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Immunomodulating properties of
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 medigraphic Artemisa
en lnea

Revista Alergia Mxico 2005;52(3):105-12

Artculo original

Immunomodulating properties of Minthostachys verticillata on human

lymphocytes and basophils

Mara Laura Gonzlez Pereyra,* LN Cariddi,* F Ybarra,* MC Isola,** MS Demo,* L Sabini,* AM Maldonado*

RESUMEN
Antecedentes: Minthostachys verticillata (Griseb) Epling es una hierba medicinal originaria de Sudamrica que se utiliza como digestivo,
antiespasmdico, antiinflamatorio y broncodilatador en asma. Est demostrada la actividad antimicrobiana in vitro de esta hierba en
cepas de estafilococos y sus efectos antivirales en el VHS-1 y la cepa RC/79 del VPr.
Objetivo: determinar la capacidad inmunomoduladora de la decoccin y el aceite esencial de M. verticillata. Como un estudio comple-
mentario, se identificaron los principales componentes del aceite esencial.
Materiales y mtodos: se determin la actividad linfoproliferativa de ambos derivados vegetales y se compar con la expansin celular
inducida por PHA, Pokeweed y BCG en estudios citomorfolgicos. Se realiz un cultivo control sin estmulo. El recuento de clusters y
colonias de linfocitos se realiz con el mtodo descrito por Lange en 1989. Entre las clulas proliferadas, la subpoblacin de LT CD8+ se
caracteriz por inmunofluorescencia directa. La capacidad de las fracciones vegetales para producir degranulacin in vitro se prob al
utilizar basfilos provenientes de individuos no alrgicos y alrgicos a hongos ambientales. Se identificaron los principales componentes
del aceite esencial de M. verticillata mediante cromatografa gaseosa.
Resultados: los derivados de M. verticillata alcanzaron mayor proliferacin que los cultivos sin estmulo, demostraron actividad mitognica
e indujeron la formacin de colonias y clusters de linfocitos de manera similar a PHA, Pokeweed y BCG. Luego de la estimulacin con los
derivados, 40% de las clulas proliferadas fueron LT CD8+. La decoccin y el aceite esencial no alcanzaron el ndice mnimo de degranulacin
de basfilos en individuos alrgicos y no alrgicos, al menos en las concentraciones probadas. El anlisis por cromatografa gaseosa
revel pulegona y mentona como los componentes mayoritarios.
Conclusiones: los derivados de M. verticillata tuvieron efecto mitognico en LT e indujeron formacin significativa de clusters y colonias.
Las concentraciones utilizadas no causaron degranulacin de basfilos. Se asume que los derivados de M. verticillata induciran la
desviacin Th1 en cultivos celulares de pacientes alrgicos, lo cual disminuira las reacciones de hipersensibilidad. Algunos de los
componentes del aceite esencial, revelados por cromatografa gaseosa, podran ser los responsables de la actividad biolgica de estos
productos.
Palabras clave: Minthostachys verticillata, actividad mitognica, degranulacin de basfilos.

ABSTRACT

Background: Minthostachys verticillata (Griseb.) Epling is a South American traditional medicinal herb used as digestive, anti-spasmodic,
anti-inflammatory and bronchial dilator agent among other uses. Its anti-microbial activity against staphylococcal strains and its anti-viral
properties against HVS-1 and strain RC/79 of PrV have been demonstrated.
Objective: To determine the immunomodulating ability of M. verticillata decoction and essential oil. As a complementary study, the main
constituents of the essential oil were identified.
Materials and methods: Lymphocyte-proliferating activity of both vegetal derivatives was tested and compared with cellular expansion
induced by PHA, Pokeweed, CGB in cytomorphological study. A non-stimulate culture was used as control reference. The score of
lymphocyte clusters and colonies was performed using the method described by Lange. Among proliferated cells, LT CD8+ subpopulation
was characterized by direct immunofluorescence. The in vitro degranulant ability of the vegetal fractions was tested on basophils from
allergic and non-allergic individuals sensitized to environmental fungi. Essential oil components of M. verticillata were identified by gas
chromatography technique.
Results: M. verticillata derivatives reached higher proliferation levels compared to non-stimulated cultures, showed mitogenic activity and
induced cluster and colony formation similar to PHA, Pokeweed and CGB. Cells that proliferated after stimulation with derivatives showed
40% of LT CD8+. Tested concentrations of decoction and essential oil did not reach minimum degranulation indexes over basophils, from
both allergic and non-allergic individuals. Gas chromatography analysis revealed the presence of pulegone and menthone as the main
constituents.
Conclusions: M. verticillata derivatives were mitogenic over LT, inducing significant cluster and colony formation. There was no evidence
of degranulating ability over basophils at the concentrations tested. We assume that the derivatives from M. verticillata would induce Th1
deviation in cellular cultures from allergic patients, which would diminish hypersensitivity reactions. Some of the compounds of the
essential oil revealed by gas chromatography analysis may be responsible of the biological activity of these products.
Key words: Minthostachys verticillata, mitogenic activity, basophil degranulation.

Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005 105

Gonzlez Pereyra ML y col.
I
mmunomodulating phytomedicines include a
group of natural molecules capable of increasing
an organisms immune response. Many of these
purified substances, as well as plant extracts,
decoctions and other vegetal derivatives have
demonstrated immunomodulating activity. Examples
of these are: Hippophae rhamnoides,1 Boerhavia diffusa,2
Emblica officinalis, Evolvulus alsinoides,3 Perilla frutescens
(L.) Britton,4 among others.
Although active principles of plants have not been
all characterized, many superior plant decoctions are
known to have different activities such as inhibiting
bacterial growth, increasing the proliferative response
of lymphocytes and diminishing respiratory sympto-
matology caused by hypersensitivity reactions.5
Essential or volatile oils are products of intense
fragrance, extracted from aromatic plants. Although
they are mainly used in food and cosmetic industry,
many of them have other tested activities such as
antimicrobial properties.6 Numerous investigations
report antiviral and antibacterial activity of terpenoid
substances present in the essential oil fraction,
extracted from aromatic plants.7
Minthostachys verticillata (Griseb.) Epling, also
known as Minthostachys mollis and traditionally named
peperina, is an aromatic bush from the Labiatae
family that grows widely in the central and north-
western region of Argentina. It is mainly used in
infusions as digestive, anti-spasmodic, anti-
inflammatory and bronchial dilator agent, among
others. It is also used in industry to flavor liquors, cool
drinks and mixed herbal preparations.8
The essential oil of M. verticillata and decoction have
demonstrated to have antimicrobial activity against
some staphylococcal strains, and antiviral properties
against HSV-1 and strain RC/79 of PrV.7
* Departamento de Microbiologa e Inmunologa.
** Departamento de Biologa Molecular.
Universidad Nacional de Ro Cuarto, Provincia de Crdoba,
Argentina.
Address: Mara Laura Gonzlez Pereyra. Departamento de Micro-
biologa e Inmunologa. Universidad Nacional de Ro Cuarto, Ruta
36 km 601 - 5800 Ro Cuarto, Crdoba, Argentina.
E-mail: lauragonzalezp@yahoo.com.ar, ncariddi@yahoo.com.ar,
amaldonado@exa.unrc.edu.ar
Received: January, 2005. Accepted: March, 2005.
www.revistasmedicasmexicanas.com.mx
106
The objectives of this work were to investigate and
characterize the expansion of lymphocytes from
healthy individuals, cultured in presence of M.
verticillata decoction and essential oil, using the
expansion induced by PHA, Pokeweed and CGB as
control reference. The ability of these vegetal fractions,
used at different concentrations to induce or inhibit in
vitro basophil degranulation, was tested using human
basophils from dust mite, non-allergic individuals, and
allergic patients due to environmental fungi. As a
complementary study, main constituents of the
essential oil of M. verticillata were detected by gas
chromatography.
MATERIALS AND METHODS
Vegetal material
Green leaves and thin stems of Minthostachys verticillata
were used. They were collected from Santa Rosa de
Calamuchita hills, in the province of Crdoba,
Argentina, in April 2003.
The plant was identified by Dr. Margarita Grosso,
professor of elaborado
pdf the Botanical Department
en of Universidad
medigraphic
Nacional de Ro Cuarto (UNRC) and a voucher
specimen was stored in RCV (Ro Cuarto Vasculares)
herbarium under exsiccate N 1955. Washed and dried
material was grinded and stored in glass vials at - 20
C until used.
Decoction was obtained suspending 5 g of grinded
vegetal material in 100 mL of distilled water (final
concentration: 5%). The mixture was warmed at
boiling temperature for 20 min and clarified through
N 2 Whatman paper and 0.4 mm Millipore filter. The
percolated product was fractioned in dark glass vials,
sterilized in autoclave at 115 C for 20 min and stored
at -20C until used.9
Concentration of decoction was determined
according to dry weight of the vegetal residue, retained
in the Whattman paper, and the final volume obtained
after boiling. Final concentration for this vegetal
fraction was of 104 mg/mL.
For the preparation of essential oil, 60 g of grinded
material was hydrodistilled for 3 hours in a Clevenger-
type apparatus, yielding 4.8% of oil. This was separated
from the aqueous phase by density difference, dried
over Na2SO4 and stored at -20 C until used.10
Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005
 Immunomodulating properties of Minthostachys verticillata on human lymphocytes and basophils
Identification and quantification of essential oil
compounds of M. verticillata by gas chromatography
For the identification of main constituents of essential
oil of M. verticillata, gas chromatography analysis was
performed. A Shimatzu chromatography equipped
with a DB-5 column was used. The temperature of the
column was 60 to 240 C, with a 4 C/min increase.
The temperature of the injector was of 250 C. N2 was
used as carrier gas. The identification of the com-
pounds was made comparing their retention times
against standard pure drugs injected in the same
conditions. Quantification of components present in
the essential oil sample was made by measuring the
area under each peak of the chromatogram.11
Subjects
For lymphocyte proliferation assays, 20 healthy
individuals were evaluated. Ten non-allergic subjects
and 20 allergic patients, sensitized to dust mites and
environmental fungi, were studied for basophil
degranulation tests. The mean age ranged from 1 to
20 years. According to ethics, parents of underage
children were properly informed about the studies to
perform. Ten milliliters of peripheral blood were
obtained from each patient and collected in sterile
tubes containing heparin.
Lymphocyte isolation and culture
Blood samples were diluted 1/3 with Hanks Balanced Salt
Solution (HBSS) (Sigma, St. Louis, US), placed over
Hystopaque-1077 (Sigma, St. Louis, US) and centrifuged
at 2000 rpm for 20 min at room temperature. In order to
obtain lymphocytes, the interface was collected and
washed 3 times using Roswell Park Memorial Institute
medium (RPMI) -1640 (Sigma, St. Louis, US).
Cells (2 x 105/mL) were cultured in sterile vials
containing 2.5 mL of RPMI -1640, 10% of bovine fetal
serum and 1% of antibiotics (100 g/mL streptomycin
and 100 g/mL ampicyllin). Cultures were stimulated
with 0.25 mL of different mitogens: 10 g/mL lectin
from Phytolacca Americana (Pokeweed) (Sigma, St.
Louis, US), 10 g/mL phytohemaglutinina (PHA)
(Sigma, St. Louis, US) or 10 g/mL CGB vaccine
(Statens Serum Institute, Denmark).
Cellular expansion induced by mitogens was compared
with the one induced by 0.25 mL of 416 g/mL decoction
Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005
and 0.25 mL of 0.9 mg/mL essential oil of M. verticillata.
These concentrations were selected according to results
obtained in previous studies. A control lymphocyte culture
was prepared using RPMI -1640 and no stimuli.
Cell cultures were incubated for 48 hours at 37C
with 5% CO2 for lymphocyte proliferation assay and
72 hours for lymphocytes colony and cluster score.12,13
Lymphoblast harvest and scoring
After incubation, cultures were washed with acetic acid,
methanol (1:1) solution, and centrifuged at 2500 for 10
min to separate cells from medium. Fifty-microliter
aliquots were placed on slides, fixed for 20 min in
methanol and washed 3 times with 1 mM phosphate
balanced solution (PBS). Peripheral blood mononuclear
cells were stained with May-Grunwald solution
(MERK) for 4 min and Giemsa solution (MERK) for 15
min. Blast cells were scored under optic microscope with
1000X magnification. One-hundred cells were counted
and cell proliferation percentage was calculated. Blast
cells were differentiated from lymphocytes by their
increased nuclei and cytoplasm and by their condensed
chromatin. Proliferation rates obtained with PHA,
Pokeweed, CGB, decoction and essential oil of M.
verticillata and no-stimulation were compared according
to methods described by Margni, in 1989.12
Scoring for lymphocyte clusters and colonies
Scoring of colonies was adapted from the system
described by Lange in 1989. Aggregates of 3-20 cells
were regarded as clusters and aggregates of over 30
cells were considered colonies.13
The area of each colony and cluster was measured
in pixels using the accompanying software of the
Alphamager 2000 3.31 Image Analyzer (Alpha
Innotech Corporation). The mean total area in pixels
was used for statistical estimation.
Scoring of LT CD8 (+) by direct immunofluorescence
Among cells that proliferated after being stimulated
with decoction of M. verticillata, LT CD8 (+) were
characterized by direct immunofluorescence using an
anti-human CD8 monoclonal antibody conjugated
with FITC (Sigma, St. Louis, US).
Cells were washed twice with HBSS and PBS. Ten-
microliters of diluted anti-human CD8 monoclonal
107
Gonzlez Pereyra ML y col.

antibody were added and the suspension was RESULTS

incubated at room temperature for 30 minutes. Cells
were washed twice with 0.5 mL of sodium-azide-PBS
solution and then 15 mL of glycerin buffer were added.
Slides were observed under UV light epifluorescence
microscope. Values were expressed as percentage of
LT CD8 (+).14
Human basophil degranulation test
In vitro basophil degranulation test (HBDT) was
performed in order to compare the response induced
by the specific allergen with the one induced by
decoction and essential oil of M. verticillata, in both
allergic and non-allergic (control) individuals.
Different concentrations of decoction (210 g/mL;
104 g/mL; 10.4 g/mL, and 1.04 g/mL) and
essential oil (0.96 g/mL; 0.48 g/mL; 48 g /mL and
4.8 g/mL) derived from M. verticillata were tested.
The score of basophils was carried out using a Fuch-
Rosenthal hemocytometer. Degranulation indexes of
40% or more were considered as positive results.15
Statistical analysis
All values were expressed as a mean and standard
error of mean. Parameters were compared using
Statistica 4.5 software for Windows (StatSoft, Inc 1993).
Parameters were evaluated using the paired samples
t-test. A P value of 0.05 or less was taken as statistically
significant.
M. verticillata derivatives decoction and essential oil
reached higher proliferation rates than non-stimulated
cultures (p < 0.02). Those derivatives showed mitogenic
activity similar to PHA and Pokeweed and higher than
CGB (p < 0.02). No statistically significant difference
was found between proliferation rates induced by PHA
and Pokeweed and the ones induced by decoction and
essential oil of M. verticillata (figure 1).
Decoction and essential oil derived from M. verticillata
induced cluster and colony formation in number similar
to conventional mitogens and higher than non-
stimulated cultures (p < 0.05). Cellular proliferation rates,
induced by the conventional mitogens tested, were
considered normal in all cases (figure 2).
Although there were some few and little
lymphocyte clusters in some slides from the non-
stimulated cultures observed, no colonies were found
(figure 3a). Addition of PHA, Pokeweed or CGB (figure
3b) induced both colony and cluster formation in all
slides observed. Similar results were found when using
M. verticillata
pdf fractions, decoction
elaborado en (figure 3c) and
medigraphic
essential oil (figure 3d) as stimuli.
Total area of lymphocyte colonies and clusters was
measured in pixels. A statistically significant difference
was found when comparing the mean total area values
of clusters from non-stimulated cultures with values
of stimulated cultures. Mean colony size values from

85
NS
80
75 PHA
70 Pokeweed
65
60 CGB
55 Decoction
50
45 Essential oil
40
35
30
25
20
15
10
5
0
Stimulus

Figure 1.

108 Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005

 Immunomodulating properties of Minthostachys verticillata on human lymphocytes and basophils

NS cultures stimulated with PHA, Pokeweed and CGB

PHA
differed significantly from values found in cultures
Pokew eed
8 stimulated with decoction and essential oil of M.
CGB
7 Decoction verticillata (p < 0.05) (figure 4). No statistically
Essential oil
6 significant difference was found between the mean
5 area values induced by vegetal derivatives decoction
4 and essential oil.
Direct immunofluorescence technique revealed that
3
40% of the cells that proliferated under vegetal
2
derivatives stimuli were LT CD8 (+).
1 Basophils from allergic individuals resisted a
0 maximum concentration of 104 g/mL of decoction
Cluster Colonies
and 0.48 mg/mL of essential oil derived from M.
Figure 2.
verticillata.

Figure 3.

Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005 109

Gonzlez Pereyra ML y col.
80000
70000
60000
50000
40000
30000
20000
10000
0
Cluster
Figure 4.
Gas chromatography analysis revealed pulegone
and menthone presence in 58.70 and 26.29%
respectively as the main compounds. Other terpenoid
compounds such as -pinene (0. 72%) and -pinene
(1. 25%) were found in lower quantities.
DISCUSSION
Minthostachys verticillata is an herb commonly used in
folk medicine for its digestive effects, and lately it has
also been used as anti-inflammatory and bronchial
dilator agent in asthmatic disease. Studies performed
with this plant show it has in vitro antimicrobial and
antiviral activity; its ability for stimulating the immune
system is still being studied.7,10
Numerous investigators have evaluated a variety
of medicinal plants, testing their immunomodulatory
ability by different techniques.3,4,16,17
For evaluating immunomodulatory ability of M.
verticillata, the assays performed included in vitro
stimulation of lymphocyte proliferation with products
obtained from this herb: decoction and essential oil.
After 48 hours of incubation with the vegetal fractions,
human lymphocytes responded with cellular
expansion. Similar studies performed using a variety
of plants from different countries, including Argentina,
and utilizing similar techniques have demonstrated
that plant derivatives could be used as potential
positive modulators of the immune response.18,19
Evaluation of data referred to mitogenic properties
of M. verticillata derivatives, compared with the effect
of conventional vegetal lectins, such as PHA and
110
NS
PHA
Pokeweed
CGB
Decoction
Essential oil
Colonies
Pokeweed, revealed a similar lymphoproliferative
effect. This suggests potential application of products
derived from M. verticillata, in the evaluation of cell-
mediated immune response by conventional techniques
to test immunocompetence. If possible, it would offer
the additional advantage of working with non-toxic
vegetal fractions at much lower cost compared with
PHA, Pokeweed and other conventional mitogens.
pdfIn most investigations
elaborado lymphocyte
en proliferation is
medigraphic
demonstrated by H3-Thymidine incorporation to
cellular material.3 In our studies lymphoproliferative
ability of products derived from M. verticillata was
determined by direct observation under the light
microscope. Complementary analysis of lymphoproli-
ferative ability, based in lymphocyte cluster or colony
formation, diminishes subjective aspects of the
technique, validating the results. Figures presented in
results do not show ambiguity. Methods for the
lymphocyte clusters and colonies analysis were taken
form Chow, et al. 2001,20 researchers from China, a
country with a strong tradition in the use of plants
with phytomedical properties. These authors
investigated the effects of KY88 Liver Livo, a herbal
compound containing blupeurum on human
lymphocytes and observed results identical to ours.
It is known that vegetal mitogens stimulate cellular
populations selectively. PHA stimulates preferably T
cells; Pokeweed stimulates both T cells and B cells,
while CGB stimulates specifically T cells sensitized
with Mycobacterium tuberculosis. In many cases, CGB
stimulation increases highly T cell proliferation in
magnitude, comparable to non-specific mitogens.
Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005
 Immunomodulating properties of Minthostachys verticillata on human lymphocytes and basophils
When analyzing cell proliferation in cultures
stimulated with M. verticillata decoction, direct
immunofluorescence technique revealed that vegetal
derivatives positively modulate the Th1 response,
expanding the cellular population which develops into
cytotoxic T cells. These results do not differ from those
found by other authors, that have worked with
products derived from different vegetal species with
medicinal properties.21,22
According to Hanks Balanced Saline Solution
results, degranulation induced by M. verticillata
derivatives, at concentrations far higher than those
tolerated by basophils, could be considered unspecific
since there was no information proving patients had
been naturally sensitized with the plant.15
The normality threshold (DI value under which the
test is considered negative) of these assays is slightly
higher than the values established by others authors
in their techniques. In the present studies, we took as
reference the positivity criteria of Benveniste, 1981,
who considers HBDT as positive if DI is > 40%, when
the basophil score of the control well is < 60 cells/
mm3. So, we could say the DI normality threshold must
be established individually for each technique and
particular allergen.15
M. verticillata is placed among non-toxic vegetal
drugs that are authorized for their use with medicinal
purposes, accepted as an official drug in the
Farmacopea Nacional Argentina 6th edition. A research
group from UNRC has studied the toxic effect of
essential oil of M. verticillata over Vero cells
monolayers, demonstrating a CC50 of 3.9 mg/mL.
Gas chromatography revealed the presence of
pulegone and menthone in 58.70% and 26.29%
respectively as the main constituents and other
terpenoid compounds in lower quantities. Some of
these could be responsible for the compounds
biological activity. In the near future well evaluate the
immunomodulating activity on human lymphocytes
and basophils of each identified constituent separately.
Chemical composition of decoction has not been
studied yet. However, considering water and heat as
extraction agent and factor, we assume that biological
activity of decoction is due to hydrosoluble-
thermosetting substances such as flavonoids or
isoflavones.23
Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005
CONCLUSIONS
According to our results, we can conclude that
decoction and essential oil derived from M. verticillata
showed excellent in vitro mitogenic activity and could
be used as alternative non-toxic mitogenic reagents
in diagnostic assessment of cellular immunity. In
future studies, all proliferated cellular populations
could be studied in order to confirm if they really
follow the Th1 profile. Decoction and essential oil at
concentrations equal or lower than 104 mg/mL and
0.9 mg/mL respectively, will be studied in order to
assess their potential ability for inhibiting basophil
degranulation, induced by the specific allergen. If
concentrations of vegetal constituents for inhibiting
hypersensitivity reactions were found, the pathway
through which it is done could be investigated, since
their anti-inflammatory properties are traditionally
known.
AKNOWLEDGEMENTS
This work was supported by grant (Resolution N 077/
2003 Cod C18/116 Years 2003/2005) from SECyT,
Universidad Nacional de Ro Cuarto.
REFERENCES
1. Geetha S, Sai Ram M, Singh V, et al, Anti-oxidant and
immunomodulatory properties of seabuckthorn (Hippophae
rhamnoides) an in vitro study. J Ethnopharmacol 2002;79:373-
8.
2. Mehrotra S, Mishra KP, Maurya R, et al. Immunomodulation
by ethanolic extract of Boerhavia difusa roots. Int
Immunopharmacol 2002;2:987-96.
3. Ganju L, Karan D, Chanda S, et al. Immunomodulatory effects
of agents of plant origin. Biomed Pharmacother 2003;57:296-
300.
4. Ragainskiene O, Gailys V, Jankauskiene K, et al. Common
perilla (Perilla frutescens [L.] Britton.) as a perspective
immunomodulator. Medicina (Kaunas) 2004;40(3)220-4.
5. Gmez-Flores R, Caldern CL, Scheibel LW, et al.
Immunoenhancing properties of Plantago major leaf extract.
Phytother Res 2000;14(8):617-22.
6. Cowan MM. Plant products as antimicrobial agents. Clin
Microbiol Rev 1999;12:564-82.
7. Primo V, Rovera M. Determinacin de la actividad bacteriana
y antiviral del aceite esencial de Minthostachys verticillata
(Griseb.) Epling. Rev Argent Microbiol 2001;33:113-7.
8. Nuez C, Cantero J. Las plantas medicinales del sur de la
provincia de Crdoba. Editorial de la Fundacin. Universidad
Nacional de Ro Cuarto, 2000;pp:15-30.
111
 Gonzlez Pereyra ML y col.
9. Mongelli E, Desmarchelier C, Coussia J, et al. Actividad
antimicrobiana e interaccin con el ADN de plantas medicinales
de la amazonia peruana. Rev Arg Microbiol 1996;27:199-203.
10. De Feo V, Ricciardi A, Biscardi D. Chemical composition and
antimicrobial screening of the essential oil of Minthostachys
verticillata (Griseb.) Epl. (Lamiaceae). J Essent Oil Res
1998;10:61-65.
11. Zygadlo JA, Maestri DM, Lamarque AL, et al. Essential oil
variability of Minthostachys verticillata. Biochem Syst Ecol
1996;24,4:319-23.
12. Margni R. Transformacin linfocitaria. En: Inmunologa e
inmunoqumica. 5 ed. Buenos Aires: Mdica Panamericana,
1989.
13. Lange BJ. Growth of human leukemia cells in vitro. In: Baserga
R, ed. Cell growth and division: a practical approach. Oxford:
Oxford University Press 1989;pp:61-80.
14. Edidin M. Fluorescent labeling of cell surfaces. In: Wang YL,
Taylor DL, editors. Fluorescence microscopy of living cells in
culture. Part A: fluorescence analogs, labeling cells and basic
microscopy. New York: Academic Press, 1989.
15. Grinstein M, Simkin G, Alonso MR, et al. Test de degranulacin
de basfilos humanos como un mtodo in vitro para el diag-
nstico y prevencin de reacciones adversas a drogas. Rev
Arg Asma, Alerg Immunol 1986; 3(8):765-86.
16. Puri A, Sahai R, Singh KL, et al. Immunostimulant activity of
dry fruits and plant material used in Indian traditional medical
112
View publication stats
system for mothers after child birth and invalids. J
Ethnopharmacol 2002;71:89-92.
17. Diallo D, Paulsen BS, Liljebck TH, et al. The malian medi-
cinal plant Trichilia emetica; studies on polysaccharides with
complement fixing ability. J Ethnopharmacol 2003;84:279-
87.
18. Fernndez T, Cerd-Zolezzi P, Risco E, et al. Immunomo-
dulating properties of Argentine plants with ethnomedical use.
Phytomedicine 2002;9(6):546-52.
19. Suvachittanont W, Jaranchavanapet P. Mitogenic effect of
Parkia speciosa seed lectin oh human lymphocytes. Planta
Med 2002;66(8):699-704.
20. Chow LW, Loo WT, Sham JS. Effects of an herbal compound
containing bupleurum on human lymphocytes. Hong Kong Med
J 2001;7:408-13.
21. Ko E, Rho S, Lee EJ, et al. Traditional Korean medicine (SCRT)
modulate Th1/Th2 specific cytokine production in mice CD4+
T cell. J Ethnopharmacol 2004;92(1):121-8.
22. Kim TS, Kang BY, Cho D, et al. Induction of interleukin-12
production in mouse macrophages by berberine, a
benzodioxoloquinolizine alkaloid, deviates CD4+ T cells
from a Th2 to a Th1 response. Immunology 2003;109(3):
407-14.
23. Prez-Loyola M, Segarte-Naro FR, Ponce de Len O. Nueva
composicin de origen natural con aplicacin al ultrasonido
teraputico. Rev Cubana Plant Med 2000;5(3):114-7.
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Revista Alergia Mxico Volumen 52, Nm. 3, mayo-junio, 2005