Caracterización química de los extractos

La caracterización química de los extractos se realizó mediante métodos de análisis bioquímicos con determinaciones espectrofotométricas de proteínas, flavonoides y fenoles a partir de la representación de la curva patrón correspondiente a cada técnica con la medición de cuatro repeticiones de absorbancias de cada patrón. Fueron obtenidas tres curvas de calibración, donde se graficaron valores de absorbancia de cada patrón en función de la concentración (1919. Silva A de S e, de Magalhães WT, Moreira LM, Rocha MVP, Bastos AKP. Microwave-assisted extraction of polysaccharides from Arthrospira (Spirulina) platensis using the concept of green chemistry. Algal Res [Internet]. 2018 Nov 1 [cited 2024 Nov 26];35:178-84. Available from: https://www.sciencedirect.com/science/article/pii/S2211926418301668 ).

La cuantificación de proteínas de cada extracto se realizó mediante el método de Micro-Lowry (2020. Waterborg JH. The Lowry Method for Protein Quantitation. In: Walker JM, editor. The Protein Protocols Handbook [Internet]. Totowa, NJ: Humana Press; 2009 [cited 2024 Nov 26]. p. 7-10. Available from: https://doi.org/10.1007/978-1-59745-198-7_2 ), el contenido de fenoles según el método de Folin-Ciocalteau (2121. Singleton VL, Rossi JA. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic [Internet]. 1965 Jan 1 [cited 2024 Nov 26];16(3):144-58. Available from: https://www.ajevonline.org/content/16/3/144 ) y los flavonoides se determinaron utilizando un método espectrofotométrico (2222. Quettier-Deleu C, Gressier B, Vasseur J, Dine T, Brunet C, Luyckx M, et al. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J Ethnopharmacol. 2000 Sep;72(1-2):35-42.).

Evaluación de la actividad biológica de los extractos

Para determinar si la composición de proteínas, fenoles y flavonoides influían en la actividad biológica de los extractos, se les realizó la evaluación biológica a los extractos de menor (A) y mayor (B) contenido de dichos compuestos. Para esto, se realizó un experimento en el que se embebieron semillas de arroz cv. INCA LP-7, durante 24 h, con diferentes concentraciones (5; 1; 0,5; 0,05; 0,005 mg L^-1) de los extractos A y B.

Para la germinación, las semillas se colocaron en placas Petri (20 semillas por placa y cuatro placas por tratamiento) que contenían agua destilada. Los tratamientos conformados fueron los siguientes:

Las placas se colocaron en la cámara de germinación a 28 °C por siete días, evaluándose el número de semillas germinadas por placa a las 24, 48, 72 y 144 horas, con lo que se determinó el porcentaje final de germinación y la velocidad de germinación, y a los diez días, la masa seca de las radículas (25 radículas por tratamiento, cinco muestras de cinco radículas cada una).

Para el procesamiento de los datos, se calcularon las medias, las desviaciones estándar y los intervalos de confianza a α=0,05, mediante el programa Excel.

Caracterización química de los extractos

En la Tabla 1 se muestra el contenido de proteínas, fenoles y flavonoides presentes en los extractos analizados.

Como se muestra en la Tabla 1, las mayores concentraciones de proteínas se obtuvieron cuando se empleó EtOH al 70 % (extractos 3, 4, 7, 8), independientemente de la relación masa-solvente utilizada, por lo que una menor concentración de etanol favorece la presencia de proteínas en los extractos. Nótese que con la relación masa-solvente 1:20 y una maceración de 10 días (extracto 4), que constituye el extracto más económico, se obtuvo un buen contenido de proteínas; lo que sugiere que este extracto pudiera ser utilizado para la biofortificación de los cultivos.

Con el contenido de fenoles, ocurrió algo similar, mientras que las mayores concentraciones de flavonoides se obtuvieron en los extractos obtenidos con EtOH al 90 % (extractos 1, 2, 5, 6), independientemente, de la relación masa-solvente y el tiempo de maceración.

De esto se desprende, que la extracción con EtOH 70 % favoreció el contenido de proteínas solubles y de fenoles totales de los extractos, lo cual es lógico ya que al tener una cantidad de agua mayor se favorece la polaridad. Según Cepoi y cols (2009) (2323. Cepoi L, Ludmila R, Vera M, Angela C, Chiriac T, Sadovnic D. Antioxidative activity of ethanol extracts from Spirulina platensis and Nostoc linckia measured by various methods. Analele Univ Din Oradea Fasc Biol [Internet]. 2009 Nov 1;TOM XVI. Available from: https://www.researchgate.net/publication/40422973_Antioxidative_activity_of_ethanol_extracts_from_Spirulina_platensis_and_Nostoc_linckia_measured_by_various_methods ), la extracción de compuestos antioxidantes de biomasa de Spirulina fue más efectiva con una solución de etanol al 70 %. Igualmente, la actividad antioxidante se vio favorecida en extractos de Spirulina obtenidos con el etanol como solvente, por encima de otros solventes como H₂0, metanol y éter de petróleo (2424. Varsale A, Singh V, Mali S, Parihar P, Mane R. Phytochemical analysis, antioxidant and antifungal activity of different solvent extracts of Spirulina platensis collected from Rankala Lake, Kolhapur, Maharashtra. Phytochem Anal [Internet]. 2019;10(1):36-42. Available from: https://storage.unitedwebnetwork.com/files/521/ba6ab112b0e28c164f459c61b622daf1.pdf ). Por otra parte, la extracción de compuestos fenólicos totales se vio favorecida en extractos preparados con 50 % de etanol sobre los que se prepararon con 50 % de dimetil sulfóxido (2525. Martí-Quijal FJ, Ramon-Mascarell F, Pallarés N, Ferrer E, Berrada H, Phimolsiripol Y, et al. Extraction of Antioxidant Compounds and Pigments from Spirulina (Arthrospira platensis) Assisted by Pulsed Electric Fields and the Binary Mixture of Organic Solvents and Water. Appl Sci [Internet]. 2021 Jan [cited 2024 Nov 26];11(16):7629. Available from: https://www.mdpi.com/2076-3417/11/16/7629 ).

La maceración con la solución de EtOH 90 % incrementó el contenido de flavonoides totales, lo cual pudiera ser útil para favorecer cultivos sometidos a estrés ambiental, ya que se conoce el papel de estos compuestos en incrementar la actividad antioxidante (2626. Agustina S, Aidha NN, Oktarina E, Kurniati NF. Evaluation of Antioxidant and Wound Healing Activities of Spirulina sp. Extract. Egypt J Chem [Internet]. 2021 Aug 1 [cited 2024 Nov 26];64(8):4601-10. Available from: https://ejchem.journals.ekb.eg/article_162338.html ). Otros autores han obtenido altas concentraciones de flavonoides, alcaloides y saponinas de biomasa de Spirulina con la extracción con etanol absoluto (2727. Yuniati R, Zainuri M, Kusumaningrum H. Qualitative Tests of Secondary Metabolite Compounds in Ethanol Extract of Spirulina platensis from Karimun Jawa Sea, Indonesia. Biosaintifika J Biol Biol Educ [Internet]. 2020 Dec 25 [cited 2024 Nov 26];12(3):343-9. Available from: https://journal.unnes.ac.id/nju/biosaintifika/article/view/23153 ).

Es interesante destacar que las concentraciones de los compuestos evaluados no tuvieron diferencias significativas cuando se comparan los dos tiempos de maceración empleados. El factor tiempo se hace más significativo cuando se utilizan métodos como la ultrasonicación o la extracción asistida por microondas, no tanto la maceración (2828. Kong S, Wiratni W, Budiman A. Protein Extraction from Spirulina platensis by Using Ultrasound Assisted Extraction: Effect of Solvent Types and Extraction Time. Key Eng Mater [Internet]. 2021 Jan 11;872:33-7. Available from: https://www.researchgate.net/publication/348402116_Protein_Extraction_from_Spirulina_platensis_by_Using_Ultrasound_Assisted_Extraction_Effect_of_Solvent_Types_and_Extraction_Time ).

La presencia de proteínas, fenoles, flavonoides, etc. en los extractos de Spirulina, favorece la germinación de semillas, la formación de plantas sanas y equilibradas, logran un mayor rendimiento del cultivo, mayor vida poscosecha, mayor vigor de hoja, menos incidencia de enfermedades y mayor resistencia frente a estrés por factores climáticos o sequías (2929. Rahim A, Çakir C, Ozturk M, Şahin B, Soulaimani A, Sibaoueih M, et al. Chemical characterization and nutritional value of Spirulina platensis cultivated in natural conditions of Chichaoua region (Morocco). South Afr J Bot [Internet]. 2021 Sep 1 [cited 2024 Sep 23];141:235-42. Available from: https://www.sciencedirect.com/science/article/pii/S0254629921001770 -3131. López-Padrón I, Martínez-González L, Pérez-Domínguez G, Reyes-Guerrero Y, Núñez-Vázquez M, Cabrera-Rodríguez JA. Las algas y sus usos en la agricultura. Una visión actualizada. Cultiv Trop [Internet]. 2020 Aug 5 [cited 2024 May 14];41(2):e10-e10. Available from: https://ediciones.inca.edu.cu/index.php/ediciones/article/view/1554 ). Se debe tener en cuenta, que en los extractos, como se mencionó anteriormente, hay otros muchos compuestos que no se determinaron en este trabajo y que pueden influir en la actividad biológica de los mismos.

No obstante, con esta caracterización química limitada, se decidió seleccionar los extractos 1 y 3 como los extractos de menor y mayor contenido de proteínas y fenoles respectivamente, para evaluar su actividad biológica como estimulador de la germinación de semillas de arroz.

Actividad biológica de los extractos

La velocidad y el porcentaje final de germinación de las semillas de cada uno de los tratamientos se muestran en la Tabla 2. Como se puede apreciar, hubo influencia en el porcentaje final de germinación con dos concentraciones (5 y 0,5 mg L^-1) del extracto 3 aunque no hubo diferencias significativas en la velocidad de germinación, por lo que la mayor concentración de proteínas solubles y fenoles totales que presentó dicho extracto incrementó significativamente el porcentaje final de germinación de las semillas de arroz cv. INCA LP-7.

Los resultados de la masa seca de radículas mostraron que no hubo diferencias significativas entre tratamientos; sin embargo, la inmersión de las semillas en 5 mg L^-1 y 0,05 mg L^-1 del extracto 3, incrementaron en 8,2 y 10,3 % la masa seca de las radículas, respectivamente. Nótese que el tratamiento con 5 mg L^-1 de este extracto fue uno de los que incrementó significativamente el porcentaje final de germinación; confirmando la influencia que una mayor concentración de proteínas solubles y fenoles totales ejerció en la germinación y crecimiento inicial de las plántulas de arroz.

Las microalgas y sus extractos son estimulantes naturales que aceleran la germinación de las semillas y aumentan el vigor de las plántulas cuando se emplean en dosis relativamente bajas (3232. Sivalingam KM. Isolation, identification and evaluation of Spirulina platensis for its effect on seed germination of groundnut (Arachis hypogaea L.), Wolaita Sodo, Southern Ethiopia. J. Algal Biomass Utln. 2020;11(2):34-42. Available from: https://storage.unitedwebnetwork.com/files/521/4ca5cd59fe556a59877dcb110401ee19.pdf ). Varios estudios describen sus efectos beneficiosos en el porcentaje, índice y tiempo medio de germinación, así como en la longitud de la plúmula y de la radícula. Estos resultados se atribuyen a la activación de rutas enzimáticas claves para la fisiología de la germinación. Por ejemplo, la α-amilasa es una enzima sintetizada en la capa de aleurona y su expresión génica está regulada por las giberelinas. Esta enzima es responsable de la movilización de sustancias de reserva, como el almidón, desde el endospermo para apoyar el crecimiento y diferenciación del embrión (3333. Oliveira GE, Pinho RGV, Andrade T de, Pinho ÉV de RV, Santos CD dos, Veiga AD. Physiological quality and amylase enzyme expression in maize seeds. Ciênc E Agrotecnologia [Internet]. 2013 Feb [cited 2024 Sep 23];37:40-8. Available from: https://www.scielo.br/j/cagro/a/JJpGvzwnJkQ9Zx5JLQRN9xR/?lang=en ). Para demostrar esta hipótesis, recientemente, se informaron los efectos significativos que un extracto de Spirulina platensis ejercieron en el cultivo del maní (Arachis hypogaea L.), en indicadores tales como porcentaje de germinación de las semillas, longitud de la radícula y contenidos de proteínas y carbohidratos (3232. Sivalingam KM. Isolation, identification and evaluation of Spirulina platensis for its effect on seed germination of groundnut (Arachis hypogaea L.), Wolaita Sodo, Southern Ethiopia. J. Algal Biomass Utln. 2020;11(2):34-42. Available from: https://storage.unitedwebnetwork.com/files/521/4ca5cd59fe556a59877dcb110401ee19.pdf ). En otro estudio, se informó sobre la mejora del vigor, la calidad y la germinación de semillas de frijol negro (Vigna mungo L.), correlacionado con el incremento del ácido giberélico y la actividad de la enzima α-amilasa, al ser embebidas en un extracto de Spirulina platensis al 1,5 % durante 3 horas (3434. Field Crop Research Institute, VAAS, Vietnam, Thinh NQ. Influences of seed priming with Spirulina platensisextract on seed quality properties in black gram (Vigna mungo L.). Vietnam J Sci Technol Eng [Internet]. 2021 Mar 31 [cited 2024 Nov 26];63(1):36-41. Available from: https://vietnamscience.vjst.vn/index.php/VJSTE/article/view/388/268 ). Por otra parte, se encontró que la utilización de un extracto metanólico (0,25 %) favoreció casi todos los indicadores de crecimiento, el contenido de nutrientes, los componentes de rendimiento y el nivel de fitohormonas de Lupinus luteus (3535. Shedeed ZA, Gheda S, Elsanadily S, Alharbi K, Osman MEH. Spirulina platensis Biofertilization for Enhancing Growth, Photosynthetic Capacity and Yield of Lupinus luteus. Agriculture [Internet]. 2022 Jun [cited 2024 Nov 26];12(6):781. Available from: https://www.mdpi.com/2077-0472/12/6/781 ). En otra investigación realizada se encontró que al tratar semillas de tomate con 0,2 g/100 mL de un extracto de Spirulina, se obtuvo una tasa de germinación de 86,7 % (3636. Iuliana Neag E, Stupar Z, Roman C. EFFECT OF SPIRULINA SPP. EXTRACTS ON TOMATO AND ONION SEED GERMINATION |. AGRICULTURA [Internet]. 2022 [cited 2024 Nov 26];124(3-4). Available from: https://journals.usamvcluj.ro/index.php/agricultura/article/view/14437 ), además concentraciones de 25 y 50 % de dicho extracto tuvieron un efecto positivo en la germinación de semillas y el desarrollo de plántulas de lechuga (3737. Akgül F, Akgül R. The effect of Spirulina platensis (Gomont) Geitler extracts on seed germination of Lactuca. Alınteri Zirai Bilim Derg [Internet]. 2019 Dec 31 [cited 2024 Nov 26];34(2):148-53. Available from: https://ageconsearch.umn.edu/record/296790?v=pdf ).

Los resultados obtenidos en esta investigación, aunque son preliminares aún, mostraron la importancia que tiene la concentración de etanol y el tiempo de maceración en la composición y actividad como bioestimulante agrícola de los extractos de Spirulina; por lo que es necesario continuar profundizando en este sentido, con vistas a la obtención de extractos activos biológicamente que puedan ser utilizados satisfactoriamente en la agricultura.

Chemical characterization of the extracts

The chemical characterization of the extracts was carried out by biochemical analysis methods with spectrophotometric determinations of proteins, flavonoids and phenols from the representation of the standard curve corresponding to each technique with the measurement of four repetitions of absorbances of each standard. Three calibration curves were obtained, where absorbance values of each standard were plotted as a function of concentration (1919. Silva A de S e, de Magalhães WT, Moreira LM, Rocha MVP, Bastos AKP. Microwave-assisted extraction of polysaccharides from Arthrospira (Spirulina) platensis using the concept of green chemistry. Algal Res [Internet]. 2018 Nov 1 [cited 2024 Nov 26];35:178-84. Available from: https://www.sciencedirect.com/science/article/pii/S2211926418301668 ).

The quantification of proteins of each extract was performed using the Micro-Lowry method (2020. Waterborg JH. The Lowry Method for Protein Quantitation. In: Walker JM, editor. The Protein Protocols Handbook [Internet]. Totowa, NJ: Humana Press; 2009 [cited 2024 Nov 26]. p. 7-10. Available from: https://doi.org/10.1007/978-1-59745-198-7_2 ), the phenol content according to the Folin-Ciocalteau method (2121. Singleton VL, Rossi JA. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic [Internet]. 1965 Jan 1 [cited 2024 Nov 26];16(3):144-58. Available from: https://www.ajevonline.org/content/16/3/144 ) and flavonoids were determined using a spectrophotometric method (2222. Quettier-Deleu C, Gressier B, Vasseur J, Dine T, Brunet C, Luyckx M, et al. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J Ethnopharmacol. 2000 Sep;72(1-2):35-42.).

Evaluation of the biological activity of the extracts

To determine whether the composition of proteins, phenols and flavonoids influenced the biological activity of the extracts, biological evaluation was performed on the extracts with the lowest (A) and highest (B) content of these compounds. For this purpose, an experiment was carried out in which rice seeds cv. INCA LP-7, for 24 h, with different concentrations (5; 1; 0.5; 0.05; 0.005 mg L^-1) of A and B extracts.

For germination, seeds were placed in Petri dishes (20 seeds per dish and four dishes per treatment) containing distilled water. The treatments were as follows:

The plates were placed in the germination chamber at 28 °C for seven days, evaluating the number of germinated seeds per plate at 24, 48, 72 and 144 hours, determining the final germination percentage and germination speed, and after ten days, the dry mass of the radicles (25 radicles per treatment, five samples of five radicles each).

For data processing, means, standard deviations and confidence intervals at α=0.05 were calculated using Excel software.

Chemical characterization of the extracts

Table 1 shows the content of proteins, phenols and flavonoids present in the extracts analyzed.

As shown in Table 1, the highest protein concentrations were obtained when 70 % EtOH was used (extracts 3, 4, 7, 8), regardless of the mass-solvent ratio used, so that a lower ethanol concentration favors the presence of proteins in the extracts. Note that with a mass-solvent ratio of 1:20 and a 10-day maceration (extract 4), which is the most economical extract, a good protein content was obtained, suggesting that this extract could be used for crop biofortification.

With the phenol content, something similar occurred, while the highest concentrations of flavonoids were obtained in the extracts obtained with 90 % EtOH (extracts 1, 2, 5, 6), independently of the mass-solvent ratio and maceration time.

It follows that extraction with EtOH 70 % favored the content of soluble proteins and total phenols in the extracts, which is logical since the higher amount of water favors polarity. According some authors (2323. Cepoi L, Ludmila R, Vera M, Angela C, Chiriac T, Sadovnic D. Antioxidative activity of ethanol extracts from Spirulina platensis and Nostoc linckia measured by various methods. Analele Univ Din Oradea Fasc Biol [Internet]. 2009 Nov 1;TOM XVI. Available from: https://www.researchgate.net/publication/40422973_Antioxidative_activity_of_ethanol_extracts_from_Spirulina_platensis_and_Nostoc_linckia_measured_by_various_methods ), the extraction of antioxidant compounds from Spirulina biomass was more effective with a 70 % ethanol solution. Likewise, antioxidant activity was favored in Spirulina extracts obtained with ethanol as solvent over other solvents such as H₂0, methanol and petroleum ether (2424. Varsale A, Singh V, Mali S, Parihar P, Mane R. Phytochemical analysis, antioxidant and antifungal activity of different solvent extracts of Spirulina platensis collected from Rankala Lake, Kolhapur, Maharashtra. Phytochem Anal [Internet]. 2019;10(1):36-42. Available from: https://storage.unitedwebnetwork.com/files/521/ba6ab112b0e28c164f459c61b622daf1.pdf ). On the other hand, the extraction of total phenolic compounds was favored in extracts prepared with 50 % ethanol over those prepared with 50 % dimethyl sulfoxide (2525. Martí-Quijal FJ, Ramon-Mascarell F, Pallarés N, Ferrer E, Berrada H, Phimolsiripol Y, et al. Extraction of Antioxidant Compounds and Pigments from Spirulina (Arthrospira platensis) Assisted by Pulsed Electric Fields and the Binary Mixture of Organic Solvents and Water. Appl Sci [Internet]. 2021 Jan [cited 2024 Nov 26];11(16):7629. Available from: https://www.mdpi.com/2076-3417/11/16/7629 ).

Maceration with 90 % EtOH solution increased the content of total flavonoids, which could be useful to favor crops subjected to environmental stress, since the role of these compounds in increasing antioxidant activity is known (2626. Agustina S, Aidha NN, Oktarina E, Kurniati NF. Evaluation of Antioxidant and Wound Healing Activities of Spirulina sp. Extract. Egypt J Chem [Internet]. 2021 Aug 1 [cited 2024 Nov 26];64(8):4601-10. Available from: https://ejchem.journals.ekb.eg/article_162338.html ). Other authors have obtained high concentrations of flavonoids, alkaloids and saponins from Spirulina biomass with absolute ethanol extraction (2727. Yuniati R, Zainuri M, Kusumaningrum H. Qualitative Tests of Secondary Metabolite Compounds in Ethanol Extract of Spirulina platensis from Karimun Jawa Sea, Indonesia. Biosaintifika J Biol Biol Educ [Internet]. 2020 Dec 25 [cited 2024 Nov 26];12(3):343-9. Available from: https://journal.unnes.ac.id/nju/biosaintifika/article/view/23153 ).

It is interesting to note that the concentrations of the compounds evaluated did not differ significantly when comparing the two maceration times used. The time factor becomes more significant when using methods such as ultrasonication or microwave-assisted extraction, not so much maceration (2828. Kong S, Wiratni W, Budiman A. Protein Extraction from Spirulina platensis by Using Ultrasound Assisted Extraction: Effect of Solvent Types and Extraction Time. Key Eng Mater [Internet]. 2021 Jan 11;872:33-7. Available from: https://www.researchgate.net/publication/348402116_Protein_Extraction_from_Spirulina_platensis_by_Using_Ultrasound_Assisted_Extraction_Effect_of_Solvent_Types_and_Extraction_Time ).

The presence of proteins, phenols, flavonoids, etc. in Spirulina extracts favors seed germination, the formation of healthy and balanced plants, higher crop yields, longer post-harvest life, greater leaf vigor, less incidence of diseases and greater resistance to stress due to climatic factors or drought (2929. Rahim A, Çakir C, Ozturk M, Şahin B, Soulaimani A, Sibaoueih M, et al. Chemical characterization and nutritional value of Spirulina platensis cultivated in natural conditions of Chichaoua region (Morocco). South Afr J Bot [Internet]. 2021 Sep 1 [cited 2024 Sep 23];141:235-42. Available from: https://www.sciencedirect.com/science/article/pii/S0254629921001770 -3131. López-Padrón I, Martínez-González L, Pérez-Domínguez G, Reyes-Guerrero Y, Núñez-Vázquez M, Cabrera-Rodríguez JA. Las algas y sus usos en la agricultura. Una visión actualizada. Cultiv Trop [Internet]. 2020 Aug 5 [cited 2024 May 14];41(2):e10-e10. Available from: https://ediciones.inca.edu.cu/index.php/ediciones/article/view/1554 ). It should be taken into account that in the extracts, as mentioned above, there are many other compounds that were not determined in this work and that can influence their biological activity.

Nevertheless, with this limited chemical characterization, it was decided to select extracts 1 and 3 as the extracts with the lowest and highest protein and phenol content, respectively, to evaluate their biological activity as a stimulator of rice seed germination.

Biological activity of the extracts

The speed and final percentage of seed germination for each of the treatments are shown in Table 2. As can be seen, there was an influence on the final germination percentage with two concentrations (5 and 0.5 mg L^-1) of extract 3, although there were no significant differences in the germination speed, so that the higher concentration of soluble proteins and total phenols presented by this extract significantly increased the final germination percentage of rice seeds cv. INCA LP-7.

The results of radicle dry mass showed that there were no significant differences between treatments; however, the immersion of the seeds in 5 mg L^-1 and 0.05 mg L^-1 of extract 3 increased the dry mass of the radicles by 8.2 and 10.3 %, respectively. Note that the treatment with 5 mg L^-1 of this extract was one of those that significantly increased the final germination percentage; confirming the influence that a higher concentration of soluble proteins and total phenols exerted on the germination and initial growth of rice seedlings.

Microalgae and their extracts are natural stimulants that accelerate seed germination and increase seedling vigor when used at relatively low doses (3232. Sivalingam KM. Isolation, identification and evaluation of Spirulina platensis for its effect on seed germination of groundnut (Arachis hypogaea L.), Wolaita Sodo, Southern Ethiopia. J. Algal Biomass Utln. 2020;11(2):34-42. Available from: https://storage.unitedwebnetwork.com/files/521/4ca5cd59fe556a59877dcb110401ee19.pdf ). Several studies describe their beneficial effects on germination percentage, rate and mean germination time, as well as plumule and radicle length. These results are attributed to the activation of key enzymatic pathways for germination physiology. For example, α-amylase is an enzyme synthesized in the aleurone layer and its gene expression is regulated by gibberellins. This enzyme is responsible for the mobilization of reserve substances, such as starch, from the endosperm to support embryo growth and differentiation (3333. Oliveira GE, Pinho RGV, Andrade T de, Pinho ÉV de RV, Santos CD dos, Veiga AD. Physiological quality and amylase enzyme expression in maize seeds. Ciênc E Agrotecnologia [Internet]. 2013 Feb [cited 2024 Sep 23];37:40-8. Available from: https://www.scielo.br/j/cagro/a/JJpGvzwnJkQ9Zx5JLQRN9xR/?lang=en ). To demonstrate this hypothesis, the significant effects that an extract of Spirulina platensis exerted on peanut (Arachis hypogaea L.) crop on indicators such as seed germination percentage, radicle length, and protein and carbohydrate contents were recently reported (3232. Sivalingam KM. Isolation, identification and evaluation of Spirulina platensis for its effect on seed germination of groundnut (Arachis hypogaea L.), Wolaita Sodo, Southern Ethiopia. J. Algal Biomass Utln. 2020;11(2):34-42. Available from: https://storage.unitedwebnetwork.com/files/521/4ca5cd59fe556a59877dcb110401ee19.pdf ). In another study, enhanced vigor, quality and germination of black bean (Vigna mungo L.) seeds were reported to be correlated with increased gibberellic acid and α-amylase enzyme activity, when soaked in 1.5 % Spirulina platensis extract for 3 hours (3434. Field Crop Research Institute, VAAS, Vietnam, Thinh NQ. Influences of seed priming with Spirulina platensisextract on seed quality properties in black gram (Vigna mungo L.). Vietnam J Sci Technol Eng [Internet]. 2021 Mar 31 [cited 2024 Nov 26];63(1):36-41. Available from: https://vietnamscience.vjst.vn/index.php/VJSTE/article/view/388/268 ). On the other hand, it was found that the use of a methanolic extract (0.25 %) favored almost all growth indicators, nutrient content, yield components and phytohormone level of Lupinus luteus (3535. Shedeed ZA, Gheda S, Elsanadily S, Alharbi K, Osman MEH. Spirulina platensis Biofertilization for Enhancing Growth, Photosynthetic Capacity and Yield of Lupinus luteus. Agriculture [Internet]. 2022 Jun [cited 2024 Nov 26];12(6):781. Available from: https://www.mdpi.com/2077-0472/12/6/781 ). In another investigation, it was found that when tomato seeds were treated with 0.2 g 100 mL of Spirulina extract, a germination rate of 86.7 % was obtained (3636. Iuliana Neag E, Stupar Z, Roman C. EFFECT OF SPIRULINA SPP. EXTRACTS ON TOMATO AND ONION SEED GERMINATION |. AGRICULTURA [Internet]. 2022 [cited 2024 Nov 26];124(3-4). Available from: https://journals.usamvcluj.ro/index.php/agricultura/article/view/14437 ), and concentrations of 25 and 50 % of this extract had a positive effect on seed germination and the development of lettuce seedlings (3737. Akgül F, Akgül R. The effect of Spirulina platensis (Gomont) Geitler extracts on seed germination of Lactuca. Alınteri Zirai Bilim Derg [Internet]. 2019 Dec 31 [cited 2024 Nov 26];34(2):148-53. Available from: https://ageconsearch.umn.edu/record/296790?v=pdf ).

The results obtained in this research, although still preliminary, showed the importance of ethanol concentration and maceration time in the composition and activity as agricultural biostimulant of Spirulina extracts; therefore, it is necessary to continue deepening in this sense, with a view to obtaining biologically active extracts that can be satisfactorily used in agriculture.