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Antitumor and immunomodulatory effects of Justicia spicigera Schltdl

(Acanthaceae)

Article  in  Journal of ethnopharmacology · March 2012

DOI: 10.1016/j.jep.2012.03.036 · Source: PubMed

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 Journal of Ethnopharmacology 141 (2012) 888–894

Contents lists available at SciVerse ScienceDirect

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

Antitumor and immunomodulatory effects of Justicia spicigera Schltdl

(Acanthaceae)
Angel Josabad Alonso-Castro a,b,∗ , Elizabeth Ortiz-Sánchez b , Fabiola Domínguez c ,
Victor Arana-Argáez d , Maria del Carmen Juárez-Vázquez b , Marco Chávez e,f ,
Candy Carranza-Álvarez g , Octavio Gaspar-Ramírez h , Guillermo Espinosa-Reyes h ,
Gabriela López-Toledo b , Rolffy Ortiz-Andrade d , Alejandro García-Carrancá b,i,∗
a
Facultad de Química, Universidad Nacional Autónoma de México, D.F., Mexico
b
Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, Secretaría de Salud, México, D.F., Mexico
c
Centro de Investigación Biomédica de Oriente, IMSS, Metepec, Puebla, Mexico
d
Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico
e
Unidad de Investigación Médica en Farmacología de Productos Naturales, Pediatría, CMN Siglo XXI, IMSS, D.F., Mexico
f
Unidad de Investigación en Enfermedades Neurológicas, Especialidades, CMN Siglo XXI, IMSS, D.F., Mexico
g
Unidad Académica Multidisciplinaria de la Zona Huasteca, Universidad Autónoma de San Luis Potosí, Ciudad Valles, San Luis Potosí, Mexico
h
Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
i
Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Medicinal plants are an important source of antitumor compounds. This
Received 26 November 2011 study evaluated the acute toxicity in vitro and in vivo, as well as the cytotoxic, antitumor and immunomod-
Received in revised form 4 February 2012 ulatory effects of ethanolic extracts of Justicia spicigera leaves (JSE).
Accepted 17 March 2012
Materials and methods: The in vitro and in vivo toxicity of JSE was evaluated with comet assay in peripheral
Available online 28 March 2012
blood mononuclear cells (PBMC) and acute toxicity in mice, according to the Lorke procedure, respec-
tively. The apoptotic effect of JSE on human cancer cells and human noncancerous cells was evaluated
Keywords:
using flow cytometry with annexin-Alexa 488/propidium iodide. Also, different doses of JSE were injected
Justicia spicigera
Cancer
intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 18 days. The growth and
Antitumor weight of tumors were measured. The in vitro immunomodulatory effects of JSE were evaluated estimat-
Apoptosis ing the effects of JSE on the phagocytosis of the yeast Saccharomyces cerevisiae, NO production and H2 O2
Toxicity release in macrophages, as well as the proliferation of splenocytes and NK activity.
Immunomodulatory Results: The comet assay showed that only JSE tested at 200 and 1000 ␮g/ml induced a significantly DNA
damage in PBMC, compared to untreated cells, whereas the LD50 was >5000 mg/kg by intraperitoneal
route (i.p.) and by oral route. JSE showed pro-apoptotic (Annexin/PI) effects by 35% against HeLa cells,
but lack toxic effects against human normal cells.
JSE administrated at 10, 50 and 100 mg/kg i.p. inhibited the tumor growth by 28%, 41% and 53%,
respectively, in mice bearing HeLa tumor. JSE stimulated, in a concentration dependent manner, the
phagocytosis of Saccharomyces cerevisiae yeasts, the NO production and H2 O2 release by human differ-
entiated macrophages. In addition, JSE stimulated the proliferation of murine splenocytes and induced
the NK cell activity.
Conclusion: Justicia spicigera shows low toxic effects in vitro and in vivo, exerts apoptotic effects on HeLa
cells, has antitumor effects in mice bearing HeLa tumor and induces immunomodulatory activities in vitro.

© 2012 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: CDDP, cisplatin; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; JSE, Justicia spicigera ethanolic extract; KM, kaempferitrin; LPS,
lipopolysaccharides; MTT, thiazolyl blue tetrazolium bromide; NK, natural killer cells; NO, nitric oxide; PBMC, peripheral blood mononuclear cells; PMA, phorbol 12-myristate
13-acetate.
∗ Corresponding authors at: Av. San Fernando No. 22, Col. Sección XVI, Tlalpan, 14080 México, D.F., Mexico. Tel.: +52 55 56280433; fax: +52 55 54854371.
E-mail addresses: [email protected] (A.J. Alonso-Castro), [email protected] (A. García-Carrancá).

0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2012.03.036
 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894 889

1. Introduction 2.2. Cells and culture conditions

The efficacy of antitumor therapies would improve if it ben- Human cervical carcinoma cells (HeLa) and the human immor-
efits immune response against tumor development (Mitchell, talized keratinocytes (HaCaT), used as normal cells, obtained
2003). However, many of antitumoral drugs are known to produce from ATCC (Manassas, VA, USA), were maintained in DMEM sup-
immunosuppression. Therefore there is a worldwide need to inves- plemented with 7% FBS and antibiotics (100 units/ml penicillin
tigate antitumor agents with low toxic effects and with effects that and 100 pg/ml streptomycin). Peripheral blood mononuclear cells
improve host defense mechanisms against tumor growth. (PBMC), obtained from healthy volunteers as described (Yañez
Plants have represented an important source for anti-cancer et al., 2004), were used as normal cells in toxic assays and as effec-
drugs, and México possesses a great diversity of flora with potential tor cells in immunomodulatory assays. PBMC were seeded in RPMI
for searching new compounds for cancer treatment (Alonso-Castro medium supplemented with 10% FBS and antibiotics (100 units/ml
et al., 2011). penicillin and 100 pg/ml streptomycin). All the procedures carried
Justicia spicigera Schltdl (Acanthaceae), native from México and out in this study were approved by the Research Ethic Committee
extending into South America, commonly known as muicle or from Instituto Nacional de Cancerología (Distrito Federal, México).
mohuite, is an evergreen shrub with tubular orange flowers. In All cell cultures were grown at 37 ◦ C and 5% CO2 .
Mexican traditional medicine, Justicia spicigera is used as immunos-
timulatory (Herrera-Arellano et al., 2009) and for the empirical
2.3. Animals
treatment of cervical cancer (Márquez et al., 1999). However, the
toxic and immunomodulatory effects of this plant are unknown.
In this study, C57BL/6 and CD-1 mice were obtained from
Thus, it is important to evaluate the toxicology of natural products
Instituto de Investigaciones Biomedicas, Universidad Nacional
to support their safety on further clinic studies.
Autonoma de Mexico, whereas athymic mice nu/nu were from
Previous reports indicated that Justicia spicigera exerted toxic
Instituto Nacional de Ciencias Médicas y Nutrición Salvador
effects against the human cancer cells TF-1, CaLo, InBl, T47D and
Subirán, México. The in vivo experiments were performed follow-
HeLa (Caceres-Cortes et al., 2001; Vega-Avila et al., 2009). Recently,
ing the NIH Guide for Treatment and Care for Laboratory Animals
we showed that ethanolic extracts of Justicia spicigera leaves had
and by the Mexican Official Norm for Animal Care and Handing
high cytotoxic effects against HeLa cells (IC50 = 17 ␮g/ml), and stim-
(NOM-062-ZOO-1999). The mice, having free access to food and
ulated the proliferation of human peripheral blood mononuclear
water, were housed in cages with filtered air in a climate and light
cells in a concentration dependent manner, and tested at 100 ␮g/ml
controlled room with a 12 h light/dark cycle.
showed a similar potency as compared to phytohemagglutinin
1 ␮g/ml (Jacobo-Salcedo et al., 2011).
Previous phytochemical determination of ethanol extracts of 2.4. Preparation of Justicia spicigera extract and quantification of
Justicia spicigera leaves revealed the presence of kaempherol-3,7- kaempferitrin
bisrhamnoside (kaempferitrin) (Euler and Alam, 1982; Domínguez
et al., 1990). Samples of Justicia spicigera collected in Ciudad Valles, San, Luis
This work demonstrates, for first time, that an ethanolic extract Potosí, México on August 2010 were identified by a specialist (J.
of Justicia spicigera (JSE) exerts low toxic effects in vitro and in vivo, Garcia-Perez, UASLP) and preserved at the herbarium Isidro Pala-
induces antitumor and immunomodulatory effects. cios of Instituto de Investigación de Zonas Desérticas, Universidad
Autónoma de San Luis Potosí (SLPM) for future reference with
voucher number 46450.
2. Materials and methods Powdered dried leaves of Justicia spicigera (10 g) were extracted
with ethanol using a Soxtherm apparatus (Soxtherm automatic,
2.1. Materials Gerhadt, Germany) for 2 h. The extract was filtered and concen-
trated under reduced pressure to dryness and the residue was
Dulbecco’s modified Eagle medium (DMEM), RPMI medium and dissolved in DMSO and protected from light. The detection and
fetal bovine serum (FBS) were from GIBCO BRL (Grand Island, NY, quantification of KM in JSE were performed by reversed-phase
USA). Kaempferitrin (KM) obtained from ChromaDex (Laguna Hills, HPLC method in a waters 2795 (Waters Corp., Milford, MA, USA)
CA, USA) was 98% purity according to the manufacturer. Cisplatin instrument equipped with autosampler and a 996 photodiode array
(CDDP) was from Accord Farma (Distrito Federal, México). detector, Kromasil column C-18 150 mm × 4.6 mm (Metachem

Fig. 1. HPLC elution pattern of kaempferitrin from Justicia spicigera ethanolic extract.
890 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894

Technologies Inc.) with injection volume 5 ␮l, flow 0.4 ml/min.

The mobile phase was acetic acid (HPLC grade Merck Dormstadt, (A)
8
Germany) 2% in water and the stationary phase was acetonitrile b b
(HPLC grade Merck, Dormstadt, Germany). Retention times and UV b
spectra of KM were compared to those of high-purity commer-

Olive Tail Moment

6
cial standards. The calibration plot was obtained in the range of
concentrations 124–496 ␮g/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. 4

2.5. Comet assay a a

a
2 a
Peripheral blood mononuclear cells (PBMC) were treated in the
presence of vehicle (DMSO 0.1%), with different concentrations
of JSE (10, 50, 200 and 1000 ␮g/ml) or sodium arsenite NaAsO2
65 ng/ml (positive control) during 5 h. DNA damage in PBMC was 0
Vehicle NaAsO2 0 50 200 400 1000
assessed by comet assay (Singh et al., 1988). The extent of DNA
migration was analyzed in 100 cells (50 randomly selected cell JSE concentration ( g/ml)
nucleoids by duplicate) using an epifluorescent microscope (Nikon Treatments
LABOPHOT-X2), whereas the olive tail moment [(tail mean − head
mean) × tail %DNA/100] was determined using Komet 4.0 Image
Analysis and Data Capture Software (Komet, versión 4; Kinetic (B)
Imaging Ltd., Merseyside, UK). The comet image magnification was 30
200×. DNA migration was measured by image analysis (Image Plus-
b
Pro Plus Version 3.0) as the length of the comet image (total image b
b
Tail lenght ( m)

length). The total image length included the tail and the nucleus of
the comet and was reported in ␮m ± SD.
20

2.6. Acute toxicity test

a
a
Six weeks old female CD-1 mice, weighing 25–30 g, were used. a a
The acute toxicity of the JSE, administrated intraperitoneal or intra- 10

gastric in mice, was estimated according to Lorke method (1983).

Mice were observed daily during 14 days for mortality, behavioral
changes and other toxic signs.
0
2.7. Apoptosis assay Vehicle NaAsO2 0 50 200 400 1000

JSE concentration ( g/ml)

HeLa or HaCaT cells were seeded in 60 mm culture plates at Treatments
8 × 104 cells/plate. After 24 h, control vehicle (DMSO 0.01%), IC50 of
JSE or CDDP was added to cells during 48 h. Then, cells were stained Fig. 2. JSE exerts low toxic effects in vitro. Peripheral blood mononuclear cells
with annexin-Alexa 488 and propidium iodide (PI) contained in the (PBMC) were obtained from healthy volunteers, and treated in the presence of
Vybrant Apoptosis Assay Kit no. 2 (Invitrogen, Carlsbad, CA, USA). vehicle (DMSO 0.1%), different concentrations (10, 50, 200 and 1000 ␮g/ml) of JSE
or sodium arsenite NaAsO2 65 ng/ml (positive control) during 5 h. DNA damage in
The apoptosis assay was carried as described by Ortiz-Sánchez
PBMC was assessed by comet assay, represented as olive tail moment (A) and tail
et al. (2009) using FACScan analytic flow cytometer (BD Bioscience, length (B), taken as the mean value of the total image length in 100 cells per treat-
Mountain View, CA, USA). The level of apoptosis of cells treated with ment. Results represent the mean ± standard deviation (SD) of three independent
JSE or CDDP is calculated by the subtraction of the total percent- experiments in quadruplicate. Lowercase letters indicate significant differences
according to ANOVA test (P ≤ 0.05), followed by post hoc Tukey tests.
age of apoptosis of untreated cells from the percentage of apoptosis
of cells treated with JSE or CDDP. The software Weasel cytometry
analysis software v.2.6.1 (Walter and Eliza Hall Institute of Medical
2.9. Phagocytosis assay
Research, Parkville, Victoria, Australia) was used for data analysis.

The phagocytosis assay was carried out as described previously

2.8. Antitumor assay
(Esteban et al., 1998).
PBMC were seeded in 24-well plates at 5 × 105 cells/well
The antitumor assay was carried out as described by Alonso-
and cultured overnight in the presence of phorbol 12-myristate
Castro et al. (2012). Six weeks old athymic nu/nu mice, weighing
13-acetate (PMA) 50 ng/ml to permit the differentiation into
20–23 g, were used. Mice were injected subcutaneously in the back
macrophages. Then, non adherent cells were removed and vari-
with HeLa cells (1.5 × 106 ). Nine days after tumor implantation,
ous concentrations of JSE (1–200 ␮g/ml) or LPS 1 ␮g/ml was added.
groups of five mice received doses of JSE from 10 to 100 mg/kg dis-
After 24 h, cells were co-cultured with Saccharomyces cerevisiae
solved in 0.1 ml of 0.9% saline solution, injected intraperitoneally,
yeasts (5 × 106 yeast/well) and labeled with 100 ␮g/ml of pro-
daily over 15 days. The control animals received 0.1 ml of the vehi-
pidium iodide. After 90 min, non-ingested labeling yeasts were
cle solution. Tumors were measured with a Vernier caliper, and
removed and the amount of Saccharomyces cerevisiae phagocytosed
their size in mm3 was calculated as follows (Looney et al., 1971):
by macrophages was determinate measuring the cellular fluores-
length × width × height cent intensity using a Cell Lab Quanta SC (Beckman Coulter) flow
Tumor volume =
2 cytometer.
 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894 891

Fig. 3. JSE induces apoptosis in HeLa cells. HeLa or HaCaT cells were treated with vehicle (DMSO 0.01%), JSE 17 ␮g/ml or CDDP 4 ␮g/ml during 48 h. The apoptosis assay was
carried out as described in Section 2, recording 10,000 events. The percentage of cells in each quadrant is localized in the corner. The cells in the right quadrants (Annexin
V+ /PI− and Annexin V+ /PI+ ) are considered as apoptotic cells. Data are representative of four independent experiments in duplicate.

2.10. Nitric oxide (NO) production Na2 EDTA 0.1 mM). After centrifugation at 1500 rpm for 5 min, the
pelleted cells were washed in PBS, resuspended and cultured in
Nitric oxide (NO) production was determined based on Griess RPMI medium with 7% FBS, penicillin 100 IU/ml, streptomycin
reaction (Green et al., 1981). Human macrophages, differentiated 100 ␮g/ml. Cells were counted with a hemocytometer by try-
as described above, were co-cultured with Saccharomyces cerevisiae pan blue dye exclusion technique. Cell viability exceeded 95%.
yeasts (5 × 106 yeast/well) and treated with various concentrations Murine splenocytes were seeded in 96 well plates at a density
of JSE (1–200 ␮g/ml) or LPS 1 ␮g/ml. After 24 h of incubation, the of 2 × 105 cells/well. After 24 h of incubation, JSE concentrations
plate was centrifuged for 10 min at 300 × g and the cell culture between 1 and 200 ␮g/mL were added to the cells. Positive controls
supernatants were mixed with equal volume of Griess reagent received lipopolysaccharides (LPS) 1 ␮g/ml. After 48 h of treatment,
(Sigma). The absorbance was spectrophotometrically measured at the MTT assay was performed and the relative viability was calcu-
540 nm and the NO concentration was determined by a standard lated as described (Jacobo-Salcedo et al., 2011).
curve of NaNO2 (5–60 ␮M) and expressed as ␮M.
2.13. NK cell activity
2.11. Hydrogen peroxide release
The activity of natural killer (NK) cells was measured as previ-
Hydrogen peroxide release by macrophages was determined ously described (Tu et al., 2008) with some modifications. Briefly,
according to Pick and Mizel (1981). Cell culture supernatants K562 cells (human erythromyeloblastoid leukemia) used as target
(100 ␮l) of monocyte–macrophages co-cultured with Saccha- cells, were seeded in 96-well plates at a density of 2 × 104 cells/well
romyces cerevisiae were mixed with an equal volume of fresh phenol in RPMI medium. After 24 h, murine splenocytes, used as the effec-
red solution (5.5 mM dextrose, 0.056 g phenol red and 8.5 U/ml tor cells, were added at 1 × 106 cells/well to give effector/target
Type I HRP in DPBS) in 96-well plate and incubated for 3 h. Reaction cells a ratio of 50:1. After incubation for 4 h, cells were treated
was stopped adding 10 ␮l of NaOH 1 N solution. The absorbance was with JSE concentrations between 1 and 200 ␮g/ml during 48 h.
spectrophotometrically measured at 620 nm and the H2 O2 con- Then, cells were subjected to MTT assay (Jacobo-Salcedo et al.,
centration determined by standard curve of H2 O2 (5–40 ␮M) and 2011). NK cell activity was calculated as follows: NK activity
expressed as ␮M. (%) = (ODT − (ODS − ODE))/ODT × 100% where ODT is optical den-
sity value of target cells control, ODS is optical density value of test
2.12. Isolation of murine splenocytes and proliferation assay samples and ODE is optical density value of effector cells control.

Six weeks old male C57BL/6 mice were sacrificed and their 2.14. Statistical analysis
spleens were removed aseptically and passed through a stain-
less mesh on ice. The cells were incubated at 37 ◦ C for 5 min Experimental values are expressed as the mean ± the standard
with erythrocytes lysis buffer (NH4 Cl 150 mM, KHCO3 10 mM, deviation of at least three experiments in triplicate. Data were
892 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894

analyzed using one-way ANOVA, followed by post hoc Tukey tests. (A)
The level of P ≤ 0.05 was used to determine statistical significance. Control
All calculations were performed using the Graph Pad Prisma V.3 CDDP 1 mg/kg
600 JSE 10 mg/kg
software system (GraphPad Software, San Diego, CA, USA). JSE 50 mg/kg
JSE 100 mg/kg

Tumor size (mm3)

3. Results

400
3.1. KM quantitation

HPLC analysis showed that KM was the major component in

JSE (Fig. 1). The content of this compound in the extract was
200
12.75 mg/g. Therefore, JSE concentrations assayed in vitro con-
tained 7.7 ␮M KM (JSE 10 ␮g/ml), 13.1 ␮M KM (JSE 17 ␮g/ml),
38.5 ␮M KM (JSE 50 ␮g/ml), 77 ␮M KM (JSE 100 ␮g/ml) and 154 ␮M
KM (JSE 200 ␮g/ml). The JSE concentrations assayed in vivo con-
tained 19.1 ␮M KM (JSE 10 mg/kg), 95.5 ␮M KM (JSE 50 mg/kg) and 0
0 3 6 9 12 15 18
191 ␮M KM (JSE 100 mg/kg).
Treatment (days)
3.2. JSE exerts low toxic effects in vitro and in vivo (B) 800

a
In comet assay, the tail length and the olive tail moment in
cells treated with JSE at 200 and 1000 ␮g/ml were significantly
higher and with the same potency than the positive control NaAsO2 600

Tumor weight (mg)

65 ng/ml (P ≤ 0.05), whereas lower JSE concentrations did not show c
DNA damage (Fig. 2A and B). The LD50 estimated by the Lorke
method (1983) was >5000 mg/kg i.p. and >5000 mg/kg o.p. At
400 d
doses of 1600 mg/kg or higher, neurological deficit was observed
in the mice only in the first 4 h after treatment. The symptoms e
included immobility, dizziness and sedation. Also, JSE at doses up
b
to 1000 mg/kg decreased significantly body weight, whereas lower 200
concentrations had no such effect (results not shown). On the con-
trary, lower dose increases body weight as much as vehicle treated
mice (results not shown).
0
3.3. JSE induces apoptosis in HeLa cells Control
CDDP JSE JSE JSE
1 mg/kg 10 mg/kg 50 mg/kg 100 mg/kg
Previously, we showed that Justicia spicigera had the highest Treatments
cytotoxic activity on HeLa cells (IC50 = 17 ␮g/ml) (Jacobo-Salcedo
et al., 2011). To analyze whether JSE has the potential effect to Fig. 4. JSE inhibits the growth and decreases tumor weight in nu/nu mice bear-
ing HeLa tumor. Mice were injected with HeLa cells (1 × 106 ) and 8 days after
induce apoptosis in cancer cell lines, HeLa cells were treated with tumor implantation were treated intraperitoneally with varying doses of JSE
JSE and analyzed by flow cytometry. JSE induced 35% of apopto- (10–100 mg/kg) or CDDP1 mg/kg daily over a period of 18 days. Tumors were
sis (Annexin+ /PI− plus Annexin+ /PI+ ) compared to untreated cells, measured (A) and weighed (B) as described in Section 2. Data are representa-
whereas CDDP induced 54% of apoptosis (Fig. 3). In human nor- tive of three independent experiments in quintuplicate. Results represent the
mean ± standard deviation (SD). Lowercase letters indicate significant differences
mal HaCaT cells, JSE and CDDP induced 7% and 56%, respectively, of
according to ANOVA test (P ≤ 0.05), followed by post hoc Tukey tests.
apoptosis (Fig. 3).

3.4. JSE exerts antitumor effects a higher potency than LPS 1 ␮g/ml (Fig. 5B). In murine spleno-
cytes, JSE stimulated the proliferation by 127% (100 ␮g/ml) and
After 15 days of treatment, JSE administrated at 10, 50 and 134% (200 ␮g/ml), compared to untreated cells (Fig. 5C). Further-
100 mg/kg i.p. inhibited the growth of the HeLa cell tumors sig- more, JSE induced the NK cell activity by 9.9% and 10.7% added at
nificantly (P ≤ 0.05) by 28%, 41% and 53%, whereas CDDP 1 mg/kg 100 ␮g/ml and 200 ␮g/ml, respectively (Fig. 5D).
inhibited the tumor growth by 63% compared to untreated mice
(Fig. 4A). Furthermore, JSE inhibited significantly (P ≤ 0.05) tumoral 4. Discussion
weight in mice by 24% (10 mg/kg) and 44% (50 mg/kg) and 51%
(100 mg/kg) whereas CDDP 1 mg/kg inhibited tumor weight by 67% In Mexican traditional medicine, leaves of Justicia spicigera are
as compared to untreated mice (Fig. 4B). used as immunostimulatory (Herrera-Arellano et al., 2009) and for
the empirical treatment of cervical cancer (Márquez et al., 1999).
3.5. JSE induces immunomodulatory effects in vitro However, the antitumor effects of Justicia spicigera as well as its
toxic and immunomodulatory effects are still unknown.
We found that JSE stimulated the phagocytosis of Saccharomyces Previously it was shown the presence of kaempferitrin in
cerevisiae cells by human differentiated macrophages, in a con- ethanol extracts of Justicia spicigera (Euler and Alam, 1982;
centration dependent manner, and tested at concentrations of Domínguez et al., 1990). In this study, we showed that KM was the
100 ␮g/ml and 200 ␮g/ml induced the phagocytosis with higher most abundant compound in JSE and that its content (12.75 mg/g)
potency than the positive control LPS 1 ␮g/ml (Fig. 5A). JSE induced was 2.3-fold and 5.9-fold lower than those reported for the medic-
the NO production and H2 O2 release in human macrophages in a inal plants Hibiscus cannabinus and Cinnamomum osmophloeum,
concentration dependent manner, and tested at 200 ␮g/ml showed respectively (Rho et al., 2010; Lin et al., 2011). However, a more
 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894 893

160 25 25
(A) c c (B) NO
140 b HO f'
b b
20 20
d

NO production ( M)
120

H 2O2 release ( M)
a b'
a
% Phagocytosis

100
15 e' 15

80 b
b
10 b d' 10
60

40 c c'
5 5
a a
20 a' a'

0 0
Control Vehicle LPS 10 50 100 200 Control Vehicle LPS 10 50 100 200

JSE ( g/ml) JSE ( g/ml)

14
(C) b
(D) c
140 b
b 12
c
120 a a
10
a a % NK activity
100 b
% Viability

8 b
80
6
60

4
40

20 2
a a
0 0
Control Vehicle LPS 10 50 100 200 Control Vehicle 10 50 100 200
JSE ( g/ml) JSE ( g/ml)

Fig. 5. JSE exerts immunomodulatory effects in vitro. PBMC differentiated into macrophages, with MPA, and co-cultured with Saccharomyces cerevisiae yeast were treated
with DMSO 0.01% (vehicle), LPS 1 ␮g/ml (positive control) or indicated concentrations of JSE for 24 h. The amount of Saccharomyces cerevisiae phagocytosed was measured by
flow cytometry (A), and NO production and H2 O2 release were quantified from the cell culture medium supernatants (B). Murine splenocytes were treated with LPS 1 ␮g/ml
or various concentrations of JSE for 48 h. Cell proliferation was measured with the MTT assay (C). K562 cells cultured in the presence of murine splenocytes were treated
with various concentrations of JSE for 48 h. NK activity was measured with the MTT assay and calculated as described in Section 2 (D). Results represent the mean ± standard
deviation (SD) of three independent experiments in quadruplicate. Lowercase letters indicate significant differences according to ANOVA test (P ≤ 0.05), followed by post hoc
Tukey tests.

efficient protocol to extract kaempferitrin from Justicia spicigera is Apoptosis is recognized as an efficient strategy for cancer
currently being performed in our laboratory. The HPLC results also chemotherapy and an indicator for cancer treatment and preven-
showed additional unidentified compounds with retention times tion. Previously, we showed that Justicia spicigera showed strong
of 9.8 min and 11.9–12.4 min as constituents of JSE. Experiments cytotoxic effects against HeLa cells (Jacobo-Salcedo et al., 2011).
currently in course in our group seek to identify these JSE com- In this study, we showed that JSE exerted cytotoxic effects and
ponents.Toxicological studies with natural products are needed to induced 35% apoptosis in HeLa cells, compared to untreated cells.
provide security in future clinical studies. In this study, we first eval- Although JSE showed lower pro-apoptotic potency on HeLa cells
uated the in vitro toxic effects of JSE using the comet assay. Only JSE compared to CDDP, this plant extract did not display toxic effects on
tested at concentrations of 400 and 1000 ␮g/ml showed significant the normal cells HaCaT (7% of apoptosis). In contrast, CDDP showed
(P ≤ 0.05) toxic effects. Then we evaluated the in vivo toxic effects of high toxic effects on normal cells (56% of apoptosis). The results
JSE by the acute toxicity test according to the Lorke method (1983). suggest that Justicia spicigera exerts cytotoxic effects against cancer
The results showed that the LD50 for JSE was >5000 mg/kg i.p. and cells with low toxic effects on human normal cells.
>5000 mg/kg o.p. However, the chronic toxicity study should be The JSE doses used to evaluate antitumor effects were selected
carried out in order to validate JSE safety on long term use. The based on their lack of toxic effects, as evaluated in acute toxic-
relative species Justicia pectoralis showed a LD50 of 3531 mg/kg o.p ity test and on preliminary studies carried out in our laboratory.
in Swiss albino mice and no symptoms of toxicity were reported Although the capability of JSE 100 mg/kg to decrease tumor weight
(Lagarto Parra et al., 2001). The results regarding the toxicological was 1.5-fold lower compared to CDDP 1 mg/kg, this plant extract
effects of JSE evaluated in vitro and in vivo suggest that this plant did not have any effect on body weight in mice (results not
might be considered as low toxic. shown). In contrast, CDDP reduced body weight by 11% (results
894 A.J. Alonso-Castro et al. / Journal of Ethnopharmacology 141 (2012) 888–894

not shown). In this study, JSE showed a tumor growth inhibition Alonso-Castro, A.J., Ortiz-Sánchez, E., Dominguez, F., López-Toledo, G., Chávez,
by 53%, which indicates that Justicia spicigera might be an impor- M., Ortiz-Tello, A.J., Garcia-Carranca, A., 2012. Antitumor effect of Cro-
ton lechleri Mull. Arg. (Euphorbiaceae). Journal of Ethnopharmacology 140,
tant source of antitumor compounds. It is highly desirable to find 438–442.
new antitumor agents with same or similar potency than drugs Caceres-Cortes, J.R., Cantu-Garza, F.A., Mendoza-Mata, M.T., Chavez-Gonzalez, M.A.,
currently used in chemotherapy, but with lower toxic effects. Jus- Ramos-Mandujano, G., Zambrano-Ramirez, I.R., 2001. Cytotoxic activity of Jus-
ticia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in cell
ticia spicigera showed a similar antitumor potency compared to cycle dependent fashion. Phytotherapy Research 15, 691–697.
CDDP, but showed much lower toxic effects in vitro and in vivo Domínguez, X., Achenbach, H., González, C.C., Ferré-D’Amare, A.R., 1990. Estudio
than CDDP. químico del muitle (Justicia spicigera). Revista Latinoamericana de Química 21,
142–143.
Immunosuppression is one of the main obstacles in chemo- and
Esteban, M.A., Mulero, V., Muñoz, J., Meseguer, J., 1998. Methodological aspects of
radiotherapy. Therefore, it is necessary to find new antitumor drugs assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream
which can potentiate the immune system. Activated macrophages (Sparus aurata L.) by flow cytometry and electron microscopy. Cell and Tissue
Research 293, 133–141.
play an important role in the immune system against tumor growth
Euler, K.L., Alam, M., 1982. Isolation of kaempferitrin from Justicia spicigera. Journal
due to their ability of phagocytosis, synthesis and release of NO of Natural Products 45, 220–221.
and H2 O2 , which are considered cytotoxic agents toward certain Green, L.C., Luzuriaga, K.R., Wagner, D.A., Rand, W., Istfan, N., Young, V.R., Tan-
tumors (Mavier and Edgington, 1984). The NO and H2 O2 pro- nenbaum, S.R., 1981. Nitrate biosynthesis in man. Proceedings of the National
Academy of Sciences of the United States of America 78, 7764–7768.
duced by macrophages inhibit mitochondrial respiration and DNA Herrera-Arellano, A., Jaime-Delgado, M., Herrera-Alvarez, S., Oaxaca-Navarro, J.,
replication which affects essential enzymes in target cells (Hibbs Salazar-Martinez, E., 2009. Uso de terapia alternativa/complementaria en
et al., 1987). Spleen is involved in fighting the systemic infections, pacientes seropositivos a VIH. Revista Médica del Instituto Mexicano del Seguro
Social 47, 651–658.
whereas NK cells play a critical role in immune surveillance by the Hibbs Jr., J.B., Vavrin, Z., Taintor, R.R., 1987. l-Arginine is required for expression
secretion of cytokines such as IFN-␥ (Randy and Venkataraman, of the activated macrophage effector mechanism causing selective metabolic
2002). Previously, we showed that Justicia spicigera ethanol extracts inhibition in target cells. Journal of Immunology 138, 550–565.
Jacobo-Salcedo, M.d.R., Alonso-Castro, A.J., Salazar-Olivo, L.A., Carranza-Álvarez, C.,
tested at 100 ␮g/ml stimulated the proliferation of human periph- Gonzalez-Espindola, L.A., Dominguez, F., Maciel-Torres, S.P., Garcia-Lujan, C.,
eral blood mononuclear cells with similar potency as compared to Gonzalez-Martinez M.d.R., Gomez-Sanchez, M., Estrada-Castillon, E., Zapata-
phytohemagglutinin 1 ␮g/ml (Jacobo-Salcedo et al., 2011). In this Bustos, R., Medellin-Milan, P., Garcia-Carranca, A., 2011. Antimicrobial and
cytotoxic effects of Mexican medicinal plants. Natural Product Communications
study, we showed that Justicia spicigera stimulates the phagocy-
6, 1925–1928.
tosis, NO production and H2 O2 release with higher potency than Lagarto Parra, A., Silva Yhebra, R., Guerra Sardiñas, I., Iglesias Buela, L., 2001. Com-
LPS in human differentiated macrophages. Also, Justicia spicigera parative study of the assay of Artemia salina L. and the estimate of the medium
lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts.
stimulates the proliferation of murine splenocytes and induces the
Phytomedicine 8, 395–400.
activity of natural killer cells. Thus, Justicia spicigera exerts signif- Lin, T.Y., Liao, J.W., Chang, S.T., Wang, S.Y., 2011. Antidyslipidemic activity of hot-
icant immunomodulatory effects on immune responses mediated water extracts from leaves of Cinnamomum osmophloeum Kaneh. Phytotherapy
by macrophages, splenocytes and NK cells. Research 25, 1317–1322.
Looney, W.B., Mayo, A.A., Janners, M.Y., Mellon, J.G., Allen, P., Salak, D., Morris, H.P.,
In summary, this study shows that Justicia spicigera is a good 1971. Cell proliferation and tumor growth in hepatomas 3924A. Cancer Research
source of the flavonoid kaempferitrin. In addition, Justicia spicigera 31, 821–825.
exerts low toxic effects in vitro and in vivo, induces high toxic effects Lorke, D., 1983. A new approach to partial acute toxicity testing. Archives of Toxi-
cology 54, 275–287.
in vitro and in vivo against HeLa cells and exerts immunomodula- Márquez, A., Lara-Ochoa, F., Esquivel-Rodriguez, B., Mata, R., 1999. Plantas Medici-
tory activities in vitro. Thus, Justicia spicigera could be a good source nales de México II Composición, usos y actividad biológica. Universidad Nacional
of potential anticancer drugs due to its low toxicity, antitumor Autónoma de México, Distrito Federal, México, p. 178.
Mavier, P., Edgington, T.S., 1984. Human monocyte-mediated tumor cytotoxicity. I.
effects and immunomodulatory activities. Demonstration of an oxygen-dependent myeloperoxidase-independent mech-
Experiments are currently being carried out in our laboratory anism. Journal of Immunology 132, 1980–1986.
to isolate and evaluate active compound(s) from JSE responsible of Mitchell, M.S., 2003. Immunotherapy as part of combinations for the treatment of
cancer. International Immunopharmacology 3, 1051–1059.
toxic effects on HeLa cells in vitro and in vivo. Future results will
Ortiz-Sánchez, E., Daniels, T.R., Helguera, G., Martinez-Maza, O., Bonavida, B.,
describe the molecular mechanism of action by which JSE and its Penichet, M.L., 2009. Enhanced cytotoxicity of an anti-transferrin receptor
active compound(s) exert their cytotoxic effects on cancer cells. In IgG3-avidin fusion protein in combination with gambogic acid against human
malignant hematopoietic cells: functional relevance of iron, the receptor, and
addition, further experiments will evaluate the capability of Jus-
reactive oxygen species. Leukemia 23, 59–70.
ticia spicigera to stimulate immune system in an in vivo model. Pick, E., Mizel, D., 1981. Rapid microassays for the measurement of superoxide
Nevertheless, it is needed to realize more studies to elucidate the and hydrogen peroxide production by macrophages in culture using an auto-
immunomodulatory mechanisms. matic enzyme immunoassay reader. Journal of Immunological Methods 46,
2111–2126.
Randy, R.B., Venkataraman, S., 2002. Natural killer T (NKT) cells and their
Acknowledgments role in antitumor immunity. Critical Reviews in Oncology/Hematology 41,
287–298.
Rho, H.S., Ahn, S.M., Lee, B.C., Kim, M.K., Ghimeray, A.K., Jin, C.W., Cho, D.H., 2010.
AJAC (174493), GLT (199976) and OGR (205072) were endowed
Changes in flavonoid content and tyrosinase inhibitory activity in kenaf leaf
with graduate fellowships from CONACYT. We acknowledge gen- extract after far-infrared treatment. Bioorganic and Medicinal Chemical Letters
erous grant support from Instituto de Ciencia y Tecnología del 20, 7534–7536.
Gobierno del Distrito Federal (ICyT-GDF; GI/PIFUTP08-142 to AGC), Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for
quantitation of low levels of DNA damage in individual cells. Experimental Cell
and Consejo Nacional de Ciencia y Tecnología-México (CONACYT- Research 175, 184–191.
México grant 127822 to AGC). We thank Miriam Guido-Jimenez Tu, J., Sun, H.X., Ye, Y.P., 2008. Immunomodulatory and antitumor activity
and Rocio Mendez-Martinez for their technical assistance. of triterpenoid fractions from the rhizomes of Astilbe chinensis. Journal of
Ethnopharmacology 119, 266–271.
Vega-Avila, E., Espejo-Serna, A., Alarcon-Aguilar, F., Velazco-Lesama, R., 2009. Cyto-
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