Antiprotozoal activities of Vincetoxicum stocksii and Carum copticum

The antileishmanial and antimalarial activities of different fractions of Vincetoxicum stocksii, Carum copticum, Gentiana olivierii and Zygophyllum fabago were determined. Amongst these, V. stocksii and C. copticum showed moderate to good antimalarial activity. The ethyl acetate fractions of C. copticum and hexane fraction of V. stocksii showed 94 % and 52% inhibition of Plasmodium falciparum (D6 Clone) respectively. The hexane ethyl acetate fraction of C. copticum V. stocksi exhibited antileishmanial activity against, Leishmania donovani promastigotes with IC50 of 30 μg/mL and 51 g/mL, respectively. Article Info Received: 23 August 2011 Accepted: 26 August 2011 Available Online: 10 September 2011


Introduction
The parasitic diseases such as malaria and leishmaniasis have a high death rate in developing countries.Every year more than 500 million people, mostly those living in the world's poorest countries are at risk of malaria (Mansoor, 2003).Approximately 350 million people living in tropical and sub-tropical countries, suffer from this disease (Bharate, 2007).Therefore, there is a need to develop effective antimalarial and antileishmanial drugs for the treatment of these diseases.
The extract was filtered and concentrated under reduced pressure in a rotary evaporator at 40ºC, to obtain thick crude extract, which yielded C. copticum (23.4 g), V. stocksii (20.7 g), G. olivieri (27.5 g) and Z. fabago (22.8 g).The crude ethanolic extract of these plants was evaluated for antimalarial and antileishmanial activity.
Ethanol extract of V. stocksii was dissolved in water and partitioned first with hexane (Fraction AVH, 3.9 g) and then with ethyl acetate (Fraction AVE, 5.3 g) and finally with butanol (Fraction AVB, 9.8 g).The activity of each fraction was determined for antimalarial and antileishmanial assay.
Antileishmanial activity: Antileishmanial activity of the extracts of the plants was tested in vitro against a culture of Leishmania donovani promastigotes grown at 26ºC, in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Chem.Co.).A 3-day-old culture was diluted to 5 x 10 5 promastigotes/mL.Extract dilutions were prepared in cell suspension in 96-well plates, which were incubated at 26 ºC for 48 hours.Almar blue assay was used to determine the growth of L. donovani promastigotes (Mikus and Steverding, 2000).Fluostar Galaxy plate reader (BMG Lab Technologies) was used to measure standard fluorescence at excitation wavelength of 544 nm and emission wavelength of 590 nm.Amphotericin B and pentamidine were used as the standard antileishmanial agents.IC50 and IC90 values were computed from dose-response curves generated by plotting percent growth versus drug concentration.

Assay for in vitro antimalarial activity:
The plasmodial LDH Activity is the bases for the determination of antimalarial activity.A red blood cells suspension was infected with D6 or W2 strains of P. falciparum D6 Clone (200 L, with 2%hematocrit and 2%parasitemia, in RPMI 1640 medium, with 60 g/mL amikacin and 10% human serum) was added to the wells of a 96 well plate containing 10 g/mL of test samples diluted in medium at various concentrations.The plate was kept in a modular incubation chamber (Billups-Rothenberg, CA) flushed with 90%N2, 5%O2, and 5%CO2 gas mixture, and incubated at 37ºC, for 72 hours.Malstat TM reagent was used to determine parasitic LDH activity (Flow Inc., Portland, OR) as described by elsewhere (Makler and Hinrichs, 1993).Incubation mixture (20 L) was mixed with 100 L of the Malstat TM reagent at room temperature and incubated for 30 min.Then twenty microliters of of NBT/PES (Sigma, St. Louis, MO) 1:1 mixture was added.The plate was further incubated for 1 hour in the dark.Hundred microliter of a 5%acetic acid solution was then added to stop the reaction.EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont) was used to read the plate at 650 nm.IC50 values were computed from the dose-response curves.Chloroquine and artemisinin were used as the drug controls in each assay.DMSO (0.25%) was used as vehicle for control.

Results and Discussion
Ethanol extracts of all the four plants were evaluated for in vitro antileishmanial and antimalarial, activities.
Only the extracts of C. copticum and V. stocksii showed moderate to mild antileishmanial activities with IC90 values of 60 µg/mL and 70 µg/mL respectively.IC50 of 50 µg/mL was detected for V. stocksii (Table I).No such activity was shown by G. olivieri and Z. fabago.C. copticum showed good antimalarial activity, whereas, moderate activity was shown by V. stocksii.Poor activity was shown by G. olivieri and no such activity was observed by Z. fabago.Therefore, further fractionation of the extracts of the two active medicinal plants i.e., C. copticum and V. stocksii were carried out.
The most potent antilimalarial activity (94%) was exhibited by fraction ACCE-3 and ACCHE-2 (90%) of C. copticum (Table II).The significant antimalarial activity of ACCE-2 and ACCHE-3 indicates that the active compounds must be present in the less-polar fractions  ACCE-3).The chemical composition of the essential oil from dry seeds of C. copticum was found to have thymol (41.3%), α-terpinolene (17.5%) and ρcymene (11.8%) as the major constituents of the oil and have shown to exhibit strong insecticidal activity.The mortalities of the insect species reached 100% at concentrations higher than 185.2 μL/L and 12-hours exposure time (Sahaf, 2007).The findings indicate that C. copticum oil had potential antiprotozoal activities.
Since good antiprotozoal activity was shown by Vincetoxicum stocksii, so its ethanol extract was dissolved in water and partitioned first with hexane (Fraction AVH) and then with ethyl acetate (Fraction AVE) and finally with butanol (Fraction AVB) (Table III).Moderate activity was shown against P. falciparum and L. donovani by the hexane fraction of V. stocksii whereas poor or no activity was shown by the other two fractions.
Vincetoxicum is known for its poisonous as well as its medicinal properties.It has also been found to be allelopathic and active against a number of microorganisms (Zaidi and Crow, 2005).Pronounced cytotoxicity has also been reported against Candida albicans by Staerk et al. (2000).This effect is probably due to the presence of phenanthroindolizidine alkaloids reported from other species of Vincetoxicum.
In conclusion, this study demonstrates good antimalarial and moderate anitleishmanial activity of the extracts of C. copticum and V. stocksi, which parallels the traditional use of these herbs as an antiprotozoal medicine.