Duroia saccifera: in vitro germination, friable calli and identification of β-sitosterol and stigmasterol from the active extract against Mycobacterium tuberculosis

Stefhania Alzate Lozano,Priscila Cristina Bartolomeu Halicki,Julio Cezar De Souza,Cecilia Veronica Nunez,Aline Bastos Brilhante De Sousa,Marcos Gabriel Maciel Salazar,David Ribeiro Da Silva,Pedro Eduardo Almeida Da Silva,Daniela Fernandes Ramos

doi:10.1590/2175-7860202071054

Stefhania Alzate Lozano, Priscila Cristina Bartolomeu Halicki + Show 7 more

Open Access

https://doi.org/10.1590/2175-7860202071054

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Abstract

Abstract Duroia saccifera (Rubiaceae) occurs in the Amazon rainforest and their extracts showed antibacterial properties. To obtain greater quantities of active substances, leaf segments from in vitro D. saccifera seedlings were used as explants for calli induction; calli were multiplied via multiple subcultures, dried and extracted with hexane followed by ethyl acetate (EtOAc) and methanol (MeOH). As D. macrophylla had been reported to produce antimycobacterial substances, we assayed calli extracts against Mycobacterium tuberculosis (H37Rv strain). Calli EtOAc extract was active, with a minimal inhibitory concentration (MIC) of ≤ 25 mg mL-1, IC90of 19.5 mg mL-1 and minimal bactericidal concentration (MBC) of 200 mg mL-1. EtOAc extract was analyzed by Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) to determine its chemical profile, and was found to be rich in terpenes. Chromatographic fractionation of the EtOAc extract yielded a mixture of two sterols, β-sitosterol and stigmasterol (in proportion of 2:1), which were identified by 1H and 13C NMR analysis. As far as we know, this is the first report of Duroia saccifera in vitro cell culture, antituberculosis activity of calli extract and β-sitosterol and stigmasterol isolation from in vitro plant cell culture.

Highlights

Tuberculosis (TB) is a chronic contagious disease caused by Mycobacterium tuberculosis (WHO 2017)
As part of the search for new antituberculosis substances conducted by our bioprospection research group, we had previously isolated two triterpenes and eight indole alkaloids from Duroia macrophylla that were active against M. tuberculosis (H37Rv strain) (Martins et al 2013; Nunez et al 2014)
A second study reported the isolation of the triterpenes oleanoic and ursolic acid active against M. tuberculosis strain (MIC 200 μg mL−1) from D. macrophylla extracts (Martins et al 2013)

Summary

Introduction

Tuberculosis (TB) is a chronic contagious disease caused by Mycobacterium tuberculosis (WHO 2017). The drugs clinically available for TB treatment (rifampin and isoniazid) have become ineffective, as the causative microorganism has developed resistance, so causing an increase in the number of patients who have the disease in a resistant form (WHO 2017). This serious public health problem has led to a renewed initiative to search for novel effective drugs, including new natural plant products, which might potentially fight TB. This rainforest tree occurs in Brazil, Colombia, Venezuela, and Peru (Taylor et al 2007). In Brazil, it is popularly known as “cabeça-deurubu”, “puruí-da-mata” or “puruí-grande” (Cavalcante 1974), and is present in the states of Acre, Amazonas, Pará, and Rondônia, where it occurs in unflooded (“terra firme”) and adjacent seasonally-inundated forests, on both whitewater (“várzea”) and black-water (“igapó”) rivers (Taylor et al 2007)

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