Antitrypanosomal potentials of the extract and fractions of Abrus precatorius seeds

Objective:To evaluate the in vivo trypanocidal activity of the methanol extract and fractions of Abrus precatorius seeds in mice.Methods:Parasiteamia was induced unto mice by intraperitoneal injection of 1.25×105 Trypanosoma in normal saline.Five days when a high level of parasiteamia was established treatment commenced until ten days.The mice were treated with 10,20 and 40 mg/kg bt.of the extract and 5 and 10 mg/kg bt.of the fraction（F2）,respectively for 5 days.Diminazene acelurate at the dose of 3.5 mg/kg bt.for two days was used as the reference drug.The level of parasitaemia and packed cell volume（PCV） of the animals estimated. Results:At doses of 10,20 and 40 mg/kg the crude extract showed a sharp reduction in the level of parasitaemia in mice compared with the untreated group.The mice treated with F,at doses of 5 and 10 mg/kg showed a sharp reduction in the level of parasitamia to zero in day 9,and a gradual recovery from the 12th day of treatment.This effect is comparable to that of the mice treated with 7 mg/kg of standard drug diminazene aceturate.The PCV of the treated showed a gradual decrease with time,but not as much as the untreated group.Phytochemical screening revealed the presence of glycosides,alkaloids,carbohydrates,tannins and proteins in the Abrus precatorius powder while F2 was rich in alkaloids.Conclusions:This study shows that both the extract and the fractions of Abrus precatorius seeds exhibited a promising trypanocidal property.Alkaloids may be responsible for the observed activity.

In vitro-in vivo studies on anti-trypanosomal potentials of Zapoteca portoricensis

Objective:Aqueous extracts of Zapoteca portoricensis are used traditionally as antidiarrhea agent and in the treatment of diverse gastrointestinal disorders here in Nigeria specifically,the southern part.Similarly,the aqueous extract of the plant is also used traditionally as anticonvulsant,antispasmodic and in the treatment of tonsillitis.Recently too,the anti-inflammatory and antimicrobial activities of the methanol extracts of the root of Zapoteca portoricensis was reported.In this research,we are set to investigate the trypanocidal activity of Zapoteca portoricensis.Methods:The methanol extract of the root of Zapoteca portoricensis was investigated for both in vitro and in vivo trypanocidal activity following established models.In summary,phytochemical analysis was carried out on both the crude powdered root and on the methanol extract following standard procedures. The oral acute toxicity test(LD50 ) of the crude methanol extract was determined according to the method described by Lorke（1983）.Albino mice（17g-21g） of either sex were used.The methanol extract was suspended in 3%v/v tween 85 and administered orally at doses of 10 mg/kg,100 mg/kg and 1 000 mg/kg to three groups of mice（n = 3 ）.The animals were observed for 24 hours.Based on the result obtained in this initial test,doses of 4 mg/kg,6 mg/kg,and 8 mg/kg were administered to three different mice.The LD50 was calculated as the geometric mean of the lowest dose killing a mouse and the highest dose showing no death.The invivo /in-vitro antitrypanosomal evaluations were carried out in experimental animals and tissue cell culture respectively. Results:The result of the in vitro studies shows the inhibitive concentration-50（IC-50） against Trypanosoma brucei rhodesiense（T.b.rhodesiense） to be 0.372 mg/kg,while the control drug melarsoprol was 0.006 mg/kg.On Trypanosoma brucei brucei（T.cruzi）,the IC-50 is 6.42 mg/kg against 0.87 of the reference drug Benznidazole.The cytotoxicity on L-6 cells exhibited an IC-50 of 0.039 6 mg/kg against the reference drug,podophyllotoxin of 0.01 mg/kg.However,the in vivo study shows that the extract,at the administered doses,could not exhibit appreciable reduction of parasitemia and hence resulted to the death of test animals. Conclusion:The present data suggests that Zapoteca portoricensis could yield useful leads for the development of potentially potent antitrypanocides.

In-vitro and In-vivo Anti-trypanosomal Activity of Terminalia chebula Retz Dried Fruits

African trypanosomosis had caused lots of havocs to both humans and animals over a century with successes and failure in curtailing it. This study was aimed at screening medicinal plant, Terminalia chebula dried fruits against Trypanosoma evansi for trypanocidal activity. Twenty grams of powdered Terminalia chebula dried fruits was cold extracted with methanol. Obtained MPE （methanolic plant extract） was in vitro tested against Trypanosoma brucei （1 × 10^6 trypanosomes/mL of the medium in each ELISA plate wells） at concentrations （250～1,000 μg/mL） on Vero cells grown in DMEM （Debecco＇s Modified Eagle Medium） in appropriate conditions for trypanocidal activity. In-vitro cytotoxicity test of MPE of Terminalia chebula was conducted on Vero cells grown in DMEM. In-vivo assay for trypanocidal activity, each mouse was inoculated with 1 × 10^4/mL of trypanosomes and treated （48 h post inoculation） with MPE of Terminalia chebula at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 BL per mouse via intraperitoneal route to different groups of mice, 6 mice per concentration. In-vitro cytotoxicity test was done on Veto cells at concentrations （1.58～100 μg/mL） of MPE of Terminalia chebula. Results of in-vitro trypanocidal activity varied from immobilization, reduction and to the killing of the trypanosomes. At 250 μg/mL ofMPE ofTerminalia chebula dried fruits, there was significant trypanocidal activity at 4 h of incubation and trypanosomes were not detected in corresponding ELISA plate wells at 5 h of incubation, which was statistically equivalent to reference drug, diminazine aceturate （50 μL/mL） at 4 h of incubation. Results of in-vivo trypanocidal activity revealed that at concentrations （l 2.5～25 mg/kg body weight） of MPE of Terrninalia chebula, mice in these groups survived for 6 days. While at 50 and 100 to 200 mg/kg body weight, mice in these groups survived up to 7 and 8 days, respectively. In-vitro cytotoxicity test showed that all concentrations of MPE of Terminalia chenula and diminazine aceturate were cytotoxic to cells except at 1.56 μL/mL and 6.25 μL/mL. In conclusion, MPE of Terminalia chebula dried fruits possessed trypanocidal compounds. Further study （bioassay-guided purification） is required to know the full potential of Terrninalia chebula as future trypanocide candidate.

Therapeutic Activity of Partially Purified Fractions of Emblica officinalis （Syn, Phyllanthus embfica） Dried Fruits against Trypanosoma evansi

Emblica officinalis （E. oJficinalis） dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM （Dubecco＇s Modified Eagle Medium） and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations （250-1,000 μg/mL） with trypanosomes concentration （1 × 106 trypanosomes/mL in each ELISA plate well） and incubated at appropriate conditions for 72 h. In-vitro cytotoxieity of MPE of E. officinalis was determined on Vero cells at concentrations （（1.56-100 ~tg/mL）. Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE ofE. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC （higher performance liquid chromatography） with bioassay at different strata on Alsever＇s medium. In-vivo assay for trypanocidal activity, MPE and PPFs （partially purified fractions） of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 104/mL oftrypanosomes and treated （48 h post inoculation） at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 [tL per mouse via intraperitoneal route （in treating parassitemic mice） to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions ofE. officinalis was carried out with mobile phase ofacetonitdle： water （40：60） in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250μg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate （Berenil）, standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1-4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6-9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56-3.13 and 1.56-6.25 μg/mL, respectively. From this report, PPFs ofE. officinalis dried fruits demonstrated potential pathway for a new development oftrypanocide in near future if additional investigations are put in place.

Evaluation of metal nanoparticles for drug delivery systems

Diminazene aceturate is a trypanocide with unwanted toxicity and limited efficacy.It was reasoned that conjugating diminazene aceturate to functionalized nanoparticle would lower untoward toxicity while improving selectivity and therapeutic efficacy.Silver and gold nanoparticles were evaluated for their capacities to serve as carriers for diminazene aceturate.The silver and gold nanoparticles were synthesized,functionalized and coupled to diminazene aceturate following established protocols.The nanoparticle conjugates were characterized.The free diminazene aceturate and drug conjugated nanoparticles were subsequently evaluated for cytotoxicity in vitro.The characterizations by transmission electron microscopy or UV/Vis spectroscopy revealed that conjugation of diminazene aceturate to silver or gold nanoparticles was successful.Evaluation for cytotoxic actions in vitro demonstrated no significance difference between free diminazene aceturate and the conjugates.Our data suggest that surface modified metal nanoparticles could be optimized for drug delivery systems.