Inhibitory effects of (+)-spectaline and iso-6-spectaline from Senna spectabilis on the growth and ultrastructure of human-infective species Trypanosoma brucei rhodesiense bloodstream form

Highlights

• (+)-spectaline and iso-6-spectaline were isolated from the leaves of Senna spectabilis.

• The piperidine alkaloids have inhibitory effects on Trypanosoma brucei rhodesiense.

• These compounds induced autophagic cell death in the parasite.

• Ultrastructural changes include formation of autophagosomes, disorganizations of kinetoplast, and swelling of mitochondria.

Abstract

In our ongoing work searching for new trypanocidal lead compounds from Malaysian plants, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis (sin. Cassia spectabilis). Analysis of the ¹H and ¹³C NMR spectra showed that 1 and 2 presented analytical and spectroscopic data in full agreement with those published in the literature. All compounds were screened in vitro against Trypanosoma brucei rhodesiense in comparison to the standard drug pentamidine. Compound 1 and 2 inhibited growth of T. b. rhodesiense with an IC₅₀ value of 0.41 ± 0.01 μM and 0.71 ± 0.01 μM, without toxic effect on L6 cells with associated a selectivity index of 134.92 and 123.74, respectively. These data show that piperidine alkaloids constitute a class of natural products that feature a broad spectrum of biological activities, and are potential templates for the development of new trypanocidal drugs. To our knowledge, the compounds are being reported for the first time to have inhibitory effects on T. b. rhodesiense. The ultrastructural alterations in the trypanosome induced by 1 and 2, leading to programmed cell death were characterized using electron microscopy. These alterations include wrinkling of the trypanosome surface, formation of autophagic vacuoles, disorganization of kinetoplast, and swelling of the mitochondria. These findings evidence a possible autophagic cell death.

Introduction

Human African Trypanosomiasis (HAT) or also known as African sleeping sickness is a vector-born parasitic disease caused by tsetse-fly mediated transmission of Trypanosoma brucei rhodesiense (East Africa) and Trypanosoma brucei gambiense (West Africa). This disease demonstrates a main cause of morbidity and mortality in Sub-Saharan Africa that affects millions of people with an estimated number of actual cases of around 20,000 in 2015 (WHO, 2017). Current treatment for HAT infection predominantly relies on the use of pentamidine and suramin in first stage treatment, and melarsoprol, eflornithine and nifurtimox in second stage treatment. However, the use of these drugs have several drawbacks including high toxicity, emerging drug resistance and impractical administration regimes, a situation that poses significant challenges in poorly equipped areas with inadequate medical facilities and resources (Lim et al., 2016).

In the last decade, phytotherapy has received great attention in the search for alternatives to chemotherapy in parasitic control. Plants and their derived secondary metabolites are an attractive source of lead compounds because they have numerous bioactive molecules which may be effective against kinetoplastid protozoa such as, alkaloids, phenolics, glycosides, and terpenes (Gehrig and Efferth, 2008). No new, safe and reliable trypanocidal compounds have been introduced in the past thirty years (Mbaya and Ibrahim, 2011). It is crucial to search for new and safer drug candidates for future HAT drug development, including those derived from natural product and plant sources.

As part of concerted campaign to discover new treatments in collaboration with Drugs for Neglected Diseases initiative (DNDi), we embarked on a high-throughput screening (HTS) program to search for potential natural compounds against HAT from Malaysian flora. A total of 300 methanolic crude extracts of plant samples from reserved forests of Peninsula Malaysia including S. spectabilis were screened for their potential inhibitory effect on the growth of T. b. rhodesiense STIB 900 by whole cell viability based HTS. We discovered that the methanol extract of S. spectabilis leaves exhibited anti-trypanosomal activity towards T. b. rhodesiense with an IC₅₀ value of 1.54 ± 0.28 μg/mL. According to the DNDi screening protocol, anti-trypanosomal activity of plant extracts is scored into three categories: active (IC₅₀ < 1.56 μg/mL), moderately active (1.56 μg/mL < IC₅₀ < 12.5 μg/mL), and inactive (IC₅₀ > 12.5 μg/mL). Pure compounds are considered active with an IC₅₀ < 0.5 μg/mL, IC₅₀ in between 0.5 and 5 μg/mL is designated as moderately active, and IC₅₀ > 5 μg/mL is classified as inactive (Ioset et al., 2009). Although Senna genus is well known for its anti-microbial activities, to the best of our knowledge, this is the first report to show that S. spectabilis possessed anti-trypanosomal property, particularly on T. b. rhodesiense. Herein, we present the bioassay-guided isolation of active compounds from the leaves of S. spectabilis that inhibit the growth of T. b. rhodesiense. Two known piperidine alkaloids namely, (+)-spectaline (1) and its isomer iso-6-spectaline (2) were isolated from the leaves of S. spectabilis with the greatest IC₅₀ values of 0.41 ± 0.01 μM and 0.71 ± 0.01 μM, respectively. Compound 1 and 2 showed no toxic effect on L6 cells with associated selectivity index of 134.92 and 123.74, respectively. In view of their selectively active anti-trypanosomal activity, we also investigated the cellular and ultrastructural aspects of these compounds on T. b. rhodesiense bloodstream form.

Senna spectabilis (Fabaceae) is a wooden flowering plant native to Central and South America. It has also been found to be naturalized in Africa and Asia and widely cultivated as ornamental plants. Traditionally, S. spectabilis is used as anti-inflammatory, analgesic, laxative, purgative, anti-microbial, and anti-ulcerogenic agents in Brazilian folk medicine (Viegas et al., 2004, Silva et al., 2009, Silva et al., 2011). In Africa, S. spectabilis is traditionally used to treat constipation, insomnia, anxiety, epilepsy, malaria, dysentery, and cephalalgia (Bum et al., 2010). In Asia, particularly in Ayurvedic and Thai medicines, the leaves of S. spectabilis are commonly used for the treatment of fever, headaches and various skin infections such as ringworm, eczema and scabies (Singh et al., 2013). The seed of S. spectabilis is also reported as aperients, anti-asthma and diuretic agents, or to improve visual activity in Traditional Chinese Medicine (Singh et al., 2013). In addition, a previous study showed that the flower extract of S. spectabilis exhibited anti-leishmanial properties against Leishmania major. Its dichloromethane and n-butanol fractions together with a mixture of (−)-cassine/(−)-spectaline (∼7:3) were found to possess inhibitory activity against the parasite L. major promastigotes with IC₅₀ values of 0.6, 1.6 and 24.9 μg/mL, respectively (de Albuquerque Melo et al., 2014).