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

Mexican diabetics for the empirical treatment of diabetes
Type 2 diabetes (T2DM), the cause of about 5% of all deaths (Argáez-López et al., 2003). J. spicigera (JS) Schltdl (Acanthaceae)
globally each year, affects more than 220 million people worldwide is an evergreen shrub with tubular orange flowers that grows in
and it is expected to rise to more than double in 2030 (World Health hot climates, native from México to South America.
Organization (WHO), 2011). T2DM has expensive implications for JS is widely distributed in the Yucatan peninsula and is known as
health systems in Mexico (Arredondo and de Icaza, 2009) and other lool chak, ts’i’its and yichkaan; in Huasteca Potosina (San Luis Potosı́,
countries. The lack of awareness of this disease combined with Mexico) it is commonly known as muicle or muite. This specie of
insufficient access to health services can lead to complications such Justicia is described as erect or scandent perennial herbs or
as atherosclerosis, cardiac dysfunction, retinopathy, neuropathy, subshrubs. Leaves present cystoliths and are petiolate with a leaf
nephropathy and blindness (World Health Organization (WHO), margin that is usually entire. Inflorescences are in spikes or panicles
2011). Therefore, there is a worldwide interest for new drugs that cimas, and the species rarely has solitary, terminal, or axillary flowers.
can help to reverse disease progression. 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
n
Corresponding author. Tel.: þ52 999 9225711. stamens, a capsule with four seeds, and a basal sterile portion
E-mail address: rolffy@uady.mx (R. Ortiz-Andrade). (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 Desérticas, Universidad Autónoma de San Luis Potosı́ (SLPM) for
an infusion and is indicated for the treatment of chronic head- future reference with the voucher number 46450.
aches, hypertension and epilepsy, so as for the treatment of Dried leaves of J. spicigera (40 g) were extracted with ethanol
ailments related to the digestive system, such as stomach pain, using a Soxtherm apparatus (Soxtherm automatic, Gerhadt,
diarrhea, dysentery as well as for constipation. In addition to this, Germany) for 3 h. The extract (JSE) was filtered and evaporated
is used as decoction of the leaves or branches (and sometimes the under vacuum and then lyophilized (Free Zone 2.5 Labconco,
flower) for cases of skin diseases such as erysipelas (skin infection USA). The extract was dissolved in methanol and filtered through
caused by the itch mite), syphilis, tumors, or difficult-cure a 0.45 mm nylon membrane filter (Gelman Science) before use. For
pimples. It’s also indicated for use in fever, kidney infection, bioassays, extract was dissolved in DMSO and preserved at room
anemia, so as anti-inflammatory, for dizziness and sleep, and for temperature protected from light until use on cell cultures.
some respiratory ailments such as cough, bronchitis and con-
stipation. There are also reports that both the infusion of leafs, as 2.3. Quantitation of kaempferitrin in Justicia spicigera
decoction of aerial parts are used in Mexican traditional medicine
are widely used for the empirical treatment of diabetes (Graham, The detection and quantification of KM in JSE were performed
1990; Braz et al., 2002; Arellano-Rodrı́guez et al., 2003; Meckes by reversed-phase HPLC method in a waters 2795 (Waters Corp.,
et al., 2004; Vega-Avila et al., 2009; Andrade-Cetto and Heinrich, Milford, MA, USA) instrument equipped with quaternary pump,
2005; Johnson et al., 2006). The indigenous Teenek from Huasteca autosampler and a 996 photodiode array detector, and Waters
Potosina (San Luis Potosi, Mexico) make a tea by boiling 20 g of JS Millenium 32 software for peak identification and integration.
leaves with 1 l of water. The tea is taken 3 times a day before Kromasil column C-18 with a 150  4.6 mm i.d 0.45 mm
every meal (personal communication). However, there is no (Metachem Technologies Inc) with injection volume 5 ml, flow
scientific evidence about the antidiabetic properties of JS. 0.4 ml/min. The mobile phase was acetic acid (HPLC grade Merck
Previous phytochemical studies with ethanol extracts of Dormstadt, Germany) 2% in water and the stationary phase was
J. spicigera leaves shown the isolation of the flavone kaem- acetonitrile (Merck, Dormstadt, Germany HPLC grade), gradient %
pherol-3,7- bisrhamnoside (kaempferitrin; KM) (Euler and Alam, B 10 to 70 in 20 min. Retention times and UV spectra of KM was
1982; Domı́nguez et al., 1990). KM has showed antidiabetic compared to those of high-purity commercial standards. The
properties in vitro and in vivo assays (De Sousa et al., 2004; calibration plot was obtained in the range of concentrations 124
Tzeng et al., 2009; Jorge et al., 2004; Vishnu-Prasad et al., 2009). 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. Materials and methods
2.4. Cell viability assay
2.1. Reagents, cell lines and chemicals
Murine and human preadipocytes were seeded as described
Murine 3T3-F442A preadipocytes and adult cat serum were a previously in 96-well microplates (Alonso-Castro et al., 2011).
gift from Dr. W. Kuri-Harcuch (CINVESTAV, México). Human After 24 h of incubation, ethanol extract of J. spicigera (JSE) at
normal subcutaneous preadipocytes were obtained as previously concentrations ranging from 0.1 to 250 mg/ml were added to the
described (Herrera-Herrera et al., 2009). Dulbecco’s modified cells. After 48 h, MTT assay was carried out (Alonso-Castro et al.,
Eagle medium (DMEM), Leibovitz L15 medium and fetal bovine 2011). The optical density (O.D.) was measured at 590 nm in an
serum (FBS) were purchase from GIBCO BRL (Grand Island, NY, ELISA reader (Biorad Laboratories, CA, USA) using wells without
USA) whereas calf serum was from HyClone (Logan, UT, USA). cells as a blank. The viability of treated cells was estimated from
Human Tumor Necrosis Factor alpha (TNF-a) and 2-[N-(7-nitro- the relative growth as follows:
benz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG)
were obtained from Peprotech (London, UK) to Invitrogen control O:D:sample O:D:
relative viability ¼  100
(Carlsbad, CA, USA), respectively. Rosiglitazone (RGZ), from Cayman control O:D:
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- 2.5. 2-NBDG uptake assay
turer. All other chemicals were from Sigma.
ABTS (2,2-azinobis-(3-ethyl-benzothiazoline)-6-sulfonic acid), Murine 3T3-F442A preadipocytes were differentiated into adi-
DPPH (2,2-diphenyl-1-picrylhydrazyl) and the water soluble ana- pocytes with DMEM containing 10% FBS, 5 mg/ml insulin and 1 mM
logue of vitamin E (Trolox; 6-hydroxy-2,5,7,8-tetramethylchro- D-biotin, whereas human preadipocytes were differentiated into
man-2-carboxylic acid) were purchase from Sigma (St Louis, human adipocytes with L15 containing 5% FBS, insulin 5 mg/ml, RGZ
MO, USA). 1 mM, dexamethasone 100 nM, tri-iodothyronine 0.2 nM, and 3-iso-
As positive controls in pharmacological evaluations were used butyl-1-methylxanthine 25 mM. Murine 3T3-F442A or human adi-
Glibenclamide (Silanes Laboratories, México City, México), Repa- pocytes were seeded on 96-well fluorescence plates and incubated
glinide (Prandins, Sanfer Laboratories, México City, México) and for 60 min with PBS containing BSA 1 mg/ml and 80 mM of the
Saxagliptin (Onglyzas, AstraZeneca/Bristol–Myers Squibb, México fluorescent glucose analog 2-NBDG (Alonso-Castro et al., 2011) in
City, Mexico) which were solubilized in saline (0.9% NaCl the presence of different concentrations of ethanol extract of JSE
solution). (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
2.2. Plant material and extraction evaluated on 3T3-F442A and human adipocytes pre-incubated with
TNF-a 10 ng/ml for 7 day to induce insulin-resistance (Hotamisligil
Samples of J. spicigera collected in Ciudad Valles, San, Luis Potosı́, et al., 1994). After incubation, free 2-NBDG was washed out from
México (211580 43.9000 N and 981580 31.4400 W) on August 2010 were cultures and fluorescence retained in cell monolayers was measured
identified by a specialist (J. Garcia-Perez) and preserved at the with a Tecan-GENios fluorescence microplate reader (Tecan, Salzburg,
herbarium Isidro Palacios of Instituto de Investigación de Zonas Austria). Values of 2-NBDG incorporation in the absence of insulin
 R. Ortiz-Andrade et al. / Journal of Ethnopharmacology 143 (2012) 455–462 457

were subtracted from those obtained with insulin 100 nM to NOM-062-ZOO-1999 Mexico) and were approved by the Research
establish 100% of specific 2-NBDG uptake. Bioethics Committee of CIR-UADY.

2.6. Antioxidant assays 2.7.2. Oral glucose tolerance test (OGTT)

A protocol previously described was used (Ortiz-Andrade et al.,
2.6.1. ABTS radical scavenging assay 2007). Male Wistar normoglycaemic rats were used in the
For ABTS assay, the procedure followed the method of Erel experiment. Rats were deprived of food for 16 h before experi-
(2004) with slight modification. Sodium acetate was dissolved in mentation, but allowed free access to tap water throughout the
100 ml deionized water to prepare a buffer solution (30 mM, pH experiment. All experiments were carried out using five animals
3.6). ABTS (10 mM) was dissolved in 100 ml of buffer solution and per group. JSE was dissolved in physiological saline solution (0.9%
27.8 ml of H2O2 solution (35% v/v) to obtained the concentration NaCl solution, ISS) and was administered orally by intragastric
2 mM, allowing them to react for 12–16 h at room temperature in route at 10 mg/kg (group 2), 50 mg/kg (group 3) and 100 mg/kg
the darkness before used to obtain ABTS radical cation (ABTS þ ) (group 4), respectively. Glibenclamide (10 mg/kg), Repaglinide
solution. The assay were performed in 96-well plates, containing (4 mg/kg) and Saxagliptin (10 mg/kg) were used as hypoglycemic
200 ml of sodium acetate buffer (0.4 M, pH 5.8), 5 ml of sample reference drugs (groups 5–7). Control rats received the vehicle in
solution (0–800 mg/ml) or Trolox standard curve (0–100 mg/ml) the same volume (0.5 ml/100 g) by the same route (group 1).
and 20 ml of ABTS þ solution to each well. After initial mixing for Ten minutes after administration of test samples, a dose of 2 g/kg
10 min, the absorbance was measured at 734 nm using spectro- of glucose solution was administered orally to each rat. Blood
photometer (Multiskan Ascent, Thermo Labsystems). All assays samples were collected from the tail tip at 0 (before oral
were performed in triplicate at least three times each. The administration) and 0.5, 1, 2, 3 and 4 h after glucose administra-
ABTS þ scavenging capacity of the extract was compared with tion. Blood glucose concentration was estimated by enzymatic
that of Trolox and calculated from the following equation: glucose–oxidase method using a commercial glucometer
(Accu-Chek Active, Roches) which was calibrated and standar-
Abs controlAbs Sample dized before and between measuring the glucoses. The percen-
ABTS scavenging activity ð%Þ ¼  100
Abs control tage variation of glycemia for each group was calculated in
where Abs control was the absorbance of the blank and Abs relation to initial (0 h) level, according to:
sample was the absorbance in the presence of the samples and Gx G0
standard. EC50 value is the concentration of the sample required % Variation of glycemia ¼  100
G0
to scavenge 50% ABTS radical cation.
where G0 were initial glycemia values and Gx were the glycemia
values at þ0.5, þ1, þ2, þ3 and þ4 h, respectively. (Verspohl,
2.6.2. DPPH radical scavenging assay 2002; Ortiz-Andrade et al., 2005).
The complementary study for the antioxidant capacity of the
JSE extract was confirmed by the DPPH scavenging assay accord- 2.7.3. Oral glucose tolerance test (OGTT) in T2DM model
ing to the method of Burda and Oleszek (2001) with slight Streptozotocin (STZ) was dissolved in citrate buffer (0.1 M, pH
modification. The DPPH stock solution was prepared by dissolving 4.5) and nicotinamide (NDA) in physiological saline solution (ISS).
7.5 mg DPPH with 250 ml methanol solution (0.1 mM). Different Diabetes was induced in overnight fasted rats (16 h) by a single
concentrations (0–800 mg/ml) of the sample solution or standard intraperitoneal (i.p.) injection of 65 mg/kg STZ 15 min after i.p.
Trolox (0–100 mg/ml) were mixed with equal volume of metha- administration of NDA 100 mg/kg (Masiello et al., 1998; Ortiz-
nol. The assays were performed in 96-well plates, containing Andrade et al., 2008). After fifteen days, T2DM was confirmed by
175 ml of DPPH stock solution and 25 ml of sample solution or the elevated plasmatic glucose levels, and considering for this
Trolox standard curve to each well. After incubation for 30 min, studies the animals with fasted plasmatic glucose concentration
the absorbance was measured at l ¼517 nm using spectrophot- above 150 mg/dl.
ometer (Multiskan Ascent, Thermo Labsystems). All assays were The protocol described above was used to evaluate the anti-
performed in triplicate at least three times each. The ability to diabetic effect of JSE. 16 h before the experiments, rats were
scavenge DPPH radical was calculated by the following equation: fasted with free access to water. JSE was dissolve in isotonic saline
Abs controlAbs Sample solution (ISS) and was administered orally by intragastric route at
DPPH radical scavenging activity ð%Þ ¼  100
Abs control 100 mg/kg (group 2). Glibenclamide (10 mg/kg) was used as
hypoglycemic reference drug (group 3). Control rats received
where Abs control was the absorbance of the blank and Abs
the vehicle in the same volume (0.5 ml/100 g) by the same route
sample was the absorbance in the presence of the samples or
(group 1). Ten minutes after administration of test samples, a
standard. EC50 value is the concentration of the sample required
dose of 2 g/kg of glucose solution was administered to each rat.
to scavenge 50% DPPH free radical.
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-
2.7. Pharmacological evaluations tion. Blood glucose concentration was estimated by enzymatic
glucose–oxidase method using a commercial glucometer (Accu-
2.7.1. Animals Chek Active, Roches) which was calibrated and standardized
Male Wistar rats ten weeks old and weight 250–300 g were before and between measuring the glucoses. The percentage
obtained from the Animal House of the Centro de Investigaciones variation of glycemia for each group was calculated in relation
Regionales ‘‘Dr. Hideyo Noguchi’’, Universidad Autónoma de Yucatán to initial (0 h) level using the formulae described above.
(CIR-UADY). Rats were fed with rodent diet and water ad libitum and
were housed under standard laboratory conditions maintained at 2.8. Statistical analysis
room temperature and at photoperiod of 12 h day/night cycle,
2573 1C and humidity of 50–65% during thirty days before experi- For cell viability and 2-NBDG uptake assays so as for antiox-
ment. All animal procedures were conducted in accordance with the idants assays, experimental values are expressed as mean 7
Federal Regulations for Animal Experimentation and Care (SAGARPA, 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 JSE ( g/ml) - 10 50 100 250
herbal substance to the native herbal drug preparation (DER native) Treatments
was 14.5:1. The HPLC analysis showed that KM was major compo-
Fig. 2. Effect of J. spicigera extract on 3T3 murine and human preadipocytes
nent in JSE (Fig. 1). The content of KM in JSE was 69.65 mg/g. 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
3.2. JSE lacks toxic effects on fibroblasts 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
We test the toxic effects of J. spicigera on the proliferation of by MTT test (see Materials and methods). Results represent the mean 7 standard
the normal cells murine and human fibroblasts. JSE did not affect deviation (SD) of three independent experiments in cuadruplicate.
significantly (pZ0.05) murine fibroblasts viability, compared to
untreated cells, at any of the tested concentrations. On the 10 mg/ml and 50 mg/ml did it by 96% and 115%, respectively
contrary, on human cells only JSE 250 mg/ml decrease viability (Fig. 3A; white bars).
(26%) significantly (pr0.05), whereas lower JSE concentrations The effects of JSE were also assayed on the 2-NBDG uptake in
lacked toxic effects (Fig. 2). murine and human adipocytes made insulin-resistant by TNF-a
treatment (see Materials and methods) to further evaluate the
3.3. JSE stimulates 2-NBDG uptake in insulin-sensitive and insulin- antidiabetic potential of this plant. In murine diabetic-like adipo-
resistant murine and human adipocytes 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
To determine whether JSE stimulate the glucose uptake by murine cells (Fig. 3B; gray bars), whereas in human diabetic-like
adipose cells, the 2-NBDG uptake by terminally differentiated adipocytes JSE increased by 52% (1 mg/ml), 85% (10 mg/ml) and
murine 3T3-F442A and human subcutaneous adipocytes was 99% (50 mg/ml) (Fig. 3B; striped white bars).
assayed in the presence of JSE non-toxic concentrations. JSE
stimulated 2-NBDG uptake in both cell types in a concentration- 3.4. Antioxidant in vitro assays of JSE
dependent manner. In insulin-sensitive 3T3-F442A adipocytes,
JSE 1 mg/ml stimulated the 2-NBDG uptake by 35% compared to 3.4.1. Radical scavenging activity against ABTS
the insulin 100 nM whereas JSE 10 mg/ml and 50 mg/ml promoted ABTS a stable free radical with the characteristic absorption at
the 2-NBDG uptake by 67% and 127%, respectively (Fig. 3 and 4A; 734 nm was used to study the radical scavenging effect of JSE. The
black bars). In insulin-sensitive human adipocytes, JSE 1 mg/ml results demonstrated that JSE reacted with ABTS at different
increased the 2-NBDG incorporation by 46%, whereas JSE 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

** 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
100
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
0 when compared with the vehicle group (Table 2 and 3, po0.01).
The glibenclamide (10 mg/kg), repaglinide (4 mg/kg) and saxa-
0 1 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).
Time after administration (h)
In the normoglycemic rats experiment (Table 2), it was
Fig. 4. Effect of 100 mg/kg of JSE on blood glucose levels after a single oral observed that reduction in blood glucose concentration at all
administration of 2 g/kg of glucose in STZ–NDA diabetic induced male Wistar rats. doses, started at the 0.5 h causing a reduction ranging from 25.6
Each plot represents the means7 S.E.M. (n¼ 5;** P o 0.01).
to 29.9% with average 27.7% of blood glucose levels when
compared with vehicle group. Only JSE 100 mg/kg reduced
by measuring the reduction of radical cation generated by significantly (p o0.01) blood glucose levels by 27% (1 h), 39.5%
ABTS þ . JSE showed a maximum decolourization of 71.21 71.4% (2 h) and 45.8% (3 h), which was the more significant reduction
at a maximum concentration of 800 mg/ml with the EC50 value showed in normoglycemic rats (Table 2). Lower doses (10 and
180 mg/ml (Table 1). 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.
3.4.2. Radical scavenging activity using DPPH Due that only JSE 100 mg/kg exerted significant differences in
The DPPH approach is widely applied to measure the antiox- blood glucose levels in all times in normoglycemic rats, this JSE
idant properties of compounds. DPPH is an organic nitrogen radical dose was tested in STZ-induced diabetic rats. The glibenclamide
with ultraviolet–visible absorption in the range 515–520 nm, treated group (5 mg/kg) showed a significant reduction of
and the color of its solution fades upon reduction. The samples blood glucose levels from 0.5 to 4 h (po0.01). JSE showed
was tested against this radical at different concentrations significant (p o0.01) decrease in blood glucose levels by 155%
(0–800 mg/ml) and the readings were observed by decreasing (0.5 h) and 185% (1.0 h), whereas at 2.0, 3.0 and 4.0 h JSE did not
the absorbance taken as a measure indicates the extent of radical show significant difference when compared with vehicle group
scavenging propriety. The ethanolic extracts of J. spicigera showed (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) 455–462 461

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