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Leishmanicidal, Cytotoxic and Antifungal Activity of Medicinal Plants Used

Against Cutaneous Diseases in Mayan Traditional Medicine

Article · March 2015

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1 1793

Leishmanicidal, Cytotoxic and Antifungal Activity

of Medicinal Plants Used Against Cutaneous
Diseases in Mayan Traditional Medicine
Marina Vera-Ku
Autonomous University of Yucatan, Regional Research Center ―Dr. Hideyo Noguchi‖, Zoonosis department,
Mérida, Yucatán, México.
Email: mari77100@gmail.com
Martha Mena-Reynoso
Autonomous University of Yucatán, Faculty of Chemistry, Pharmacology Department, Mérida, Yuc. Mexico.
Email: martha.mena@correo.uady.mx
Diego Alpuche-Aguilar
Autonomous University of Yucatán, Faculty of Chemistry, Pharmacology Department, Mérida, Yuc. México.
Email: dgaa_12_879@hotmail.com
Rubi Gamboa-León
Autonomous University of Yucatan, Regional Research Center ―Dr. Hideyo Noguchi‖, Parasitology Department,
Merida, Yuc., México.
Email: miriamrubi@yahoo.com.mx
Miguel E. Rosado-Vallado
Autonomous University of Yucatan, Regional Research Center ―Dr. Hideyo Noguchi‖, Parasitology Department,
Merida, Yuc., México.
Email: rvallado@correo.uady.mx
ABSTRACT
Skin diseases are treated by the Mayan healers using no simple way to classify skin problems, due to their
several plants. Nine species currently used were selected, complex aetiology, however, symptoms caused by cancers,
for this study. Extracts of the roots, stems and leaves were infectious agents such as viruses, bacteria, fungi and
tested against several pathogen agents such as Aspergillus protozoa are often treated with Mayan medicine, which
niger, Trichophytum mentagrophytes; Candida albicans, made our research more challenging. Under this premise,
Saccharomyces cerevisiae; Leishmania L. mexicana, and we looked for the species used in Mayan traditional
the cytotoxic activity was tested using the cell lines medicine for the treatment of cutaneous diseases, however,
MDCK, L-929 and Hep-2. The root extracts of Bonellia the ―Chiclero sore‖ (chech or perhaps, taacan) caused by
albiflora and Cnidoscolus souzae had significant activity Leishmania L. mexicana, have been clearly identified
against A. niger, S. cerevisiae and C. albicans among the Mayan and have several traditional treatments
(MIC=<1000, <500, and 3.90 µg/ml respectively). The such as baths prepared with fresh macerations, decoctions
leaves extract of B. albiflora and Platymiscium yucatanum or fresh crushed plasters [2].
were significantly active against L. mexicana (IC50 = 0.50 The leishmaniases have an important impact worldwide,
and 2.56 µg/ml respectively). This study contributes to with 0.7 and 1.2 million of visceral and cutaneous
confirm the importance of the preservation of the leishmaniasis cases respectively each year and there are
traditional medicine as a guide to new possible treatments estimated 20,000 to 40,000 leishmaniasis deaths per year
for Leishmaniasis, and also asserts the importance of the [3].
protection of species of restricted distribution (endemic)
from Yucatan. In Mexico, the Yucatan peninsula is recognized as an
endemic area for L. mexicana, which causes localized
Keywords - Mayan traditional medicine, screening cutaneous lesions [4]. Chemotherapy with antimonials is
medicinal plants, antifungal activity, Leishmania the first choice of treatment for leishmaniasis; however,
mexicana, leishmanicidal activity, cytotoxicity. the treatment is prolonged, expensive, and not very
effective [5, 6]. Despite the Mayan classification of other
1. INTRODUCTION cutaneous diseases is not according to the etiological agent,
In the Yucatan peninsula there is a rich knowledge related the clinical manifestations gave us a hint about their
to the medicinal use of several plants since ancestral times possible origin [7, 8]. Cutaneous leishmaniasis is
[1] to cure some of the most common health problems in transmitted by the sandfly of the Lutzomya genera, There
the population. In the work developed by Roys [2], there is are about 20 species of Leishmania that may cause
a registry of Mayan traditional remedies to treat all kind of cutaneous leishmaniasis, .characterized by the appearing
diseases, however the most abundant of those are used to of a sore that the develops in an ulcer at the bite site, here
treat diarrheal, respiratory and cutaneous diseases. There is the amastigotes do not spread and the ulcers become

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1
visible either a few days or several months after the initial
bite, sometimes these ulcers heal spontaneously. The
diffuse cutaneous leishmaniasis type manifests itself when
the amastigote spreads through the skin in those
individuals with a defective immunity of T-cells. This type
of infection responds very poorly to drugs and therefore
causes ulcers in the entire host's body [9].
On the other side, the cutaneous diseases caused by fungi
and the rise of AIDS bring more complexity to the
problem, since there are also a large number of
opportunistic fungal infections associated to the disease.
This brought an interest in the development of new
antifungal treatments [10]. Some mycoses are produced by
cutaneous fungi belonging either to the Epidermophyton,
Microsporum or Trichophyton kinds, which are universal
and represent the most frequent mycoses in humans,
however one of the fungal genera of importance is
Candida, since they are one of the ten principal leading
causes of nosocomial infections in Mexico since 1999 [11]
and in those patients with candidemia, the attributable
mortality rate is 57% [12].
The existent treatments against such fungal diseases are
myconazole, clotrimazole, griseofulvin and itraconazole
which have several undesirable secondary effects [13-16].
As a mortality cause, cancer occupies the third place
worldwide [17, 18], and the second in Mexico [19, 20].
Surgery, radiation and chemotherapy are the treatment
against cancer [17] and their side effects are well known.
According to Mata Pinzón et al., [21], in traditional
medicine, this disease is considered as a nosological entity
which has symptoms such as blisters, abscesses,
ulcerations and wounds resistant to conventional
treatments and hard to heal. Considering this definition
together with the conceptualization of traditional medicine,
we should be sensitive to the differences and pay attention
to the traditional criteria [22].
Under this basis, we want to contribute to the search for
new alternative treatments against leishmaniasis, mycoses
and cancer. To select the plants for this study, a
bibliography search and ethnobotanical research
(interviews) were made, focusing the search in plants used
to treat skin diseases with fungal, protozoal and cancerous-
like symptoms. The interviews were performed according
to ethical considerations. The Mayan healers agreed to
share their knowledge because ―God gave it to them to
help others (Don Juan Bautista Kob, personal
communication), because it must be communicated to
preserve it, and because it is not right to be selfish with
knowledge‖ (María de Jesús Jiménez Chalé, 2007,
personal communication).
The obtained results were limited to plants that are
considered of restricted distribution (i.e. endemic or
cuasiendemic to the Yucatan peninsula), since they are an
important and unique natural resource [23].
1.1 Plant material
Nine plants were selected to develop this study and were
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1794
chosen according to the following criteria: a) plants should
be used in the Mayan traditional medicine, b) must be used
in the treatment of cutaneous diseases, c) must be endemic
to the Yucatan peninsula, d) the species must not have
been already studied previously for their antifungal,
cytotoxic or leishmanicidal activity according to the
NAPRALERT database (table 1).
The plants were scrutinized, identified and collected by the
authors and expert botanists, and voucher specimens were
prepared for each plant. They were later deposited at the
herbarium ―U Najil Tikin Xiw‖ of the Center of Scientific
Research of Yucatan (CICY) and also to the ―Alfredo
Barrera Marín‖ of The Autonomous University of Yucatan
(UADY).
The plants collected during this research were are listed in
alphabetical order in Table 1, and scientific names of plant
species were confirmed according to the ―Centro de
Investigación Científica de Yucatán (CICY), flora de la
península de Yucatán‖ database (website:
http://www.cicy.mx/sitios/flora%20digital/indice_busqued
a.php), the Missouri botanical garden database for tropicos
(website: http://www.tropicos.org/) and the plant list
(website http://www.theplantlist.org/). All mentioned
databases were searched in September, 2014.
1.2 Extract preparation
The collected plants were separated into leaves, stems and
roots, and then dried, and ground in a blade mill and
weighed. Then, 500 g of the grinded material of each plant
part were extracted using a Soxhlet equipment, and
methanol as extracting solvent for 24 hours. The 27
resulting crude extracts were concentrated with a rotary
evaporator and the resulting material was dried to constant
weight to obtain the yielding. The average yielding for
leaves was 32.46%, the stems were 10.87% and the roots
were 7.99%.
1.3 Biological evaluation
A search of the selected plants was made in the
NAPRALERT database and no previous reports about
their biological activities, ethnobotanical uses, or isolation
of components were found [24]. All the tests were
performed twice by triplicate and some of the standardized
methods and protocols as described by Pacciaroni et al.
[25].
1.3.1 Antifungal activity screening test
Saccharomyces cerevisiae (ATCC-287) and the
pathogenic yeast and fungi Candida albicans (ATCC
10231), Aspergillus niger (ATCC 16888) and
Trichophytum mentagrophytes (ATCC 9129) strains were
used to test antifungal activity. Yeasts were cultured either
for assay or for in YM broth Difco®. For subculture of
filamentous fungus Aspergillus niger (ATCC-16888) and
Trichophytum mentagrophytes (ATCC-9129) we used
Saboraud agar; and for the assay they were cultured in
Nutritive broth Difco®.
 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1 1795

Table1. Species of the selected plants, extracts abbreviations, Mayan name and Traditional uses.

Scientific name Family Collection Mayan Mayan Traditional use

(Abbreviation) number name
Acacia gaumeri Blake Fabaceae MVK-53 Catzim Used to make charcoal, mellific,
(AG) for wood and construction,
forage; medicinal for chills,
diarrhea, hemorrhoids b, d
Acalypha alopecuroides Jacq. Euphorbiaceae MVK-4 X-Mizbil Treatment for small-pox like
(AA) infections a
Cnidoscolus souzae Mc Vaugh Euphorbiaceae MVK-1 Tzintzin For the treatment of rheumatism,
(CS) chay retention of the afterbirth,
hemorrhoids and inflamed
protuberances, infection of the
gums, jaundice and biliousness,
falling or staggering a, pellagra e
Echites yucatanensis Millsp. Apocynaceae MVK-2 Kalis aak' c Snake bites, wound healing and
ex Stand. treatment of warts d
(EY)
Havardia albicans Britton and Fabaceae G. Hdz-3 Chukum Used for construction, as
Rose combustible, treating leather,
(HA) mellific, dye and for the
treatment of mouth sores, vaginal
cancer, diarrhea, insect bites and
dysentery b
Bonellia albiflora Lundell Primulaceae MVK-50 Sak k' inche' Poison and treatment of chronic
(BA) sores on the legs, and pustules a
Lonchocarpus rugosus Benth. Fabaceae MVK-52 Kanalzin, Anti-coughing, for treatment of
(LR) Kan-zin tuberculosis and asthma a, sores e
Neea choriophylla Standley Nictaginaceae MVK-3 Ta'tsi' c Against the bad winds and skin
(NC) infections d
Platymiscium yucatanum Fabaceae MVK-51 Zubín, Abdominal pain, snake bite a, bad
Standley Zubin-che' sores d
(PY)
Source: a[2]; b[26]; c[27], dMayan healers’ interviewe.

A solution of each methanolic extract was prepared to a
concentration of 4000 µg/mL using dimethylsulfoxide
(DMSO), as a positive control a solution of 50 μg/ml of
ketoconazole was used for the filamentous fungi, and 25
μg/ml were used for the yeasts. Two negative controls
were used: DMSO to a final concentration of 3.125 %, and
YM broth as growth control.
In a 96 wells microplate, an extract concentration of 1000
µg/mL was used on the first well, and then diluted to 500
µg/ml (1:2, v/v), until a final concentration of 0.49 µg/ml.
Strains were subcultured 24 h before the test in order to
obtain a metabolically active fungal culture. Then, 100 µl
of the culture were inoculated in 20 ml of YM broth for
yeasts and Nutritive broth for filamentous fungi. Both
were incubated until they reached turbidity of 0.5 of the
McFarland nephelometer which corresponds to 1.5-
2.0×106 UFC/ml. 100 µl of the solution were deposited in
each well to obtain a final concentration of 1×105 UFC/ml
for filamentous fungi and 1×103 UFC/ml for yeasts. The
plates were incubated for seven days for filamentous fungi,
and for 48 hours for yeasts. Later, there were added 75 µl
of the reagent (p-iodo-nitro-fenil-tetrazolium-violet) to
achieve concentration of 0.01% which was added to each
well and incubated in a shaker-water bath at 37°C for at
least 30 min. A change in color from clear to red-violet
indicates microorganism’s metabolic activity, which
makes it evident that the crude methanolic extract, at that
concentration, was not able to inhibit the fungus
development; also, the color absence indicates that the
extract had no effect against the fungus. All extracts were
tested by triplicate and the minimum Inhibitory
concentration (MIC) was detected. When the extracts
reached an inhibitory value ≤ 500 μg/ml, MIC was
determined, and endpoint was defined as the lowest
concentration of extract resulting in a total inhibition of
visual growth compared to the growth control wells
containing no antifungal [28].

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1
1.3.2 Leishmanicidal activity screening test
 Leishmania culture.
The Leishmania L. mexicana (strain: Hd18-
(MHET/MX/97/Hd18) was used to test the leishmanicidal
activity. The parasites were cultured in TC-199 culture
media supplemented with fetal bovine serum (FBS),
penicillin (100 UI/ml), and streptomycin (100 µg/ml).
After harvesting the parasites they were washed with
RPMI media (with antibiotics). The resulting pellet was
re-suspended in 1ml of media. An aliquot of 10 µL was
mixed with 90 µL of a 2% solution of formaldehyde
(dilution 1:10 v/v). The parasites were counted in a
Neubauer chamber.
 Leishmanicidal activity assay
The assay was performed as previously described by
Peraza-Sánchez et al. [7], in a 96 wells plate. The extracts
were dissolved using DMSO and supplemented culture
media. Each extract dissolution was sterilized with a
millipore filter (0.45 µm particle size mesh) and each well
was dosified with 100 µl of the corresponding extract
solution, to a final concentration of 1, 10, 100 µg/ml. then,
100 µl of the supplemented culture media containing 1×10
6 parasites/ml were added. DMSO final concentration on
any well was not over 0.5% since it would inhibit the
Leishmania promastigote growth. Amphotericin-B was
used as positive control to a final concentration of 1.0
μg/mL, growth control was RPMI supplemented media
and the negative control was DMSO up to a final
concentration of 0.5%.
For assay Leishmania promastigotes in logarithmic growth
phase were used. They were transferred to a plate and
incubated to 24 ºC for six days (early lag phase). The
leishmanicidal activity was determined by counting the
parasites in a Neubauer chamber on the third and sixth
days and then compared to the controls. The results were
registered as growth inhibition percentages and the
medium Inhibitory concentration (IC50) was determined
with the PROBIT 1.5 statistic program, and ANOVA test
was used to establish statistical significant differences
between test and control.
1.3.3 Cytotoxic activity screening test
 Cell culture
To measure the toxicity of the extracts to normal cells, the
strains MDCK (normal cells of canine kidney) and L-929
(normal cells of mouse conjunctive tissue, fibroblastic
morphology) were used, along with the cancer cell line
Hep-2 (human carcinoma of larynx) to provide an insight
to the activity of the extracts.
The normal mouse fibroblast cell line Sigma L-929 was
obtained from national dealer’s in vitro trade, the cell lines
MDCK (ATCC-CCL-34) and Hep-2 (ATCC-CCL-23),
were gracefully donated by Dr. Rosa Esther Moo Puc,
from the Yucatán Medical Research Unit, Medical Unit of
High Specialty, Medical Center Ignacio García Téllez,
Mexican Institute of Social Security (IMSS).
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1796
The MDCK and L-929 cell lines were cultured in the
Dulbecco’s Modified Eagle Medium (with GlutaMAX™ I,
high glucose, 110 mg/L sodium pyruvate, and pyridoxine
HCl). And the Hep-2 cell line was cultured in the
Advanced D-MEM (with D-glucose at 4500 mg/L, NEEA,
Sodium pyruvate at 110 mg/L, without L-glutamine). Both
media were also supplemented with 10 % of FBS,
Amphotericin B (5 μg/mL) as antifungal, and ceftazide
(100 μg/mL) as antibacterial. The cells were cultivated in
an incubator at 95% humidity, 37 ºC and 5 % CO2. The
culture was renewed whenever the cells reached 80 to 90%
of confluence. The cells were washed twice with
phosphate saline buffer (PSB) pH 7.2 and harvested by
adding Trypsin-EDTA and incubating them for 5 to 10
minutes. After inactivating the enzyme with 10 ml of
DMEM media an aliquot of 1×105 cells/ml was added to
the cell culture flask containing 10ml of the supplemented
corresponding media [29].
 Cytotoxic activity bioassay
This assay was performed according to the National
Cancer Institute (NCI) standards [30-32]. The cell lines
were cultured in DMEM-advanced and DMEM
supplemented media and incubated under the standard
conditions previously mentioned (section 2.3.3.1).
To have a successful bioassay the cells must be adapted
and growing in the test plate [26]; for this effect, 100 µl of
supplemented media culture containing 1×106 cells/ml
were added to a 96 wells plate and incubated for 24 to 48
hours.
Afterwards, the culture media was changed and 150 µl
were added to the cells attached to the plate.
The 27 methanolic crude extracts were dissolved in 100%
DMSO to a concentration of 10 mg/ml; from this solution,
10 µl (100 µg) were taken and diluted 1:10 v/v in culture
media (final concentration 1 µg/µl). From this dilution, 10
µl were put in the first well and then serial dilutions were
made to obtain four final extract concentrations (50, 25,
12.5, 6.25 μg/ml). Then the plate was incubated for 48 to
72 hours. The assay was performed in triplicates twice and
Vincristine was used as a positive control in four different
concentrations (0.5, 0.25, 0.125 and 0.0625 μg/ml).
DMSO was used as negative control to the above
mentioned concentrations (v/v percentage) and as a growth
control, culture media was used. After the incubation
period, the culture media was removed and 100 μl of PBS
were added to the MDCK and Hep-2 cell lines and
DMEM for the L-929. Then the 10 μl of each MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide) and PMS (Phenazine Methosulfate) reagents
were added and the plates were incubated for 1- 2 hours at
37ºC. Viable cells gave a violet precipitate. Following
incubation, the content of the wells was discarded and 100
μl of 2-propanol was added to the wells and the
microplates were left to room temperature for one hour.
The absorbance was read in a Multiskan™ ELISA reader
with a 540 and 620 nm wave length filter. The inhibition
of cellular growth was determined by comparing them to
the controls.
 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1 1797

2. RESULTS AND DISCUSSION sakurasosaponin as the main metabolite responsible for the
antifungal activity. In a work performed by Buitimea-
2.1 Antifungal activity Cantúa and collaborators [35] Jacquinia macrocarpa (i.e.
Bonellia macrocarpa) was tested against phytopatogenic
The extracts were considered active when their minimum
fungi of maize and the active fractions showed chitinase
inhibitory concentration (MIC) was less or equal to 1000
activity against polymeric extracts from fungi and
μg/ml [28]. According to the results shown in Table 2,
inhibited fungal β-1,3-glucanase activity acting as
only the root extract of C. souzae is active against C.
competitive inhibitors The authors established the
albicans and the root extract of B. albiflora is marginally
possibility that the antifungal fractions inhibit the β-1,3-
active against A. niger. If we compare our results to those
glucanase activity affecting β-1,3-glucan synthesis,
obtained by Silva and collaborators [33] we can see that in
causing the production of a defective cell wall. These
general, filamentous fungi are not as susceptible to plant
defects in the cell wall may allow the antifungal fractions
extracts as bacteria. Based on a previous screening work
to hydrolyze chitin causing delay in fungal growth. These
by Vera-Ku [26], García Sosa and collaborators [34] have
findings give us a hint in the possible mechanism of action
performed a bioassay-guided purification of the
of our extract, and will help us plan a more extensive
methanolic extract of the roots of Jacquinia flammea (i.e.
phytochemical and biological evaluation of B. albiflora.
Bonellia flammea) showed moderate antifungal activity
The most active extract was CSR which was selectively
against dermatophytes and very strong antifungal activity
aggressive against C. albicans. The leaves extract
against Colletotrichum gloeosporioide, a phytopahogenic
belonging to B. albiflora (BAH) was selectively active
fungi, using a combination of vacuum-liquid
against the S. cerevisiae yeast, and the root extract (BAR)
chromatography and high performance liquid
was active against Aspergillus niger.
chromatography, allowed the identification of

Table 2. Minimum Inhibitory Concentration (MIC) of methanolic extracts from Leaves (H), stems (T), and roots (R)
against the yeasts and filamentous fungi.
Minimum inhibitory concentration (μg/ml)
Extracts
a
C. a S. c A. n T. m
b
CSR 3.90 >1000 >1000 >1000
BAH >1000 500 >1000 >1000
BAT >1000 1000 >1000 >1000
BAR >1000 500 1000 >1000
Myconazole c 25 25 50 50
Vehicle d >1000 >1000 >1000 >1000
e
Culture Media >1000 >1000 >1000 >1000
a
C.a = Candida albicans; S.c = Saccharomyces cerevisiae; A.n = Aspergillus niger; T.m = Trichophyton
mentagrophytes; bnot active; cpositive control, dnegative control egrowth control. The active extracts are highlighted in
bold characters.
2.2 Leishmanicidal activity many of those plants are referred as poisons. This is the
case of B. albiflora. This plant was cytotoxic (Table 4),
The extracts tested against Leishmania, with an IC50 ≥100 and because it showed stronger activity than the control
µg/ml were not included in the table, and for the selection against L. mexicana, (Table 3), we selected the leaves
of plants for further study we considered of interest those (BAH) extract to develop further in vivo studies.
extracts with an IC50 of ≤20 µg/ml. Therefore, from the
methanolic evaluated extracts the ones with most activity Also of interest are the leaves extract of Platymiscium
against Leishmania promastigotes were those from the yucatanum (IC50 = 2.56 μg/ml) and the stems extract of C.
leaves of B. albiflora (IC50 = 0.50), the leaves of P. souzae (IC50 = 6.70 μg/ml) which showed good
yucatanum (IC50 = 2.56) and the stems of C. souzae (IC50 leishmanicidal activity and no cytotoxic activity (Table 3).
= 6.70). The crude extract activity of PYH (2.56 μg/ml) was close
to the Amphotericin-B control (1.0 μg/ml), and it is
Usually, medicinal plants used against cutaneous diseases probable that the active metabolite (s) could have an even
are toxic, but since they are used externally, toxicity is not stronger activity.
a problem for the remedies of the Mayan healers, despite

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1 1798

Table 3. Medium Inhibitory Concentration (IC50) and selectivity index (SI) of methanolic extracts from Leaves (H),
stems (T), and roots (R) against Leishmania L. mexicana LV4 strain.
Leishmania L. mexicana LV4 SI
Species Extracts
strain. IC50 (µg/ml) IC50 L-929/IC50LV4
Cnidoscolus souzae CST 6.70* 25.9
CSR 87.08 2.9
Echites yucatanensis EYH 17.96* 8.6
EYT 75.50 1.2
EYR 92.21 2.0
Bonellia albiflora BAH 0.50* 52.6
BAT 99.48 0.28
Lonchocarpus rugosus LRH 33.65
LRT 10.31* 12.8
Platymiscium yucatanum PYH 2.56* 145.6
Amphotericin-B a 1.0 -
Vehicle b - -
Culture Mediac >1000 -
a
positive control, b negative control, c growth control. H = Leaves, T = Stems, R = Root. The active extracts are
highlighted in bold characters. *Statistical difference: P ≤ 0.05 (ANOVA).

The ANOVA statistical program was run to all active
extracts (CST, BAH, PYH) and there was a significant
difference (P ≤ 0.05) between the tested extract
concentrations and the negative control.
According to our findings, it looks that the extracts of
some plants do not match completely with their
ethnobotanical use, and here we justify the importance of
testing the biological activity of plants reported as
medicinal, even when their traditional use does not match
the screening technique test.
Since the activities of P. yucatanum and B. albiflora were
detected only in the leaves extracts, we consider these
plants as renewable sources of extracts, and therefore we
selected those plants for further studies that include testing
the leishmanicidal activity against the promastigote stage
[7, 36, 37] and a set of in vivo tests are currently being
developed [38].
2.3 Cytotoxic activity
Four extract concentrations were tested (50, 25, 12.5, 6.25
μg/ml), and the cell viability and growth inhibition were
established. The correlation index was used to obtain the
IC50 concentrations using the Probit program and to
calculate the confidence interval (95%).
The extracts were considered active when they showed an
IC50 ≤ 20 µg/ml, according to the National Cancer Institute
criteria [31, 32]. In the Table 4, we listed the IC50 of the
extracts and their confidence interval (95%) respect to the
cell lines MDCK (kidney), L-929 (subcutaneous
connective tissue) and Hep-2 (epidermis carcinoma).
Only the extracts of B. albiflora showed activity against
two normal cellular lines (MDCK and L-929) and the
leaves and root extracts (BAH and BAR respectively)
were active against the cancer line Hep-2 (IC50 = 18.893,
and 19.156 μg/ml respectively).
Since the simpler approach to detect promising extracts
from ancestral knowledge, would be based on the
ethnobotanical information, we screened nine plants used
against skin problems by the Mayan traditional medicine.
From the first screening against fungi, we found the C.
souzae root extract (CSR) as a promising source of new
metabolites for the treatment of candidiasis (MIC = 3.9
μg/ml), Its crude extract was six times more active than
the myconazole control (MIC = 25 μg/ml) and the stems
extract (CST) was active against the Leishmania L.
mexicana LV4 strain (IC50 = 6.7 µg/ml), it was not
cytotoxic against normal cell lines, but slightly cytotoxic
to the cencerous line Hep-2, with a selectivity index of 4.2.
To date there are no other studies to our knowledge, about
this species. The CSR extract will be considered a leading
extract for further studies and for the development of new
products with antifungal activity. Since this plant is very
common in the Yucatan peninsula is our responsibility to
preserve and study this species for a better understanding
of its activity against C. albicans.
The leaves and root extracts of B. albiflora were active
against S. cerevisiae and the root extract (BAR) was
slightly active against A. niger (MIC = 1000 µg/ml) and
the leaves extract was particularly active against
Leishmania (IC50 = 0.5 µg/ml). This plant was also
slightly cytotoxic, however the selectivity index between
normal cell line L-929 and cancer cell line Hep-2 was
1.393 for the BAH extract and 1.203 for the BAR extract,
which was similar to that of vincristine: 1.213.

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1 1799

Table 4. Medium Inhibitory concentration (IC50) of the methanolic extracts against MDCK, L-929 and Hep-2 cell lines.
Medium Inhibitory Concentration
Species Extract IC50 (μg/ml)
MDCKa CI (95%) L-929b CI (95%) Hep-2c CI (95%)
AGH 41.077 (40.95-41.207) 57.438 (57.305-57.568) 116.332 (115.60-117.043)
A. gaumeri AGT 86.357 (85.914-86.804) 75.18 (74.84-75.527) 91.264 (90.909-91.62)
AGR 54.954 (54.662-55.24) 114.842 (114.153 -115.54) 121.060 (120.15-121.964)
AAH 35.645 (35.598-35.693) 64.447 (64.236-64.669) 56.689 (56.514-56.865)
A. alopecuroides AAT 56.559 (56.327-56.792) 127.879 (126.782-129.011) 89.392 (89.026-89.764)
AAR 62.03 (61.805-62.257) 125.879 (124.82-127.162) 56.702 (56.577-56.839)
BAH 26.473 (26.422-26.527) 26.333 (26.302-26.363) 18.893 (18.868 -18.920)
B. albiflora BAT 29.054 (29.028-29.075) 28.093 (28.062-28.129) 24.110 (24.075-24.147)
BAR 20.573 (20.548-20.594) 23.057 (23.036-23.081) 19.156 (19.134-19.180)
CSH 2896.01 (2772.732-3026.936) 3119.61 (2859.33-3415.56) 51.547 (51.363-51.728)
C. souzae CST 257.573 254.633-260.627) 173.500 (171.747-175.272) 89.228 (88.749-89.709)
CSR 337.21 332.166 – 342.331) 252.058 (249.118-255.092) 60.256 (60.115-60.409)
EYH 269.153 (265.836-272.599) 155.06 (153.762-156.345) 87.619 (87.223-88.038)
E. yucatanensis EYT 48.239 (48.136-48.338) 107.77 (107.022-108.51) 92.427 (91.962-92.895)
EYR 75.128 (74.874-75.376) 71.532 (71.221-71.858) 153.462 (152.19-154.783)
HAH 27.746 (27.714-27.784) 49.888 (49.812-49.958) 95.345 (94.892-95.82)
H. albicans HAT 34.222 (34.140-34.299) 38.842 (38.762-38.924) 80.742 (80.415-81.059)
HAR 62.287 (62.012-62.579) 27.791 (27.679-27.906) 133.506 (132.66 -134.38)
LRH 149.830 (148.675 -151.024) 200.171 (198.314-202.025) 75.928 (75.481-76.397)
L. rugosus LRT 291.273 (287.280-295.420) 132.099 (130.808-133.403) 60.884 (60.668-61.09)
LRR 1483.884 (1436.379-1533.503) 804.266 (785.773-823.464) 182.642 (181.092-184.24)
NCH 61.108 60.947-61.268) 111.712 (111.135-112.303) 187.413 (185.085-189.80)
N. choriophylla NCT 45.909 (45.798-46.027) 144.245 (143.293-145.23) 362.911 (356.553-369.42)
NCR 47.195 (47.084-47.299) 132.130 (131.277-132.991) 295.325 (290.47-300.257)
PYH 42.894 (42.805-42.980) 372.649 (366.991-378.492) 94.798 (94.35-95.258)
P. yucatanum PYT 38.797 (38.713-38.880) 169.356 (167.907-170.859) 157.616 (156.415-158.85)
PYR 34.316 (34.264-34.365) 101.883 (101.349-102.426) 102.329 (101.857-102.83)
Vincristinea 0.097 (0.097-0.097) 0.131 (0.131-0.132) 0.108 (0.108-0.109)
Vehicleb - - -
Culture Mediac - - -
Cell lines = MDCK, L-929, Hep-2; positive control a, negative control b, Growth control c, CI95%) = 95% confidence
Interval.

B. albiflora, also known as Muy-ak, has been reported to
be used as poison and for the cure of chronic sores on the
legs and pustules [2]. Hitherto, there is no other
information to our knowledge regarding the biological
activity or ethnobotanical use of this endemic plant.
However, [26] reported a phenotypically similar species,
Bonellia flammea (syn. Jacquinia flammea), which
showed radical scavenger, β-carotene protective activity,
inhibitory activity against Bacillus subtilis and antifungal
activity against C. albicans, S. cerevisiae, Criptococcus
neoformans, Aspergillus flavus, Microsporum canis,
Microsporum gypseum, Trichophyton mentagrophytes,
Trichophyton rubrum, and Epidermophyton floccosum.
Additionally, it displayed a strong cytotoxic activity
against a panel of more than 60 cell lines used by the NCI
for screening extracts [26]. Based on this information,
Bonellia albiflora was selected for further cytotoxicity and
antifungal testing and also Cnidoscolus souzae, since it
showed a good potential for the search of new active
substances with antifungal properties since it had a
particularly strong activity against C. albicans, with a
lower MIC than the positive control used.
In general, most extracts were not cytotoxic, which we
consider very positive since this characteristic is ideal for
antifungals, however the extracts of the Bonellia albiflora
leaves (IC50 = 18.89 μg/ml) and root (IC50 = 19.16 μg/ml)
against Hep-2 cell line show that the activity is linked to a
cytotoxic effect that interferes with the functionality of
cells. That explains also the recommendation of the Mayan
healers (María de Jesús Jiménez-Chalé and Felipa Cetina
Moo, 2008) that becase of the poisonous nature of this
plant, it must be used only topically.
Finally it is known that potent extracts not necessarily
result in good drugs, and crucial parameters along the drug
discovery process such as the selectivity index, and other
physicochemical properties relate to desirable
pharmacological qualities. In this case, of the species
tested, over half were active, and all but one (P.
yucatanum) displayed a remarkable SI of 145.6 for the

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 International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.48, Issue.1
Leishmania promastigotes (Table 3) as indicated by a lack
of cytotoxicity (IC50 = 372.649 μg/ml) against cultured L-
929 cells. As the SI demonstrates the differential activity
of an extract, the greater the SI value is, the more selective
it is. An SI value less than 2 indicates general toxicity of
the pure compound [39].
The results of this preliminary investigation support the
traditional knowledge of Mayan healers, and justify the
ethnomedical use of these plants as a promising method,
specifically, in leishmaniasis therapy, to result in new
leads for drug discovery.
3. CONCLUSIONS
This work strongly contributes to the knowledge of the
skin diseases healing properties of medicinal plants used
by the Mayan healers of the Yucatan Peninsula by
establishing the antifungal and leishmanicidal activities of
nine endemic medicinal plants.
CST, EYH, BAH and PYH (IC50 ≤ 10 µg/ml) extracts are
a potential source for future research of new active
metabolites for the treatment of leishmaniasis.
Our results give preliminary support the use of these plants
in the Mayan traditional medicine as a useful tool for
finding new promising extracts in the search for new
antifungal and leishmanicidal drugs, given the importance
of continue for the search towards new alternative
treatments of fungal diseases and leishmaniasis, and the
validation of their traditional use.
None of the tested extracts showed activity against the
filamentous fungi T. mentagrophytes, however, It is
important to point out that in traditional medicine most of
the plants are applied fresh, or as a decoction for a bath.
Usually, the composition of the healing remedy is very
different of the crude extract used for the tests which
broadens our field of study. Also, we believe that the
ethnobotanical search, and the plants screening are very
valuable tools to identify a particularly interesting species
such as C. souzae, responsible for the best activity against
C. albicans, and which is not reported in the literature as
used traditionally to treat fungal diseases, but was included
in the study because the Mayan Healer María de Jesús
Jiménez-Chalé said she used the latex to heal ―pellagra‖;
despite this term is medically associated to the lack of B3
vitamin (niacin), she associate the appearing of skin scales
and itching from fungal infections with this term.
Our results raised important questions such as: are we
following the right path to find new antifungals? Maybe
we should be testing potions instead of plants! [40] But if
we do so, we would be missing great extracts to develop in
new medicines. We know that the answer is not easy, and
we must be able to collaborate, be flexible, not selfish, and
help each other to solve the medical problems of mankind.
Conflict of Interest
Our research was conducted in the absence of any
commercial or financial relationships that could be
construed as a potential conflict of interest.
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1800
ACKNOWLEDGEMENT
We would like to aknowledge to the Mayan healers: María
de Jesús Jiménez Chalé, Felipa Cetina Moo, Juan Bautista
Kob, Jacinto Tzab for their generosity in sharing their
invaluable ancestral knowledge.
Also to the Autonomous University of Yucatan, for the
grant, PRIORI, SISTPROY No. FQUI-05-004, to develop
this project.
Also we would like to thank the Lic. Humberto Guerrero
López, Tech. Paulino Simá (CICY) and Dr. Gonzalo Mena
for their assistance in the collection and identification of
the selected plants. Also thanks to Dr. Zulema Cantillo-
Ciau for her kind assistance during the preparation of the
plant extracts, to QFB. Mirza Mut-Martín for her valuable
help in the development of the Leishmanicidal activity
assay, and special thanks to Dr. Rosa Moo for her kind
assistance and valuable supervision of the cytotoxicity
assay. We also appreciate the kind assistance of MSc.
Ashley King during the revision of his paper.
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