Research Article
Ricardo Galeano Chena
Ricardo Galeano Chena
Corresponding
Author
Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Asunción, Paraguay.
E-mail: ricar.gal.08@gmail.com;
Tel.:+0590 48 30 59/ 0690996254
Laura Chaparro Aguilera
Laura Chaparro Aguilera
Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, Paraguay.
Gerardo Cebrián-Torrejón
Gerardo Cebrián-Torrejón
Corresponding
Authors
COVACHIM-M2E Laboratory EA 3592,
University of the Antilles, Fouillole Campus, UFR SEN, Department of Chemistry,
Pointe-à-Pitre Cedex, France.
E-mail: gerardo.cebrian-torrejon@univ-antilles.fr;
Tel.:+0590 48 30 59/ 0690996254
Fátima Piris da Motta
Fátima Piris da Motta
Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Asunción, Paraguay.
Abstract
Cannabis sativa L., commonly known as Marijuana, is a plant traditionally
used to satisfy ritual, medicinal and textile needs. Modern medicine has used
drugs derived from Cannabis for the
treatment of diverse symptomatologies that affect human beings such as
spasticity produced by multiple sclerosis and convulsions, among others. Despite
its medicinal background, its recreational use is one of the determining
factors that lead to the criminalization of its cultivation, in addition to the
growing market for its illegal trafficking. In view of the need to open the
debate and scientific research on the plant, the Paraguayan Congress passed the
law that creates a national program for the study and medical and scientific
research on the medicinal use of C. sativa and its derivatives. The objective of the present
work was to evaluate the extraction yields and in vitro antifungal activity
against mycelia of Alternaria solani,
the pathogen causing early blight in tomato crops. The treatment prepared from
the ethanolic extract of C. sativa with a final concentration of 10000 ppm
produced a 35% inhibition of the mycelium of A. solani while the positive control, consisting of a commercial
antifungal commonly used against the fungus, produced 100% fungal inhibition.
Although the result obtained with the plant extract could be considered as a
first study of this as a raw material for the creation of biocidal substances
for application in the agricultural field, it is necessary to develop more
studies on the target pathogen considering different concentrations of the
plant extract until reaching a value that can approximate the results exhibited
by the commercial fungicide.
Abstract Keywords
Alternaria
solani, Cannabis sativa, agricultural,
antifungal, Paraguayan, Marijuana.
1. Introduction
Cannabis sativa L. (Fig. 1a), commonly known as Marijuana, is a
vegetable used since different times by man as a source to satisfy his ritual,
textile and medicinal needs [1]. It is a
cosmopolitan plant, cultivated for certain purposes although it can also be
found wild, its seed possesses nutritional qualities as it contains fats,
fibers and proteins [2]. In recent
decades, modern medicine has used medicines derived from the plant for the
treatment of various symptomatologies that affect humans such as spasticity
produced by multiple sclerosis, control of nausea and vomiting derived from
chemotherapeutic treatments and convulsions, among others [3]. In
Paraguay, the controversy surrounding the plant is generated because its
cultivation is illicit [4].
Cannabis is a plant with potential biological activity on
certain test organisms, this quality could be due to the complex interaction
between its constituent metabolites [5-6]. Tetrahydrocannabinol (Fig. 1b), known as THC, is one of the predominant
metabolites found in the Cannabis plant
being responsible for generating psychotropic effects in the consumer, altering
the central nervous system [2-3].
Promising biological activities of crude resin obtained
from South American varieties of cannabis as well as isolates of its key
components have been reported on bacterial and fungal strains [7], results
that stimulate the evaluation of the behavior of marijuana-derived substances
cultivated in these latitudes of the world.
Despite the historical background on various applications
of Cannabis, its recreational use is
one of the determining factors leading to the criminalization of its
cultivation and related relationships, another triggering phenomenon is the
growing illegal trafficking market of the plant, Paraguay being one of the
largest producers in South America [8]. However,
in recent years, through the debate between key actors in the field of illicit
drugs and pioneering state initiatives in certain countries around the world,
the legal bases have been established on issues related to the regulation of
the supply, cultivation, possession and domestic consumption of the plant, as
well as controls by ad hoc entities
and scientific research based on it [9-10].
The Congress of Paraguay enacted law number 6007 that "Creates the national program for the study and medical and scientific research of the medicinal use of the Cannabis plant and its derivatives" [11] and its regulatory decrees for the controlled production of the plant as well as the one that establishes the general conditions for the production and industrialization of Cannabis, granting powers of application, control and supervision to governmental entities competent in the matter [12]. The current regulation is a great step forward in the incipient path to research with Cannabis focused on different areas of knowledge such as agriculture or medicine. Following this initiatives, we focused our attention on the application of Cannabis as a biopesticide against Alternaria solani Sorauer (Figure 1c). This genus of fungi presents a pathogenic potential that affects the agricultural sector causing early blight disease [13] in addition to presenting a high incidence in certain crops compared to other fungi and producing certain metabolites that are toxic to humans [14-15]. In addition, the emergence of pests resistant to synthetic biopesticides used in modern agricultural practices generates the need to explore novel and environmentally friendly alternatives in the fight against phytopathogens, a scenario that favors research on extracts of natural origin with pesticidal potential [16]. Current legislation in Paraguay opens the way for scientific research with the Cannabis plant and led this research with the objective of evaluating the extraction yield and in vitro antifungal activity of Marijuana extract on mycelia of Alternaria solani, in order to have background information that will allow the generation of new knowledge based on Cannabis, in addition to generating an approach that promotes critical thinking about the plant or its potential derivatives, trying to banish the idea that the plant can only be used for illicit purposes.
2.1 The
plant materials
The plant studied was collected in the city of Caaguazú
(Paraguay) as part of an operation carried out by the National Anti-Drug
Secretariat - SENAD "Cutting and destruction of marijuana
plantation". The sample was collected in April 2020. To ensure the
identity of the collected plant species, taxonomic identification was carried
out under the guidance of a botanist (Ms. Fátima Piris da Motta) from the
Botany area of the Faculty of Exact and Natural Sciences of the National
University of Asunción - FACEN UNA.
2.2 Extractive
work
The processes of drying, grinding and maceration of the
sample were carried out in the SENAD forensic laboratory, excluding stems and
lower leaves, leaving the buds and upper leaves of the plant for the
preparation of the extract.
The plant was ground with the help of an electric mill
until a fine powder was obtained. Once the powder was obtained, it was
macerated with absolute ethanol (EtOH).
The maceration was carried out during one week, making
intermediate agitations on the preparation so that the effect of the solvent
was as homogeneous as possible. Once the liquid extract was obtained, it was
filtered by gravity to separate the solid particles from the solution, after
which the solvent was evaporated with a rotary evaporator.
Figure 1. (a) Picture of Cannabis sativa,
(b) Structure of tetrahydrocannabinol (THC) ,
(c) Picture of culture of Alternaria solani
2.3 Antifungal activity
The isolates of Alternaria
solani used in this experiment were provided by the Phytomycology
laboratory of the Plant Protection area of the Faculty of Agricultural Sciences
of the National University of Asuncion - FCA UNA.
The antifungal activity assays were established using a
completely randomized design. Sterilized
potato-dextrose-agar (PDA) and medium technique poisoned with the treatment to
be tested [17] were used as culture medium. 0.1 mL of antibiotic
composed of Oxytetracycline was added to the PDA medium in order to inhibit
undesirable bacterial growth during the development of the experiment, then 100
mL of the medium was mixed with the plant extract to obtain a final
concentration of 10000 ppm. From this stock concentration, solutions of
concentrations of 5000 ppm, 2500 ppm and 1250 ppm respectively were prepared,
the culture medium being the solvent. A commercial reference antifungal
Acronis® (thiophanate-methyl) was used as a negative control and the positive
control was the pathogen in an untreated culture medium. The solutions of
different concentrations, as well as the controls were added in triplicate in
Petri dishes and allowed to stand for twenty-four hours at room temperature of
35ºC to check the sterility of the experimental units, after confirming the
viability of the treatments, the pathogen was seeded, extracting a disc of
purified Alternaria mycelium of 0.5 mm in diameter with the help of a sterile
metal punch and placed in the center of each plate. After seeding was completed
in the test treatments, the plates were kept at a temperature of 35°C. Measurement
of Alternaria growth diameter began forty-eight hours after sowing, measuring
the halo every twenty-four hours and ending once the positive control
completely covered the Petri dish containing it, for a total period of one
hundred and sixty-eight hours. The
fungal growth, recorded at 168 h from the start of incubation, in the presence
of treatments at different concentrations of Cannabis sativa shown in Table 1. The efficacy of each concentration of the Cannabis
extract-based treatment on mycelia was evaluated according to Abbot's formula
and classified according to the scale established by the International
Organization for Biological Control OIBL [18].
(i)
Percentage
of inhibition (%) = Control colony growth - colony growth concentration x 100
Table 1. Means of fungal growth, recorded at 168 h from
the start of incubation, in the presence of treatments at
different concentrations of Cannabis sativa.
Treatment |
n |
Means of fungal growth (cm) |
SD |
10000ppm |
3 |
0.4833 |
0.0058 |
5000ppm |
3 |
0.5183 |
0.0115 |
2500ppm |
3 |
0.5500 |
0.0050 |
1250ppm |
3 |
0.5567 |
0.0029 |
Control |
3 |
0.6000 |
0.0050 |
3 |
0.0000 |
0.0000 |
Control colony growth : Measurements of Alternaria spp. growth diameters were evaluated using INFOSTAT® software by applying the ANOVA test with a 5% error and then Tukey's test to identify statistically different groups.
3. Results and discussion
The response of Alternaria solani; in terms of inhibition against the different concentrations of the ethanolic extract of Cannabis was associated with the toxicity scale (Table 2).
Table 2. Toxicity level classification of treatments based on Cannabis sativa extract against Alternaria solani
Concentration
of EtOH Cannabis extracts |
Percentage of inhibition of Alternaria |
Scale
of |
10000 ppm |
35.10 |
slightly toxic |
5000 ppm |
25.35 |
Innocuous |
2500 ppm |
15.97 |
Innocuous |
1250 ppm |
13.92 |
Innocuous |
CP |
100.00 |
Toxic |
CN |
0.00 |
Innocuous |
CN: Pathogen in the absence of treatment
CP: Pathogen with commercial fungicide treatment *[18].
For the extraction of Cannabis, we followed the work of
Moreno-Sanz et al. [19], they evaluated the biological activities of Cannabis on cell lines involved in
hypoxia and inflammation processes and found different plant activities using
ethanol and hexane as working solvents respectively. In the present work, we
decided to work with ethanol extracts and the yield of extraction was
0.4%.
For the antifungal activity, the 10000 ppm concentration
of Cannabis extract produced the
highest percentage inhibition of Alternaria
solani mycelia. This finding is congruent with that obtained by Bashir et al. [13] where
higher concentration treatments prepared with 20% Cannabis sativa extracts were tested in vitro against Alternaria
solani.
The greatest
challenge of this study was to obtain authorization for the collection of the
plant. Since it is an illicit plant, whose cultivation and possession are penalized
in the country, they come from crop eradication operations carried out by law
enforcement authorities concerning the control of narcotic substances.
As a limitation within the work, the availability of the plant to obtain the extract to be tested. Access to this type of sample is subject to cultivar eradication operations carried out by law enforcement authorities in anti-drug matters. This makes it difficult to obtain homogeneous plant samples in terms of growth stage, climatic and agricultural conditions at the time of collection. In Paraguay, this is the first work carried out with an extract obtained from C. sativa of illicit origin, thus building the basis for future research with the plant.
4. Conclusions
The extract obtained from buds and upper leaves of the Cannabis sativa plant collected in a rural area of Paraguay showed modest antifungal activity against Alternaria solani. This could be a precedent for further research using extracts of the plant in order to design biocidal agents based on it and broaden the spectrum of natural substances for agricultural pest control. Future studies should focus on the highest concentration tested in this experiment as a starting point. It is necessary to have a significant amount of raw material in order to guarantee the obtaining of crude extracts to be tested, or to optimize the extraction process using techniques other than maceration, and the behavior of the marijuana extract should also be evaluated in vitro and in vivo experiments against other fungal strains and varieties of phytopathogens such as bacteria, viruses and parasites. In spite of the scarce background available on the use of the plant as an antifungal, this work aims to create a precedent to promote the study of marijuana with potential applications in the agricultural field.
List
of abbreviations
THC: Tetrahydrocannabinol
SENAD : National Anti-Drug Secretariat
UNA : National University of Asunción
FACEN : Faculty of Exact and Natural Sciences
FCA : Faculty of Agricultural Sciences
OIBL: International Organization for Biological
Control
PDA : Potato-dextrose-agar
EtOH : Ethanol
ANOVA : Analysis of variance
Ethical approval
Attached is Fiscal Note No. 285 issued by Attorney Osvaldo Esteban García Giménez, fiscal agent of the Specialized Unit in the Fight against Drug Trafficking - V Department -Caaguazú, authorizing the use of plant samples collected during the Caaguazú- Canindeyú operation carried out in Campos Morombi on April 4, 2020 in the Republic of Paraguay for the purposes set forth in notes dated March 16 and April 12, 2021 respectively sent by Lic. Mario Ricardo Galeano Chena, Director of the Specialized Forensic Directorate - DFE of the National Anti-Drug Secretariat - SENAD, Paraguay. In addition, enclosed is a copy of the legal proceeding act of delivery of samples by the intervening authorities and a favorable opinion issued by the Directorate of Legal and Juridical Affairs of the National Anti-Drug Secretariat - SENAD for the realization of the research project.
Authors’ contributions
Drafting and revision of the manuscript, field sampling, execution of experiments, revision of statistical analysis, management for obtaining legal permits for plant use, R.G; Literature review, Drafting and revision of the manuscript, statistical analysis, execution of experiments, L.C.; Taxonomic identification of the plant, revision of the manuscript, F.P.; Drafting and revision of the manuscript, G.C.
Acknowledgements
We would like to thank Dr. Adans Colman, professor at the Universidad Nacional de Asuncion for providing space and materials for the in vitro experiments. We also thank the Faculties of Agricultural Sciences and Exact and Natural Sciences of the Universidad Nacional de Asuncion. This work was part of the post-graduate thesis to obtain the degree of Master Scientiae in Organic Chemistry with Emphasis in Medicinal Phytochemistry and Bioactive Synthetics, FACEN UNA.
Funding
No external funds received
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
Conflicts of interest
The authors of this work declare that there is no conflict of interest.
References
1.
Boninia, S.; Premolia,
M.; Tambarob, S.; Kumarc, A.; Maccarinellia, G.; Memoa, M.; Mastinu A. Cannabis
sativa: A comprehensive ethnopharmacological review of a medicinal plant with a
long history. J. Ethnopharmacol.. 2018, 227, 300–315.
https://doi.org/10.1016/j.jep.2018.09.004
2.
Alonso, J.I.; Sánchez, M.; Torija, E.
Evolución histórica de la clasificación taxonómica del cáñamo Historical
evolution of taxonomic classification of hemp. Bol. R. Soc. Esp. Hist. Nat. 2021,115,
147–154. https://doi.org/10.29077/bol.115.e04.alonso
3.
MacCallum, C.A.; Russo, E.B. Practical considerations
in medical cannabis administration and dosing. Eur. J. Intern. Med. 2018, 49,
12–19. https://doi.org/10.1016/j.ejim.2018.01.004
4.
Presidency of the Republic of Paraguay,
"Ley No 1340/88 - Modifica y actualiza la Ley No 357/1972. Que reprime el
tráfico ilícito de estupefacientes y drogas peligrosas y otros delitos afines y
establece medidas de prevención y recuperación de farmacodependientes.
1988, Available online: https://www.mspbs.gov.py/dependencias/dnvs/adjunto/04944e-leyN13401988quereprimeeltraficoilicito.pdf
5.
Khan, B. A.; Warner, P.; Wang, H. Antibacterial
Properties of Hemp and Other Natural Fibre Plants: A Review. BioResources.
2014, 9, 3642–3659. https://doi.org/10.15376/biores.9.2.3642-3659
6.
Andre, C.M.; Hausman, J.; Guerriero, G. Cannabis
sativa : The Plant of the
Thousand and One. Molecules. 2016, 7, 1–17.
https://doi.org/10.3389/fpls.2016.00019
7.
Vozza, M.E.; Mendieta, J.R.; Villamonte, M.D.;
Colman, S.L.; Nercessian, D. Antifungal
and antibacterial activities of Cannabis sativa L. resins. J. Ethnopharmacol.
2024, 318, 116839. https://doi.org/10.1016/j.jep.2023.116839
8.
Program of Assistance Against Transnational
Organized Crime. Latin American Threat Assessment and Documentation Tool.
Transnational Organized Crime Threat Assessment in Latin America. 2021, Available online:
https://elpaccto.eu/wp-content/uploads/2021/07/Documento-web-2.MB_.pdf
9.
Álvarez, N.; Pose, N.; Luján, C. The
international politics of cannabis regulation in Uruguay. An analysis of the
Uruguayan response to the challenges and opportunities of the international
drug regime. Desafíos, 2017, 29, 19–59.
10.
Transnational Institute. Cannabis
regulation in Europe: a report on the Netherlands. 2019,
Available online: https://www.tni.org/es/publicacion/la-regulacion-del-cannabis-en-europa-informe-sobre-los-paises-bajos.
11.
Presidency of the Republic of Paraguay. Law
No 6007/2017 Which Creates the National Program for the Study and Medical and
Scientific Research of the Medicinal use of the Cannabis plant and its
derivatives. 2017. Available online:
https://www.bacn.gov.py/leyes-paraguayas/9911/ley-n-6007-crea-el-programa-nacional-para-el-estudio-y-la-investigacion-medica-y-cientifica-del-uso-medicinal-de-la-planta-de-cannabis-y-sus-derivados.
12.
Presidency of the Republic of Paraguay.
Decree 2725/2019 Whereby the general conditions for the production of
industrial hemp (non-psychoactive cannabis) are established. 2020. Available
online: https://baselegal.com.py/docs/aeeb9ada-33e4-11eb-a564-525400c761ca/text.
13.
Bashir, A.; Kan, M.T.; Raees, A.; Mehmood, B.; Yunas, M.T.;
Rehman, H.M.U.; Hussain, S. Efficiency of Selected Botanicals Against (Alternaria Solani) Causing Early Blight
Disease on Tomato in Azad Jammu and Kashmir. Pak. J. Phytopathol. 2020, 32,
179–186. https://doi.org/10.33866/phytopathol.030.02.0587
14.
Trigos, A.; Ramírez, K.; Salinas, A.
Presence of phytopathogenic fungi in fruits and vegetables and their relation
to food safety. Rev. Mex. Micol. 2008, 28, 125-129.
15.
Abrunhosa, L.; Morales, H.; Soares, C.; Calado,
T. Vila-Chã, A.S.; Pereira, M.;
Venâncio, A. Mycotoxins detected in food products in Portugal: A Review. Rev.
Bio Ciencias. 2012, 2, 5–31.
16.
Mesa, V.A.M.; Marín, P.A.; Ocampo, O.;
Calle, J.; Monsalve, Z. Fungicides from plant extracts: an alternative in the
integrated management of phytopathogenic fungi. J. Agric. Res. 2019, 45, 23-30.
17.
Fuentes,Y.M.O.; Chavez, E.C.; Flores,
J.L.L.; Camacho, S.H.; Ortiz,J.C.D. In vitro evaluation of the antifungal
activity of four methanolic plant extracts for the control of three species of
fusarium spp. Phyton (B. Aires). 2012, 81, 69-73.
https://doi.org/10.32604/phyton.2012.81.069
18.
Gonzale, L.C.; Garcia , B.L.M.; Nicao,
M.E.L.; Fernandez, A. R.; Arbernal M.G.S. In vitro effect of seven chemical
fungicides on Beauveria Bassiana. Fitosanidad. 2011,15, 31-38.
19.
Sanz, G.M.; Vera, C.F.; Carnerero, C.S.; Roura,
X.N.; Baena,V.S.M. Biological Activity of Cannabis sativa L. extracts
critically depends on solvent polarity and decarboxylation. Separations. 2020,
7, 1–16. https://doi.org/10.3390/separations7040056.
This work is licensed under the
Creative Commons Attribution
4.0
License (CC BY-NC 4.0).
Abstract
Cannabis sativa L., commonly known as Marijuana, is a plant traditionally
used to satisfy ritual, medicinal and textile needs. Modern medicine has used
drugs derived from Cannabis for the
treatment of diverse symptomatologies that affect human beings such as
spasticity produced by multiple sclerosis and convulsions, among others. Despite
its medicinal background, its recreational use is one of the determining
factors that lead to the criminalization of its cultivation, in addition to the
growing market for its illegal trafficking. In view of the need to open the
debate and scientific research on the plant, the Paraguayan Congress passed the
law that creates a national program for the study and medical and scientific
research on the medicinal use of C. sativa and its derivatives. The objective of the present
work was to evaluate the extraction yields and in vitro antifungal activity
against mycelia of Alternaria solani,
the pathogen causing early blight in tomato crops. The treatment prepared from
the ethanolic extract of C. sativa with a final concentration of 10000 ppm
produced a 35% inhibition of the mycelium of A. solani while the positive control, consisting of a commercial
antifungal commonly used against the fungus, produced 100% fungal inhibition.
Although the result obtained with the plant extract could be considered as a
first study of this as a raw material for the creation of biocidal substances
for application in the agricultural field, it is necessary to develop more
studies on the target pathogen considering different concentrations of the
plant extract until reaching a value that can approximate the results exhibited
by the commercial fungicide.
Abstract Keywords
Alternaria
solani, Cannabis sativa, agricultural,
antifungal, Paraguayan, Marijuana.
This work is licensed under the
Creative Commons Attribution
4.0
License (CC BY-NC 4.0).
Editor-in-Chief
Prof. Dr. Radosław Kowalski
This work is licensed under the
Creative Commons Attribution 4.0
License.(CC BY-NC 4.0).