Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside

Jeferson Gustavo Henn; Luiza Steffens; Nathalia Denise De Moura Sperotto; Betânia De Souza Ponce; Rodrigo Moisés Veríssimo; Fernanda Brião Menezes Boaretto; Gustavo Hassemer; Valéria Flores Péres; Helena Schirmer; Jaqueline Nascimento Picada; Jenifer Saffi; Dinara Jaqueline Moura

doi:10.1016/j.jep.2018.10.003

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside

Jeferson Gustavo Henn, Luiza Steffens, Nathalia Denise De Moura Sperotto, Betânia De Souza Ponce, Rodrigo Moisés Veríssimo, Fernanda Brião Menezes Boaretto, Gustavo Hassemer, Valéria Flores Péres, Helena Schirmer, Jaqueline Nascimento Picada, Jenifer Saffi, Dinara Jaqueline Moura

6 Citations (Scopus)

Abstract

Ethnopharmacological relevance: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. Aims of the study: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. Materials and methods: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (2²) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. Results: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. Conclusion: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

Original language	English
Journal	Journal of Ethnopharmacology
Volume	229
Pages (from-to)	145-156
ISSN	0378-8741
DOIs	https://doi.org/10.1016/j.jep.2018.10.003
Publication status	Published - 30 Jan 2019

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10.1016/j.jep.2018.10.003

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Henn, J. G., Steffens, L., De Moura Sperotto, N. D., De Souza Ponce, B., Veríssimo, R. M., Boaretto, F. B. M., Hassemer, G., Péres, V. F., Schirmer, H., Picada, J. N., Saffi, J., & Moura, D. J. (2019). Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside. Journal of Ethnopharmacology, 229, 145-156. https://doi.org/10.1016/j.jep.2018.10.003

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside. / Henn, Jeferson Gustavo; Steffens, Luiza; De Moura Sperotto, Nathalia Denise et al.

In: Journal of Ethnopharmacology, Vol. 229, 30.01.2019, p. 145-156.

Research output: Contribution to journal › Journal article › Research › peer-review

Henn, JG, Steffens, L, De Moura Sperotto, ND, De Souza Ponce, B, Veríssimo, RM, Boaretto, FBM, Hassemer, G, Péres, VF, Schirmer, H, Picada, JN, Saffi, J & Moura, DJ 2019, 'Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside', Journal of Ethnopharmacology, vol. 229, pp. 145-156. https://doi.org/10.1016/j.jep.2018.10.003

@article{24c6b84bba044e73b76fdb464d2c7cb3,

title = "Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside",

abstract = "Ethnopharmacological relevance: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. Aims of the study: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. Materials and methods: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. Results: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. Conclusion: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.",

author = "Henn, {Jeferson Gustavo} and Luiza Steffens and {De Moura Sperotto}, {Nathalia Denise} and {De Souza Ponce}, Bet{\^a}nia and Ver{\'i}ssimo, {Rodrigo Mois{\'e}s} and Boaretto, {Fernanda Bri{\~a}o Menezes} and Gustavo Hassemer and P{\'e}res, {Val{\'e}ria Flores} and Helena Schirmer and Picada, {Jaqueline Nascimento} and Jenifer Saffi and Moura, {Dinara Jaqueline}",

year = "2019",

month = jan,

day = "30",

doi = "10.1016/j.jep.2018.10.003",

language = "English",

volume = "229",

pages = "145--156",

journal = "Journal of Ethnopharmacology",

issn = "0378-8741",

publisher = "Elsevier Ireland Ltd",

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TY - JOUR

T1 - Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside

AU - Henn, Jeferson Gustavo

AU - Steffens, Luiza

AU - De Moura Sperotto, Nathalia Denise

AU - De Souza Ponce, Betânia

AU - Veríssimo, Rodrigo Moisés

AU - Boaretto, Fernanda Brião Menezes

AU - Hassemer, Gustavo

AU - Péres, Valéria Flores

AU - Schirmer, Helena

AU - Picada, Jaqueline Nascimento

AU - Saffi, Jenifer

AU - Moura, Dinara Jaqueline

PY - 2019/1/30

Y1 - 2019/1/30

N2 - Ethnopharmacological relevance: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. Aims of the study: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. Materials and methods: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. Results: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. Conclusion: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

AB - Ethnopharmacological relevance: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. Aims of the study: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. Materials and methods: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. Results: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. Conclusion: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

U2 - 10.1016/j.jep.2018.10.003

DO - 10.1016/j.jep.2018.10.003

M3 - Journal article

C2 - 30316886

SN - 0378-8741

VL - 229

SP - 145

EP - 156

JO - Journal of Ethnopharmacology

JF - Journal of Ethnopharmacology

ER -

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside

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