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Introduction

The use of natural products for medicinal purposes is one of the oldest practices by humans, showing an important role in global health, and symbolizing the main source of many ethnic groups (Veiga Junior, Pinto, & Maciel, 2005). However, the pharmacological properties of natural products are not fully explained (Veiga Junior et al., 2005). Therefore, the population use plants for phytotherapy purposes, which is necessary scientific research regarding to their benefits actions. Such studies allow the discovery of new drugs and attest the biological effects of medicinal plants.

The biological and pharmacological properties of plants can be attributed to the presence of chemical compounds on their crude extracts (Kasote, Katyare, Hegde, & Bae, 2015). Some plants are rich in polyphenols compounds (Metodiewa, Kochman, & Karolczak, 1997), which can be responsible for the antioxidant property showed by plants extracts. The antioxidant activity intensifies the therapeutic effects of plants, through their ability to act against some oxidant agents, promoting health maintenance, as well as in the prevention of diseases (Smina, Mathew, Janardhanan, & Devasagayam, 2011).

Under oxidative stress, the presence of free radicals causes tissue damage, like as lipoperoxidation of cell membranes, activation of proinflammatory agents and aeroallergen sensitization (Diaz-Sanchez, Garcia, Wang, Jyrala, & Saxon, 1999; Halliwell & Gutteridge, 1990). Oxidative stress is associated with many diseases such as rhinosinusitis (Kinnula, 2005).

In the literature, it is shown that natural products also have antimicrobial activity (Nascimento, Locatelli, Freitas, & Silva, 2000). The search for active compounds with different mechanisms of inhibition of microorganism growth is increasing due to the high incidence of resistant microorganisms in clinical microbiology in Brazil (Montelli & Levy, 1991). Luffa operculata (L.) Cong, commonly known as sponge cucumber or wild loofa, is used in Brazil in the treatment of rhinosinusitis (Diretrizes Brasileiras de Rinossinusites, 2008). This plant is traditionally used by inhaling the fruit aqueous extract, which can promote the discharge of mucus and relief from nasal obstructions (Diretrizes Brasileiras de Rinossinusites, 2008).

The pharmacological mechanisms of action of this plant is not completely explained, however its nasal decongestant properties were attributed to the presence of cucurbitacins and glycosides, which promote the exudation and improve the ciliary motility, together with the emollient effect of saponins (Lorenzi & Matos, 2002). Different classes of chemical compounds have already been identified in the sponge cucumber, such as 2,3-dicaffeoylglycaric acid, a substance which is related to the antibacterial activity of the extracts. Besides that, some flavonoids, saponins, free steroids and phenolic acids have been isolated from this species (Heiser & Schilling, 1988; Lorenzi & Matos, 2002).

Previous studies have demonstrated that the use of Luffa operculata proved to be safe and effective in the treatment of rhinosinusitis, as well as revealed antibacterial potential (Passali et al., 2015; Scalia, Dolci, Ueda, & Sassagawa, 2015). In this sense, the aim of the present work was to evaluate the chemical composition, antioxidant and antimicrobial activities of Luffa operculata extracts.

Material and methods

Plant extract preparations

The dried material (fruit) and the extract (hydroalcoholic 65%) of Luffa operculata 10% (COM) for the nasal use with the indication of use for sinusitis and rhinitis were obtained commercially in the city of Ponta Grossa, Brazil. The aqueous extract (INF) was obtained by infusion by covering and soaking the leaves with boiling water for 30 minutes, filtered and cooled to room temperature. It was also prepared the hydroalcoholic extract 65% (HEtOH) and ethanolic (EtOH) extract by maceration, addition of the extractor liquid in light shelter and occasional agitation for 7 days. The standardized concentration was 10% when compared to the commercially obtained extract.

Determination of total phenolic compounds

Total phenolic content of the Luffa operculata extracts was determined by using Folin-Ciocalteu’s reagent (Singleton, Orthofer, & Lamuela-Raventós, 1999). 5 µL of samples of each extract were mixed with 10% (v/v) Folin-Ciocalteu reagent and 7.5% (m/v) aqueous solution of Na₂CO₃. The absorbance was measured at 765 nm after 15 minutes of incubation at 45ºC. Total phenolic content of each extract was determined from the analytical curve of gallic acid standard and the results were expressed as milligram of gallic acid per gram of extract (mg GA g^-1).

Determination of total flavonoid contents

Total flavonoid content of the Luffa operculata extracts was determined by complexation with aluminum ion Al(III) (Kosalec, Bakmaz, Pepeljnjak, & Vladimir-Knezević, 2004). 5 µL samples of each extract were mixed with 10% (w/v) AlCl₃ and 0.1 mol L^-1 CH₃COOK. The absorbance was measured at 420 nm. Total flavonoid content for each extract was determined from the analytical curve of quercetin standard and the results were expressed as milligram of quercetin per gram of extract (mg QER g^-1).

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) radical scavenging activity

Antioxidant activity of Luffa operculata extracts was evaluated by ABTS^•+ method as described by (Pellegrini, Re, Yang, & Rice-Evans, 1999) by using ascorbic acid as standard. First, 20 µL of samples of extracts at different concentrations (1–100 µg mL^-1) were mixed with sodium phosphate buffer (pH 7.4) and ABTS^•+ solution. After 30 minutes of incubation in the absence of light, the absorbance was measured at 734 nm. The results were expressed as IC₅₀ values.

2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) radical scavenging activity

Antioxidant activity of the Luffa operculata extracts was also evaluated by DPPH^• method (Brand-Williams, Cuvelier, & Berset, 1995) by using ascorbic acid as standard compound. Samples containing 20 µL of extracts at different concentrations (1–100 µg mL^-1) were mixed with DPPH^• ethanolic solution, followed by the addition of ethanol. The absorbance was measured at 518 nm after 30 minutes of incubation in the absence of light. All the results were expressed as IC₅₀ values.

Antimicrobial activity

An initial screening of the antibacterial potential for Luffa operculata extract was performed by testing the inhibition of a gram positive (S. aureus), gram negative (E. coli) and yeast fungus (C. albicans). Antimicrobial activity of Luffa operculata was analyzed using the minimum inhibitory concentration technique (MIC), determinate by microdilution technique that was adapted from the Clinical Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2014). The strains of Staphylococcus aureus ATCC 25923, Escherichia coli 25922 e Candida albicans 10231 were used. The extracts were diluted in 2.5 % DMSO and in Muller-Hinton broth for bacteria and in Muller-Hinton broth with 2% of glucose for yeast fungus, in the final concentrations of 2000-625 µg mL^-1. The strains were prepared in sterile physiological solution, which contained 1.0-2.0 x 10⁸ UFC mL^-1 for bacteria and 1.0-2.0 x 10⁶ UFC mL^-1 for yeast, equal to the MacFarland’s scale 0.5 standard. Thereafter, it was diluted (1:10) and 50 µL of the microorganisms and added to the antimicrobial activity test. Next, the plaques were incubated at 35ºC for 24 hours. After incubation, the optic density (equal to the turbidity produced by the growth of microorganisms in broth) was measured at 630 nm.

Statistical analysis

The results were expressed as mean±standard deviation for IC₅₀ values and total content of phenolics and flavonoids. Tukey and Bonferroni tests were performed for antioxidant and antimicrobial activities. The software GraphPad Prism 5.0 was used for statistical analysis.

Results

Total phenolic compounds and flavonoids

The total content of phenolics and flavonoids are observed in Table 1. It can be verified that the HEtOH and INF samples exhibited a similar content of polyphenols, being the COM sample the one with lowest polyphenols quantities.

Antioxidant activity

Table 1 shows the IC₅₀ values obtained in the ABTS^·+ and DPPH^· methods for the extracts. Of the analyzed extracts, HEtOH extract exhibited the highest antioxidant activity against ABTS^·+ and DPPH^·. However, it presented a significant difference (p < 0.05) to the same concentration extract commercially obtained. The IC₅₀ values observed for the COM extract were higher, which means a higher concentration of the extract is necessary to reach an antioxidant activity, suggesting a weaker antioxidant activity. As for the INF extract, no significant difference was found (p < 0.05), indicating a potential activity close to the HEtOH extract, and a good antioxidant activity. The EtOH also had a significant difference compared to other extracts.

Antimicrobial activity

Table 2 shows the percentage of inhibition for microorganism growth. The HEtOH extract showed a MIC of 1.000 µg mL^-1 for E. coli, 500 µg mL^-1 for S. aureus and 2.000 µg mL^-1 for C. albicans, being S. aureus the most sensitive pathogen to the extract, and C. albicans the most resistant. While the other extracts did not present inhibition of 100% of the microorganisms and under a 2.000 µg mL^-1 concentration, all extracts reached an inhibition of at least 50 % (IC₅₀).

Discussion

Within plants, there is a variety of different classes of phenolic compounds with distinct polarities and chemical properties. Therefore, an adequate selection of solvents plays an essential role in determining the nature and quantity of extracted compounds (Bhebhe, Füller, Chipurura, & Muchuweti, 2016). From our results (Table 1), it was observed that HEtOH presented more phenolic and flavonoid levels than the EtOH extract. This behavior is in accordance with some works reported in literature (Pérez-Jiménez & Saura-Calixto, 2006), since it is known that the addition of water creates an increase in polarity, which facilitate the extraction of the mentioned compounds.

The presence of polyphenols in the extracts is in accordance with previous studies, that have identified the presence of these compounds in Luffa operculate (Schilling & Heiser, 1981; Silva, Costa, Souza, Lopes, & Ueda, 2018). In the COM sample, a lower level of these compounds was observed, even with the same concentration as HEtOH, which could be explained by deficient inspection and quality control of the natural products (Newman & Cragg, 2016), allowing for errors in the indicated concentration, as well as an incorrect storage, causing degradation of the compounds, a process which has been described in the literature by (Devlin & Harris, 1984).

Antioxidant activity by using ABTS^·+ and DPPH^· scavenging activities were employed in order to verify the free radical sequestration capacity of natural products (Alves, David, David, Bahia, & Aguiar, 2010) and these methods are correlated with the total content of phenolic compounds and flavonoids (Waterman & Mole, 1994). In this work, it was demonstrated that the extracts which showed the highest content of polyphenols on their chemical composition exhibited the lowest IC₅₀ values on these methods.

HEtOH extract showed a higher antioxidant activity, with the lowest IC₅₀ values levels, in the ABTS^·+ and DPPH^· assays (Table 1) indicating a superior antioxidant activity to the one found in Luffa cylindrica (IC₅₀ of 26.46 µg mL^-1 for DPPH^·; (Sharma et al., 2012) and Luffa acutangula Var. amara (IC₅₀ of 43.76 µg mL^-1 and 84.00 µg mL^-1 for ABTS^·+ and DPPH^·, respectively; (Kalaskar & Surana, 2014). However, the COM sample presented a significant difference (p < 0.05), with higher IC₅₀ values. Higher concentrations of extract are necessary to reach the same antioxidant effect, and therefore, a lower antioxidant potential.

Generally, the extracts presented a good activity in scavenging free radicals, indicating a potential pharmacological activity in preventing oxidative stress and worsening, or development of inflammatory diseases such as rhinosinusitis (Heffner & Repine, 1989; Metodiewa et al., 1997). It has been demonstrated that antioxidants tended to improve and reduce inflammation in patients with respiratory diseases after treatment with these compounds. This can be explained probably due to the decrease in inflammatory stimuli released by free radicals mediation (Valko et al., 2007). Although the initial results are promising and indicate that the employment of such compounds may be useful in the treatment of inflammatory respiratory diseases, it is necessary further investigations to validate its use (Barnes, 2008).

An initial screening of the antibacterial potential for Luffa operculata extract was performed by testing the inhibition of a gram positive (S. aureus), gram negative (E. coli) and yeast fungus (C. albicans). Differently from what happens to antibiotics, there are few instances reported in the literature for a potential mechanism of action to natural products. The compounds isolated from plants are substances which could act on the intermediate metabolism, activating enzymes, altering the activity of inhibitors that influence the nutrients in the medium, interfering in enzymatic processes at a nuclear or ribosomal level, and causing changes to membranes or even interfering with secondary metabolism (Cowan, 1999). The crude extracts have a promising antimicrobial activity, which can be related to synergism between phytochemical constituents of plants (Delgado-Adámez, Fernández-León, Velardo-Micharet, & González-Gómez, 2012; Lee & Lee, 2010).

The compounds present in the Luffa operculata extracts suggest a relative antimicrobial activity, with a good response against S. aureus, especially for HEtOH, with a MIC of 500 μg mL^-1, while for E. coli a MIC of 1.000 μg mL^-1 was observed. The greater sensitivity of S. aureus is in accordance to literature, which indicates a greater sensitivity of this bacterium to secondary metabolites (Ferreira et al., 2010). The double membrane present in gram-negative bacteria makes up a complex casing, responsible for the lower sensitivity of these microorganisms to plant extracts (Francescato, Deuschle, Mallmann, Alves, & Heinzmann, 2007), as well as lower antifungal activity in natural compounds (Estevez-Braun, Estevez-Reyes, Moujir, Ravelo, & Gonzalez, 1994).

The results obtained for the extracts COM is in accordance with other essays that used commercial extract of Luffa operculate (Scalia et al., 2015). In general, the results demonstrated a biological potential of inhibition of bacteria and fungi for the extracts, which may be further explored with more tests employing additional microorganisms.

Conclusion

It was possible to observe that HEtOH was the extract that presented the best results for the applied tests, while the COM showed worse performance. It is considered that the present in vitro study contributed to the expansion of the knowledge regarding the antioxidant and antimicrobial activities of the Luffa operculata extracts. It showed promising potential for new studies and possible application as antioxidant or antimicrobial of natural origin, besides generating a screening of potential order of activity of the extracts. Thus, hey may be important sources of compounds for biological activities.

Acknowledgements

Researchers are grateful to the Foundation of Support to Technological and Scientific Development of Paraná – Fundação Araucária.

References

Alves, C. Q., David, J. M., David, J. P., Bahia, M. V, & Aguiar, R. M. (2010). Métodos para determinação de atividade antioxidante in vitro em substratos orgânicos. Quimica Nova, 33(10), 2202-2210. DOI: 10.1590/S0100-40422010001000033

Barnes, P. J. (2008). Future treatments for chronic obstructive pulmonary disease and its comorbidities. Proceedings of the American Thoracic Society, 5(8), 857-864. DOI: 10.1513/pats.200807-069TH

Bhebhe, M., Füller, T. N., Chipurura, B., & Muchuweti, M. (2016). Effect of Solvent Type on Total Phenolic Content and Free Radical Scavenging Activity of Black Tea and Herbal Infusions. Food Analytical Methods, 9(4), 1060-1067. DOI: 10.1007/s12161-015-0270-z

Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT - Food Science and Technology, 28(1), 25-30. DOI: 10.1016/S0023-6438(95)80008-5

Clinical and Laboratory Standards Institute. (2014). Performance Standards for Antimicrobial Susceptibility Testing;Twenty-Fourth Informational Supplement. Wayne, PA: CLSI

Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12(4), 564-582. DOI: 10.1128/cmr.12.4.564

Delgado-Adámez, J., Fernández-León, M. F., Velardo-Micharet, B., & González-Gómez, D. (2012). In vitro assays of the antibacterial and antioxidant activity of aqueous leaf extracts from different Prunus salicina Lindl. cultivars. Food and Chemical Toxicology, 50(7), 2481-2486. DOI: 10.1016/j.fct.2012.02.024

Devlin, H. R., & Harris, I. J. (1984). Mechanism of the oxidation of aqueous phenol with dissolved oxygen. Industrial & Engineering Chemistry Fundamentals, 23(4), 387-392. DOI: 10.1021/i100016a002

Diaz-Sanchez, D., Garcia, M. P., Wang, M., Jyrala, M., & Saxon, A. (1999). Nasal challenge with diesel exhaust particles can induce sensitization to a neoallergen in the human mucosa. Journal of Allergy and Clinical Immunology, 104(6), 1183-1188. DOI: 10.1016/S0091-6749(99)70011-4

Diretrizes Brasileiras de Rinossinusites. (2008). Revista Brasileira de Otorrinolaringologia, 74(2), 6-59. DOI: 10.1590/S0034-72992008000700002

Estevez-Braun, A., Estevez-Reyes, R., Moujir, L. M., Ravelo, A. G., & Gonzalez, A. G. (1994). Antibiotic activity and absolute configuration of ss-heptadeca-2(2),9(z)-diene-4,6-diyne- 1 ,%diol from bupleurum salicifolium. Journal of Natural Products, 57(8), 1178–1182. DOI: 10.1021/np50110a009

Ferreira, S. B., Palmeira, J. D., Souza, J. H., Almeida, J. M., Pereira, M. C., Pequeno, A. S., … Catão, R. M. R. (2010). Avaliação da atividade antimicrobiana in vitro do extrato hidroalcóolico de Stryphnodendron adstringens (Mart.) Coville sobre isolados ambulatoriais de Staphylococcus aureus. Revista Brasileira de Análises Clínicas, 42(1), 27–31.

Francescato, L. N., Deuschle, R. A. N., Mallmann, C. A., Alves, S. H., & Heinzmann, B. M. (2007). Atividade antimicrobiana de Senecio heterotrichius DC. (Asteraceae). Brazilian Journal of Pharmaceutical Sciences, 43(2), 239-245. DOI: 10.1590/S1516-93322007000200010

Halliwell, B., & Gutteridge, J. M. C. (1990). Role of free radicals and catalytic metal ions in human disease: An overview. Methods in Enzymology, 186, 1-85. DOI: 10.1016/0076-6879(90)86093-B

Heffner, J. E., & Repine, J. E. (1989). Pulmonary Strategies of Antioxidant Defense. American Review of Respiratory Disease, 140(2), 531-554. DOI: 10.1164/ajrccm/140.2.531

Heiser, C. B., & Schilling, E. E. (1988). Phylogeny and Distribution of Luffa (Cucurbitaceae). Biotropica, 20(3), 185-191. DOI: 10.2307/2388233

Kalaskar, M. G., & Surana, S. J. (2014). Free radical scavenging, immunomodulatory activity and chemical composition of luffa acutangula var. amara (cucurbitaceae) pericarp. Journal of the Chilean Chemical Society, 59(1), 2299-2302. DOI: 10.4067/S0717-97072014000100012

Kasote, D. M., Katyare, S. S., Hegde, M. V., & Bae, H. (2015). Significance of antioxidant potential of plants and its relevance to therapeutic applications. International Journal of Biological Sciences, 11(8), 982-991. DOI: 10.7150/ijbs.12096

Kinnula, V. L. (2005). Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases. Thorax, 60(8), 693-700. DOI: 10.1136/thx.2004.037473

Kosalec, I., Bakmaz, M., Pepeljnjak, S., & Vladimir-Knezević, S. (2004). Quantitative analysis of the flavonoids in raw propolis from northern Croatia. Acta Pharmaceutica (Zagreb, Croatia), 54(1), 65-72. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15050046

Lee, O. H., & Lee, B. Y. (2010). Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresource Technology, 101(10), 3751-3754. DOI: 10.1016/j.biortech.2009.12.052

Lorenzi, H., & Matos, F. J. A. (2002). Plantas medicinais no Brasil: nativas e exóticas cultivadas. Nova Odessa, SP: Instituto Plantarum.

Metodiewa, D., Kochman, A., & Karolczak, S. (1997). Evidence for antiradical and antioxidant properties of four biologically active N,N-diethylamioethyl ethers of flavanone oximes: A comparison with natural polyphenolic flavonoid (rutin) action. Biochemistry and Molecular Biology International, 41(5), 1067-1075. DOI: 10.1080/15216549700202141

Montelli, A. C., & Levy, C. E. (1991). Sistema COBA - Aspectos relativos aos dados dos laboratórios de referência. Revista de Microbiologia, 22, 197–205.

Nascimento, G. G. F., Locatelli, J., Freitas, P. C., & Silva, G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, 31(4), 247-256. DOI: 10.1590/S1517-83822000000400003

Newman, D. J., & Cragg, G. M. (2016). Natural Products as Sources of New Drugs from 1981 to 2014. Journal of Natural Products, 79(3), 629-661. DOI: 10.1021/acs.jnatprod.5b01055

Passali, D., Loglisci, M., Passali, G. C., Cassano, P., Rodriguez, H. A., & Bellussi, L. M. (2015). A prospective open-label study to assess the efficacy and safety of a herbal medicinal product (Sinupret) in patients with acute rhinosinusitis. ORL; Journal for Oto-rhino-Laryngology and its Related Specialties, 77(1), 27-32. DOI: 10.1159/000370123

Pellegrini, N., Ke, R., Yang, M., & Rice-Evans, C. (1999). Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,2’-azinobis(3-ethylenebenzothiazoline- 6-sulfonic acid radical Cation decolorization assay. Methods in Enzymology, 299, 379-389. DOI: 10.1016/S0076-6879(99)99037-7

Pérez-Jiménez, J., & Saura-Calixto, F. (2006). Effect of solvent and certain food constituents on different antioxidant capacity assays. Food Research International, 39(7), 791-800. DOI: 10.1016/j.foodres.2006.02.003

Scalia, R. A., Dolci, J. E. L., Ueda, S. M. Y., & Sassagawa, S. M. (2015). In vitro antimicrobial activity of Luffa operculata. Brazilian Journal of Otorhinolaryngology, 81(4), 422-430. DOI: 10.1016/j.bjorl.2014.07.015

Schilling, E. E., & Heiser Jr., C. B. (1981). Flavonoids and the Systematics of Luffa. Biochemical Systematics and Ecology, 9(4), 263-265. DOI: 10.1016/0305-1978(81)90006-5

Sharma, N. K., Sangh, P., Priyanka, Jha, K. K., Singh, H. K., & Shrivastava, A. K. (2012). Free radical scavenging activity of methanolic extract of Luffa cylindrica leaves. International Journal of Green Pharmacy, 6(3), 231-236. DOI: 10.4103/0973-8258.104938

Silva, L., Costa, H. O., Souza, F. C., Lopes, E. M. C., & Ueda, S. M. Y. (2018). Preclinical evaluation of Luffa operculata Cogn. and its main active principle in the treatment of bacterial rhinosinusitis. Brazilian Journal of Otorhinolaryngology, 84(1), 82-88. DOI: 10.1016/j.bjorl.2016.11.004

Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 299, 152-178. DOI: 10.1016/S0076-6879(99)99017-1

Smina, T. P., Mathew, J., Janardhanan, K. K., & Devasagayam, T. P. A. (2011). Antioxidant activity and toxicity profile of total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst occurring in South India. Environmental Toxicology and Pharmacology, 32(3), 438-446. DOI: 10.1016/j.etap.2011.08.011

Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology, 39(1), 44-84. DOI: 10.1016/j.biocel.2006.07.001

Veiga Junior, V. F. V, Pinto, A. C., & Maciel, M. A. M. (2005). Plantas Medicinais: cura segura? Química Nova, 28(3), 519-528. DOI: 10.1590/S0100-40422005000300026

Waterman, P. G., & Mole, S. (1994). Analysis of phenolic plant metabolites. Oxford, UK: Blackwell Scientific Publications.

Author notes

josevellosa@yahoo.com.br

IR