bouvardia ternifolia: potential implications
in Alzheimer’s disease
Giovanni Garc´ıa-Morales • Maira Huerta-Reyes • Manase´ s Gonza´ lez-Cortazar •

Alejandro Zamilpa • Enrique Jime´ nez-Ferrer • Rau´ l Silva-Garc´ıa •

Rube´n Roma´ n-Ramos • Arturo Aguilar-Rojas

Received: 5 August 2014 / Accepted: 27 February 2015

The Pharmaceutical Society of Korea 2015

Abstract Bouvardia ternifolia has been used medicinally
to treat inflammation. In the present study, we investigate
the anti-Alzheimer’s potential effect of the hydroalcoholic
extract of B. ternifolia through evaluation of anti-inflam-
matory and antioxidant activities, quantification of the
percentage inhibition of acetylcholinesterase activity, pro-
tection effect against b-amyloid fibrillar-induce neuro-
toxicity, and the identification of the main constituents. Our
results show that B. ternifolia extract and ethyl acetate
fraction induced anti-inflammatory effects by reducing in-
flammation by [70 %, while antioxidant test revealed
significant IC50 values for flavonoid content fraction
(30.67 ± 2.09 lg/ml) and ethyl acetate fraction (42.66 ±
0.93 lg/ml). The maximum inhibition of acetyl-
cholinesterase was exhibited by scopoletin content fraction
(38.43 ± 3.94 %), while ethyl acetate fraction exerted
neuroprotective effect against b-amyloid peptide
(83.97 ± 5.03 %). Phytochemical analysis, showed the
presence of 3-O-quercetin glucopyranoside (415 mg/g),
rutin (229.9 mg/g), ursolic and oleanolic acid (54 and
20.8 mg/g respectively), 3-O-quercetin rhamnopyranoside
(12.8 mg/g), chlorogenic acid (9.5 mg/g), and scopoletin
(1.38 mg/g). Our findings support the use of B. ternifolia
since the extract induced significant neuroprotection against
b-amyloid peptide, anti-inflammatory, antioxidant and anti-
acetylcholinesterase effects that could be attributed to its
contents of polyphenols, coumarins, and triterpenes, and
encourage further studies for development of this extract as
therapeutic agent in treatment of Alzheimer’s disease.
Keywords Bouvardia ternifolia Alzheimer’s disease
Polyphenols Cumarins Triterpens

G. Garc´ıa-Morales M. Huerta-Reyes R. Roma´n-Ramos

M. Gonza´lez-Cortazar A. Zamilpa
E. Jime´nez-Ferrer A. Aguilar-Rojas
Centro de Investigacio´ n Biome´dica del Sur, Instituto
Mexicano del Seguro Social (IMSS), Xochitepec, Morelos,
Mexico
G. Garc´ıa-Morales R. Roma´n-Ramos
Divisio´ n de Ciencias Biolo´ gicas y de la Salud
(DCBS), Universidad Auto´ noma Metropolitana-
Iztapalapa (UAM-
Iztapalapa), Mexico, DF, Mexico
Departamento de Ciencias de la Salud, DCBS, UAM-Iztapalapa,
Mexico, DF, Mexico
A. Aguilar-Rojas (&)
Unidad de Investigacio´ n Me´dica en Medicina Reproductiva,
Unidad Me´dica de Alta Especialidad (UMAE) en Ginecolog´ıa
Obstetricia No. 4 ‘‘Dr. Luis Castelazo Ayala’’, IMSS, Av. R´ıo
Magdalena No. 289, Col. Tizapa´n San A´ ngel, Delegacio´ n A´
lvaro
Obrego´ n, 01090 Mexico, DF, Mexico
e-mail: arturolation@yahoo.com

M. Huerta-Reyes
Unidad de Investigacio´ n Me´dica en Farmacolog´ıa, Hospital
de Especialidades, Centro Me´dico Nacional Siglo XXI
(CMN- SXXI), IMSS, Mexico, DF, Mexico

R. Silva-Garc´ıa
Unidad de Investigacio´ n Me´dica en Inmunolog´ıa, Hospital
de
Pediatr´ıa, CMN-SXXI, IMSS, Mexico, DF, Mexico 123
123
Anti-Alzheimer’s diseases effects of B. ternifolia
 G. Garc´ıa-Morales et
al.
Introduction O-Tetradecanoyl

The plant species Bouvardia ternifolia (Cav.) Schldtl.

(Rubiaceae) is distributed in Mesoamerica, Mexico, and
the United States. B. ternifolia is popularly known as
‘‘trompetilla’’ and has been used medicinally to treat
in-
toxication produced by stings from snake bites, scorpion
bites, or bee stings, as well as headache and inflammation
(Argueta-Villamar et al. 1994). The pharmacological ac-
tivities of the B. ternifolia extract as an antitumoral (Jolad
et al. 1977), cytotoxic (Tobey et al. 1978) and as antivenom
in scorpion poisoning (Jimenez-Ferrer et al. 2005) have
been described. On the other hand, preliminary screening
studies also revealed that the hydroalcoholic extract of B.
ternifolia exhibited competitive inhibition of the enzyme
acetylcholinesterase, a key molecule for treatment of Alz-
heimer’s disease (AD) (Herrera-Ruiz et al. 2012).
AD is a progressive disease leading to the degeneration
of cholinergic neurons, loss of cholinergic neurotransmis-
sion, and the formation of abnormal aggregates of b-
amyloid (Ab) protein fragments throughout the brain
(Senol et al. 2011). Although neuronal loss is the main
neuropathologic feature underlying the symptoms of AD,
the accumulation of Ab may initiate the inflammatory
process (Lee et al. 2010), as well as the production of
reactive oxygen species (ROS) and oxidative injury
(Wollen 2010). In conjunction, all of these aspects promote
profound neural damage and the typical symptomatology
of AD (Francis et al. 1999; Weiner et al. 2012).
At present, there is no cure for AD. The few agents ap-
proved by the U.S. Federal Drug Administration (FDA) for
the treatment of AD have only modest effectiveness in terms
of modifying clinical symptoms, and none appear to affect
disease progression or prevention (Pasinetti 2012). In
addition to their low therapeutic effectiveness, these drugs
can cause serious side effects (Dunn et al. 2000; Alfirevic
et al. 2007).
Because AD is a genetically complex, slowly
progressive, and irreversible neurodegenerative disease of
the brain, it has been considered that a multitarget approach
may provide more therapeutic value (Wollen 2010; Hampel
et al. 2011). Because the capacity for inhibiting
acetylcholinesterase, as well as its traditional uses that
suggest the possible ability to modulate some neurological
diseases, we extended these observations to further
investigate the anti-inflammatory and antioxidant activities
of B. ternifolia, as well as to quantify the percentage
inhibition of acetylcholinesterase, evaluate the protective
ef- fects against Ab-induced neurotoxicity, and to identify
the main constituents in the extract. For this purpose, the
an- tioxidant activity of the hydroalcoholic extract of B.
ternifolia, as well as several fractions derived from it, was
determined by the inhibition of thiobarbituric acid reactive
substances (TBARS) formation in rat brain homogenate,
while anti-in- flammatory activity was measured by 12-
 Anti-Alzheimer’s diseases effects of B. ternifolia
phorbol-13-acetate (TPA)-induced ear edema assay in
mice. The percentage of inhibition on the
acetylcholinesterase was determined by the Ellman’s assay
while the protective effect against Ab-induced
neurotoxicity was evaluated by neural viability in SH-
SY5Y cell determined by XTT. HPLC and other
chromatographic methods were employed for chemical
characterization of the active extract.

Materials and methods

Plant material and preparation of the extract

The aerial parts of B. ternifolia (Cav.) Schltdl. were col-

lected in the state of Morelos, Mexico (Latitude:
19 010 37.3800 N, Longitude: 99 120 38.7600 WE). No
specific
permissions were required for this location and the field
studies no involve endangered or protected species. Abigail
Aguilar, M.Sc., curator of the Mexican Institute of Social
Security Medicinal Herbarium (IMSSM), identified the
botanical material employed. A voucher specimen was
deposited at the IMSSM with number 15,649. The dried
and powdered plant material (1700 g) was extracted by
maceration with 7.5 L of an ethanol solution (60 %) per kg
of plant material for 24 h (93) at room temperature. After
filtration, the solvent was removed under reduced pressure
distillation (Heidolph Instruments). A semisolid extract
(BtHA) (278.2 g) was finally lyophilized and stored at 4 C
in centrifuge tubes until needed.

Evaluation of anti-inflammatory activity

Animal experiments were carried out in strict accordance

with the Mexican Regulations of Experimental Animal
Care (NOM-062-ZOO-1999). The protocol was approved
by the Institutional Research Committee and the Ethics
Committee of the Mexican Institute of the Social Security
(IMSS) (Registry number R-2010-1701-57). Animals were
anesthetized with veterinary pentobarbital sodium, and all
efforts were made to minimize suffering. Animals were
maintained at 23 ± 2 C on 12/12 h light–dark cycles with
free access to food and water.
The assay of 12-O-Tetradecanoylphorbol-13-acetate
(TPA)-induced ear edema in mice was performed as previ-
ously reported (De Young et al. 1989; Recio et al. 1994).
Prior to the assays, the anti-inflammatory reaction progress
curve of BtHA extract was analyzed at increasing concen-
trations. This data shown that 6.5 mg of this were the
highest amount of extract able to inhibit edematous
response. All the curves of anti-inflammatory activity were
performed at this dose. The following conditions were used
for the present experiment. Male ICR-strain mice weighing
28–30 g (Har- lan, S.A. de C.V.) were anesthetized
with veterinary
G. Garc´ıa-Morales et
al.
Anti-Alzheimer’s diseases effects of B. ternifolia
pentobarbital sodium (60 mg/ml) (Pfizer, Inc.). To promote
inflammation, 2.5 lg TPA in ethanol (Sigma-Aldrich Che-
mical Co.) was applied to both sides of the mouse’s right
ear. The left ear received ethanol and was used as
reference. After
15 min of TPA treatment, 6.5 mg of the test fractions or
80 lg of Dexamethasone in acetone (Sigma) as anti-in-
flammatory (positive control) were applied to both sides of
the right ear. The control group received only ethanol.
Treatments and control were administered at 10 ll of final
volume. 4 h later, the animals were sacrificed by cervical
dislocation and a plug was removed from each ear. The
edematous response was measured as the difference in
weight between the two plugs after treatment. Percentage
inhibition of edema was calculated with the following
equation: % = [(edema A - edema B)/edema A] 9
100. Edema A = edema induced by TPA alone, and
edema B = edema induced by TPA plus sample.
Evaluation of antioxidant activity
Antioxidant activity of the B. ternifolia fractions was as-
sayed by lipid peroxidation of brain homogenates and was
quantified by the contents of TBARS (Ohkawa et al. 1979;
Ng et al. 2000). Quercetin and a-Tocopherol compounds at
1 lg/ml were utilized as references and an inhibitor-free
sample was employed as control. The antioxidant activity
of the B. ternifolia extract and fractions was evaluated at 10
and 100 lg/ml as previously was shown (Gonza´ lez et
al.
2009). As oxidative stress promoter, 10 lM of FeSO4
(Sigma) were added to each sample. Concentration of
TBARS was calculated by interpolation in a standard curve
of tetramethoxypropane (TMP) as previously reported
(Esterbauer and Cheeseman 1990). Final results were ex-
pressed as nmol of TBARS per mg of protein. Increasing
concentrations (10–1000 lg/ml) of BtHA, BtAcOEt,
BtC1N, and Flav3 were employed in displacement curves
of the lipid peroxidation assay. Quercetin and a-Toco-
pherol (0–100 lg/ml) were used as references (Gonza
´lez et al. 2009). The inhibition ratio (%) was calculated
fol- lowing the formula: Inhibition ratio (%) = (C -
E)/ C 9 100 %, where C was the absorbance of control
group and E, the absorbance of test group.
Determination of acetylcholinesterase percent
inhibition
The anti-acetylcholinesterase activity of B. ternifolia
extract was determined spectrophotometrically following
the method described by Ellman (Ellman et al. 1961),
using acetyl- cholinesterase from rat brain homogenate
and acetylcholine iodide as substrate. All assays were
performed in a final vol- ume of 3.0 ml in the presence
of 0.1 M phosphate-buffer
solution, pH 8.0, 333 mM 5,50 -dithiobis(2-nitrobenzoics)
acid (DTNB) and 0.0125 units/ml of enzyme. One unit (U)
of enzymatic activity was defined as the amount of rat
brain homogenate (ll) and increasing 0.001 absorbance at
412 nm in this condition (Chen and Kubo 2002). The
reactions were measured on the spectrophotometer Lambda
25 (Perkin- Elmer) at 412 nm for 3 min every 20 s.
Samples in absences of inhibitor as negative control or in
presences of 0.01 mM Tacrine (9-Amino-1,2,3,4-
tetrahydroacridine hydrochlo- ridehydrate) (Sigma) as
positive control, were employed.
Prior to the study of anticholinesterase activity, the
Michaelis constant (Km) and maximum velocity (Vm)
values were determined. For this proposes, increasing
concentrations of acetylcholine iodide were added and data
were analyzed by non-linear regression analysis. The anti-
acetylcholinesterase activity of the BtHA was evaluated at
increasing concentrations of this extract. Data from con-
centration-inhibition experiments of BtHA, were calculated
by non-linear regression analysis and allowed us to cal-
culate the Half maximal Inhibitory concentrations (IC50).
The rest of the inhibition curves were carried out em-
ploying the IC50 data calculated (0.04 mg/ml of extract and
fractions) as well as the Km value of acetylcholine iodide
(1.86 mM) as substrate.
All the assays were carried out in triplicate and per-
formed at least four times in independent experiments. The
results were reported as percentage of inhibition following
the equation: % Inhibition = 100 (1 - Vm1/Vm2), where
Vm1 was the maximum velocity values of control group
and Vm2, the maximum velocity values of test group.
Evaluation of protective effect against Ab-induced
neurotoxicity
Preparation of the peptide
The Ab1–42 peptide (Sigma) was dissolved in
hexafluoroiso- propanol (Sigma) to 1 mg/ml and incubated
at room tem- perature for 24 h. The hexafluoroisopropanol
was eliminated by vacuum in a Speed Vac and the resulting
unaggregated Ab1–42 film, was stored at -20 C until use.
The Ab1–42 ag- gregations to fibrillar forms were prepared
according with the method of Dahlgren et al. 2002.
Cell culture
Human neuronal cell line (SH-SY5Y) was purchased from
American Type Culture Collection (ATCC, Manassas, VA,
USA). Cell line was routinely grown at 37 C in a hu-
midified incubator with 5 % CO2 in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with antibiotics,
10 % fetal bovine serum and 2 mM glutamine (Invitrogen).
G. Garc´ıa-Morales et
al.
Anti-Alzheimer’s diseases effects of B. ternifolia
Cell viability assay
The protective effect of B. ternifolia in AD was evaluated
by the quantification of the cell viability of the neurob-
lastoma cells, SH-SY5Y. Neurons were treated with in-
creasing concentrations of fibrillar preparations of Ab1–42
(Dahlgren et al. 2002; Patel and Good 2007) in presence of
the extract or fractions of B. ternifolia. Previously at the
assays, the effective dose (ED50) of the extract and frac-
tions were determined. Briefly, SH-SY5Y cells were plated
3
(5 9 10 cell/well) in 96-well plates in 100 ll of media.
24 h later, the cells were rinsed with PBS. Vehicle or
peptide (0.001–100 lM) were prepared in serum-free
medium and added to the cell in presence of extract or
fractions of B. ternifolia (6.25 lg/well). The treated cells
were incubated for 24 h in routinely growth conditions.
Cell viability was determinate by the Cell Proliferation Kit
II (XTT) (Roche Applied Science) following the
manufacturer’s protocol. Viability was expressed as per-
centage of control [(absorbance of treated neurons/ab-
sorbance of untreated neurons) 9 100)] (Tarozzi et al.
2010). Assays were performed in triplicate incubations
from three independent experiments. Data were analyzed
by one-way ANOVA followed by Dunnet´ts test for com-
parisons against control. Values of p B 0.05 (*) and
p B 0.001 (***) were considered statistically significant.
Phytochemical analysis of BtHA
Purification of the main compounds of BtHA was per-
formed by successive chromatography methods (Fig. 1)
whereas the identification of the isolated compounds was
1 13
carried on by spectroscopic ( H NMR, C NMR, HMQC),
spectrometric (EI) and HPLC techniques. The quantifica-
tion of the identified compounds was achieved by HPLC
using calibration curves. Experimental conditions for iso-
lation, identification and quantification of compounds de-
tected in BtHA extract are described: 26 g of BtHA were
fractioned by a bipartition process with ethyl acetate/water,
obtaining BtAcOEt fraction and BtAq fraction. The BtAq
fraction was dissolved in methanol to obtain a supernatant
(BtMeOH) and a precipitate (BtPP) (Fig. 1). 4 g of BtA-
cOEt were subjected to an open chromatography column
previously packed with silica gel (mesh 70-230) (Merck).
The elution solvent consisted of an n-hexane/ethyl acetate/
methanol gradient mixture, in which we obtained six
fractions: Fr-A(100:0:0); Fr-B(75:25:0); Fr-C(5:5:0); Fr-
D(5:5:1); Fr-E(5:5:2.5), and Fr-F(0:0:100) (Fig. 1). From
the subsequent fractionation of Fr-A, Fr-B, and Fr-C by
normal open chromatography column, we detected ursolic
(1) and oleanolic acid triterpens (2) (Fig. 1).
On the other hand, fractions Fr-D, Fr-E, and Fr-F were
grouped and purified by reversed phase (Polygoprep 60-50
C18) (Macherey–Nagel). Gradient mixture of water/ace-
tonitrile was used as mobile phase to obtain four fractions:
BtC1M(100:0); BtC1N(80:20); BtC1Q(70:30), and
BtFlav3(50:50) (Fig. 1). The fraction BtC1M was mainly
constituted of the polyphenolic compound chlorogenic acid
(3) which experimental spectroscopic and spectrometric
data of the compound isolated where according with pre-
vious reports (Sari 2010). Regarding the BtFlav3 fraction,
its principally content was the flavonol 3-O-quercetin
rhamnopyranoside (4), following by 3-O-quercetin glu-
copyranoside (5), and rutin (6). In the case of compounds 4
and 5, the identification was achieved by comparison be-
tween the experimental data obtained by spectroscopic and
spectrometric data of the isolated compounds and the lit-
erature (Kombal and Glasl 1995; Yen et al. 2009). The
identification of compound 6 was determined by HPLC
analysis when experimental peak overlapped with the
commercial standard, although the detection wavelength
was scanned at 190–400 nm. In the remaining fraction
BtC1N, we identified the coumarin scopoletin (7) by
overlapping between the experimental peak and the com-
mercial standard (190–400 nm scanned wavelength).
In order to isolate and quantified these triterpens, we
followed the method described by Nagafuji (Nagafuji et al.
2004). Briefly, 20 g of BtHA were suspended in 60 %
methanol and centrifuged (at 3000 rpm, 50 min, 25 C) to
obtain a supernatant (BtS/NHA) and a precipitate (pp)
(Fig. 1). Then, 2 g of precipitate were recovered, sus-
pended in 60 % methanol solution, and passed through a
Diaion HP-20 column (Merck & Co., Inc) (BtFR1). The
methanol solution was passed through the same column
(BtFR2), where a spontaneous precipitate (BtPPF2) and a
supernatant (BtS/NF2) were obtained. Finally, the ethyl
acetate solution was treated in the same way (BtFR3)
(Fig. 1). An abundant mixture of ursolic and oleanolic acid
was detected in BtPPF2 and BtS/NF2 which experimental
spectroscopic data and HPLC analysis were found to be in
good agreement with the values reported by
Razborsˇek et al. (2008).
The HPLC analysis of BtHA was conducted on a Waters
2695 liquid chromatographer equipped with a Waters 2996
photodiode array detector in order to quantify the com-
pounds detected. Separation was carried out using a RP
C-18 Superspher (Merck) HLPC column (120 9 4 mm;
5 lm) with the following solvent ratios for the mobile
phase where solvent A is water and solvent B is acetoni-
trile: A:B = 100:0 (0 9 1 min); 90:10 (2–4 min); 80:20
(5–9 min); 70:30 (10–15 min); 60:40 (16–18 min); 40:60
(19–20 min); 0:100 (21–30 min), and 100:0 (31–32 min).
Sample injection volume was 10 ll with a 1 ml/min flow
rate. The detection wavelength was scanned at
190–400 nm. Quantification of the main compounds was
achieved using calibration curves (linear regression where
 G. Garc´ıa-Morales et
al.

Fig. 1 Schematic representation of the fractionation of the hydroalcoholic extract of Bouvardia ternifolia. Black boxes are the fractions tested.
Dashes boxes are the chemical compounds identified
Anti-Alzheimer’s diseases effects of B. ternifolia
2
r [ 0.9807), which were separately constructed with
pure standards. The HPLC-solvents and HPLC-references
were purchased from Sigma or Merck.
Statistical analysis
All data were analyzed using the GraphPad prism software
(GraphPad Software) and represented as mean ± Standard
error of the mean (SEM). Data were evaluated by one-way
ANOVA followed by Dunnett’s test for comparisons
against control. Values of p B 0.05 or higher were con-
sidered statistically significant. IC50 were estimated by
means of a linear regression equation.
Results
Evaluation of anti-inflammatory activity
BtHA extract (3.09 ± 0.74 mg) and fractions BtAcOEt
(1.48 ± 0.19 mg) (Fig. 2a), BtC1M (1.95 ± 0.26 mg),
BtC1N (1.25 ± 0.37 mg) (Fig. 2b), BtPPF2 (0.76 ±
0.12 mg), and BtF3 (1.71 ± 0.45 mg) (Fig. 2c) show a
significant anti-inflammatory effect by reducing inflam-
mation by [70 % in the TPA-induced ear edema assay. In
contrast, no anti-inflammatory effects were detected in
fractions BtMeOH, BtPP (Fig. 2a), BtC1Q, BtFlav3
(Fig. 2b), BtS/NHA, BtFR1, and BtS/NF2 (Fig. 2c) when
compared with the Dexamethasone-treated control (0.97 ±
0.35 mg).
Evaluation of antioxidant activity
BtHA showed lipid peroxidation protection at 100 lg/ml
(8.79 ± 1.7 nM/mg protein), as well as the BtAcOEt
fraction (2.68 ± 0.46 nM/mg protein) (Fig. 3a) and
BtC1M (2.65 ± 0.19 nM/mg protein) (Fig. 3b), in com-
parison with the control. However, 10-lg/ml concentra-
tions of these same fractions did not show significant
effects. In Fig. 3b, the antioxidant effect of fractions
BtC1N and BtFlav3 was dose-dependent. BtC1N showed
6.67 ± 0.49 nM/mg proteins of TBARS at 10 lg/ml and
0.64 ± 0.04 nM/mg protein at 100 lg/ml, while in the case
of BtFlav3, the latter showed 6.02 ± 0.44 nM/mg proteins
123
Fig. 2 Evaluation of Bouvardia ternifolia as anti-inflammatory by
(TPA)-induced ear edema in mice. BtHA extract and fractions
BtAcOEt (a), BtC1M, BtC1N (b), BtPPF2, and BtF3 (c) show a
significant anti-inflammatory effect by reducing inflammation by
Fig. 3 Antioxidant activities of Bouvardia ternifolia evaluated by
TBARS assays. BtHA and BtAcOEt fraction showed lipid peroxida-
tion protection at 100 lg/ml (a) as well as BtC1M fraction (b), in
comparison with the control. b The antioxidant effect of fractions
BtC1N and BtFlav3 was dose-dependent. The remaining fractions,
BtMeOH, BtPP (a), BtC1Q (b), and BtS/NHA, BtFR1, BtS/NF2,
of TBARS production at 10 lg/ml and 0.34 ± 0.02 nM/mg
protein at 100 lg/ml. Both fractions exhibited a higher
antioxidant effect than the references: Quercetin (0.98 ±
[70 % when compared with the Dexamethasone-treated control. In
contrast, no anti-inflammatory effects were detected in the remaining
fractions. Results represent mean ± SEM. ***p B 0.001 compared to
TPA. Each value represents the mean of six observations
BtPPF2, and BtF3 (c) did not show protection against lipid
peroxidation. Data are shown as nmoles of TBARS per mg of
protein. Each value represents the mean of four observations ± SEM.
The value p B 0.05 (*) and p B 0.01 (**) were considered as
significant difference with respect to the standard
0.20 nM/mg protein), and a-Tocopherol (1.46 ± 0.56 nM/
mg protein) (Fig. 3b). The remaining fractions, BtMeOH,
BtPP (Fig. 3a), BtC1Q (Fig. 3b), and BtS/NHA, BtFR1,
BtS/NF2, BtPPF2, and BtF3 (Fig. 3c) did not show pro-
tection against lipid peroxidation.
On the other hand, the IC50 values of fractions BtFlav3
(30.67 ± 2.09 lg/ml), BtAcOEt (42.66 ± 0.93 lg/ml),
and BtC1N (59.94 ± 10 lg/ml) exhibited an antioxidant
effect in contrast to the BtHA crude extract (356.21 ±
22.89 lg/ml) (Fig. 4). However, these IC50 values were
greater than those exhibited by references Quercetin
(0.50 ± 0.01 lg/ml) and a-Tocopherol (2.92 ± 0.93 lg/
ml) (Fig. 4).
Determination of percentages of inhibition
on acetylcholinesterase
BtHA extract (12.26 ± 2.53 %) and fraction BtAcOEt
(13.82 ± 2.04 %) showed significant inhibition on acetyl-
cholinesterase enzyme when compared to the control free
on inhibitor (Fig. 5a), while BtMeOH and BtPP did not
show significant effect. In Fig. 5b, the inhibitory effect of
fractions BtC1M (38.43 ± 3.94 %) and BtFlav3 (34.08 ±
3.52 %) on the enzyme was significant, and in the case of
fraction BtC1M the percentages of inhibition even com-
parable with Tacrine (40.61 ± 2.28 %). The remaining
fractions, BtC1N, BtC1Q (Fig. 5b), and BtS/NHA, BtFR1,
BtS/NF2, BtPPF2, and BtF3 (Fig. 5c) did not show inhi-
bition on the acetylcholinesterase enzyme.
Fig. 4 Displacements assays. Data shown percentage of inhibition of
lipid peroxidation (%) by increasing concentrations of the BtHA,
BtAcOEt, BtC1N and BtFlav3. Quercetin and a-Tocopherol were
employed as references. Data represents the mean of 4 assays ± SEM
Protective effect of Bouvardia ternifolia in Alzheimer’s
disease
The protective effect of B. ternifolia against Ab1–42 fib-
rillar-induce neurotoxicity in terms of neuronal viability in
SH-SY5Y cell was evaluated. The toxic effects of in-
creasing concentrations (0.001–100 lM) of fibrillar form
of Ab1–42 were determined by using the XTT assays. When
b-amyloid peptide was tested individually, a maximal
neurotoxicity was observed at 1.0, 10 and 100 lM of
peptide (84.63 ± 2.80, 69.98 ± 5.47 and 38.39 ± 4.06 %
of neuronal viability, respectively) (Fig. 6). On the other
hand, 6.5 lg/well of BtAcOEt fraction was added to b-
amyloid treated SH-SY5Y cells. This fraction was able to
revert significantly the fibrillar peptide toxicity at 10 and
100 lM of peptide (83.97 ± 5.03 and 69.33 ± 4.56 % of
neuronal viability, respectively) (Fig. 6). In the case of
BtHA extract, it was observed an improving in neuronal
protection at 10 and 100 lM of peptide (88.90 ± 5.26 and
49.77 ± 3.98 % of neuronal viability, respectively); how-
ever, it did not shown significantly decreased of Ab-in-
duced cell death with respect to the control (p B 0.05)
(Fig. 6). Fractions BtMeOH, BtPP, BtCIM, BtC1N,
BtC1Q, BTFlav3, BtS/NHA, BtFR1, BtS/NF2, BtPPF2 and
BtFR3 did not shown significant statically differences with
respect to the control.
Phytochemical analysis of BtHA
The chemical compounds detected and isolated from the
active BtHA extract were identified as polyphenols
(chlorogenic acid, 3-O-quercetin rhamnopyranoside, 3-O-
quercetin glucopyranoside, rutin), triterpens (ursolic and
oleanolic acid) and a cumarin (scopoletin). The quantita-
tive analysis showed that 3-O-quercetin glucopyranoside
was the main compound found in BtHA, since the quan-
tification analysis showed as the most abundant (415 mg/g
of extract) (Table 1). The quantification values obtained for
the rest of the compounds are showed in Table 1.
Discussion
At present, cholinesterase inhibitors are one of the most
prescribed drug classes for AD treatment (Senol et al.
2011); however, these drugs cause many side effects (Dunn
et al. 2000; Alfirevic et al. 2007). In consequence, finding
new sources for the safest and most effective drugs against
this disease becomes relevant. Recent evidence suggests
that inflammatory responses and antioxidant properties
may contribute significantly to the progression and
chronicity of AD (Heneka and O’Banion 2007). Therefore,
in the current study was shown the properties of the B.
123
Fig. 5 Anti-acetylcholinesterase activity of Bouvardia ternifolia.
BtHA extract and fraction BtAcOEt showed significant inhibition
on acetylcholinesterase enzyme when compared to the control free on
inhibitor (a), in contrast with BtMeOH and BtPP fractions. b The
inhibitory effect of fractions BtC1M and BtFlav3 on the enzyme was
significant. The remaining fractions, BtC1N, BtC1Q (b), and BtS/
Fig. 6 Neuronal viability of SH-SY5Y cells treated with increasing
concentrations of fibrillar preparations of Ab1–42 in absence or
presence of Bouvardia ternifolia extract or fractions (6.25 lg/well).
Cell viability was expressed as percentage of control ± Standard
error of the mean (SEM) from three independent experiments.
Statistically significant differences compared to controls are shown
[p \ 0.05 (*), p B 0.001 (***)]
ternifolia extract in an in vitro AD model which comprised
the anti-inflammatory, the antioxidant, the percentage of
inhibition on acetylcholinesterase enzyme, and additional-
ly, the neuroprotective effects of this extract, supporting its
multi-target action.
In the present study, among the BtHA extract and the
fractions derived, BtAcOEt is the only fraction that
NHA, BtFR1, BtS/NF2, BtPPF2, and BtF3 (c) did not show inhibition
on the acetylcholinesterase enzyme. Each value represents the mean
of six observations ± SEM. The value p B 0.05 (*) and p B 0.01
(**) were considered as significant difference with respect to the
standard
maintained significantly activity in the entire biological test
considered at the in vitro AD model. In opposite fashion,
although BtHA and BtC1M shown anti-inflammatory, an-
tioxidant, and inhibitory properties on the percentage of
inhibition on acetylcholinesterase enzyme, they were un-
able to prevent neural damage. The fraction BtFlav3 was
able to show antioxidant effect and acetylcholinesterase
inhibition, but this fraction not exhibited effect in the anti-
inflammatory test, as well as the fraction BtC1N, which
exerted anti-inflammatory and antioxidant effects, but not
inhibitory effect on the acetylcholinesterase. Thus,
although BtAcOEt is a fraction derivate from BtHA, it is
also the direct result of removing the most polar fraction
from the whole crude extract. In this way, as many reports
indicate (Lin et al. 2007; Deharo and Ginsburg 2011;
Rosoanaivo et al., 2011), it is well known fact in phyto-
chemistry that crude plant extracts, that include multiple
and diverse compounds (Kurapati et al. 2013), are usually
more powerful medicines than pure isolated compounds.
This effect may be due to the multiple actions of complex
mixtures, but could also arise from synergistic interactions
among their components (Fernandez et al. 2005).
The chemical analysis of BtHA performed in the present
contribution revealed polyphenolic compounds as the ma-
joritary content. Among them, 3-O-quercetin glucopyra-
noside is the majority constituent and could be considered
as the main element involved in the anti-inflammatory and
antioxidant effects observed. This is consistent with find-
ings in which quercetin and its glucopyranoside derivatives
are one of the most abundant polyphenols found in
Table 1 Chemical compounds isolated and identified in the active
hydroalcoholic extract of Bouvardia ternifolia (BtHA)
Compound Quantity present
in BtHA (mg/g of extract)
Ursolic acid 54
Oleanolic acid 20.8
Chlorogenic acid 9.5
3-O-quercetin rhamnopyranoside 12.8
3-O-quercetin glucopyranoside 415
Rutin 229.9
Scopoletin 1.38
vegetables and fruits with potent anti-inflammatory and
antioxidant properties (Materska 2008). On the other hand,
protection against lipid peroxidation and high bioavail-
ability in the digestive tract has been shown for both
quercetin and glucopyranoside derivatives (Wagner et al.
2006).
The remainder of the polyphenols detected may also be
involved in the biological effects observed: rutin, in par-
ticular, has been described as a multifunctional agent that
can inhibit the specific pathogenic processes involved in
AD, such as Ab aggregation, prevent mitochondrial dam-
age, reduce ROS production, reduce the generation of
Nitric oxide (NO), diminish the activity of inducible Nitric
oxide synthase (iNOS), and decrease the production of
proinflammatory cytokines (Pu et al. 2007; Wang et al.
2012). In the same line, chlorogenic acid is able to regulate
inflammation by the increase of proinflammatory factors
such as Interleukin-6 (IL-6) and Tumor necrosis factor-
alpha (TNF-a) (Du et al. 2013) and by inhibition of
Prostaglandin E2 (PGE2) production (Shan et al. 2009).
The antioxidant activities of chlorogenic acid are also ob-
served in its inhibiting the formation of ROS or its scav-
enging of the latter (Morishita and Ohnishi 2001).
Interestingly, in this work, we also found that fraction
BtC1M, which mainly content is chlorogenic acid, was
able to inhibit acetylcholinesterase in values comparable to
control (Tacrine). This information is in agreement with
Kwon and collaborators, who reported the inhibitory ac-
tivity on acetylcholinesterase by chlorogenic acid in a dose
dependent-manner (Kwon et al. 2010). Thus, the potential
neuroprotective effects of the chemical compounds present
in BtHA permit us to suggest this plant as an alternative
therapeutic agent against AD.
The fact that polyphenols are the main compounds de-
tected in BtHA does not preclude their possible active in-
volvement in the anti-inflammatory and antioxidant
activities of the remaining compounds identified in BtHA,
such as coumarin and the triterpenes. This is the case of
fraction BtC1N, where scopoletin was identified as the
main compound. This fraction exerted high anti-
inflammatory and antioxidant activities ([90 %) which
are in agreement with previous reports of the biological
properties of this coumarin (Kim et al. 2004; Chang et al.
2012). Scopoletin is a coumarin that exerts inhibitory ef-
fects on the production of PGE2, TNF-a, IL-1b, and IL-6 in
macrophages (Kim et al. 2004). Likewise, the triterpenes
and ursolic and oleanolic acids (mainly concentrated in
fraction BtPPF2) showed potent anti-inflammatory activity
in the present study that is consistent with the literature
(Liu 1995). Some reports indicate that ursolic acid also
reduces Ab-induced oxidative damage, such as free radical
formation and lipid peroxidation, in in vitro assay systems,
in turn also reducing the production of proinflammatory
cytokines, leading to a neuroprotective effect against Ab
(Yoo and Park 2012; Yoon et al. 2014). Therefore, taking
all of this information together, there is the possibility that
the neuroprotector properties exerted by BtHA may be due
to the anti-inflammatory, the antioxidant and the inhibitory
on acetylcholinesterase effects that could be attributed to
its contents of polyphenols, coumarins, and triterpenes. The
establishment of the precise role and combination of these
active components requires further investigation.
Conclusion
The hydroalcoholic extract of B. ternifolia, which contains
polyphenol compounds such as, 3-O-quercetin glucopyra-
noside, 3-O-quercetin rhamnopyranoside, rutin, and
chlorogenic acid, as well as coumarin, scopoletin, and the
triterpenes ursolic and oleanolic acid, is able to induce
significant anti-inflammatory and antioxidant effects, as
well as inhibition on acetylcholinesterase enzyme, and
conferring neuroprotection against b-amyloid peptide for
AD. These data support its use in traditional medicine and
encourages future studies for its development as a
therapeutic agent in the treatment of AD.
Acknowledgments This study was supported by grant R-2010-
1701-57 (to A. A.-R.) from the FIS-Instituto Mexicano del Seguro
Social, Me´xico. G. Garc´ıa-Morales is grateful for a doctoral
fellow- ship from CONACyT (Exp. No. 212,779). The authors wish
to thank Jonathan Ordun˜ o, Arturo Pe´rez (CIBIS-IMSS), and
Antonio Nieto- Camacho (Instituto de Qu´ımica-UNAM) for
technical assistance.
Conflict of interest The authors declare that there is no conflict of
interests regarding the publication of this article.
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