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
Graphical abstract
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).
Section snippets
General experimental procedures
Melting points were recorded on a SMP40 automatic melting point apparatus (Stuart) and were uncorrected. The absorption spectra in the region of infrared (IR) were recorded on a FT-IR C103470 spectrometer (Perkin Elmer) employing the ATR technique. Optical rotations were measured on a polarimeter P300 (KRÜSS) equipped with a sodium lamp (λ = 589 nm) at 20 °C using DCM as a solvent. The electrospray ionization mass spectra (ESI-MS) were measured on a UHPLC Accela™ LCQ Duo Mass Spectrometer
Bioassay-guided isolation and identification of anti-trypanosomal compounds and their inhibitory activity against T. b. rhodesiense
According to the screening protocol, anti-trypanosomal activity of plant extracts was 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). When goes to the compound level, IC50 < 0.5 μg/mL was classified as active, IC50 in between 0.5 and 5 μg/mL was designated as moderately active, and IC50 > 5 μg/mL is classified as inactive (Ioset et al., 2009). In this study, methanol extract of S. spectabilis leaves
Discussion
Several species of Senna have been reported to accumulate phenolic, flavonoids, anthraquinones, and estilbenoid compounds with diverse biological and pharmacological properties (Table 2). In 2014, de Albuquerque Melo and colleagues reported that together with its homologue (−)-cassine, (−)-spectaline in the flowers of S. spectabilis showed significant anti-leishmanial activity. The mixture of (−)-cassine/(−)-spectaline exhibited potential activity against the parasite Leishmania major with IC50
Conclusions
This study reports the two piperidine alkaloids, (+)-spectaline (1) and iso-6-spectaline (2) isolated from the leaves of S. spectabilis showed significant trypanocidal activities against T. b. rhodesiense, without toxicity against L6 cells. Treatment with these compounds evidence a possible autophagic cell death. According to these data, the alkaloids were chemical hits and potential candidates for further investigations. Further elucidation of the molecular events linked to the autophagic PCD
Conflicts of interest
We wish to confirm that there are no known conflicts of interest associated with this publication.
Acknowledgements
This research was supported by grants from Ministry of Science, Technology and Innovation (MOSTI) of Malaysia (02-05-20-SF0005). We thank Swiss Tropical and Public Health Institute (Swiss TPH), Basel for providing the strain of T. b. rhodesiense STIB 900 used in this study. Nelson Jeng-Yeou Chear, is a recipient of USM Fellowship sponsored by the Universiti Sains Malaysia, Malaysia.
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