Antiprotozoal activities of Vincetoxicum stocksii and Carum copticum
Abstract
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 mg/mL, respectively.
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 subtropical 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 natives of Balochistan from centuries have been using more than 400 species of medicinal plants. The medicinally important plants Carum copticum Linn. Syn (Umbelliferae), Vincetoxicum stocksii (Asclepiadaceae) Ali & Khatoon, Gentiana olivieri Griseb (Gentianaceae) and Zygophyllum fabago (Zygophyllaceae) are widely distributed in Pakistan. C. copticum is used for curing diarrhea, amebiasis, dyspepsia and killing worms in stomach and other stomach disorders. It is also used as an antihelmintic, antispasmosadic and antiseptic agent (Sahaf, 2007). It is traditionally used, as antileishmanial herb Vincetoxicum stocksii is used by the natives as poultice for the treatment of wounds, injuries and external cancers (Staerk et al., 2000). G. olivieri is used in traditional use folk medicine for the treatment of skin diseases, abscesses, ulcer, blood pressure (Mansoor et al., 1998) and fever caused by malaria. Z. fabago is used to treat skin diseases, septic, injuries and external wounds (Zaidi, 2006) and Leishmania. In this paper the antimalarial and antileishmanial effects of these four medicinal plants were determined.
Materials and Methods
Plant material
The whole plant of C. copticum, V. stocksii, G. olivieri and Z. fabago (2 kg each) were collected in June 2006 from the mountains of Quetta. These were identified and a voucher specimen of all the plants were deposited in the herbarium of Botanical Garden, University of Balochistan Quetta. Plants were cleaned, dried in shade, ground, packed and then sent to USA for analysis. These were soaked in ethanol and extracted repeatedly 3-4 times at room temperature. 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.
Preparation of fractions
Only biologically active plant extracts were subjected to further fractionation. The crude ethanol extract of C. copticum was fractionated by vacuum liquid chromatography. Elution was initiated by hexane (Fraction ACCH-1 4.6 g), followed by hexane/ethyl acetate (Fraction ACCHE-2, 2.8 g), ethyl acetate (Fraction ACCE-3, 1.9 g), ethyl acetate/acetone (Fraction ACCEA-4, 2.5 g), acetone (Fraction ACCA-5, 2.8 g), acetone/methanol (Fraction ACCAM-6, 2.7 g), methanol (Fraction AccM-7, 3.5 g) and finally with water (ACCW-8, 2.1 g).
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 105 promastigotes/mL. Extract dilutions were prepared in cell suspension in 96-well plates, which were incubated at 26ºC 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 mL, with 2% hematocrit and 2% parasitemia, in RPMI 1640 medium, with 60 mg/mL amikacin and 10% human serum) was added to the wells of a 96 well plate containing 10 mg/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. MalstatTM reagent was used to determine parasitic LDH activity (Flow Inc., Portland, OR) as described by elsewhere (Makler and Hinrichs, 1993). Incubation mixture (20 mL) was mixed with 100 mL of the MalstatTM reagent at room temperature and incubated for 30 min. Then twenty microliters of of NBT/PES (Sigma, USA) 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.
Result 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 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.
Table I: Antiprotozoal activity of the ethanol extracts of Gentiana olivieri, Carum copticum, Zygophylum fabago and Vincetoxicum stocksii
| Sample Name | P. falciparum | L. donovani | L. donovani |
|---|---|---|---|
| % Inhibition | IC50 (µg/mL) | IC90 (µg/mL) | |
| Gentiana olivieri | 28 | Inactive | Inactive |
| Carum copticum | 60 | 22 | 60 |
| Zygophyllum fabago | Inactive | Inactive | Inactive |
| Vincetoxicum stocksii | 40 | 50 | 70 |
| Pentamidine | Not tested | 1.2 | 6 |
| Amphotericin B | Not tested | 0.3 | 0.7 |
| Chloroquine | 99.2 | Not tested | Not tested |
| Artemisinin | 98.8 | Not tested | Not tested |
| IC50 and IC90 are the sample concentrations that kill 50 and 90% Leishmanial cells compared to the solvent controls; Test = 3 concentrations: 100, 20, 4 µg/mL | |||
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 of hexane/ethyl acetate (Fraction ACCHE-2) and ethyl acetate (ACCE-3). The chemical composition of the essential oil from dry seeds of C. copticum was found to have thymol (41.3%), alpha-terpinolene (17.5%) and alpha-cymene (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 uL/L and 12 hours exposure time (Sahaf, 2007). The findings indicate that C. copticum oil had potential antiprotozoal activities.
Table II: In vitro antimalarial and antileishmanial activities of fractions of Carum copticum
| Samples Name | P. falciparum | L. donovani | |
|---|---|---|---|
| %Inhibition | IC50 (µg/mL) | IC90 (µg/mL) | |
| Carum copticum (ACCH-1) | 0 | 90 | Inactive |
| Carum copticum (ACCHE-2) | 90 | 30 | 90 |
| Carum copticum (ACCE-3) | 94 | 50 | 90 |
| Carum copticum (ACCEA-4) | 82 | 50 | 98 |
| Pentamidine | Not tested | 2.1 | 6.2 |
| Amphotericin B | Not tested | 0.09 | 0.2 |
| Chloroquine | 99 | Not tested | Not tested |
| Artemisinin | 99.4 | Not tested | Not tested |
| IC50 and IC90, are the concentrations that affords 50 and 90%inhibition of leishmanial growth compared to the solvent controls; Test = 3 concentrations; Carum copticum concentration: 100, 20, 4 µg/mL | |||
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.
Table III: In vitro antimalarial and antileishmanial activity of the fractions of Vincetoxicum stocksii
| Samples Name | P. falciparum | L. donovani | |
|---|---|---|---|
| % Inhibition | IC50 (µg/mL) | IC90 (µg/ mL) | |
| Vincetoxicum stocksii AVH | 52 | 51 | 90 |
| Vincetoxicum stocksii AVE | 21 | Inactive* | Inactive* |
| Vincetoxicum stocksii AVB | 15 | Inactive* | Inactive* |
| Pentamidine | Not tested | 1.2 | 5.2 |
| Amphotericin B | Not tested | 0.5 | 0.9 |
| Chloroquine | 99.6 | Not tested | Not tested |
| Artemisinin | 99 | Not tested | Not tested |
| IC50 and IC90, are the concentrations that affords 50 and 90% inhibition of leishmanial growth compared to the solvent controls; *at 40 µg/mL | |||
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.
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.
Acknowledgment
Special thanks to Dr. M. T. Hamman and Shabana I. Khan for providing facilities and guidance during this study.
References
Bharate SB, Khan SI, Yunus NA, Chauthe SK, Jacob MR, Tekwani BL, Khan IA, Singh IP. Antiprotozoal and antimicrobial activities of O-alkylated and formylated acylphloroglucinols. Bioorg Med Chem. 2007; 15: 87-96.
Makler MT, Hinrichs DJ. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am J Trop Med Hyg. 1993; 48: 205-09.
Mansoor A, Samad A, Zaidi MI. Hypotensive effect of G. olivieri and its alkaloid gentianine in rats. Pharm Pharmacol Comm. 1998; 4: 229-30.
Mansoor A. Toxicological evaluation of the extracts and pure compounds of G. olivieri. Pakistan J Biol Sci. 2003; 6: 1949-50.
Mikus J, Steverding D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye alamar blue. Parasitol Int. 2000; 48, 265-69.
Sahaf BZ, Moharramipour S, Meshkatalsadat MH. Chemical constituents and fumigant toxicity of essential oil from Carum copticum against two stored product beetles. Insect Sci. 2007; 14: 213–18.
Staerk D, Christensen J, Lemmich E, Duus JØ, Olsen CE, Janoezewsk JW. Cytotoxic activity of some phenanthrol-indolizidine N-oxide alkaloids from Cynanchum vincetoxicum. J Nat Prod. 2000; 63: 1584-86.
Zaidi MI. Phytotoxicity and insecticidal activity of Vincetoxicum stocksii. In: Plant ecology and biodiversity research. Barak SK (ed). New Delhi, Regency Publishers, 2006, pp 193-97.
Zaidi MA, Crow SA Jr. Biologically active traditional medicinal herbs from Balochistan, Pakistan. J Ethnopharmacol. 2005; 96: 331-34.
