Journal of Ethnopharmacology 143 (2012) 455–462

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antidiabetic effects of Justicia spicigera Schltdl (Acanthaceae)

Rolffy Ortiz-Andrade a,n, Angel Cabañas-Wuan a, Vı́ctor E. Arana-Argáez a,
Angel Josabad Alonso-Castro b,c, Rocio Zapata-Bustos d, Luis A. Salazar-Olivo d, Fabiola Domı́nguez e,
Marco Chávez f,g, Candy Carranza-Álvarez h, Alejandro Garcı́a-Carrancá c,i
a
Facultad de Quı́mica, Universidad Autónoma de Yucatán, Mérida, Yucatán, México
b
Facultad de Quı́mica, Universidad Nacional Autónoma de México, D.F, México
c
Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerologı́a, Secretarı́a de Salud, México, D.F., México
d
División de Biologı́a Molecular, Instituto Potosino de Investigación Cientı́fica y Tecnológica, San Luis Potosı́, SLP, México
e
Centro de Investigación Biomédica de Oriente, IMSS, Metepec, Puebla, México
f
Unidad de Investigación Médica en Farmacologı́a de Productos Naturales, Pediatrı́a, CMN Siglo XXI, IMSS, D.F., México
g
Unidad de Investigación en Enfermedades Neurológicas, Especialidades, CMN Siglo XXI, IMSS, D.F., México
h
Unidad Académica Multidisciplinaria Zona Huasteca, Universidad Autónoma de San Luis Potosı́, Ciudad Valles, San Luis Potosı́, México
i
Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., México

a r t i c l e i n f o abstract

Article history: Ethnopharmacological importance: Justicia spicigera is a plant species used for the Teenak (Huesteca
Received 1 May 2012 Potosina) and Mayan (Yucatan peninsula) indigenous for the empirical treatment of diabetes, infections
Received in revised form and as stimulant.
19 June 2012
Aim of the study: To evaluate the cytotoxicity, antioxidant and antidiabetic properties of J. spicigera.
Accepted 25 June 2012
Available online 20 July 2012
Materials and methods: The effects of ethanolic extracts of J. spicigera (JSE) on the glucose uptake in
insulin-sensitive and insulin-resistant murine 3T3-F442A and human subcutaneous adipocytes was
Keywords: evaluated. The antioxidant activities of the extract of JSE was determined by ABTS and DPPH methods.
Justicia spicigera Additionally, it was evaluated the antidiabetic properties of JSE on T2DM model.
Glucose uptake
Results: JSE stimulated 2-NBDG uptake by insulin-sensitive and insulin-resistant human and murine
Cytotoxic
adipocytes in a concentration-dependent manner with higher potency than rosiglitazone 1 mM.
Antioxidant
Diabetes JSE showed antioxidant effects in vitro and induced glucose lowering effects in normoglycemic and
STZ-induced diabetic rats.
Conclusion: The antidiabetic effects of administration of J. spicigera are related to the stimulation of
glucose uptake in both insulin-sensitive and insulin-resistant murine and human adipocytes and this
evidence justify its empirical use in Traditional Medicine. In addition, J. spicigera exerts glucose
lowering effects in normoglycemic and STZ-induced diabetic rats.
& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The use of medicinal plants is a common practice among

Type 2 diabetes (T2DM), the cause of about 5% of all deaths
globally each year, affects more than 220 million people worldwide
and it is expected to rise to more than double in 2030 (World Health
Organization (WHO), 2011). T2DM has expensive implications for
health systems in Mexico (Arredondo and de Icaza, 2009) and other
countries. The lack of awareness of this disease combined with
insufficient access to health services can lead to complications such
as atherosclerosis, cardiac dysfunction, retinopathy, neuropathy,
nephropathy and blindness (World Health Organization (WHO),
2011). Therefore, there is a worldwide interest for new drugs that
can help to reverse disease progression.
n
Corresponding author. Tel.: þ52 999 9225711.
E-mail address: rolffy@uady.mx (R. Ortiz-Andrade).
Mexican diabetics for the empirical treatment of diabetes
(Argáez-López et al., 2003). J. spicigera (JS) Schltdl (Acanthaceae)
is an evergreen shrub with tubular orange flowers that grows in
hot climates, native from México to South America.
JS is widely distributed in the Yucatan peninsula and is known as
lool chak, ts’i’its and yichkaan; in Huasteca Potosina (San Luis Potosı́,
Mexico) it is commonly known as muicle or muite. This specie of
Justicia is described as erect or scandent perennial herbs or
subshrubs. Leaves present cystoliths and are petiolate with a leaf
margin that is usually entire. Inflorescences are in spikes or panicles
cimas, and the species rarely has solitary, terminal, or axillary flowers.
The bracts and bracteoles are usually conspicuous and imbricate. This
can be easily recognized by their bilabial corolla, with a posterior lip
that is generally two-lobed, an anterior lip that is three lobed, two
stamens, a capsule with four seeds, and a basal sterile portion
(Graham, 1990; Braz et al., 2002; Arellano-Rodrı́guez et al., 2003).

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.06.043
456 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462
Traditionally, in the Yucatan peninsula, the leaves are used as
an infusion and is indicated for the treatment of chronic head-
aches, hypertension and epilepsy, so as for the treatment of
ailments related to the digestive system, such as stomach pain,
diarrhea, dysentery as well as for constipation. In addition to this,
is used as decoction of the leaves or branches (and sometimes the
flower) for cases of skin diseases such as erysipelas (skin infection
caused by the itch mite), syphilis, tumors, or difficult-cure
pimples. It’s also indicated for use in fever, kidney infection,
anemia, so as anti-inflammatory, for dizziness and sleep, and for
some respiratory ailments such as cough, bronchitis and con-
stipation. There are also reports that both the infusion of leafs, as
decoction of aerial parts are used in Mexican traditional medicine
are widely used for the empirical treatment of diabetes (Graham,
1990; Braz et al., 2002; Arellano-Rodrı́guez et al., 2003; Meckes
et al., 2004; Vega-Avila et al., 2009; Andrade-Cetto and Heinrich,
2005; Johnson et al., 2006). The indigenous Teenek from Huasteca
Potosina (San Luis Potosi, Mexico) make a tea by boiling 20 g of JS
leaves with 1 l of water. The tea is taken 3 times a day before
every meal (personal communication). However, there is no
scientific evidence about the antidiabetic properties of JS.
Previous phytochemical studies with ethanol extracts of
J. spicigera leaves shown the isolation of the flavone kaem-
pherol-3,7- bisrhamnoside (kaempferitrin; KM) (Euler and Alam,
1982; Domı́nguez et al., 1990). KM has showed antidiabetic
properties in vitro and in vivo assays (De Sousa et al., 2004;
Tzeng et al., 2009; Jorge et al., 2004; Vishnu-Prasad et al., 2009).
2. Materials and methods
2.1. Reagents, cell lines and chemicals
Murine 3T3-F442A preadipocytes and adult cat serum were a
gift from Dr. W. Kuri-Harcuch (CINVESTAV, México). Human
normal subcutaneous preadipocytes were obtained as previously
described (Herrera-Herrera et al., 2009). Dulbecco’s modified
Eagle medium (DMEM), Leibovitz L15 medium and fetal bovine
serum (FBS) were purchase from GIBCO BRL (Grand Island, NY,
USA) whereas calf serum was from HyClone (Logan, UT, USA).
Human Tumor Necrosis Factor alpha (TNF-a) and 2-[N-(7-nitro-
benz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG)
were obtained from Peprotech (London, UK) to Invitrogen
(Carlsbad, CA, USA), respectively. Rosiglitazone (RGZ), from Cayman
Chem. (Ann Arbor, MI, USA), was 98% purity according to the
manufacturer. Kaempferitrin (KM) obtained from ChromaDex
(Laguna Hills, CA, USA) was 98% purity according to the manufac-
turer. All other chemicals were from Sigma.
ABTS (2,2-azinobis-(3-ethyl-benzothiazoline)-6-sulfonic acid),
DPPH (2,2-diphenyl-1-picrylhydrazyl) and the water soluble ana-
logue of vitamin E (Trolox; 6-hydroxy-2,5,7,8-tetramethylchro-
man-2-carboxylic acid) were purchase from Sigma (St Louis,
MO, USA).
As positive controls in pharmacological evaluations were used
Glibenclamide (Silanes Laboratories, México City, México), Repa-
glinide (Prandins, Sanfer Laboratories, México City, México) and
Saxagliptin (Onglyzas, AstraZeneca/Bristol–Myers Squibb, México
City, Mexico) which were solubilized in saline (0.9% NaCl
solution).
2.2. Plant material and extraction
Samples of J. spicigera collected in Ciudad Valles, San, Luis Potosı́,
México (211580 43.9000 N and 981580 31.4400 W) on August 2010 were
identified by a specialist (J. Garcia-Perez) and preserved at the
herbarium Isidro Palacios of Instituto de Investigación de Zonas
Desérticas, Universidad Autónoma de San Luis Potosı́ (SLPM) for
future reference with the voucher number 46450.
Dried leaves of J. spicigera (40 g) were extracted with ethanol
using a Soxtherm apparatus (Soxtherm automatic, Gerhadt,
Germany) for 3 h. The extract (JSE) was filtered and evaporated
under vacuum and then lyophilized (Free Zone 2.5 Labconco,
USA). The extract was dissolved in methanol and filtered through
a 0.45 mm nylon membrane filter (Gelman Science) before use. For
bioassays, extract was dissolved in DMSO and preserved at room
temperature protected from light until use on cell cultures.
2.3. Quantitation of kaempferitrin in Justicia spicigera
The detection and quantification of KM in JSE were performed
by reversed-phase HPLC method in a waters 2795 (Waters Corp.,
Milford, MA, USA) instrument equipped with quaternary pump,
autosampler and a 996 photodiode array detector, and Waters
Millenium 32 software for peak identification and integration.
Kromasil column C-18 with a 150  4.6 mm i.d 0.45 mm
(Metachem Technologies Inc) with injection volume 5 ml, flow
0.4 ml/min. The mobile phase was acetic acid (HPLC grade Merck
Dormstadt, Germany) 2% in water and the stationary phase was
acetonitrile (Merck, Dormstadt, Germany HPLC grade), gradient %
B 10 to 70 in 20 min. Retention times and UV spectra of KM was
compared to those of high-purity commercial standards. The
calibration plot was obtained in the range of concentrations 124
to 496 mg/ml. The concentration of kaempferitrin in the extracts
was calculated from calibration plot. Maximum of absorbance
was 260 nm with retention time of 10.54 min.
2.4. Cell viability assay
Murine and human preadipocytes were seeded as described
previously in 96-well microplates (Alonso-Castro et al., 2011).
After 24 h of incubation, ethanol extract of J. spicigera (JSE) at
concentrations ranging from 0.1 to 250 mg/ml were added to the
cells. After 48 h, MTT assay was carried out (Alonso-Castro et al.,
2011). The optical density (O.D.) was measured at 590 nm in an
ELISA reader (Biorad Laboratories, CA, USA) using wells without
cells as a blank. The viability of treated cells was estimated from
the relative growth as follows:
control O:D:sample O:D:
relative viability ¼  100
control O:D:
2.5. 2-NBDG uptake assay
Murine 3T3-F442A preadipocytes were differentiated into adi-
pocytes with DMEM containing 10% FBS, 5 mg/ml insulin and 1 mM
D-biotin, whereas human preadipocytes were differentiated into
human adipocytes with L15 containing 5% FBS, insulin 5 mg/ml, RGZ
1 mM, dexamethasone 100 nM, tri-iodothyronine 0.2 nM, and 3-iso-
butyl-1-methylxanthine 25 mM. Murine 3T3-F442A or human adi-
pocytes were seeded on 96-well fluorescence plates and incubated
for 60 min with PBS containing BSA 1 mg/ml and 80 mM of the
fluorescent glucose analog 2-NBDG (Alonso-Castro et al., 2011) in
the presence of different concentrations of ethanol extract of JSE
(1 to 50 mg/ml). Control cultures were treated with insulin 100 nM
or RGZ 10 mM. JSE effects on 2-NBDG incorporation were also
evaluated on 3T3-F442A and human adipocytes pre-incubated with
TNF-a 10 ng/ml for 7 day to induce insulin-resistance (Hotamisligil
et al., 1994). After incubation, free 2-NBDG was washed out from
cultures and fluorescence retained in cell monolayers was measured
with a Tecan-GENios fluorescence microplate reader (Tecan, Salzburg,
Austria). Values of 2-NBDG incorporation in the absence of insulin
 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462
were subtracted from those obtained with insulin 100 nM to
establish 100% of specific 2-NBDG uptake.
2.6. Antioxidant assays
2.6.1. ABTS radical scavenging assay
For ABTS assay, the procedure followed the method of Erel
(2004) with slight modification. Sodium acetate was dissolved in
100 ml deionized water to prepare a buffer solution (30 mM, pH
3.6). ABTS (10 mM) was dissolved in 100 ml of buffer solution and
27.8 ml of H2O2 solution (35% v/v) to obtained the concentration
2 mM, allowing them to react for 12–16 h at room temperature in
the darkness before used to obtain ABTS radical cation (ABTS þ )
solution. The assay were performed in 96-well plates, containing
200 ml of sodium acetate buffer (0.4 M, pH 5.8), 5 ml of sample
solution (0–800 mg/ml) or Trolox standard curve (0–100 mg/ml)
and 20 ml of ABTS þ solution to each well. After initial mixing for
10 min, the absorbance was measured at 734 nm using spectro-
photometer (Multiskan Ascent, Thermo Labsystems). All assays
were performed in triplicate at least three times each. The
ABTS þ scavenging capacity of the extract was compared with
that of Trolox and calculated from the following equation:
Abs controlAbs Sample
ABTS scavenging activity ð%Þ ¼  100
Abs control
where Abs control was the absorbance of the blank and Abs
sample was the absorbance in the presence of the samples and
standard. EC50 value is the concentration of the sample required
to scavenge 50% ABTS radical cation.
2.6.2. DPPH radical scavenging assay
The complementary study for the antioxidant capacity of the
JSE extract was confirmed by the DPPH scavenging assay accord-
ing to the method of Burda and Oleszek (2001) with slight
modification. The DPPH stock solution was prepared by dissolving
7.5 mg DPPH with 250 ml methanol solution (0.1 mM). Different
concentrations (0–800 mg/ml) of the sample solution or standard
Trolox (0–100 mg/ml) were mixed with equal volume of metha-
nol. The assays were performed in 96-well plates, containing
175 ml of DPPH stock solution and 25 ml of sample solution or
Trolox standard curve to each well. After incubation for 30 min,
the absorbance was measured at l ¼517 nm using spectrophot-
ometer (Multiskan Ascent, Thermo Labsystems). All assays were
performed in triplicate at least three times each. The ability to
scavenge DPPH radical was calculated by the following equation:
Abs controlAbs Sample
DPPH radical scavenging activity ð%Þ ¼  100
Abs control
where Abs control was the absorbance of the blank and Abs
sample was the absorbance in the presence of the samples or
standard. EC50 value is the concentration of the sample required
to scavenge 50% DPPH free radical.
2.7. Pharmacological evaluations
2.7.1. Animals
Male Wistar rats ten weeks old and weight 250–300 g were
obtained from the Animal House of the Centro de Investigaciones
Regionales ‘‘Dr. Hideyo Noguchi’’, Universidad Autónoma de Yucatán
(CIR-UADY). Rats were fed with rodent diet and water ad libitum and
were housed under standard laboratory conditions maintained at
room temperature and at photoperiod of 12 h day/night cycle,
2573 1C and humidity of 50–65% during thirty days before experi-
ment. All animal procedures were conducted in accordance with the
Federal Regulations for Animal Experimentation and Care (SAGARPA,
457
NOM-062-ZOO-1999 Mexico) and were approved by the Research
Bioethics Committee of CIR-UADY.
2.7.2. Oral glucose tolerance test (OGTT)
A protocol previously described was used (Ortiz-Andrade et al.,
2007). Male Wistar normoglycaemic rats were used in the
experiment. Rats were deprived of food for 16 h before experi-
mentation, but allowed free access to tap water throughout the
experiment. All experiments were carried out using five animals
per group. JSE was dissolved in physiological saline solution (0.9%
NaCl solution, ISS) and was administered orally by intragastric
route at 10 mg/kg (group 2), 50 mg/kg (group 3) and 100 mg/kg
(group 4), respectively. Glibenclamide (10 mg/kg), Repaglinide
(4 mg/kg) and Saxagliptin (10 mg/kg) were used as hypoglycemic
reference drugs (groups 5–7). Control rats received the vehicle in
the same volume (0.5 ml/100 g) by the same route (group 1).
Ten minutes after administration of test samples, a dose of 2 g/kg
of glucose solution was administered orally to each rat. Blood
samples were collected from the tail tip at 0 (before oral
administration) and 0.5, 1, 2, 3 and 4 h after glucose administra-
tion. Blood glucose concentration was estimated by enzymatic
glucose–oxidase method using a commercial glucometer
(Accu-Chek Active, Roches) which was calibrated and standar-
dized before and between measuring the glucoses. The percen-
tage variation of glycemia for each group was calculated in
relation to initial (0 h) level, according to:
Gx G0
% Variation of glycemia ¼  100
G0
where G0 were initial glycemia values and Gx were the glycemia
values at þ0.5, þ1, þ2, þ3 and þ4 h, respectively. (Verspohl,
2002; Ortiz-Andrade et al., 2005).
2.7.3. Oral glucose tolerance test (OGTT) in T2DM model
Streptozotocin (STZ) was dissolved in citrate buffer (0.1 M, pH
4.5) and nicotinamide (NDA) in physiological saline solution (ISS).
Diabetes was induced in overnight fasted rats (16 h) by a single
intraperitoneal (i.p.) injection of 65 mg/kg STZ 15 min after i.p.
administration of NDA 100 mg/kg (Masiello et al., 1998; Ortiz-
Andrade et al., 2008). After fifteen days, T2DM was confirmed by
the elevated plasmatic glucose levels, and considering for this
studies the animals with fasted plasmatic glucose concentration
above 150 mg/dl.
The protocol described above was used to evaluate the anti-
diabetic effect of JSE. 16 h before the experiments, rats were
fasted with free access to water. JSE was dissolve in isotonic saline
solution (ISS) and was administered orally by intragastric route at
100 mg/kg (group 2). Glibenclamide (10 mg/kg) was used as
hypoglycemic reference drug (group 3). Control rats received
the vehicle in the same volume (0.5 ml/100 g) by the same route
(group 1). Ten minutes after administration of test samples, a
dose of 2 g/kg of glucose solution was administered to each rat.
Blood samples were collected from the tail tip at 0 (before oral
administration) and 0.5, 1, 2, 3 and 4 h after glucose administra-
tion. Blood glucose concentration was estimated by enzymatic
glucose–oxidase method using a commercial glucometer (Accu-
Chek Active, Roches) which was calibrated and standardized
before and between measuring the glucoses. The percentage
variation of glycemia for each group was calculated in relation
to initial (0 h) level using the formulae described above.
2.8. Statistical analysis
For cell viability and 2-NBDG uptake assays so as for antiox-
idants assays, experimental values are expressed as mean 7
standard deviations of at least three experiments in triplicate.
458 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462

1.20

1.00

0.80 Kaempferitrin

AU
0.60

0.40

0.20

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Minutes

Fig. 1. Fig. 1 HPLC elution pattern of kaempferitrin from J. spicigera ethanolic extract.

Data were analyzed by using one-way ANOVA and the level of

120
p r0.05 was used as criterion of statistical significance. All a a
calculations were done using the JMP 5.1 program (SAS a a' a'
a'
Institute Inc). 100
For in vivo pharmacological evaluations, experimental values
Relative viability (%)
b b'
b
are expressed as mean 7standard error of mean (S.E.M.) and all 80 b'
calculations were done using MicrocalTM Origin 8.0 (Microcal
Software Inc., USA). Statistical analysis was performed by one-
60
way analysis of variance (ANOVA) with post hoc Tukey test
(sample versus control). Values of p r0.05 or p r0.01 were
considered to be statically significant. 40

20
3. Results

3.1. Plant extraction and kaempferitrin quantitation 0

BM + + + + +

The vaporization of JSE produced 2.76 g. Thus, the ratio of the
herbal substance to the native herbal drug preparation (DER native)
was 14.5:1. The HPLC analysis showed that KM was major compo-
nent in JSE (Fig. 1). The content of KM in JSE was 69.65 mg/g.
3.2. JSE lacks toxic effects on fibroblasts
We test the toxic effects of J. spicigera on the proliferation of
the normal cells murine and human fibroblasts. JSE did not affect
significantly (pZ0.05) murine fibroblasts viability, compared to
untreated cells, at any of the tested concentrations. On the
contrary, on human cells only JSE 250 mg/ml decrease viability
(26%) significantly (pr0.05), whereas lower JSE concentrations
lacked toxic effects (Fig. 2).
3.3. JSE stimulates 2-NBDG uptake in insulin-sensitive and insulin-
resistant murine and human adipocytes
To determine whether JSE stimulate the glucose uptake by
adipose cells, the 2-NBDG uptake by terminally differentiated
murine 3T3-F442A and human subcutaneous adipocytes was
assayed in the presence of JSE non-toxic concentrations. JSE
stimulated 2-NBDG uptake in both cell types in a concentration-
dependent manner. In insulin-sensitive 3T3-F442A adipocytes,
JSE 1 mg/ml stimulated the 2-NBDG uptake by 35% compared to
the insulin 100 nM whereas JSE 10 mg/ml and 50 mg/ml promoted
the 2-NBDG uptake by 67% and 127%, respectively (Fig. 3 and 4A;
black bars). In insulin-sensitive human adipocytes, JSE 1 mg/ml
increased the 2-NBDG incorporation by 46%, whereas JSE
JSE ( g/ml) - 10 50 100 250
Treatments
Fig. 2. Effect of J. spicigera extract on 3T3 murine and human preadipocytes
viability. Murine 3T3-F442A and human normal subcutaneous preadipocytes were
inoculated in 96-well culture plates (5  103 cells/well) in DMEM with 7% calf
serum or L15 medium supplemented with 5% fetal bovine serum, respectively
(basal medium; BM). After 1 day, cultures were fed with their respective BM
added with different JSE concentrations. After 2 day, cell viability was determined
by MTT test (see Materials and methods). Results represent the mean 7 standard
deviation (SD) of three independent experiments in cuadruplicate.
10 mg/ml and 50 mg/ml did it by 96% and 115%, respectively
(Fig. 3A; white bars).
The effects of JSE were also assayed on the 2-NBDG uptake in
murine and human adipocytes made insulin-resistant by TNF-a
treatment (see Materials and methods) to further evaluate the
antidiabetic potential of this plant. In murine diabetic-like adipo-
cytes, JSE induced the 2-NBDG uptake by 42% (1 mg/ml), 72%
(10 mg/ml) and 137% (50 mg/ml) respect to insulin-sensitive
murine cells (Fig. 3B; gray bars), whereas in human diabetic-like
adipocytes JSE increased by 52% (1 mg/ml), 85% (10 mg/ml) and
99% (50 mg/ml) (Fig. 3B; striped white bars).
3.4. Antioxidant in vitro assays of JSE
3.4.1. Radical scavenging activity against ABTS
ABTS a stable free radical with the characteristic absorption at
734 nm was used to study the radical scavenging effect of JSE. The
results demonstrated that JSE reacted with ABTS at different
concentrations (0–800 mg/ml) and the readings were observed
 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462 459

175 175

d
150 150
2-NBDglucose uptake (%) e d
125 b' 125
b' b
b b' b c'
b' c' c'
100 100 c' c
c
d'
75 d 75

b'
c'
50 50 b
c

25 25

a a' a a' a a'

0 0
INS (100 nM) - + - - - - - INS (100 nM) - + - - - - -
JSE ( g/ml) - - 1 10 25 50 - JSE ( g/ml) - - 1 10 25 50 -
RGZ (10 M) - - - - - - + RGZ (10 M) - - - - - - +

Treatments

Fig. 3. Effect of JSE on the 2-NBDG uptake in normal and diabetic-like murine and human adipocytes. Insulin-sensitive (A) and insulin-resistant (B), 3T3-F442A and human
adipocytes were incubated for 60 min with PBS/BSA containing 80 mM of 2-NBDG and the indicated concentrations of JSE. Control treatments were incubated with insulin
100 nM (INS) or Rosiglitazone 10 mM (RGZ). After incubation, free 2-NBDG was cleared from cultures and fluorescence associated to cell monolayers was measured in a
fluorescence reader. Results represent the mean 7SD of three independent experiments in triplicate.

400
ISS 5 mL/Kg a maximum decolourization of 81.11 72.3% at a maximum
Glibenclamide 5 mg/Kg concentration of 800 mg/ml with the EC50 value 100 mg/ml
EJs 100 mg/kg (Table 1).
300
Variation of glucose (%)

3.5. Hypoglycemic and antidiabetic effect of JS

200 The ethanolic extract of J. spicigera leaves (JSE), exerted a

**
**
100
** **
0 when compared with the vehicle group (Table 2 and 3, po0.01).
0 1
Time after administration (h)
Fig. 4. Effect of 100 mg/kg of JSE on blood glucose levels after a single oral
administration of 2 g/kg of glucose in STZ–NDA diabetic induced male Wistar rats.
Each plot represents the means7 S.E.M. (n¼ 5;** P o 0.01).
by measuring the reduction of radical cation generated by
ABTS þ . JSE showed a maximum decolourization of 71.21 71.4%
at a maximum concentration of 800 mg/ml with the EC50 value
180 mg/ml (Table 1).
3.4.2. Radical scavenging activity using DPPH
The DPPH approach is widely applied to measure the antiox-
idant properties of compounds. DPPH is an organic nitrogen radical
with ultraviolet–visible absorption in the range 515–520 nm,
and the color of its solution fades upon reduction. The samples
was tested against this radical at different concentrations
(0–800 mg/ml) and the readings were observed by decreasing
the absorbance taken as a measure indicates the extent of radical
scavenging propriety. The ethanolic extracts of J. spicigera showed
significant decrease in blood glucose levels in a dose-dependent
manner in normoglycemic rats, when administrated orally in a
single dose for each group (10, 50 or 100 mg/kg) after a single oral
ingestion of glucose. JSE produced reduction in plasma glucose
** concentration from 0.5 h and until 4.0 h after administration of
JSE in all the different doses and there was significant difference
The glibenclamide (10 mg/kg), repaglinide (4 mg/kg) and saxa-
2 3 4
gliptin (10 mg/kg) treated group also showed a significant reduc-
tion of blood glucose levels from 0.5 to 4 h (p o0.01).
In the normoglycemic rats experiment (Table 2), it was
observed that reduction in blood glucose concentration at all
doses, started at the 0.5 h causing a reduction ranging from 25.6
to 29.9% with average 27.7% of blood glucose levels when
compared with vehicle group. Only JSE 100 mg/kg reduced
significantly (p o0.01) blood glucose levels by 27% (1 h), 39.5%
(2 h) and 45.8% (3 h), which was the more significant reduction
showed in normoglycemic rats (Table 2). Lower doses (10 and
50 mg/kg) lacked glucose lowering effects. At time of 4.0 h the
effect of JSE 100 mg/kg was significant, but more moderated with
40.8% of reduction in blood glucose levels.
Due that only JSE 100 mg/kg exerted significant differences in
blood glucose levels in all times in normoglycemic rats, this JSE
dose was tested in STZ-induced diabetic rats. The glibenclamide
treated group (5 mg/kg) showed a significant reduction of
blood glucose levels from 0.5 to 4 h (po0.01). JSE showed
significant (p o0.01) decrease in blood glucose levels by 155%
(0.5 h) and 185% (1.0 h), whereas at 2.0, 3.0 and 4.0 h JSE did not
show significant difference when compared with vehicle group
(p 40.05).
460 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462

Table 1
in vitro antioxidant activity of JSE at different concentrations in ABTS and DPPH assays.

ABTS assay DPPH assay

a
JSE (lg/ml) RSA (%) Trolox (lg/ml) RSA (%) JSE (lg/ml) RSA (%) Trolox (lg/ml) RSA (%)

0 0 0 0 0 0 0 0
50 17.13 7 1.3 20 19.27 71.4 50 25.75 7 0.9 20 21.49 7 1.1
100 33.00 7 0.9 40 23.42 70.7 100 51.47 7 1.4n 40 41.75 7 0.9
200 55.31 7 2.1n 60 58.037 1.1 200 64.18 7 2.1n 60 52.24 7 2.3
400 64.54 7 1.8n 80 72.63 72.3 400 76.22 7 1.8n 80 63.34 7 2.9
800 71.21 7 1.4n 100 79.15 72.1 800 81.11 7 2.3n 100 77.64 7 1.2
EC50(mg/ml) 180 86 100 51

Values are presented as means7 SEM (n ¼5).

a
RSA: Radical scavenging activity (%).
n
p o 0.05.

Table 2
Hypoglycemic effect of intragastric administration of J. spicigera ethanol extract (JSE at 10, 50 and 100 mg/kg), glybenclamide (10 mg/kg), repaglinide (4 mg/kg), saxagliptin
(10 mg/kg) and vehicle (ISS) in normoglycemic rats.

Time (h) ISS (A) JSE Glybenclamide Repaglinide Saxagliptin

10 mg/kg (B) 50 mg/kg (C) 100 mg/kg (D) 10 mg/kg (E) 4 mg/kg (F) 10 mg/kg (G)

a a a c c
0.5 69.99 78.18 42.85 7 5.55 40.14 7 6.82 44.37 7 5.03  15.12 7 1.74 14.74 7 4.95 25.4 7 7.5b
1.0 102.46 7 9.28 60.95 75.06a 64.68 7 1.23a 62.65 7 7.39a  29.55 7 6.32c 13.61 7 8.31c 29.2 7 7.7c
2.0 59.53 76.41 17.06 76.41a 12.90 7 4.57a 11.32 7 5.50a  44.49 7 3.73c  12.81 75.35c 1.6 7 7.7b
3.0 16.94 71.03 10.46 75.20 8.30 7 7.83 0.30 74.25a  42.49 7 2.98c  29.14 73.59c  13.4 76.6b
4.0 32.24 75.83  5.417 6.05a  12.22 7 6.59a  9.17 76.76a  54.72 7 5.53c  12.66 75.36b  12.0 78.5b

Statistical analysis was by a one-way ANOVA with Tukey’s post-hoc test. Values are expressed as mean of % variation of glycemia 7 S.E.M., and n¼5 for all groups;
the negative value (  ) indicates a decrease in glycemia compared with time 0.
a
p o 0.01 when is less compared with A, but not with E–G.
b
po 0.01 when is less compared with A, but not with B–D.
c
po 0.01 when is less compared with A–D.

Table 3 and Cinnamomum osmophloeum, respectively (Ferreres et al.,

Antidiabetic effect of intragastric administration of J. spicigera ethanol extract (JSE 2012; Rho et al., 2010). The results indicate that we performed
at 100 mg/kg), glybenclamide (10 mg/kg) and vehicle (ISS) in STZ–NDA diabetic
an efficient protocol to extract kaempferitrin from J. spicigera. The
induced rats.
findings also suggest that J. spicigera is an important source of
Time (h) ISS (A) JSE Glybenclamide kaempferitrin.
It has been reported that some polyphenols might interact
100 mg/kg (B) 5 mg/kg (C) with MTT (Wisman et al., 2008) and thus affect cell viability
0.5 292.97 7 37.57 137.81 715.52 a
52.88 7 9.81b
results. The major component of JSE, kaempferitrin (1 to 50 mM)
1.0 333.36 735.47 148.58 723.18a 52.19 7 6.90b lacks toxic effects on murine macrophages, peripheral blood
2.0 162.24 734.71 105.56 7 15.12 43.76 7 10.75b mononuclear cells and human keratinocytes (unpublished
3.0 77.13 7 34.78 68.10 7 18.08 23.20 713.49 results). Therefore, kaempferitrin might not interfere with cell
4.0 45.35 7 10.52  5.09 7 16.70 5.581 7 23.90
viability assay.
Statistical analysis was by a one-way ANOVA with Tukey’s post-hoc test. Values To gain insight into the mechanisms mediating the antidia-
are expressed as mean of % variation of glycemia 7S.E.M., and n ¼5 for all groups; betic properties of J. spicigera, the effects of nontoxic concentra-
the negative value (  ) indicates a decrease in glycemia compared with time 0. tions of JSE on glucose uptake in murine and human adipocytes
a
p o 0.01 when is less compared with A. were evaluated. JSE stimulated 2-NBDG uptake in insulin-
b
po 0.01when is less compared with A and B. sensitive murine 3T3-F442A and human subcutaneous adipocytes,
in a concentration-dependent manner. The induction of 2-NBDG
uptake by JSE 25 mg/ml showed a similar (p Z0.05) potency than
4. Discussion insulin 100 nM or RGZ 10 mM but a higher (pr0.05) potency,
tested at 50 mg/ml, than insulin or RGZ. JSE also stimulated the
In Mexican traditional medicine, J. spicigera leaves are widely 2-NBDG uptake in murine and human adipocytes made resistant
used for the empirical treatment of diabetes (Andrade-Cetto and to insulin by pretreatment with TNF-a. In such diabetic-like cells,
Heinrich, 2005; Johnson et al., 2006). However, there is JSE showed a higher (pr0.05) potency to RGZ when assayed at
no scientific evidence regarding the antidiabetic properties of concentrations higher than10 mg/ml. These results suggest that
J. spicigera. stimulation of glucose uptake in insulin-targeted tissues is one of
Previously it was shown the presence of kaempferitrin in the mechanisms by which J. spicigera exerts its antidiabetic
ethanol extracts of J. spicigera (Euler and Alam, 1982; effects. Experiments are being carried out in our laboratory to
Domı́nguez et al., 1990). In this study, we showed that KM was elucidate the mechanisms by which J. spicigera stimulates glucose
the most abundant compound in JSE (Fig. 1) and that its content uptake in insulin resistant cells.
(69.65 mg/g) was 2.3-fold and 124-fold higher than those Oxidative stress plays an important role in the pathogenesis
reported for the medicinal plants Bauhinia fortificata (antidiabetic) of various diseases such as TD2M (Tiwari, et al., 2009).
 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462
Oxidative stress is initiated by reactive oxygen species (ROS), and
these radicals are formed by a one electron reduction process of
molecular oxygen (O2). ROS can easily initiate the lipid peroxida-
tion to the cell membrane lipids, causing damage of the cell
membrane of phospholipids, lipoprotein by propagating a chain
reaction cycle (Ferretti and Bacchetti, 2011). Antioxidants
defense systems have coevolved with aerobic metabolism to
counteract oxidative damage from ROS (Puertollano et al.,
2011). Recent investigations have shown that the antioxidant
properties of plants could be correlated with oxidative stress
defense present on different human diseases (Chen et al., 2011;
Conforti et al., 2008). In this study, the antioxidant activity of
J. spicigera was then chemically determined by in vitro assay
DPPH and ABTS scavenging activity. Scavenging DPPH is related
to the inhibition of lipid peroxidation and DPPH radical involves
a hydrogen atom transfer process (Rekka and Kourounakis, 1991;
Kaviarasan et al., 2007). In this assay, the antioxidant activity on
ABTS and DPPH radical of J. spicigera may be attributed to a direct
role in trapping free radicals by donating hydrogen atoms. Thus,
the therapeutic benefits of J. spicigera may also be partly due to
its ability to decrease the generation of reactive oxygen species, a
process that results in oxidative stress and aggravates the
diabetic state.
The JSE doses used in this study were selected based on their lack
of toxic effects, as evaluated in acute toxicity test and on preliminary
studies carried out in our laboratory (Alonso-Castro et al., 2012). It is
important to point out that in all in vivo experiments the hypogly-
cemic activities of JSE were similar to those produced by the positive
control glibenclamide, which causes hypoglycemia by enhancing
insulin secretion in pancreatic b-cells. However, the antidiabetic
actions of JSE could be linked to more than one mechanism because
of JSE showed that stimulates 2-NBDG uptake in insulin-sensitive
murine 3T3-F442A and human subcutaneous adipocytes in a con-
centration-dependent manner. These possible mechanisms might
involve: (a) the modulating insulin secretion and/or insulin action,
(b) the involvement of an antioxidant system, (c) enhancement
of b-cell glucose metabolism, (d) activation of enzyme system
generating cyclic AMP or (e) phospholipids derived messenger.
Further experiments are necessary in order to elucidate the mole-
cular mechanisms by which JSE exerts its hypoglycemic effects
in STZ-induced diabetic rats. Moreover, in future communications
we will also evaluate the antidiabetic effects of J. spicigera in other
in vitro models such as muscle and liver cell lines. In addition,
kaempferitrin has been reported to exert antidiabetic effects in vitro
and in vivo (De Sousa et al., 2004). Thus, we hypothesize that the
antidiabetic effects of JSE might be due to the high quantities of
kaempferitrin present in JSE.
The results provide a scientific validation of the empirical use
of the J. spicigera on diabetes. Also, our results show a new
biological effect for J. spicigera and suggest that this plant might
be highly promising for the development of new T2DM therapies.
In summary, this study demonstrates that an ethanolic extract of
J. spicigera leaves (JSE) stimulates the glucose uptake in insulin-
sensitive and insulin-resistant adipose cells, exerts antioxidant
effects, and decreases glucose levels in normoglycemic rats and
STZ-induced diabetic rats.
Acknowledgements
AJAC (174493) and RZB (233280) were endowed with graduate
fellowships from CONACYT. Thank to Merck Sharp & Dohme,
México, for providing Indinavir. This work was partially supported
by CONACYT, México (Grant SALUD-2009-01-114435 to LSO).
461
References
Alonso-Castro, A.J., Zapata-Bustos, R., Domı́nguez, F., Garcia-Carranca, A., Salazar-
Olivo, L.A., 2011. Magnolia dealbata Zucc and its active principles honokiol and
magnolol stimulate glucose uptake in murine and human adipocytes using the
insulin-signaling pathway. Phytomedicine 18, 926–933.
Alonso-Castro, A.J., Ortiz-Sanchez, E., Dominguez, F., Arana-Argáez, V., MdC,
Juárez-Vázquez, Chávez, M., Carranza-Álvarez, C., Gaspar-Ramı́rez, O., Espi-
nosa-Reyes, G., López-Toledo, G., Ortiz-Andrade, R., Garcı́a-Carrancá, A., 2012.
Antitumor and immunomodulatory effects of Justicia spicigera Schltdl
(Acanthaceae). Journal of Ethnopharmacology 141, 888–894.
Andrade-Cetto, A., Heinrich, M., 2005. Mexican plants with hypoglycaemic effect
used in the treatment of diabetes. Journal of Ethnopharmacology 93, 248–325.
Arellano-Rodrı́guez, J.A., Flores Guido, J.S., Tun, J., Garrido y, M.M., Cruz, Bojórquez,
2003. Nomenclatura, forma de vida, uso, manejo y distribución de las especies
vegetales de la penı́nsula de Yucatán. Etnoflora Yucatanense 20, 5–6.
Argáez-López, N., Wacher, N.H., Kumate-Rodrı́guez, J., Cruz, M., Talavera, J., Rivera-
Arce, E., Lozoya, X., 2003. DIMSS Study Group, 2003. The use of complemen-
tary and alternative medicine therapies in type 2 diabetic patients in Mexico.
Diabetes Care 26 (8), 2470–2471.
Arredondo, A., de Icaza, E., 2009. Financial requirements for the treatment of
diabetes in Latin America: implications for the health system and for patients
in Mexico. Diabetologia 52, 1693–1695.
Braz, D.M., Carvalho-Okano, R.M., Kameyama, C., 2002. Acanthaceae da reserva
florestal mata do paraı́so, Vic- osa, Minas Gerais. Revista Brasileira Botanica 25,
495–504.
Burda, S., Oleszek, W., 2001. Antioxidant and antiradical activities of flavonoids.
Journal of Agriculture Food Chemistry 49 (6), 2774–2779.
Chen, Y., Huang, B., He, J., Han, L., Zhan, Y., Wang, Y., 2011. in vitro and in vivo
antioxidant effects of the ethanolic extract of Swertia chirayita. Journal of
Ethnopharmacology 136 (2), 309–315.
Conforti, F., Sosa, S., Marrelli, M., Menichini, F., Statti, G.A., Uzunov, D., Tubaro, A.,
Menichini, F., Loggia, R., 2008. in vivo anti-inflammatory and in vitro anti-
oxidant activities of Mediterranean dietary plants. Journal of Ethnopharma-
cology 116 (1), 144–151 28.
De Sousa, E., Zanatta, L., Seifriz, I., Creczynski-Pasa, T.B., Pizzolatti, M.G.,
Szpoganicz, B., Silva, F.R., 2004. Hypoglycemic effect and antioxidant potential
of kaempferol-3,7-O-(alpha)-dirhamnoside from Bauhinia forficata leaves.
Journal of Natural Products 67, 829–832.
Domı́nguez, X., Achenbach, H., González, C.C., Ferré-D’Amare, A.R., 1990. Estudio
quı́mico del muitle (Justicia spicigera). Revista Latinoamericana de Quı́mica 21,
142–143.
Erel, O., 2004. A novel automated direct measurement method for total antiox-
idant capacity using a new generation, more stable ABTS radical cation.
Clinical Biochemistry 37 (4), 277–285.
Euler, K.L., Alam, M., 1982. Isolation of kaempferitrin from Justicia spicigera. Journal
of Natural Products 45, 220–221.
Ferreres, F., Gil-Izquierdo, A., Vinholes, J., Silva, S.T., Valenta~ o, P., Andrade, P.B.,
2012. Bauhinia forficata Link authenticity using flavonoids profile: relation
with their biological properties. Food Chemistry 134 (2), 894–904.
Ferretti, G., Bacchetti, T., 2011. Peroxidation of lipoproteins in multiple sclerosis.
Journal of Neurology Science 311 (1-2), 92–97.
Graham, V.A., 1990. Delimitation and infra-generic classification of Justicia
(Acanthaceae). Kew Bulletin 43, 551–624.
Herrera-Herrera, M.L., Zapata-Bustos, R., Salazar-Olivo, L.A., 2009. Simplified
culture techniques for growth and differentiation of murine and human pre-
adipocytes for translational applications. Cytotherapy 11, 52–60.
Hotamisligil, G.S., Murray, D.L., Choy, L.N., Spiegelman, B.M., 1994. Tumor necrosis
factor alpha inhibits signaling from the insulin receptor. Proceedings of the
National Academy of Sciences USA 91, 4854–4858.
Johnson, L., Strich, H., Taylor, A., Timmermann, B., Malone, D., Teufel-Shone, N.,
Drummond, R., Woosley, R., Pereira, E., Martinez, A., 2006. Use of herbal
remedies by diabetic Hispanic women in the southwestern United States.
Phytotherapy Research 20, 250–255.
Jorge, A.P., Horst, H., de Sousa, E., Pizzolatti, M.G., Silva, F.R., 2004. Insulinomimetic
effects of kaempferitrin on glycaemia and on 14C-glucose uptake in rat soleus
muscle. Chemico-Biological Interactions. 149 (2-3), 89–96.
Kaviarasan, S., Naik, G.H., Gangabhagirathi, R., Anuradha, C.V., Priyadarsini, K.I.,
2007. in vitro studies on antiradical and antioxidant activities of fenugreek
(Trigonella foenum graecum) seeds. Food Chemistry 103 (1), 31–37.
Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D.,
Novelli, M., Ribes, G., 1998. Experimental NIDDM: development of a new
model in adults rats administered streptozotocin and nicotinamide. Diabetes
47, 224–229.
Meckes, M., David-Rivera, A.D., Nava-Aguilar, V., Jimenez, A., 2004. Activity of
some Mexican medicinal plant extracts on carrageenan-induced rat paw
edema. Phytomedicine 11, 446–451.
Ortiz-Andrade, R.R., Sánchez-Salgado, J.C., Navarrete-Vázquez, G., Webster, S.P., Binnie,
M., Garcı́a-Jiménez, S., León-Rivera, I., Cigarroa-Vázquez, P., Villalobos-Molina, R.,
Estrada-Soto, S., 2008. Antidiabetic and toxicological evaluations of naringenin in
normoglycaemic and NIDDM rat models and its implications on extra-pancreatic
glucose regulation. Diabetes, Obesity and Metabolism 10, 1097–1104.
Ortiz-Andrade, R.R., Ramirez-Avila, G., Garcia-Jimenez, S., Villlobos-Molina, R.,
Estrada-Soto, S., 2007. a-Glucosidase inhibitory activity of the methanolic
462 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462
extract from Tournefortia hartwegiana: an anti-hyperglycemic agent. Journal of
Ethnopharmacology 109, 48–53.
Ortiz-Andrade, R.R., Rodrı́guez-López, V., Garduño-Ramı́rez, M.L., Castillo- España,
P., Estrada-Soto, S., 2005. Anti-diabetic effect on alloxanized and normogly-
cemic rats and some pharmacological evaluations of Tournefortia hartwegiana.
Journal of Ethnopharmacology 101, 37–42.
Puertollano, M.A., Puertollano, E., de Cienfuegos, G.Á., de Pablo, M.A., 2011. Dietary
antioxidants: immunity and host defense. Current Topics in Medicinal Chem-
istry 11 (14), 1752–1766.
Rekka, E., Kourounakis, P.N., 1991. Effect of hydroxyethyl rutosides and related
compounds on lipid peroxidation and free radical scavenging activity. Some
structural aspects. Journal of Pharmacy and Pharmacology 43 (7), 486–491.
Rho, H.S., Ahn, S.M., Lee, B.C., Kim, M.K., Ghimeray, A.K., Jin, C.W., Cho, D.H., 2010.
Changes in flavonoid content and tyrosinase inhibitory activity in kenaf leaf
extract after far-infrared treatment. Bioorganic and Medicinal Chemical
Letters 20, 7534–7536.
Tiwari, A.K., Prasad, P., Kumar, B.K.T., Ammini, K.M., Gupta, A.C., Gupta, R., A., 2009.
Oxidative stress pathway genes and chronic renal insufficiency in Asian
Indians with type 2 diabetes. Journal of Diabetes Complications 23 (2),
102–111.
Tzeng, Y.M., Chen, K., Rao, Y.K., Lee, M.J., 2009. Kaempferitrin activates the insulin
signaling pathway and stimulates secretion of adiponectin in 3T3-L1 adipo-
cytes. Molecular and Cellular Pharmacology 607 (1-3), 27–34.
Vega-Avila, E., Espejo-Serna, A., Alarcon-Aguilar, F., Velazco-Lesama, R., 2009.
Cytotoxic activity of four Mexican medicinal plants. Proceedings of Western
Pharmacology Society 52, 78–82.
Verspohl, E.J., 2002. Recommended testing in diabetes research. Planta Medica 68,
581–590.
Vishnu-Prasad, C.N., Suma-Mohan, S., Banerji, A., Gopalakrishnapillai, A., 2009.
Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1
adipocytes. Biochemical and Biophysical Research Communications 380 (1),
39–43.
Wisman, K.N., Perkins, A.A., Jeffers, M.D., Hagerman, A.E., 2008. Accurate assess-
ment of the bioactivities of redox-active polyphenolics in cell culture. Journal
of Agricultural and Food Chemistry 56, 7831–7837.
World Health Organization (WHO) 2011. Prevalence data of diabetes worldwide.
Available at /http://www.who.int/mediacentre/factsheets/fs312/en/index.
htmlS. Accessed 16.02.12.