Antitrichomonal activity of Acanthospermum hispidum D . C . ( Asteraceae )

Acanthospermum hispidum (Asteraceae), used ethnomedically in the treatment of inflammatory conditions and fever, was evaluated for antiprotozoal activities such as trypanocidal and antiplasmodial effects. This study was carried out to investigate the anti-trichomonal potential of the plant. The airdried leaf was extracted successively with petroleum ether, chloroform, ethylacetate and methanol using the soxhlet extraction method. Bioactivity-guided fractionation of the most active extract was carried out using the vacuum liquid chromatographic technique for antitrichomonal activity using Trichomonas gallinae in vitro. The ethyl acetate extract (A3) was the most active extract with LC50-LC90 values of 0.58-1.06 and 0.58-1.05 mg/ml at 24 and 48 h, respectively. Subfraction C7 had the highest antitrichomonal activity with 0.25-0.66 and 0.25-0.54 mg/ml at 24 and 48 h, respectively comparable to the activity of metronidazole at 0.20-0.39 and 0.16-0.36 mg/ml at 24 and 48 h, respectively. A. hispidum possessed antitrichomonal activity which resided in the chloroform portion of the ethyl acetate extract of the plant.


INTRODUCTION
Diseases such as trypanosomiasis and trichomoniasis are taking their toll in terms of mortality and morbidity on human and animal populations in the developing countries.Available data showed that the annual incidence of trichomoniasis is more than 170 million cases worldwide (WHO, 1995).Trichomoniasis encompasses a broad range of symptoms ranging from a state of severe inflammation and irritation with a frothy malodorous discharge to a relatively asymptomatic carrier state (Swygard et al., 2004).The emergence of drug-resistant strains and dose-limiting toxic effects of existing drugs have complicated the treatment of parasitic protozoan diseases.Medicinal plants are a reservoir of bioactive compounds and therefore, effort is focused on them for potentially useful anti-infective agents.Acanthospermum hispidum DC (Asteraceae) is commonly called Bristly starburr, bristly tee or hispid starburr has its synonym as A. humile.
The plant has been reported to possess sesquiterpene lactones such as acanthospermal B, acanthospermal B epoxide, hispidunolide A and B (Herz and Kalyanaraman, 1975;Jakupovic et al., 1986;Kraus et al., 1994;Cartagena et al., 2000;Arena et al., 2011).In addition, glycosides and flavonoids have been reported to be present in the aerial part of the plant (Nair et al., 1976;Edewor and Olajire, 2011).A. hispidum has been shown to be a potentially useful plant in the treatment of protozoan infections; though, the antitrichomonal activity had not been reported.This study was carried out to investigate the activity of various extracts and fractions of A. hispidum on a protozoan parasite, Trichomonas gallinae.

Plant collection and preparation
A. hispidum D. C. (Asteraceae) was collected at Ile-Ife, Nigeria in July.Plant authentication was done by Dr. H. C. Illoh of the Botany Department, Obafemi Awolowo University and compared with herbarium specimen IFE 5986.The leaf was oven-dried at 40°C, powdered using the grinding machine (Christy Norris) and stored appropriately in an amber-coloured bottle until required.

Plant extraction
The powdered leaf of A. hispidum (2.0 kg) was successively extracted with petroleum ether, chloroform, ethyl acetate and methanol for 48 h each using the soxhlet extraction method.The extracts were concentrated in vacuo at 35°C to give petroleum ether (A 1 ), chloroform (A 2 ), ethyl acetate (A 3 ) and methanol (A 4 ) extracts.The yields obtained were 0.95, 2.63, 1.00 and 1.27%, respectively.

Parasite
T. gallinae parasites were isolated from the mouth and upper crop of Columba livia (local pigeon) using sterile cotton-tipped swab sticks immersed in physiological saline solution (Narcisi and Secor, 1996).The parasites were cultured in egg slant tubes suspended in LE medium and incubated vertically at 37°C until ready for use (Omisore et al., 2005).

Antitrichomonal bioassay
For each extract/fraction, 50 µl of 10 4 organisms/ml of T. gallinae parasites was added to 150 µl of test extract/fraction in a sterile 96microwell flat bottom plate (Nunc) with metronidazole and DMSO-LE medium as positive and negative controls, respectively.The plates were incubated at 37°C.At 24 and 48 h, surviving (motile) parasites were counted per ml with the aid of a microscope.At least each concentration was done in triplicate analyses.

Data and statistical analysis
The percentage mortality of the parasites was calculated as 100 × [100-(A/B)], where A is the number of motile organisms in the test groups and B is the number of motile organisms in the negative control group.The LC 50 and LC 90 values were derived from the respective percentage mortality values using Microsoft Excel (2007) and subjected to statistical analysis using the one-way analysis of variance (ANOVA) followed by the post-hoc Dunnett test (Graphpad Instat, 2003).

RESULTS
The results are presented in Tables 1 to 3. The LC 50 and LC 90 values of A 1 , A 2 and A 3 reduced non-significantly (P>0.05) over time from 24 to 48 h.The activities of A 1 , A 2 and A 3 were comparable to the positive control, metronidazole; however, A 4 was significantly different [F  (4, 10) 3.96, P = 0.035] from metronidazole at 24 h (Table 1).A 3 gave the highest activity as it had the lowest LC 50 and LC 90 values at 24 and 48 h.Thus, A 3 (22 g) was fractionated using VLC and sixteen eluates were obtained which were bulked into 7 fractions (B 1-7 ) according to their TLC profiles.At 24 h, all the fractions except B 4 and B 7 had similar activity compared with metronidazole while only the LC 50 of B 7 was significantly different [F(8, 18) 3.099, P = 0.022] at 24 h from the mother extract, A 3 (Table 2).At 48 h, the LC 50 values of B 2 , B 4 and B 7 were significantly different from metronidazole while only B 4 was different from A 3 [F (8, 18) = 3.624, P = 0.011).All the fractions except B 1 were significantly different from metronidazole when their LC 90 values were compared.Although, B 1-3 and B 5-6 had comparable activities, B 2 (2.9 g), which had the highest weight, was further purified.Eight bulked fractions were obtained (C 1-8 ) according to their TLC characteristics.At 24 h, all the sub-fractions except C 5 , C 6 and C 7 were significantly different [F (9, 20) = 5.52, P = 0.0007] from metronidazole while at 48 h, only C 6 and C 7 were comparable [F (9, 20) 41.46, P<0.0001] to metronidazole.However, they were significantly different [F (9, 20) 34.29, P<0.0001) from A 3 (Table 3).Thus, the activities of the two sub-fractions were significantly comparable to metronidazole at both time points and showed better activity than A3.

DISCUSSION
The extracts and fractions of A. hispidum gave moderate to remarkable levels of mortality when tested on the protozoa, T. gallinae.The activity of A1, A2 and A3 implies the bioactive component may be non-polar while the polar methanolic extract exhibited minimal activity when compared with metronidazole.It thus, appeared that the bioactive component (s) is/are relatively apolar.In addition, it seemed the extracts exhibited biostatic action on the protozoa which was not sustained after 24 h.The increasing resistance to metronidazole in the treatment of trichomoniasis, with the various adverse effects observed in the use of the drug, has led to the search for bioactive agents in medicinal plants with potential antitrichomonal activity.Trichomonad species readily obtained for laboratory study are T. muris in mice and rats and T. gallinae from crop of pigeons (Smyth, 1996).T. gallinae was used because of its availability and morphological similarity to T. vaginalis T. vaginalis, the causative parasite for human trichomoniasis.Since the duration of survival and growth rate is inversely proportional to inoculum density, trichomonads can sometimes overgrow the media and die off within 36 to 48 h, thus the choice of the time points.The most active subfractions are from the chloroform portion of the ethyl acetate extract.Deepa et al. (2004) reported that the ethyl acetate extract of A. hispidum possessed antibacterial and antifungal activities comparable to ciprofloxacin and clotrimazole, respectively.Non-polar compounds have been found effective as potential agents in the treatment of trichomoniasis.The pentacyclic triterpenoid, hederagenin, was reported as the antitrichomonal component of Cussonia holtsi (Araliaceae) with an IC 50 of 2.8 µM (He et al., 2003).In addition, bartericins A and B as well as isobavachalcone (isolated from Dorstenia barteri) were reportedly active at 0.121 to 31.25 µg/ml, against T. gallinarum (Omisore et al., 2005).The antibacterial and antimalarial activities of A. hispidum have been ascribed to Acanthospermal B and other sesquiterpene lactones (Arena et al., 2011;Ganfon et al., 2012).It is therefore possible that the putative antitrichomonal constituent of A. hispidum belongs to the class of sesquiterpene lactones which abound in the plant.
Sesquiterpene lactones (SQLs) have been reported from 10 families of flowering plants; with the greatest numbers derived from the Asteraceae.The α-methylene γ-lactone moiety of this group of compounds is very reactive with the thiol groups of important biological components such as enzymes which make SQLs have diverse biological activity.Further studies on the most active subfractions may reveal a more active compound than the standard drug, metronidazole.

Conclusion
Bioactivity-directed purification of the leaf of A. hispidum using anti-trichomonal assay yielded subfractions C 6 and C 7 which had activity comparable to metronidazole, the positive control and had better activity than the mother ethyl acetate extract.The study further showed the potential usefulness of A. hispidum in treating protozoal infections.

Table 2 .
Antitrichomonal activity of the fractions of the ethyl acetate extract (A 3 ) of A. hispidum at 24 and 48 h.
† Significantly different from A 3 .

Table 3 .
Antitrichomonal activity of subfractions of B 2 using Trichomonas gallinae at 24 and 48 h.