In vitro trypanocidal activity of comparative extraction of Terminalia bellirica (Combretaceae) dried fruits with solvents of different polarities against Trypanosma evansi.
P Shaba, N Pandey, O Sharma, R Rao, R Singh
antitrypanosomal, cytotoxicity, dried fruits
P Shaba, N Pandey, O Sharma, R Rao, R Singh. In vitro trypanocidal activity of comparative extraction of Terminalia bellirica (Combretaceae) dried fruits with solvents of different polarities against Trypanosma evansi. . The Internet Journal of Veterinary Medicine. 2008 Volume 6 Number 1.
In Africa, the estimated losses as a result of trypanosomosis in agricultural production amounted to 3 billion pounds annually (Hursey, 2000). In addition, it is estimated that 50 million cattle are at risk of being infected with trypanosomes leading to more than 3 million livestock deaths yearly, looses in calving, reduction in livestock number, drop in meat and milk off take and reduced work efficiency of draft animals and profitability of mixed farming (Mahmoud and Gray, 1980; Hursey,2000; Seed,2001)..
Human African trypanosomosis (HAT) is caused by
Chemotherapy and chemoprophylaxis are the only means of combating the menace of the disease. Chemotherapy of trypamosomosis is faced with problems such as limited choice of trypanocides in the market, high cost, toxicity, and emergence of drug-resistant trypanosome strains that have been reported (Freiburghaus
Thus, new approach and trypanocides are highly needed to combat trypanosomes. Based on this,
Materials and methods
Silica gel-G for thin layer chromatography (TLC), solvents (hexane, chloroform, methanol and aqueous) for extraction of plant materials and development /analysis of TLC plates, vanillin for spray and iodine for detection of bioactive constituents These were purchased from
Dried fruits of
Preparation of extract
Twenty grams of
The following solvent systems were tested for a suitable solvent to be used in developing TLC plates according to the method of Stahl, 1969.
Chloroform / hexane / acetic acid (50:50:1
Chloroform / ethyl acetate / acetic acid (50:50:1)
Methanol and chloroform (20: 80)
Thin layer chromatography (TLC) plates
This was done according to the method of Stahl, 1969. Aliquots from extractions were applied on TLC plates and developed in appropriate solvent system.
Swiss albino mice (20-30g) of either sex were obtained from Animal Research Laboratory Section of Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly maintained in standard environmental conditions and fed on a standard diet prepared by the institute with water
In vitro tryponocidal activity
Extracts at concentrations (250-1000 µg/ml) were added to a high parasitemic blood from mouse diluted with Alsever solution to obtain a final parasite concentration of 1x106 parasites/ml. The suspension (100 ml of medium with trypanosomes) was added at rate of 1: 1 to test extracts with inactivated bovine serum at 58 °C for 1 h. incubated at 37 °C under 5% CO2 for 5 h (Talakal
After incubation for antitrypanosomal activity was completed, contents of wells with reduced and apparently killed parasites from MPE of
:: Stock of test MPE was solubilized in 1% dimethylsuphoxide (DMSO) The concentration in the experiment had no deleterious effect by it self on host cells or parasites.1% DMSO in distilled water was used as control (Young, 2000)
Cytotoxic effect of the plant extract was determined according to the method described by Sidwell and Hoffman (1997). Vero cell line was grown as stated above but was not supplemented with fetal calf serum. Confluent monolayer of Vero cell was treated with serial dilutions of test MPE (1.56-100 µg/ml) in triplicate and incubated under same conditions described previously. After 24h of incubation, the culture plate was observed for evidence of cytotoxic effects such as distortion, detachment, swelling and sloughing of cells. The plate was incubated for 72 h and observed daily. It was repeated thrice.
In each case, after the 72 h of incubation, the culture media of the incubated Vero cells were discarded. The adhered cells were stained with a drop of crystal violet in phosphate buffered solution. The plate was incubated for24 hours at 37°C in an ordinary incubator. The plate was observed for cytotoxic effects under inverted microscope.
Results of trypanocidal activity were expressed as mean ± SEM. Statistical significance was determined by Sigma Stat (Jandel), USA.
The results of trypanocidal activity of
Bioassay status: significant reduction of trypanosomes average counts from concentrations (250-1000 µg/ml and but no complete killing of trypanosomes in any well throughout hours of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.
Bioassay status: significant reduction of trypanosomes counts from concentrations (250-1000 µg/ml and but no complete killing of trypanosomes in any well through out hours of observation. An average mean trypanosomes parasites count of 37.67± 0.58 is statistically critical value.
Bioassay status: significant reduction of trypanosomes counts from concentration of 250 µg/ml and complete killing of trypanosomes at same concentration at 4th hour of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.
Bioassay status: significant reduction of trypanosomes counts from concentration of 250 µg/ml and complete killing of trypanosomes at same concentration at 5th hour of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.
Same concentrations were used for both extracts and diminazine aceturate (Berenil)
Methanolic extract was chosen for cytotoxicity test due to its maximum trypanocidal activity
Methanol extracted more of bioactive constituents than other solvents as observed on TLC plates in accordance to the mobility of applied aliquots of extracts on the plates (plates not shown) Solvent system methanol/chloroform (20: 80) was suitable in development of TLC plates. Also, methanolic extract exhibited antitrypanosomal activity than other solvents. Antitrypanosomal activity ranged from immobilization and killing of trypanosomes. At 250 µg/ml, trypanosomes in methanolic extract were apparently killed at 4 h which was equivalent to diminazine atceturate (50 µg/ml). This results are comparable to comparative antitrypanosomal activity of
In conclusion, results of current investigation shows a positive promise of trypanocidal compounds depicted by different degrees of extraction of bioactive constituents by solvents with corresponding antirypanosomal activity. Further studies (e.g. bioassay-guided purification and
Financial contributions by India and Nigeria governments towards the research, invaluable advice/inputs by Dr. A. K Mishra, Principal Scientist, contributions by other scientists and technical staff, Division of Parasitology IVRI, Izatnagar and IVRI, Regional station Palampur, India are highly acknowledged.