In vitro free radical scavenging and antibacterial activity with plant extracts of ethnobotanical use in banana production

1. Introduction

Banana (Musa sp.) is one of the four most economical and important crops worldwide (FAO, 2023); however, its productivity is at risk because of the negative impacts of pests and diseases, most of which exhibit complex mechanisms to escape the traditional control strategies (Abdoussalami et al., 2023). Direct losses in agricultural productivity caused by pathogens, animals, and weeds range between 20 and 40%, whether caused by infection or during transportation and storage (Loranger-Merciris et al., 2023). The most important and dangerous losses have been caused by phytopathogenic bacteria. These include epidemics of bacterial wilt that affect a wide range of crops, including banana. Such epidemics are caused by species from the genera Ralstonia and Xanthomonas (Bhatt et al., 2024). Ralstonia solanacearum is identified as the second most important phytopathogenic bacteria from a scientific and economic point-of-view after Pseudomonas syringae (Wang et al., 2023). The incidence and level of damage caused by phytopathogenic organisms in banana crops like R. solanacearum is mainly due to favorable environmental conditions for the pathogen, the susceptibility of the varieties planted, the presence of infestation foci, the quality of the seed, and the presence of monocultures at different development stages (Premabati et al., 2020). On the other hand, in the post-harvest period, agricultural products can harbor human pathogens without showing any signs of deterioration; this poses a food safety risk. The emergence of multidrug-resistant pathogens such as Escherichia coli and other Enterobacteria in the food chain is a challenge for health authorities and can result in a series of human health issues. For instance, the control of foodborne diseases should begin in the field (Tenea et al., 2022; Alegbeleye et al., 2018).

Different strains exhibiting a multidrug-resistant phenotype survive and grow in a wide range of environmental conditions, having been detected in vegetables, fruits, and meats. These strains pose a high risk to human health; therefore, increasing the population demands to increasing security and safeguarding the integrity of our food supply and extending its useful life. Hence, there is a need to research new antimicrobials to combat its growth (Elik et al., 2019). The control of diseases associated with the presence of pathogenic microorganisms in agricultural foods is a strategy aligned with international needs in reducing post-harvest losses, food security, and clean production (Mostafidi et al., 2020). In addition, to comply with the modern standards of sustainability, there is a need for a transition towards new bioproducts as bio-controllers to replace the chemically synthesized traditional products (Ngegba et al., 2022). Traditionally used synthetical chemical products are widely implemented to treat undesirable microorganisms, but they generate potentially harmful residues in plants and the environment, resulting in consequences for biodiversity, water, and human health (Miller et al., 2022). Currently, the global contributions of biological control are highlighted in “One Health” models that seek solutions to problems of planetary health (Schaffner et al., 2024). The use of antibiotics in agriculture to combat bacteria is significant, where the main authorized antibiotics are streptomycin, oxytetracycline, kasugamycin, gentamicin, and oxolinic acid (Iwu et al., 2020). The primary issue associated with their use is the phenomenon of bacterial resistance to these antimicrobials, in particular to streptomycin (Haynes et al., 2020). Therefore, there is a primary need to find new, effective, safe, and affordable antibacterial drugs for the farming industry (De Winne and Peersman, 2021). The development of agricultural-derived control products and coatings for food with antimicrobial and antioxidant properties could strengthen production chains in the market. It is necessary to improve the quality of products, reduce the economic losses of farms due to product deterioration, and to mitigate potential losses due to climate change (Wang and Teplitski., 2023). Furthermore, implementing these biocontrol agents will contribute to sustainability and the protection of the health of consumers and the ecosystems associated with the production of agricultural products. It will also help avoid or reduce the use of agrochemicals (Lenzi et al., 2021). Plant extracts are of great pharmacological and agricultural interest due to their ability to reduce microbial growth in plants and control spoilage in many foods, improving their shelf life without compromising quality, managing to satisfy the consumer demand for safe and secure food (Nafees et al., 2023).

Since nature provides novel secondary metabolites, it offers a great opportunity to evaluate a wide variety of plant extracts in order to find new bioactive substances (Newman and Cragg, 2020). It is estimated that there are 2,404 species of medicinal plants currently used in Colombia, of which only 206 species have more than three documented references of their therapeutic or agricultural use (Diazgranados et al., 2022; Bernal et al., 2011). Despite these figures, biodiversity is suffering serious deterioration. Unfortunately, there is still a lack of investigation and conservation policies for the development of added value products from many of the species reported in the lists of threatened flora (Kor and Diazgranados, 2023). In Antioquia, the families of plants with medicinal use best represented are, according to the number of species, Asteraceae, Lamiaceae, Solanaceae, Amaranthaceae, Apiaceae, Fabaceae, and Urticaceae. These are associated with almost all types of use or medical categories (Fonnegra-Gómez and Villa-Londoño, 2011).

To assess the potential use given to plants in Antioquia, the objective of this research was to determine the in vitro antioxidant and antibacterial potential of 12 traditionally used plants, selected for ethnobotanical uses on the campus of the University of Antioquia. The biological activity of ethanolic extracts was evaluated on a phytopathogenic bacterium isolated from banana crops and four pathogenic microbial strains from fresh agricultural foods, as potential sources in the development of bio-controllers in the agrobiotechnology industry for banana cultivation.

2. Materials and Methods

2.6 Spectroscopic analysis

One of the most active extracts, Garcinia madruno (Kunth) Hammel (MDÑ), was selected for column chromatographic separation and subsequent isolation and identification of the active metabolite by NMR. The spectra were acquired at ¹H NMR, Bruker Ascend II HD 600 MHz. The selected samples were dissolved in deuterated chloroform (CDCl₃) for data acquisition. Spectroscopic data: the compound was determined as friedelan-3-one with a molecular formula C₃₀H₅₀O and spectroscopic data of ¹H-RMN-500MHz δ (CHCD₃ –d1) (ppm): 1.96-1.74 (2H, add, H-1); 2.40-2.37(2H, add, H-1); 2.24 (1H, Hq, H-4 ) ; 1.74, 1.28 (2H,d, H-6); 1.74, 1.36 (2H, m, H-7); 1,38 (1H, dd, H-7); 1,57 (1H, m, H-10); 1.41, 1.26 (2H, m, H-11); 1.35, 1.32 (2H, m, H-12); ); 1,56 (1H, m, H-15); 1.37,1.22 (2H, m, H-16); 1.50,1.31 (2H, m, H-21); 1.51, 0.95 (2H, m, H-22); 0.88 (s, 3H, H-23); 0.72 (s, 1H, H-24); 0.87 (s, 3H, H-25); 1.00 (s, 3H, H-26); 1.05 (s, 3H, H-27); 1.18 (s, 3H, H-28); 1.007 (s, 3H, H-29); 1.25 (s, 3H, H-30); 0,95 (s, 3H, H-30). The data were compared in the literature, finding that this compound has been reported by Abdullahi in Odeh et al., 2016; Igoli and Gray, 2008 (Fig. 1).

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Fig. 1.

Chemical structure of Friedel-3-one.

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3. Results and Discussion

The results obtained from the 13 ethanolic extracts on the capacity of decolorization assay of the radical cation (DPPH) reporting inhibitory concentration 50 (IC₅₀), and results of the susceptibility of each extract on R. solanacearum and on Enterobacteriaceae reported in inhibition zones in millimeters, have been presented in Table 1. To the best of our knowledge, this is the first report on the antibacterial activity of the 12 evaluated plants on R. solanacearum. The vehicle control (100% dimethyl sulfoxide) produced no inhibitory effect with any species, and gentamicin was a negative control.

Regarding free radical scavenging of 13 ethanolic extracts evaluated, an adequate concentration-response relationship was observed, and the results were compared with the positive control using Ascorbic acid, IC₅₀= 53.91 ± 0.3 ppm with an inhibition of 98% as a reference for the (DPPH) radical cation assay. IC₅₀ values > 100 ppm of the extracts have been presented in Fig. 2 and the percentages of inhibition in all the concentrations evaluated (1000 – 31.25ppm) for each active extract have been presented in Fig. 3. The three species that presented the best free radical scavenging for free radical scavenging were Z. martinicense with IC₅₀ values ​​of 27.29 ± 0.8 ppm, G. madruno with IC₅₀ of 10.56± 0.7 ppm, and the most active P. niruri L. with IC₅₀ of 3.79± 0.4 ppm. P. niruri L. showed a notable DPPH inhibition capacity with respect to the other species and the control, in agreement with a previous report by Deepsikha Swargiary 2024, who reported for this species fractions and phenolic compounds that capture free radicals, in addition to the hyperglycemic properties of this plant evaluated through the induction of SIRT1 and the translocation of GLUT4 in in vitro and in vivo models (Swargiary et al., 2024). Another species that presented greater free radical scavenging was G. madruno with IC₅₀ of 10.56 ± 0.7 ppm, which is consistent with what was reported by Ramírez et al., 2019 showing the high free radical scavenging (DPPH) of the ethyl acetate fraction with values ​​of 83052 ± 3385 mg AAE/100 g for the leaves of this plant, indicating that G. madruno could be a source of metabolites with antioxidant activity (Ramírez et al., 2019). Regarding the antioxidant activity of Z. martinicense, this is the first report of antiradical activity for this species; only anti-inflammatory and pharmacological activity have been reported in vivo models (Mutinda et al., 2023). It is worth highlighting the protective properties of the extracts against oxidative stress caused by microorganisms. This indicates the potential of these extracts to role as to regulate oxidative stress in the plant and as a natural protector post-harvest. These species can be further studied to explore their reducing potential, given that the methods to measure antioxidant activity consider aspects such as: intervention of mechanisms, free radical trapping, catalytic decomposition, pro-oxidant suppression, trapping percentage, and effective concentration (moles of free radicals trapped per mole of antioxidant).

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Fig. 2.

IC₅₀ values ​​> 100 in µg/mL of the promising extracts. Significant diff. among means (P < 0.05).

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Fig. 3.

IC₅₀ values ​​> 100 in µg/mL of the promising extracts. Significant diff. among means (P < 0.05).

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Fig. 4 shows inhibition zones (mm) of experimental bacteria of the different extracts on R. Solanacearus (Fig. 4a) and on Enterobacteriaceae (Fig. 4b). Experimental growth of in vitro cultures of R. Solanacearus of plant extracts have been depicted in Fig. 5, and experimental growth of plant extracts in vitro cultures of Bacillus cereus in Fig. 6. The ethanolic extracts that presented inhibition zones greater than 15 mm at the highest and lowest concentration evaluated of 400 and 200 µg/disc on R. Solanacearus were 1AEM, 4ECM3, 7IPM, 9MDÑ, 11ZTXU, and 12PDBS. No previous reports of antibacterial activity on the isolates of R. Solanacearus of these plants exist. The most active species were Garcinia madruno with inhibition zones of 25 and 21 mm, followed by Phyllanthus niruri L. with inhibition zones of 20 and 14 mm, and Enterolobium cyclocarpum (Jacq.) Griseb. with inhibition zones of 17 and 14 mm. The species analyzed in this work have presented activity in different studies reported in literature, with interesting activities in traditional medicine mainly due to their activities as analgesics, fungicides, bactericides, and in the control of parasites (Brito et al., 2022; Auranwiwat et al., 2014; Seenivasan et al., 2022; Gamal El-Din et al., 2017). Nevertheless, this work is the first report of the effectiveness of 12 ethnobotanical extracts with antibacterial activity on banana diseases caused by R. solanacearum. It is a Gram-bacterium with a global distribution and a wide range of hosts, which could reach more than 200, in approximately 50 different plant families (Salanoubat et al., 2002). This bacillus is characterized as a virulent, white, mucoid β-proteobacterium, due to the abundant production of extracellular polysaccharides. It is characteristic of smooth, irregular, and round bacilli and proteins related to glyoxylate metabolism (Saquicela et al., 2023). These types of bacterial diseases in plants are controlled with antibiotics such as streptomycin and oxytetracycline (Okonya et al., 2019). However, despite being the most widely used, resistance to streptomycin is spreading among bacterial phytopathogens (Bruehl et al., 2023), and the emergence of resistant strains such as Erwinia amylovora, Pseudomonas spp., and Xanthomonas campestris have prevented the control of several important diseases, increasing the need to develop new alternatives (Haynes et al., 2020; Iwu et al., 2020). For instance, G. madruno and P. niruri L. are a source of interesting metabolites to continue with studies in the development of alternatives to mitigate the impact of R. Solanacearus, which has generated large losses in many economically important crops around the world, so having and developing control alternatives would contribute significantly to protect the crops and increase food security. The study of the secondary metabolites of these species will allow us to determine the action and molecular mechanisms that act on R. Solanacearus leading to its control given that this pathogenic bacterium has a high genetic diversity and a wide range of hosts, including bananas.

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Fig. 4.

Inhibition halos of the most active ethanolic extracts of ethnobotanical species evaluated at 400 µg/disc on (a) a phytopathogenic bacteria Ralstonia solanacearum and (b) three pathogenic bacteria of fresh agricultural foods Escherichia Coli, Bacillus Subtilis, and Bacillus cereus. Note: Significance p<0.01 (**).

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Fig. 5.

Evaluation of plant extracts of Ant-Col (A-H), A(1AEM), B(3LNTM), C(4ECM3), D(7IPM), E(4CPF), F(9MDÑ), G(11ZTXU), H(12PDBS) in vitro cultures of Ralstonia Solanacearus The disc - corresponds to the negative control with DMSO. [A] = 400 µg/disk, [Aa] = 200 µg/disk.

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Fig. 6.

Evaluation of plant extracts of Ant-Col (A-H), A(1AEM), B(3LNTM), C(4ECM3), D(7IPM), E(4CPF), F(9MDÑ), G(11ZTXU), H(12PDBS) in in vitro cultures of Bacillus cereus ATCC 11778. The disc - corresponds to the negative control with DMSO. [A] = 400 µg/disk, [Aa] = 200 µg/disk.

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For the tests on Enterobacteriaceae, the extracts that presented activity with maximum inhibition zones greater than the 15 mm control were 1AEM, 3LNTM, 4ECM3, 4CPF, 5MVM, 7IPM, 9MDÑ, 10MPGH, 11ZTXU, 12PDBS. The most active species on Gram positive bacteria at 400 µg/disc were E. cyclocarpum with inhibition zones of 21mm on B. Subtilis and Z. martinicense and 25 mm on B. cereus. The most active species on Gram negative bacteria (E. coli) were T. litoralis with halos of 20 mm, P. niruri with halos of 26 mm, G. madruno with halos of 23 mm, and Z. martinicense with halos of 25 mm. The other extracts presented inhibition zones between 8 to 18 mm on all the bacterial strains evaluated. For instance, given the interesting properties of G. madruno, this species was selected for chemical studies, where a Friedelano-terpenoid compound was isolated and characterized. Called fredelan-3-one, this is the first time that this compound has been reported in this species. The elucidation of the structure was carried out by comparison with the literature of spectral characteristics by NMR. This type of friedelan compound has been isolated in other species of the same genus, such as Garcinia smeathmannii with good antimicrobial activity at a concentration of 2.5 mg/mL against S. faecalis, S. typhi, and E. coli (Komguem et al., 2005). Friedelin showed an inhibition zone of 40 and 38 mm on Gram-negative bacteria P. mirabilis, V. cholera, and K. pneumoniae, respectively, and 40 and 37 mm on Gram-positive bacteria B. cereus and S. epidermis, respectively (Singh et al., 2023). This type of friedelin compound, found in extracts of many plants, may be responsible for the broad spectrum of antimicrobial activity (Gowdu et al., 2012). The Garcinia genus, composed of approximately 240 species, is distributed in all tropical regions, and the edible fruits have high dietary value. A wide variety of Garcinia species have been protagonists for their extracts and isolated compounds, such as anthocyanins, polyphenols, benzophenones, bioflavonoids, and xanthones in the treatment of various diseases and medicinal uses as adipogenesis, diabetes, cardiovascular diseases, and cancer (Ramírez et al, 2019).

All the species selected in this work present extensive reports in the literature about their ethnobotanical uses and medicinal properties. For example, E. cyclocarpum is a tree with multiple uses, fruits serve as food for livestock, and they also contain phenolic compounds (Olmedo-Juárez et al., 2024; Pacheco et al., 2012). M. paniculata has been reported as an aromatic species in Asian countries (Dosoky et al., 2016). They are rich in nutrients and exert several medicinal properties such as antioxidant, antidiabetic, anti-inflammatory, antimicrobial, and anticancer due to several coumarin-type phytochemicals, phenylpropanoids, flavonoids, essential minerals, and trace elements (Abeysinghe et al., 2021). Essential oils showed antioxidant and antibacterial activity (Selestino et al., 2017). The species L. camara is a weed that has been used for centuries as a medicinal plant to treat asthma, chickenpox, whooping cough, headaches, colds, high blood pressure, bronchitis, and eye injuries. Studies have reported that the main constituents in this species are terpenes, pentacyclic triterpenoids, steroids, polyphenolics, flavonoids, and iridoid glycosides (Patil et al., 2023). In this work, it is found that L. camara extracts exhibit an IC₅₀ = 38.04 ± 0.9 µg/mL, with a great potential for antioxidant activity as previously reported, demonstrating its nutritional and medicinal functions (Kumar et al., 2014). On the other hand, T. litoralis exhibits an IC₅₀ =86,38± 0,6, with good free radical scavenging. This plant is characterized by the presence of biologically active indole alkaloid. In some species, more than one hundred structurally different alkaloids have been found, mainly from fruits and flowers. Besides, terpenolactones, steroids and flavonoids can also be found (Silveira et al., 2017). C. argenteum is known for its rich content of triterpenoid saponins with innumerable pharmacological effects and health benefits, its bioactive saponins, with special attention to antioxidant, antiulcer, anti-inflammatory, antimicrobial, cytotoxic, antihypercholesterolemic, and immunomodulatory activity (Baky et al., 2022). Nevertheless, this is the first report of the species regarding its antibacterial and antioxidant activities. Citrus species have been cultivated and used throughout the world and are commonly known as lemons, mandarins, oranges, and grapefruits. They are commonly used in traditional medicine and their medicinal benefits, including their antimicrobial properties, are associated with antihypertensive, anti-inflammatory, and antioxidant activities (Palangasinghe et al., 2024). Phyllanthus niruri, a medicinal plant with multiple bioactivities and uses in eliminating cooling blood, heat, strengthening digestion, promoting fluid production, and relieving cough. It also contains nutritional (vitamins, mineral elements, polysaccharides, amino acids, unsaturated free fatty acids) and functional components such as tannins, alkanes, phenolic acids, sterols, triterpenoids, flavonoids, lignans, aromatic micromolecules, and alkaloids (Ma et al., 2024). Spondias purpurea L., is a species commonly found in Malaysia, India, and Indonesia. All these species contain different classes of secondary metabolites such as essential oils, phenolics, saponins, sterols, triterpenes, amino acids, and polysaccharides. These compounds are probably responsible for numerous pharmacological benefits, including cytotoxic, antioxidant, ulcer protective, liver protective, anti-inflammatory, anti-fertility, anti-arthritis, anti-hypertension, anti-diabetic, and antimicrobial activities (Lakesmi, 2019). The fruits of M. glabra and M. emarginata have a high content of vitamin C, phenolic compounds, benzoic acid derivatives, flavonoids, anthocyanins, phenylpropanoids, and carotenoids, with biological activities as antioxidants, antitumor, antihyperglycemic and skin protection/skin whitening (Belwal et al., 2018). On the other hand, the second most active species, Z. martinicense, with IC₅₀ values ​​of 27.29 ppm, is widely known, with a long history of dual use in medicine and food (Mutinda et al., 2023). This genus includes volatile oils, alkaloids, amides, lignans, coumarins, and organic acids, which have important anti-inflammatory, analgesic, antitumor, hypoglycemic, hypolipidemic, antioxidant, and anti-infective properties, and are widely used in the fields of food and medicines (Wen et al., 2024). Schinus molle is a plant highly tolerant to drought and heat, the berries of S. molle have been used for antiseptic, antidepressant, analgesic, and antibacterial purposes, for the respiratory and urinary tract, for infections as a digestive purgative and toothache, diuretic, against rheumatism and menstrual disorders (Belhoussaine et al., 2024).

4. Conclusion

Enterolobium cyclocarpum, Murraya paniculata, Lantana camara L. Tabernaemontana litoralis Kunth., Chrysophyllum argenteum Jacq., Citrus limon (L.), Phyllanthus niruri L., Spondias purpurea, Garcinia madruno, Malpighia glabra L., Zanthoxylum martinicense, and Schinus molle L. are species presenting a valuable phytochemical resource with a diversity of secondary metabolites with multiple reports in the medical field, whose diversity and pharmacological action represent an alternative in the development of antimicrobial phytomedicines to address public health challenges. This is the first report of antimicrobial activity of 13 ethnobotanical ethanolic extract on phytopathogenic strain in R. solanacearum (Banana), exhibiting inhibition zones between 8 to 25 mm and for pathogenic bacteria inhibition ranges between 8-26 mm to 200 and 400 µg/disc, some of these with reports in the literature with activity against Enterobacteriaceae. The plant with the highest radical scavenging capacity was P. niruri with an IC₅₀ of 3.79 ± 0.4 ppm. G. madruno is the most active extract with antibacterial and antioxidant activity. In this chemical research, we achieved the isolation of a triterpene called friedelan-3-one. It is worth highlighting the protective properties of the extracts against oxidative stress caused by microorganisms. Likewise, these plants represent a very valuable resource for Latin American countries in little-explored fields of the agricultural sector, specifically in the development of bioinputs based on low-cost standardized extracts and/or enriched fractions for the control of phytopathogenic agents and food protectors. Therefore, developing products that protect the plant and control the spread of phytopathogens in crops, in addition to limiting contamination before and after harvest and the proliferation of human pathogenic enterobacteria in food, would be a strategy in the search for products aligned with international needs in reducing post-harvest losses, increasing food security and clean production, so it is necessary to continue the studies with these promising species reported in this work. Therefore, it is important to continue with the studies for the control of bacterial wilt in plants, the structure and its molecular mechanism and to know the potential of the most active plants in the development of biocontrollers and protectants for the control of bacteria in harvest and post-harvest in foods of great global importance such as banana and plantain crops.