(+)-Spectaline, a Piperidine Alkaloid from Senna spectabilis DC. Effective in Reducing the In Vitro Infection of Leishmania major

Senna spectabilis is known to have antimicrobial, laxative, antiulcerogenic, analgesic, and anti-inflammatory properties in folk medicine. Piperidine alkaloids extracted from various parts of this plant have been shown to have anticonvulsant (iso-6-spectaline), antinociceptive [(-)-spectaline] and lipid peroxidation [(+)-3-O-feruloylcassine, (-)-spectaline and (-)-3-Oacetylspectaline] activities. In the present study, the ethyl acetate extract from S. spectabilis exhibited antileishmanial activity via intracellular promastigote assay or leishmanicidal assay and was further fractionated by using bioassay-guided isolation approach. The antiprotozoal principle was isolated from the ethyl acetate portion through solvent fractionation and few series of chromatographic processes. The isolated active compound 1 was identified as (+)-spectaline on the basis of its spectral analysis (MS, 1D & 2D NMR) with EC50 value of 0.063 ± 0.005 μM for antileishmanial activity and selectivity index of 3.76.


Introduction
Leishmaniasis is considered as one of the most neglected tropical diseases (NTDs) in the world [1,2]. It is endemic in 98 countries worldwide with an estimated 1.3 million new cases and an annual mortality rate of 20,000 to 40,000 [3]. In addition, Global Burden of Disease Study 2010 showed that it results in 3.32 million DALY (disability-adjusted life years) [1] which is second to malaria [4]. Current treatments are complicated because of the intramacrophagic location of the infectious form [5]. Although more efforts to come on vaccine development for leishmaniasis, there is no registered human leishmaniasis vaccine to date and pentavalent antimony or antimonials still act as the primary therapy [6,7]. Other drugs available in the market face great challenges such as acquired resistance by certain common species, depleted immune capability in HIV co-infection patients and organs toxicities [2]. The unfavorable side effects and resistance together with the increasing number of patients intensified the demand for alternative remedies to curtail this disease [8][9][10].
Leishmania spp infections typically originate via the bite of infected Phlebotomus spp or Lutzomya spp female sandflies. On being bitten, promastigotes are released into the skin and bloodstream which are then phygocytosed by the host macrophage [2]. The promastigotes are then transformed to amastigotes and then multiply, causing host cells to be burdened with parasites and thus becoming prone to lysis. The lysis will release amastigotes which then may infect other macrophages, thus ensuring their survival in the host [8,10]. The disease is characterized by a spectrum of clinical manifestations which are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), and also rarer ones such as mucosal leishmaniasis (MCL) [6]. The varied 75:25, 65:35, 50:50, 0:100 v/v). The eluent size used was 400 mL of each eluent system to obtain 26 fractions. Based on the TLC profiles of these fractions, they were pooled into 8 final fractions  which designated as CS-EA-CC1-F1 ~ F8.  CS-EA-CC1-F8 exhibited highest antileishmanial activity and was further fractionated via  silica gel column chromatography (silica gel 60F, 70-230  The parasites were grown and maintained as reported previously [17]. THP-1 and Vero cells (ATCC) were cultured as reported previously by Gebre-Hiwot and colleagues [18] also Carbonell and colleagues [19], respectively.
Leishmanicidal assay against promastigotes. This assay was performed as described in Poorrajab and colleagues [20] with minimal modification. Briefly, 2 x 10 6 cells/mL of log phase cultured promatigotes were incubated with a serial range of drug concentrations on a 96-well plate for 48 hours at 26°C. The activity was measured via reduction of resazurin to resorufin using Alamar Blue ® . After 2 hours of incubation, the assay plates were read using an ELISA microplate reader at a wavelength of excitation of 530 nm and emission of 590 nm. The IC 50 value was determined via analysis using Excel ® and GraphPad Prism ® 5.
In vitro intracellular antileishmanial assay. The assay was carried out according to Seifert and Croft [21]. Briefly, log-phase THP-1 cells were harvested at 1.0 x 10 6 cells/mL. Cells were diluted to 2.0 x 10 5 cells/mL with the complete medium, seeded in 16-well Nunc™ Lab-Tek TM tissue culture chamber slide (Fisher Scientific) at a seeding density of 5.0x10 4 macrophages/well (100 µL) and allowed to adhere by the addition of PMA (Phorbol -12 myristate Acetate, P8139 Sigma) for 3 days at 37°C in a 5% CO 2 -95% air mixture. Macrophages were then infected with long-slender (stationary stage) L. major promastigotes at a macrophage-promastigote ratio of 1:5. Infected macrophages were maintained at 34°C in a 5% CO 2 -95% air mixture. After 48 hours, extracellular parasites were removed by substituting the overlay with new fresh RPMI-1640 medium supplemented with 1% L-glutamine. Extracts, fractions and standard reference drug with various concentrations were added and their activities or effective concentration to decrease the parasite burden (EC 50 ) were determined from the percentage of infected cells in treated cultures in relation to non-treated cultures after MeOH fixation and Giemsa staining.
Cytotoxicity assay. The cytotoxicity assay was performed as described by Konowalchuk and colleagues [22] as well as Carbonell and colleagues [19] with some modifications. Briefly, confluent monolayers of Vero cells were removed with trypsin EDTA, re-suspended to International Journal of Pharmacology, Phytochemistry and Ethnomedicine Vol. 3 approximately 1 x 10 5 cells/mL in MEM. Next, 0.1 mL of the cells was pipetted into each well of a 96-well microtiter plate. After incubation at 37ºC in 5% CO2/95% air for overnight, the cells were treated with compound 1. Amp-B was used as positive control and MEM with 1% DMSO was used as negative control. All samples were tested in triplicates. Cytotoxic activity of Vero cells was measured via fluorescence reduction of resazurin to resorufin [23] by microplate reader and IC 50 value was determined using Excel ® and GraphPad Prism ® 5.

Results and Discussion.
The leaf extracts from S. spectabilis can be used for oedema inhibition, treatment of constipation, poisoning and protozoic infections' of the gut [24]. In addition, extracts from the leaves also showed good activities against various bacteria, fungi and yeast [15,25]. Antioxidant and anticonvulsant activities have also been observed from alkaloids found in the leaf [26].
In antileishmanial area of study, a few alkaloids have been proven to be effective against extracellular promastigotes which have been extensively listed by Chan-Bacab and Pena Rodriguez [27]. In a more recent study [28], mixed compounds of major piperidine alkaloids from S. spectabilis flowers extract which consist of (-)-spectaline and (-)-casein have been claimed to be effective against promastigotes of L. major. Through intracellular amastigotes assay model, several pure alkaloids such as berberine [29], liriodenin [30], harmaline [27] and gabunine [31] have been proven for their antileishmanial activity. Table 1 shows the results on the antileishmanial activity of extracts and fractions of S. spectabilis. In the promastigote assay, IC 50 refers to the 50% inhibitory concentration to inhibit the growth of promastigotes [20], while EC 50 in the intracellular amastigote assay model means the effective concentration of the substance to decrease as much as 50% parasite burden of infected macrophage [21].
Based on the results presented in Table 1, the ethyl acetate extract (CS-EA) from the leaves of S. spectabilis showed antileishmanial activity by inhibiting the promastigotes' growth (IC 50 = 70.29 ± 0.38 µg/mL). The CS-EA extract was then further purified using silica gel column chromatography by subjecting it to series of bioassay-guided fractionations. Further fractionation results in only fraction 8 (CS-EA-CC1-F8) exhibited antileishmanial activity as much as 20% in decreasing intracellular amastigotes at 33.33 ± 0.05 µg/mL. Subsequently, CS-EA-CC1-F8 was further fractionated via silica gel column chromatography. Results showed that fraction 4 (CS-EA-CC1-F8-CC2-F4) is the most active in decreasing the intracellular amastigotes (EC 50 = 31.75 ± 10.04 µg/mL) and the fraction was further purified using flash column chromatography. As a result, pure compound was isolated from fraction 1 (CS-EA-CC1-F8-CC2-F4-CC3-F1) and was designated as compound 1 which possessed the highest activity in decreasing intracellular amastigotes (EC 50 = 20.58 ± 1.65 µg/mL). Compound 1 was identified as (+)-spectaline, a known piperidine alkaloid (Figure 1) that agrees in every respect (IR, NMR and MS) with those in literature [32] which the comparison of NMR data are summarised in Table 2 and 3. With an EC 50 value of 20.58 ± 1.65 µg/mL, compound 1 decreased the parasite burden or, it has the activity in macrophage clearance. This value seems to be more potent than activity of Amp-B against L.donovani infection on THP-1 which is around 231.02 µg/mL (0.25 µM) [33]. However, this value is less potent compared to berberine (a quaternary alkaloid found in several plant families) with an EC 50 value of 9.08 µg/mL (0.027 µM) [29]. Berberine has been used clinically and applied parenterally for the treatment of CL. However, it is topically inactive [34]. With IC 50 for cytotoxicity = 0.237 ± 0.004 µM and EC 50 in antileishmanial assay = 0.063 ± 0.005µM, (+)spectaline (Compound 1) has selectivity index of 3.76.
(+)-spectaline has been successfully isolated from the leaves of S. spectabilis [32]. However, no antileishmanial studies being done on this compound thus far. On the other hand, a mixed compounds of (-)-spectaline and (-)-cassein from the flower of S. spectabilis has been reported to have antileishmanial properties (IC 50 = 24.9 ± 1.4 µg/mL) via extracellular promastigote assay by De Albuquerque Melo and colleagues [28]. To the best of our knowledge, this is the first report on the antileishmanial activity of (+)-spectaline via intracellular amastigotes assay model.    26.0 (q) Figure 1: The chemical structures of (+)-spectaline (compound 1).