INTRODUCTION
Strongyloides stercoralis is a common, gastrointestinal parasitic nematode that naturally infects humans, other primates and canines by skin contact (Nolan et al. Reference Nolan, Nutman, Schad, Palmer, Soulsby, Torgerson and Brown2011). As many as 200 million people may be infected worldwide (Despommier et al. Reference Despommier, Gwadz, Hotez and Knirsch2005), primarily in tropical and subtropical regions (Garcia and Bruckner, Reference Garcia and Bruckner1988). In the Caribbean, where the infection is endemic (Tikasingh, Reference Tikasingh1981), with prevalence ranging from 0·1 to 23·6%, strongyloidosis is clinically the most severe nematodiasis. Chronic infections in immunocompetent individuals are typically asymptomatic, and can persist upwards of 30 years as a result of autoinfection (Gill and Bell, Reference Gill and Bell1979; Grove, Reference Grove1980). However, hyperinfections can occur and are linked to poor nutrition and immunosuppression caused by illness and/or corticosteroid treatment. The resulting overwhelming, or disseminated, strongyloidosis is often fatal (Cruz et al. Reference Cruz, Reboucas and Rocha1966; Grove Reference Grove1980; Powell et al. Reference Powell, Moss and Nagar1980; Ingra-Siegnam et al. Reference Ingra-Siegman, Kapila, Sen, Kaminski and Louria1981; Davidson et al. Reference Davidson, Fletcher and Chapman1984; Genta, Reference Genta1989; Mansfield and Schad, Reference Mansfield and Schad1992).
With transmission of S. stercoralis enhanced by poor sanitary conditions in the surroundings of infected individuals and its ability to persist in the soil under warm, moist conditions (Grove, Reference Grove1980), an effective control strategy against the disease has been met with challenges of diagnosis (Siddiqui and Berk, Reference Siddiqui and Berk2001) and treatment (Terry, Reference Terry1987). Commercial drugs developed for treating strongyloidosis exhibit unreliable effectiveness, and have other associated problems. For example, of the benzimidazoles, thiabendazole [2-(4′-thiazoyl)-benzimidazole] (Mintezol; Merck, Sharp & Dohme) was the drug of choice for treating chronic and severe strongyloidosis in the Caribbean (Grove, Reference Grove1982 Reference Grove b ), but poor efficacy, toxicity, unpleasant side effects, and high costs have deterred its use (Bezjak, Reference Bezjak1968; Gustaffson et al. Reference Gustafsson, Beerman and Abdi1987; Terry, Reference Terry1987). Despite some success intreating strongyloidosis, albendazole [5-(propylthio)-2-carbomethoxyamino-benzimidazole] (Zentel; Smith, Kline Beecham) is associated with teratogenicity and embryotoxicity in experimental animals (Gustafsson et al. Reference Gustafsson, Beerman and Abdi1987). Thus, its use has been discouraged, especially in women during pregnancy and lactation. Mebendazole [methyl 5-benzoyl-2-benzimidazole carbamate] (Vermox; Janssen) was reported to be inactive against the infective larvae in immunosuppressed dogs, and has little effect on the parasite in muscle tissue of mice (Grove, Reference Grove1982a ). Use of cambendazole ([5-isopropoxycarbonylamino-2-(4-thiazyl) benzimidazole] (Merck, Sharp & Dohme) has been banned as a result of its linkage to several fatal idiosyncratic reactions in cattle (Hogg, Reference Hogg1978; Main and Vass, Reference Main and Vass1980).
Alternatively, ivermectin [22,23-dihydroavermectin B1] (Merck, Sharp & Dohme), a broad-spectrum, commercial drug (Fisher, Reference Fisher1985; Grove, Reference Grove1989) that is a synthetic, lipophilic, macrocyclic lactone (Gustafsson et al. Reference Gustafsson, Beerman and Abdi1987; Campbell, Reference Campbell1989), bears promise as an efficacious treatment against strongyloidosis (Naquira et al. Reference Naquira, Jimenez, Guerra, Bernal, Nalin, Neu and Aziz1989; Marti et al. Reference Marti, Haji, Savioli, Chwaya, Mgeni, Ameir and Hatz1996). Cure rates of 82–88% have been reported (Naquira et al. Reference Naquira, Jimenez, Guerra, Bernal, Nalin, Neu and Aziz1989; Adenusi et al. Reference Adenusi, Oke and Adenusi2003). However, more work is needed to further confirm its efficacy (Adenusi et al. Reference Adenusi, Oke and Adenusi2003), and there are reports of emerging drug resistance against ivermectin by parasitic nematodes (Echevarria and Trindade, Reference Echevarria and Trindade1989; Jackson and Coop, Reference Jackson and Coop2000; Currie et al. Reference Currie, Harumal, McKinnon and Walton2004), including S. stercoralis (Gill et al. Reference Gill, Redwin, van Wyk and Lacey1991; Shikiya et al. Reference Shikiya, Kinjo and Uehara1992). Additionally, in Jamaica where the majority of infected individuals are of poor socio-economic status, the suggested single-dose regimen for ivermectin therapy (200 μg kg−1 body weight) at an average cost of US$32.00, is often unaffordable.
This paucity of effective drug therapy against strongyloidosis highlights the imperative for new sources of treatment that are efficacious and affordable.
Since time immemorial, medicinal plants have been used almost entirely by large percentages of human populations in the Caribbean, Africa, Asia and other tropical regions in the form of concoctions and decoctions for treating a myriad of ailments including intestinal worm infections (Ayensu, Reference Ayensu1981; Dagar and Dagar, Reference Dagar and Dagar1991; Naranjo et al. Reference Naranjo, Gil, Naranjo and Gay1995). Although there is abundant information on folklore usage of plants across the world, more effort is needed to scientifically validate these practices and folk-claims. Since Sloane's account (Sloane, Reference Sloane1725) about 60 publications have appeared on the topic of folklore medicine in the Caribbean with only a few dealing with parasitic infections (Asprey and Thornton, Reference Asprey and Thornton1953; Campbell, Reference Campbell1974; Lal et al. Reference Lal, Chandra, Raviprakash and Sabir1976; Goulart et al. Reference Goulart, Jourdan, Brazil, Brazil, Cosendey, Bar, Do Carmo and Gilbert1977; Ayensu, Reference Ayensu1981; Robinson et al. Reference Robinson, Williams, Lindo, Terry and Mansingh1990). Thus, in the search for a new, safe and effective treatment against strongyloidosis, Jamaican medicinal plants were selected based on their folklore usage against intestinal worm infections. Some were also selected on the basis of insecticidal activity, because similarities exist in neuromuscular transmission between nematodes and insects (Leake and Walker, Reference Leake and Walker1980), while others were selected based on known chemotaxonomic properties of allied species (Trease and Evans, Reference Trease and Evans1934; Williams, Reference Williams1992; Albert and Struwe, Reference Albert and Struwe1997).
MATERIALS AND METHODS
Test organism
Infection of S. stercoralis, procured from a 57-year-old female patient in Jamaica, was established in two parasite-free mongrel dogs of the same litter by cutaneous inoculation of each in the mid-dorsal neck region with 1 mL Locke's nematode saline (LNS) containing 3000 infective larvae (L3i) (Genta, Reference Genta1989). First-stage rhabditiform larvae (L1) were first detected at 53 and 70 days post-larval inoculation in the dogs. Infections were maintained by administering prednisolone (Predol retention enema, Glaxo Australia Pty. Ltd., Boronia, Vic.) daily at 0·5 mg kg−1 per os dog (Dawkins and Grove, Reference Dawkins and Grove1982; Schad et al. Reference Schad, Hellman and Muncey1984), under professional veterinary supervision. The dogs were fed and watered ad libitum. L3i 5 days old were harvested from charcoal coprocultures in LNS and used as the test organisms in all bioassays. They were gravimetrically quantified using a counter and an Alphaphot 2 compound light microscope (Nikon) so that 20 μL sub-samples contained about 50 L3i.
Assessment of larval mortality by eosin and determination of optimal concentration
The dye-exclusion technique (Halton and Arme, Reference Halton and Arme1971; Robinson and Arme, Reference Robinson and Arme1985) was used to indicate larval mortality in all bioassays. Heat-killed (test) as well as live larvae (control) were exposed to eosin (0·1, 0·3 and 1 mg mL−1) in LNS. Counts of stained and non-stained larvae in 20 μL suspensions were conducted at 10 min intervals for 1 h. Optimal eosin concentration as an indicator of larval mortality was determined by exposing live specimens to eosin (0·1, 0·3 and 1 mg mL−1) and counting the number of mobile, immobile (not stained) and dead (stained) larvae at 0, 1, 2, 3 and 6 h post incubation, and subsequently at 24 h intervals for 96 h.
Plant selection
Twenty-five plant species were selected for crude extract preparation using either whole plants or specific plant tissues such as leaves, roots, stem and/or flowers. Andrographis paniculata, Centrostachys indica, Allium sativum, Eryngium foetidum, Tamarindus indica, Carica papaya, Chenopodium ambrisoides, Ambrosia hispida, Bidens pilosa, Cucurbita pepo, Ricinus communis, Rytidophyllum tomentosum, Aloe vulgaris, Cecropia peltata, Ravina humilis, Portulaca oleracea, Punica granatum, Picrasma excelsa and Stachytarpheta jamaicensis were selected on the basis of their usage in Caribbean folklore against intestinal worms (Asprey and Thornton, Reference Asprey and Thornton1953; Adams, Reference Adams1972; Campbell, Reference Campbell1974; Honychurch, Reference Honychurch1980; Ayensu, Reference Ayensu1981). Annona muricata and Annona squamosa, which are also used in Caribbean folklore, were chosen based on demonstrated chemotaxonomic properties of allied species (Waterman, Reference Waterman1986). Finally, Cuscuta americana, Azadirachta indica, Mimosa pudica and Artocarpus altilis, besides their folklore medicinal uses, also have known insecticidal properties (Zanno et al. Reference Zanno, Muira, Nakanishi and Elder1975; Jacobson, Reference Jacobson1981, Reference Jacobson, Green and Hedin1986; Williams, 1992; Wilson, Reference Wilson1993).
Preparation of crude extracts
Each green plant (20 g) was chopped and extracted with 0·1 L methanol–water (4:1, v/v) for 5 days at room temperature. Crude extracts were washed with methanol–water (5 mL) twice and filtered. The filtrates were pooled, concentrated in vacuo and freeze-dried (3×24 h) to remove water and methanol residue (see determination of methanol content). Freeze-dried crude extracts were weighed and their yields (g) determined. All extracts were stored at −4 °C before bioassay.
Determination of methanol content in crude extracts
Carbon-13 (13C) Nuclear Magnetic Resonance (NMR) spectral analysis was used to detect methanol residue in randomly selected R. communis, A. cepa and S. jamaicensis crude extracts (100 mg) before and after freeze-drying. These were dissolved in deuterium oxide (3 mL) and filtered. 13C NMR spectral analyses of the extracts were determined on a Bruker AC 200 instrument. Sodium trimethylsilylpropanoate (TSP) was used as the internal standard. Methanol concentrations [μg CH3OH/100 mg extract] were approximated by comparing relative differences of NMR signals (13C peak heights) with that of the TSP (internal) standard.
Assessment of larval tolerance to methanol
Due to the toxicity of methanol (Cornish, Reference Cornish, Casarell and Doull1975), and its detection in freeze-dried samples, tolerance of infective larvae to the solvent was investigated in triplicate at 0 (control), 1·0, 5·0 and 10·0 μg CH3OH/100 mg in LNS in the presence of eosin (0·1 mg mL−1). The bioassay was conducted as indicated below (bioassay of crude plant extracts).
Bioassay of crude plant extracts
A stock suspension (100 mg mL−1) of each crude plant extract that contained eosin (0·2 mg mL−1) was prepared in LNS. An equal volume of LNS with L3i was added to a known volume of the stock, thereby halving its concentration along with eosin, and adjusting the number of L3i in sub-samples (20 μL) to about 50 L3i. Anthelmintic potential of each plant extract (50 mg mL−1) was investigated in vitro by counting the number of L3i that were mobile, immobile (not stained) and dead (stained with eosin) at 0, 1, 2, 3 and 6 h post-larval incubation, and at subsequent 24 h intervals for 5 days in tests and controls (triplicate). The larvae were kept in a light-free environment between counts to preserve their viability. Relative activity (RA) was calculated by dividing LT50 of the most effective extract (shortest duration to kill 50% of the L3i) by the LT50 of each extract. With E. foetidum crude extract being the most potent, a specimen of the plant with the assigned accession number 33 700 was deposited in Botany Herbarium of The University of the West Indies, Mona Campus, Jamaica, West Indies.
Preparation of E. foetidum crude extracts
Whole, green plants of E. foetidum (2·46 kg) were chopped and extracted with petrol (6 L) for approximately 36 h to yield an insoluble, oily extract (2·624 g) with a characteristic pungent odour. Petrol refers to the petroleum fraction with boiling point 60–80 °C. Plant extraction was continued with dichloromethane (5·5 L) for 48 h to yield a greenish-brown insoluble residue (5·43 g). Finally, the plant material was extracted with methanol–water (4:1), which yielded a dark brown extract (5·764 g) after freeze-drying (3×24 h).
Bioassay of emulsifier treatments
With the petrol and dichloromethane crude extracts of E. foetidum being water insoluble, an effective emulsifier to homogenize the extracts was identified accordingly. Triton X-100 and ‘Tween 20’ (1, 2 and 4 mg mL−1) in LNS with eosin (0·1 mg mL−1) were bioassayed in triplicate, respectively to assess for any larval inhibitory effect by the emulsifiers. Counts of larvae in 20 μL sub-samples were done at the same time intervals stated above. Controls had no emulsifier. With the inability of Triton X-100 (1, 2 and 4 mg mL−1) to adequately dissolve E. foetidum dichloromethane and petrol crude extracts, higher concentrations (10, 20 and 40 mg mL−1) were similarly bioassayed.
Bioassay of E. foetidum crude extracts
Stock suspensions of E. foetidum petrol, dichloromethane and methanol:water crude extracts (100 mg mL−1) in LNS with Triton-X 100 (20 mg mL−1) and eosin (0·2 mg mL−1) were diluted to 50 mg mL−1 by adding equal volumes of LNS that contained L3i and then were bioassayed. Larval mortality was assessed by counting the L3i in homogeneous sub-samples (20 μL) at similar time intervals that were used for the 25 plant crude extract bioassays.
Test for the presence of triolein and stigmasterol in E. foetidum petrol crude extract, and separation and demarcation of its compounds by column and thin layer chromatography
The petrol extract of E. foetidum was examined for the presence of triolein and stigmasterol using thin layer chromatography (TLC). The extract (1·005 g) was also subjected to TLC and gravity column chromatography, which involved the use of silica gel (230–400 mesh, 40–63 μm in diameter, Sigma) as the stationary phase and increasing concentrations of ethyl acetate in petrol as the mobile phase. Polyester backed pre-coated silica gel plates HF254+366 (0·25 mm thickness, 20×20 cm, Sigma Aldrich) with fluorescent indicator were used for TLC work. 10 mL aliquots were collected under gravity and the eluents were subjected to TLC and UV analyses to assess the separated compounds. Chromatograms were compared with the petrol crude extract and aliquots containing compounds having similar R f values were pooled and visualized by spraying with dodecamolybdophosphoric acid/ceric sulphate spray reagent and viewing under a UV lamp for active compounds. Pooled aliquots were concentrated in vacuo and yields (g) of the compounds were determined.
Bioassay to further identify locality of active compound(s) in the petrol crude extract
Triplicate 20 mg mL−1 suspensions of eight pooled 10 mL column chromatography aliquots that contained 16 compounds in the E. foetidum petrol crude extract were prepared in LNS. These also contained 20 mg mL−1 Triton X-100 and eosin (0·2 mg mL−1). Equal volumes of LNS with L3i were added, thereby halving concentrations of the compounds, emulsifier and eosin. Counts of larvae in 20 μL sub-samples were carried out at the same time intervals used for crude plant extracts. The bioassays allowed for further identification of the locality of the active compound(s), based on the aliquots that inactivated the larvae.
Purification of the anthelmintic compound, trans-2-dodecenal
Having identified the pooled aliquots of the E. foetidum petrol crude extract with greatest anthelmintic activity, purification was effected by chromatography of the extract (434 mg) using four 20×20 cm preparative thin layer chromatographic plates (0·5 mm thickness). The solvent system used consisted of ethyl acetate: petrol: acetic acid in a 4:96:1 ratio. The plates were run once in the solvent system to give two main bands with R f values of 0·12 and 0·76. The compounds were visualized by spraying with a 2,7-dichlorofluorescein solution followed by observation under a UV lamp. The high R f band contained the compound of interest. Therefore, a second set of two plates utilizing 2% ethyl acetate in petrol as the eluent was run for further purification. This purification attempt yielded 77·0 mg of trans-2-dodecenal (eryngial). This was analysed by infrared, NMR and mass spectroscopy.
Bioassay of the anthelmintic compound, trans-2-dodecenal, and its comparison with ivermectin
The 24 and 48 h 50% lethal doses (LD50) of eryngial and ivermectin (mm) were investigated in vitro using the L3i. Ivermectin was obtained from Sigma Aldrich, St. Louis, MO. The larvae were exposed to a range of dilutions of the compounds by adding known volumes of LNS with L3i to known volumes of stock suspensions of the anthelmintic compounds (10 mg mL−1 in LNS) which contained final concentrations of Triton X-100 (2 mg mL−1) and eosin (0·1 mg mL−1). Each dilution, including the control (0 mg mL−1), was prepared in triplicate. 20 μL sub-samples contained about 50 L3i. Larval mortality was assessed in a similar way to the plant crude extract bioassays.
Treatment of data
All data were corrected for control mortality using Abbot's formula (Busvine, Reference Busvine1972):
Abbot's formula:
$$\; P = \displaystyle{{{\rm} P_1 {\rm} - {\rm} C} \over {100 - C}} \times 100{\rm} $$
where, P = corrected percentage inactivation; P 1 = observed percentage inactivation; C = control percentage inactivation.
The time (h) taken by each crude extract to kill 50% of the infective larvae (LT50) was determined using Probit Analysis (Busvine, Reference Busvine1972). Fiducial limits could not be calculated from the data whenever the heterogeneity factor (g) exceeded 0·5 (Finney, Reference Finney1975). In such instances, Analysis of Covariance was used to test if significant differences existed between slopes of the control and test solutions. Probit analysis was also used to determine the 24 h LD50 values of trans-2-dodecenal and ivermectin in vitro.
RESULTS
Indicator of larval mortality and tolerance to eosin
Eosin (0·1, 0·3 and 1 mg mL−1) in LNS indicated staining (mortality) of heat killed L3i after 10 min of exposure, starting at the oesophagus and proctodeum. Staining was not observed in the live control larvae. The eosin concentrations also resulted in about 2% larval mortality (cumulative corrected percentage) after 96 h. Eosin (0·1 mg mL−1) was hence used as the indicator of larval mortality in all in vitro bioassays because it was the lowest effective concentration.
Methanol content in crude extracts
Methanol concentrations (μg/100 mg) after freeze-drying (24 h) extracts of R. communis, S. jamaicensis and A. cepo were 0·1, 0·025 and 0·1, respectively. With additional freeze-drying (2×24 h), methanol concentration in the extracts was reduced by 1·4, 2·8 and 10·0 fold, respectively (Table 1). Nuclear magnetic resonance spectra indicated reduced levels of methanol in the crude extract samples before and after refreeze-drying (Fig. 1). Methanol concentrations were calculated based on signal intensities in the spectra. The other crude extracts were similarly refreeze-dried and for the same duration (2×24 h).
Fig. 1. Partial 13C NMR spectrum of methanol–water (4:1) crude extracts of R. communis, A.cepa and S. jamaicensis (solvent, D2O) before refreeze-drying (a, c and e), and after refreeze-drying (b, d and f), respectively. X denotes signals from internal standard (TSP). NB: unmarked peaks represent other compounds in the plant extract.
Table 1. Concentration of methanol in crude extracts detected by nuclear magnetic resonance (NMR) analyses before and after additional 48 h freeze-drying (FD)
Methanol concentration [μg CH3OH/100 mg crude extract] determined by comparison of differences of NMR signals with the standard, sodium trimethylsilylpropanoate (TSP).
Larval tolerance to methanol
Analysis of Covariance revealed no significant difference between slopes of the control and test solutions of methanol for larval mortality, except that the slope of the 10 μg CH3OH/100 mg solution was significantly different from those of the control and 1 μg CH3OH/100 mg solutions (F = 5·868, F 0·05 (3) 3, 124 = 2·67, P<0·05). Methanol concentrations that were 10, 50 and 100 times greater than the highest concentration detected by NMR analyses (Table 1) had a mortality effect of approximately 10% on the L3i after 120 h. Refreeze-drying further reduced concentrations of methanol in the extracts to less than 0·07 μg CH3OH/100 mg solution (Table 1).
Larval behavioural response to crude extracts
Strongyloides stercoralis L3i were typically active in control solutions, swimming vigorously in a wiggling motion throughout the 5-day duration of each bioassay. Swimming behaviour was, however, altered in plant extracts with anthelmintic activity. Initially, the L3i were generally as active as the controls. However, with time, the darting swimming manoeuvres slowed. Eventually, the larvae stopped swimming and became immobile. Immobility was occasionally punctuated with ‘jerky’ movements. L3i mortality, which was indicated by eosin staining, was always preceded by permanent immobilization. The rate at which the sequence of events occurred, and the number of larvae that were immobilized only or killed, varied from one crude extract to another.
Anthelmintic activity of crude plant extracts
Durations (h) taken by the crude extracts (50 mg mL−1) of selected plants to kill 50% (LT50) of S. stercoralis L3i are shown in Table 2. The extracts’ LT50 values ranged from 18·9 h (E. foetidum) to 99·2 h (A. paniculata). LT50 values were not determined for crude extract suspensions of C. papaya, A. muricata, A. altilis and P. granatum, which resulted in less than 50% mortality after 120 h. Eryngium foetidum crude extract was significantly more effective at killing the L3i than the others and was therefore subjected to further scrutiny to elucidate the source of anthelmintic activity.
Table 2. 50% mortality time (LT50, h) of S. stercoralis infective larvae by 50 mg mL−1 crude extracts of selected Jamaican plants. Relative activity of E. foetidum crude extract used as reference for other crude extracts
RA = LT50 of the most effective extract divided by LT50 of each extract.
Selection of emulsifier
‘Tween’ 20 (1, 2 and 4 mg mL−1) in LNS (with eosin, 0·1 mg mL−1) was toxic to S. stercoralis L3i. Cumulative corrected percentage mortality ranged from 8·1 to 40·8% after 96 h. Additionally, ‘Tween 20’ did not produce homogeneous dispersions of E. foetidum dichloromethane and petrol extracts (100 mg mL−1) after shaking (12 h). Similar concentrations of Triton X-100, which caused less than 3% cumulative percentage larval mortality after 96 h, also did not produce homogeneous dispersions of the E. foetidum extracts. Higher concentrations of Triton X-100 (10, 20 and 40 mg mL−1) in LNS with eosin (conc. as above) resulted in less than 2% (cumulative corrected percentage) L3i mortality after 96 h and dispersed both crude extracts in the preparation of 10·0% stock suspensions. Hence, Triton X-100 (10 mg mL−1) was selected as the lowest effective concentration for homogenizing E. foetidum crude extracts.
Anthelmintic activity of E. foetidum crude extracts
The LT50 value (4·72 h) of E. foetidum petrol extract (50 mg mL−1) confirmed it to be significantly greater than that of the other extracts. After 6 h, the petrol extract killed 96% of the L3i, while the dichloromethane and methanol:water crude extracts resulted in less than 1% mortality after 120 h.
Absence of triolein and stigmasterol from E. foetidum petrol crude extract and anthelmintic activity of its compounds
Thin layer chromatography analysis revealed the absence of triolein and stigmasterol from the E. foetidum petrol crude extract. Pooled aliquots 15–35 resulted in 50% larval mortality after 20·8 h and 85·4% mortality after 48 h, while all the other pooled aliquots (1–2, 3–5, 74–83, 107–163) except for aliquots 36–48 and 49–73, killed less than 1% of the L3i after 120 h, respectively. Aliquots 36–48 and 49–73 killed approximately 21 and 12% of the L3i after 120 h, respectively. Pooled aliquots 15–35 was therefore identified as that portion of the eluent that contained the anthelmintic compound(s) in the greatest concentration. Analysis with TCL revealed the presence of the compound(s) of aliquot 15–35 in aliquots 36–48 and 49–73, albeit in smaller quantities.
‘Eryngial’, the purified anthelmintic compound from E. foetidum
The E. foetidum anthelmintic compound was characterized as a pale, yellow oil with a pungent odour; was shown to be a straight chain, unsaturated aldehyde of chemical formula C12H22O; and was assigned the name ‘eryngial’. The carbon-carbon double bond was found to be in conjugation with the carbonyl and its configuration was determined to be E. The aldehyde was slowly oxidized to the corresponding carboxylic acid in air. This was confirmed by analysis of the methyl ester of this acid. The preparation of the 2,4-dinitrophenylhydrazone derivative of the aldehyde, as well as Lemieux–von Rudloff degradation to decanoic acid, characterized as its methyl ester, confirmed the structure of eryngial as trans-2-dodecenal or E-2-dodecenal:
-
GCMs: m/z (rel. int.) 182·1 (<1) [M+], 135 (10), 121 (20), 97 (35) [M+-C6H13], 83 (76)
[M+-C7H15] (β-cleavage), 70 (98), 55 (80) [C3H3O], 43 (70) [C3H7], 41 (100), 29 (44) [C2H5];
FAB-MS: m/z (rel. int.) 200·5 (61) [M+NH4 +], 190·4 (14), 81·2 (11), 58·1 (22), 35·1 (100);
-
1H NMR: δ 0·90 (3H, t, J = 6·80 Hz), 1·31 (12 H, br s), 1·55 (2H, t, J = 7·13 Hz), 2·34
-
(2H, qd, J = 6·83, 1·44 Hz), 6·13 (1H, ddt, J = 15·83, 7·91, 1·09 Hz), 6·86 (1H, dt, J = 15·83, 6·80 Hz), 9·52 (1H, d, J = 7·91 Hz); 13C NMR: δ 14·0 (CH3), 22·6 (CH2), 27·8 (CH2), 29·16 (CH2), 29·2 (CH2), 29·3 (CH2), 29·4 (CH2), 31·8 (CH2), 32·7 (CH2), 132·9 (CH), 159·0 (CH), 194·1 (CH).
The chemical structure of trans-2-dodecenal (C12H22O) is shown (Fig. 2).
Fig. 2. The chemical structure of trans-2-dodecenal (C12H22O).
Bioassay of eryngial and comparison with ivermectin
The order of activity using 24 h LD50 (mm) for the anthelmintic compounds was: eryngial, 0·461>ivermectin, 2·251 (Table 3). The plant compound was significantly more effective at killing the L3i displaying a 4·9-fold increase in activity over ivermectin. There was however, no significant difference between the 48 h LD50 (mm) of trans-2-dodecenal (0·411) and ivermectin (0·499) (Table 3).
Table 3. In vitro dose-response data for S. stercoralis L3i after 24 and 48 h using trans-2-dodecenal and ivermectin
(mm)* – LD50 values listed as millimolar concentrations.
DISCUSSION
This study confirms the role of traditional medicine in the search for bioactive compounds of natural product origin. Folklore-claims, that E. foetidum and other selected medicinal plants possess anthelmintic activity, were confirmed by rigorous scientific scrutiny of the extracts using S. stercoralis L3i.
For the first time, based on 50 mg mL−1 T50 values (Table 2), it was shown that over 19 species of Jamaican plants belonging to different families exhibited a spectrum of activity, ranging from those that were strongly anthelmintic to those with little or no inactivation potential. For example, E. foetidum crude extract (50 mg mL−1) killed 50% of the L3i after 18·9 h, while P. granatum was ineffective at killing the larvae even after 120 h.
Eryngium foetidum crude extract was the most effective at killing the L3i based on the LT50 values and was therefore subjected to further scrutiny. For the first time, eryngial was identified and isolated as the main source of anthelmintic activity, and demonstrated acute toxicity against S. stercoralis L3i in vitro. Localization of eryngial in the plant's petrol crude extract (R f value = 0·40 in 10% EtOAc/petrol) and its chemical isolation were made possible by systematic bioassay guided fractionation and chromatography techniques.
Standardization of the methodology ensured reliable assessment of the plant extracts’ anthelmintic potential. For example, staining of dead S. stercoralis L3i, via the eosin exclusion technique (Halton and Arme, Reference Halton and Arme1971; Robinson and Arme, Reference Robinson and Arme1985), successfully indicated larval mortality throughout all bioassay work. This was based on the principle that selective cuticular semipermeability to solutes is compromised in dead organisms. The eosin exclusion technique was therefore used to facilitate differentiation of dead larvae from those that were either mobile or immobile but not dead. Hence, LT50 values of the crude extracts using S. stercoralis L3i larvae could be determined. Although concentrations of methanol up to 100 times more than amounts initially detected in crude extract samples after freeze-drying had minimal adverse effect on the L3i, refreeze-drying of the crude extracts (2×24 h) further reduced methanol content to lower quantities. Thus, any possible larval inactivation effect by methanol was nullified.
Strain-related heterogeneity of the parasite was minimized by the establishment and maintenance of infections in two mongrel dogs from the same litter using S. stercoralis L3i that were obtained from a single human host. Experimental studies by Grove (Reference Grove, Warren and Mahmoud1984) have shown variations in infectivity and duration of infection by different strains of S. stercoralis in mongrels. Some dogs were resistant to re-infection, while in others the self-cure phenomenon was absent and the infection became persistent. The likelihood of variations in the rates of larval inactivation occurring as a result of the plants’ intrinsic properties was optimized. Mongrel dogs have been used as models of human strongyloidosis for more than 60 years (Sandground, Reference Sandground1928; Grove and Northern, Reference Grove and Northern1982; Genta, Reference Genta1989).
The petrol and dichloromethane crude extracts of E. foetidum were not miscible with LNS because of their low solubility in the aqueous medium. Thus, Triton X-100 (10 mg mL−1), which is an emulsifier (Matthews, Reference Matthews2000), was used to bring about a homogeneous and stable dispersion of the extracts in the incubation solution. It was also the lowest concentration investigated and had a negligible adverse effect on the L3i. Also, the absence of triolein and stigmasterol from the E. foetidum petrol crude extract was noted because both compounds commonly occur in a wide variety of plants and possess insecticidal activity (Ito et al. Reference Ito, Kawashima, Nakahara, Nakanishi and Terahara1964; Chippendale and Mann, Reference Chippendale and Mann1972). This hinted at the possibility of a novel anthelmintic E. foetidum compound or group of compounds.
Infrared, NMR and mass spectroscopic analysis revealed eryngial to be an intensely odorous, herbaceous, volatile, non-polar and lipophilic unsaturated, long-chain fatty aldehyde. The compound's lipophilicity may partially explain its rapid rate of larval inactivation, possibly as a result of ease of diffusion across the cuticle. Many nematicides, for example the organophosphates, alkyl halides and oxime carbamates, owe their efficacy, in part, to being lipophilic (Lee and Atkinson, Reference Lee and Atkinson1965). Eryngial's ‘fast-acting’ activity, based on 24 and 48 h LD50 values, compared favourably with ivermectin in vitro and underscores its candidacy for drug development studies.
A comparison can be made between the results of this and an in vitro study by Boonmars et al. Reference Boonmars, Khunkitti and Sithithaworn2005. In that study, aqueous and alcohol extracts (2 mg mL−1) of the medicinal plant Cardiospermum halicacabum immobilized S. stercoralis L3i within 48–72 h, while ivermectin (0·25 mg mL−1) immobilized the larvae between 72 and 144 h. While the authors in this previous study did not conclusively demonstrate larval mortality or determined LD50 values of compounds tested, ivermectin (0·25 mg mL−1) taking greater than 72 h to immobilize the L3i, points in the direction of results obtained in the present study. Here, based on the 24 and 48 h LD50 values (1·97 mg mL−1 = 2·251 mm and 0·437 mg mL−1 = 0·499 mm, respectively), higher concentrations of ivermectin were required to effect larval inactivation in shorter durations.
The mode of action of eryngial against S. stercoralis is not known. However, paralysis in nematodes and other animals by drugs is primarily due to a block of neuromuscular transmitters (Holden-Dye and Walker, Reference Holden-Dye, Walker, Maricq and McIntire2007). Such drugs include pyrantel, levamisole, santonin, bephenium salts and methyridine (Lee and Atkinson, Reference Lee and Atkinson1965; Gustafsson et al. Reference Gustafsson, Beerman and Abdi1987; Atchison et al. Reference Atchison, Geary, Manning, Van de Waa and Thompson1992). Some anthelmintics exert activity by disrupting the nematodes’ energy-producing metabolic pathways (Lee and Atkinson, Reference Lee and Atkinson1965). Thus, mode of action studies to investigate eryngial's anthelmintic potential is timely.
Recent studies on eryngial highlight other properties and benefits of the compound. While E. foetidum has wide-ranging medicinal properties (Paul et al. Reference Paul, Seaforth and Tikasingh2011), its unsaturated aldehyde, eryngial, in particular, belongs to a class of compounds that are associated with inactivation of rabbit liver cytochrome P450 isoforms (Raner et al. Reference Raner, Chiang, Vaz and Coon1997); is one of the defensive secretions of the spiroboloid millipede Rhinocricus insulatus (Wheeler et al. Reference Wheeler, Meinwald, Hurst and Eisner1964); and has antibacterial activity (Kubo et al. Reference Kubo, Fujita, Kubo, Nihei and Ogura2004; Forbes and Steglich, Reference Forbes and Steglich2010). Eryngial is also closely related to cis-7-dodecenal, which possesses sex pheromone activity (Chisholm et al. Reference Chisholm, Palaniswany and Underhill1982), and may thus be further investigated for insecticidal activity against some moth species, for example, Malacosoma disstria (Hübner).
Eryngial, which is also known for its application as a food flavouring (Rowe, Reference Rowe1998), was listed in 2000 by the Japanese Flavour and Fragrance Material Association as an often-used flavouring agent in its food industry. This is not surprising, as E. foetidum is widely used throughout the Caribbean, Latin America and the Far East as a food flavouring and seasoning herb for meat and other foods (Ramcharan, Reference Ramcharan and Janik1999; Chhetri, Reference Chhetri2006; Kar and Borthakur, Reference Kar and Borthakur2007; Prasad et al. Reference Prasad, Reddy, Raza and Dutt2008). The usage of eryngial in the food industry suggests that it is safe for use in humans.
The results of this study indicate that eryngial has tremendous potential as a compound of medicinal value, and is a prime candidate for further drug development studies, including mammalian toxicity. Eryngial's simple structure coupled with its acute toxicity against S. stercoralis infective larvae in vitro would favour synthesis in the laboratory, and its development into a potentially safe and inexpensive drug for pharmacological application, especially in endemic regions. Superior efficacy of such an anthelmintic drug would be most likely due to its ability to arrest or lessen the rate of autoinfection by the L3i, the phenomenon that plays an important role in maintaining S. stercoralis infections in infected individuals. Inactivation of large numbers of infective larvae and, potentially, adult worms in the intestine would be expected to result in reduced risk of morbidity. Furthermore, based on comparable activity to ivermectin, eryngial should be scrutinized for mammalian toxicity and its potential broad-spectrum anthelmintic activity in vitro and in vivo. Eryngial may thus not only be further investigated for development into a phytopharmaceutical anthelmintic against strongyloidosis and other nematode infections, but also for its use as a microbicide and an alternative, ecologically safe insect pest control agent.
ACKNOWLEDGEMENTS
The Departments of Life Sciences and Chemistry, Faculty of Science and Technology, The UWI, Mona Campus, Jamaica, and colleagues, are acknowledged by the authors for assistance rendered during this project. The Caribbean Food and Nutrition Institute provided literature on medicinal plants used as concoctions in folklore against intestinal worms. Freeze-drying of all crude extracts was carried out at the Tropical Metabolism Research Unit in Jamaica. Dr Vincent Lopez, DVM, PhD, from Phoenix Veterinary Clinic, Jamaica, assumed professional veterinary care of the mongrel dogs.
FINANCIAL SUPPORT
Funding for this project was provided by the Scientific Research Council of Jamaica and the Board for Graduate Studies and Research, The University of the West Indies (Mona Campus).
All authors declare no competing interests.
