Achillea fragrantissima, rich in flavonoids and tannins, potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

Ibrahim M. El-Ashmawy, Naser A. Al-Wabel, Aida E. BayadDepartment of Veterinary Medicine, College of Agricultural and Veterinary Medicine, Qassim University, Kingdom of Saudi ArabiaDepartment of Pharmacology, Faculty of Veterinary Medicine, Alexandria University, EgyptVeterinary Services Center, Faculty of Veterinary Medicine, Alexandria University

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Achillea fragrantissima, rich in flavonoids and tannins, potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

Ibrahim M. El-Ashmawy1,2*, Naser A. Al-Wabel1, Aida E. Bayad3
1Department of Veterinary Medicine, College of Agricultural and Veterinary Medicine, Qassim University, Kingdom of Saudi Arabia
2Department of Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Egypt
3Veterinary Services Center, Faculty of Veterinary Medicine, Alexandria University

ABSTRACT

Objective: To evaluate activity of methanol extract of Achillea fragrantissima (meth) (A. fragrantissima) alone or in combination with diminazine aceturate (DA) against Trypanosoma evansi in experimentally infected rats. Methods: Sixty adult male Wister albino rats were divided equally into 6 groups (A-F). Rats in groups A-E were experimentally infected with T. evansi and those in group F were uninfected. The groups were treated respectively as follows: group A- with 3.5 mg/kg DA; group B- with 1 000 mg/kg meth A. fragrantissima; group C-3.5 mg/kg DA plus 500 mg/kg meth A. fragrantissima; group D-3.5 mg/kg DA plus 1 000 mg/ kg meth A. fragrantissima. Group E was left untreated. Parasitaemia, survivability, packed cell volume, hemoglobin concentration, total leucocytes count, lymphocyte count, and serum malondialdehyde and reduced glutathione (GSH) levels were estimated. Phytochemical screening of meth A. fragrantissima was also performed. Results: The phytochemical analysis of the meth A. fragrantissima indicated a higher content from polyphenolic tannins and non tannins and fl avonoids. The effi cacy percentage against trypanosomiasis in groups A to E was respectively as follows 80, 40, 90, 100, 0. The administration of meth-A. fragrantissima (1 000 mg/kg b.wt.) produced a moderate effi cacy against trypanosomiasis. Untreated rats in group E died between 25 and 30 d post infection. The rats given DA and meth A. fragrantissima combinations (C and D) showed faster and higher recovery rates than the uninfected control and groups A and B. The initial reduction in packed cell volume, hemoglobin, total leucocytes count, increases in serum malondialdehyde and decreases in GSH levels were reversed by the treatments. Conclusions: The administration of the methanol extracts of A. fragrantissima and DA combination therapy was more eff ective than each product alone in the treatment of rats infected with Trypanosoma evansi and further studies are required to isolate more active ingredients.

ARTICLE INFO

Article history:

Received in revised form 20 January 2016

Accepted 15 February 2016

Available online 20 March 2016

Achillea fragrantissima

Trypanosoma evansi

Flavonoids

Malondialdehyde

Reduced glutathione

Rats

1. Introduction

Trypanosomiasis is an important protozoan disease of domestic animals and man[1,2]. The disease is characterized by a rapid decrease in red blood cell, haemoglobin (Hb) concentration and packed cell volume (PCV) confirming that anemia is a critical feature in the pathogenesis of trypanosomiasis[2,3]. Tissue damage has been indicated in the pathophysiology of trypanosomiasis[4]. Oxidative stress plays an important role in the pathogenesis of sleeping sickness[4-8]. It has been established that infections caused by the Trypanosoma spp. alter the antioxidant defense of the host[4,6,7] and thus, increase the susceptibility of the erythrocytes to oxidative haemolysis[9,10].

Achillea fragrantissima (A. fragrantissima) is one of the Asteracaea Family and is widely used in traditional medicine forgastrointestinal disorders[11]. Authors reported its activity as carminative, anthelmintic and antiseptic to various infections for the urinary tract[12,13]. Moreover, insecticidal, rodenticidal, antiviral and antioxidant activities of A. fragrantissima were demonstrated[14-16]. Neither acute nor subchronic toxicity were noticed in mice and rats with the different extracts of A. fragrantissima[17,18]. In most countries control of trypanosomiasis relies mainly on chemotherapy and chemoprophylaxis using salts of three compounds – diminazine, homidium and isomethamidium. Many trials has been done to investigate combination therapy[4,7]. It is therefore conceivable that co-administration of diminazine with plants known to have immunostimulatory and antioxidant properties may potentiate its therapeutic activity.

2. Materials and methods

2.1. Animals

Wister albino mature male rats [(180±20) g] were obtained from the Animal House of the College of Agriculture and Veterinary Medicine, Qassim University, Kingdom of Saudi Arabia and housed at a temperature of 22℃-28℃ and relative humidity of 50%–60%, with artifi cial light from 5:00 a.m. to 4:00 p.m. Animals had free access to tap water and standard rat chow, used for the study. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication no. 85-23, revised 1996). The local ethics committee approved the study.

2.2. Preparation of plant material

The plant A. fragrantissima was collected at the fl owering stage, in and around Al-Gouf and Qassim Districts, Kingdom of Saudi Arabia. The collected plant species were identifi ed and confi rmed at the Department of Botany, College of Agricultural and Veterinary Medicine, Qassim University, Al Qassim, Kingdom of Saudi Arabia and a voucher specimen was deposited in the Department of Botany, College of Agricultural and Veterinary Medicine, Qassim University, Al Qassim, Kingdom of Saudi Arabia for further reference.

2.3. Phytochemical analysis of the methanolic extract of A. fragrantissima

Shade dried and powdered plant materials were successively extracted. About 300 g of the powdered plant were soaked in 3 000 mL methanol. It was left for 72 h, with intermittent shaking till obtain methanolic extract. The extract was fi ltered through Whatman No. 1 filter paper and concentrated until obtaining paste under vacuum using the rotary evaporator (Rotavapor R-215, B觠tchi, Switzerland), yield 16.5 % (w/w). The quantitative phytochemical constituents were analyzed using the standard methods[19-21].

2.4. Trypanosomes and inoculation of donor rats

A trypanosome strain Trypanosoma evansi (T. evansi) obtained from the infected animals (camels blood). The diluted infected blood (0.1 mL) contains 1 or 2 parasite per fi eld (microscopic fi eld), inoculated into healthy rats intraperitoneally to serve as the donor. Infection monitored every morning by microscopic examination of blood samples taken from the tail of the infected rats.

2.5. Infection of experimental rats

Blood collected by cardiac puncture with an EDTA coated syringe from the heavily infected rats and immediately diluted with physiological saline to serve as the inoculums. Healthy rats were injected intraperitoneally with 0.2 mL of the inoculums containing about 1 伊 106trypanosome cells , as described previously[22,23] and parasitaemia monitored every day by microscopic examination.

2.6. Experimental design and grouping

Six groups (A- F) of 10 rats each distributed into a well ventilated cages. All rats in group A-E were infected with 1 伊 106trypanosome cells intraperitoneally, while the group F was left as uninfected control. Rats in groups A-E experimentally infected with T. evansi and those in group F uninfected. The groups were treated respectively as follows: group A- given 3.5 mg/kg diminazine aceturate im. (Diminazene?, Montlant Pharmaceuticals, Saudi Arabia), (DA); group B- given 1 000 mg/kg meth A. fragrantissima; group C- given 3.5 mg/kg DA plus 500 mg/kg meth A. fragrantissima; group D- given 3.5 mg/kg DA plus 1 000 mg/kg meth A. fragrantissima. Group E- was left untreated. Rats in group E were left untreated as infected control. Treatments were given every morning for 5 consecutive days (The paste of the plant extract was weighed and diluted, in a gum acacia solution 1% as a vehicle and the volume given equals 10 mL/kg b. wt. by gavage).

2.7. Parasitaemia

Parasitaemia monitored every day under the microscope at 伊40 magnifi cation as described by Herbert and Lumsden[24].

2.8. Measurement of hematological parameters

Basal blood samples were taken, then every fi ve days of survivor rats under light ether anesthesia inside an anesthetic chamber to perform collection of blood samples (from the inner canthus of the eye). Approximately 10 min after the procedure, all animals showed signs of recovery. A volume of 0.5 mL was used to estimate hematological parameters including PCV, Hb, total leucocytes count (TLC) and lymphocyte count (LC) according to Dacie et al[25] and 3 mL was used to obtain serum for the measurement of lipid peroxidation (MDA) and reduced glutathione (GSH) levels.

2.9. Determination of serum MDA concentration

Serum malondialdehyde concentration was determined by the method of Draper and Hadley[26] and modified by Altuntas et al[27]. Briefly, 2.5 mL of 100 g/L trichloroacetic acid solution was added to 0.5 mL of serum in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1 000 g for 10 min, and 2 mL of the supernatant was added to 1 mL of 6.7 g/L thiobarbituric acid in a test tube and placed in a boiling water bath for 15 min. Then the solution was cooled in tap water and its absorbance measured using a UV spectrophotometer (Jenway, 6405 Model, Japan) at 532 nm. A total of 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid were used as the blank.

2.10. Determination of GSH in serum

GSH was assayed by spectophotometric technique according to the method described by Sedlak and Lindsay[28]. Briefl y, the method based on the reduction of 5,5 dithiobis (2-nitrobenzoic acid) with GSH to produce a yellow compound. The reduced chromogen directly proportional to GSH concentration and its absorption can be measured at 405 nm.

2.11. Statistical analysis

Data were analyzed by the General Linear Model procedure[29]. The Least Square Mean ± standard errors were calculated and tested for signifi cance using the t test .

3. Results

3.1. Parasitaemia

An average pre patent period of four days was recorded in all infected rats. The clinical signs observed were anorexia, starry hair coat, weakness and depression. These signs disappeared gradually following treatment in groups A, C, D while the signs remained in the untreated group E in addition to the inability to move, enlarged abdomen and death. All rats in group E died between 20-30 post infection. The effi cacy percentage against trypanosomiasis in groups A to E was respectively as follows 80, 40, 90, 100, 0. The administration of meth-A. fragrantissima (1 000 mg/kg b.wt.) produced a moderate effi cacy against trypanosomiasis. Untreated rats in group E died between 35 and 40 d post infection. The rats given DA and meth A. fragrantissima combinations (C and D) showed faster and higher recovery rates than the uninfected control and groups A and B. The administration of meth- A. fragrantissima (group B) produced a moderate effi cacy against trypanosomiasis. No relapse infection in the treated groups (Table 1).

Table 1Parasitaemia in rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

*Day of treatment. Group A: infected and treated with 3.5 mg/kg DA; Group B: infected and treated with 1 000 mg/kg of meth A. fragrantissima; Group C: infected and treated with 3.5 mg/kg DA + 500 mg/kg of meth A. fragrantissima; Group D: infected and treated with 3.5 mg/kg DA + 1 000 mg/kg of meth A. fragrantissima; Group E: infected and untreated; Group F: uninfected and untreated;

3.2. PCV

The percent of PCV of rats in the infected groups was signifi cantly lower than those of the uninfected untreated group F at day 5 post infection. The values of PCV of rats in group E 5 d post treatment was signifi cantly lower than those of the uninfected control and other treated groups. This continued till the death of all rats in group E. The values of PCV of rats in groups C, D (treated with meth A. fragrantissima + DA were significantly increased comparing with those of rats in group A (treated with DA alone) and group B (treated with meth A. fragrantissima alone) at days 5, 10, 15, 20 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 2).

Table 2Mean packed cell volume (%) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.3. Hb concentration

The Hb concentration of rats in the infected groups was signifi cantly lower than those of the uninfected untreated group F at day 5 post infection. The values of Hb concentration of rats in group E at 10 d post treatment was signifi cantly lower than those of the uninfected control and other treated groups. This continued till the death of all rats in group E. The values of Hb concentrationof rats in groups C, D (treated with meth A. fragrantissima + DA) were signifi cantly increased comparing with those of rats in group A (treated with DA alone) at days 15, 20 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 3).

Table 3Mean hemoglobin concentration (g/dL) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.4. TLC

The TLC of rats in the infected groups was signifi cantly lower than those of the uninfected untreated group F at day 5 post infection. The values of TLC of rats in group E 10 d post treatment was signifi cantly lower than those of the uninfected control and other treated groups. This continued till the death of all rats in group E.The values of TLC concentration of rats in groups C, D (treated with meth A. fragrantissima + DA) were significantly increased comparing with those of rats in group A (treated with DA alone) and group B (treated with meth A. fragrantissima alone) at days 5, 10, 15, 20 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 4).

Table 4Mean total leukocyte count (伊103/mm3) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.5. LC

The LC of rats in the infected groups was signifi cantly lower than those of the uninfected untreated group F at d 5 post infection. The values of LC of rats in group E 10 d post treatment was signifi cantly lower than those of the uninfected control and other treated groups. This continued till the death of all rats in group E .The values of TLC concentration of rats in group D (treated with 1 000 mg/kg meth A. fragrantissima + DA) were signifi cantly increased comparing with all treated groups at days 5, 10, 15 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 5).

Table 5Mean lymphocyte count ( %) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.6. Levels of MDA in serum

The MDA levels of rats in the infected groups were signifi cantly higher than those of the uninfected untreated group F at d 5 post infection. The values of MDA levels of rats in group E 5 d post treatment was signifi cantly higher than those of the uninfected control and other treated groups. This continued till the death of all rats in group E. The values of MDA levels concentration of rats in groups C, D(treated with meth A. fragrantissima + DA) were signifi cantly decreased comparing with those of rats in groups A and B (treated respectively with DA alone or meth A. fragrantissima 1 000 mg/kg alone) at days 5, 15 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 6).

Table 6Mean serum malondialdehyde level (nmol/ dL) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.7. Levels of GSH in serum

The GSH content of rats in the infected groups was signifi cantly lower than those of the uninfected untreated group F at d 5 post infection. The values of GSH content of rats in group E 5 d post treatment was significantly lower than those of the uninfected control and other treated groups. This continued till the death of all rats in group E. The values of GSH content of rats in groups C andD (treated with meth A. fragrantissima + DA) were significantly increased comparing with those of rats in groups A and B (treated respectively with DA alone and meth A. fragrantissima 1 000 mg/kg alone) at d 5 and 15 post treatment while not signifi cantly diff erent compared with the uninfected control group F (Table 7).

Table 7Mean serum reduced glutathione (mg/dL) of rats experimentally infected with T. evansi and treated with diminazine aceturate alone, or combination with methanolic extract of A. fragrantissima.

3.8. Phytochemical analysis of the methanolic extract of A. fragrantissima

The phytochemical constituents (mean ±SE, n= 5) present in methanolic extract of A. fragrantissima indicated respectively the presence of total phenolics, tannins, non tannin phenolics and total fl avonoids (25.57 ± 3.16) mg/g, (16.62 ± 1.45) mg/g, (8.98 ± 0.48) mg/g , (10.4 ± 0.84) mg/g of methanolic residue.

4. Discussion

Parasitaemia in rats in this experiment was associated with clinical signs of anorexia, pyrexia, depression, swollen abdomen and similar to those in mice, dogs and rabbits infected with Trypanosoma brucei (T. brucei) brucei[30,31] and in cattle infected with Trypanosoma congolense[32]. Following treatment, the clinical signs were gradually disappeared. All rats in the infected untreated group were died between 25 and 30 d post infection, due to the progress of the parasitaemia. The efficacy percentages against trypanosomiasis in groups A to E were respectively as follows 80, 40, 90, 100, 0. The administration of meth A. fragrantissima (1 000 mg/kg b.wt.) produced a moderate efficacy against trypanosomiasis. Recently, many authors recorded the effi cacy of certain plant extracts against trypanosomiasis[33-40]. The rats given DA and meth A. fragrantissima combinations (C and D) showed faster and higher recovery rates than the uninfected control and groups A and B. At the same direction, Chekwube et al[41] studied the eff ect of DA alone or in combination with either levamisole and/or vitamin C in albino rats experimentally infected with T. brucei brucei and found that levamisole and/or vitamin C combination with DA were more effective in the treatment of infected rats. da Silva et al[42] reported that selenium supplementation decreases the parasitaemia of various Trypanosoma infections and reduces important parameters associated with diseases such as anemia and parasite-induced organ damage.

The quantitative phytochemical analysis of the methanolic extract of A. fragrantissima indicated the presence of a high content from flavonoids, polyphenolics and tannins) and confirmed also from other studies[15,16]. Previous studies strongly correlate between the trypanocidal action of plant extracts and their contents of certain bioactive constituents especially fl avonoids, tannins and others. da Rocha et al[43] isolated 3 flavonoids from the roots of Arrabidaea brachypoda, compound 1 exhibited no activity toward T. cruzi, while fl avonoids 2 and 3 exhibited selective activity against these trypomastigotes. Gallic acid is a plant phenolic and well known hydrolysable tannins, Koide et al[44] reported its trypanocidal activity against T. brucei in both the long slender bloodstream forms and the procyclic forms, in vitro. Moreover, there are several studies investigated the relation of the herbal trypanocidal activity and their contents from fl avonoids, polyphenolics and tannins[45-47]. So, the presence of fl avonoids and tannins with higher concentrations may at least in part explain the observed activity against trypanosomiasis.

Our study showed the incidence of anemia (decreased PCV and Hb values), leucopenia and lymphopenia in all infected rats 5 d post infection. By 10 th day post treatment, rats treated with DA and meth A. fragrantissima (in a dose -dependent manner) combination therapy recovered early than rats treated with DA alone. It could be seen that the previous parameters of groups that received DA plus meth A. fragrantissima were not signifi cantly diff erent with the uninfected control group and were bitter than group A (DA alone). Group D shows rapid return to normal values than other treated groups. At the same direction, there was an increase in serum MDA and decrease in GSH levels in all infected rats 5 d PI. By 10 th day post treatment, rats treated with DA and meth A. fragrantissima (in a dose-dependent manner) combination therapy restored the level of GSH and diminished the level of MDA than rats treated with DA alone. It could be also seen that the previous parameters of groups that received DA plus meth A. fragrantissima were not signifi cantly diff erent with the uninfected control group and were bitter than group A (DA alone). Group D shows rapid return to normal values than other treated groups.

Moreover, it was shown that treatment with methanolic extract of A. fragrantissima reduced the lipid peroxidation, increased the level of reduced glutathione and keep the hematological parameters normal and indicated that there is a correlation between MDA levels and induction of anemia in trypanosomiasis. The proposed mechanisms are erythrophagocytosis, destruction of the erythrocytes by the immune system, erythrocyte osmotic fragility, hemodilution and erythropoietic depression[48,49]. It is[9,50] found that erythrocytes of T. brucei brucei-infected mice and Trypanosoma vivax in cattle produced signifi cantly greater amounts of by-products of lipid peroxidation than the erythrocytes of the control animals. This suggests that the infected animals may have reduced ability in the prevention of free radicals-mediated lipid peroxidation in the erythrocyte membrane. This may contribute to the pathogenesis in anemia in trypanosomiasis. It is believed that antioxidant treatment might prevent the erythrocyte destruction, since A. fragrantissima is a potent antioxidant as indicated from the present study. The chemical analysis of the methanolic extract of A. fragrantissima indicated the presence of several compounds which are potent as antioxidant activity (it is rich in fl avonoids, and polyphenolics) and confi rmed also from other studies[51,52].

The administration of fl avonoids, possessing antioxidant activity,may reduce the cellular injury caused by trypanosome-induced generation of free radicals[53]. At the same direction, treatment with a fl avonoid mixture and diminazene aceturate signifi cantly reduced trypanosome-induced increases in erythrocyte osmotic fragility and lipoperoxidative changes, suggesting possible antioxidant properties of this mixture and its therapeutic value in trypanosomosis[54]. Do Carmo et al[55] mentioned that some plant forms improved the hematological and biochemical parameters in rats experimentally infected with T. evansi.

Based on our results, we conclude that coadministration of diminazene aceturate and methanolic extract of A. fragrantissima was more active against experimentally induction of parasitaemia and more potent in prevention of PCV, Hb and GSH reduction, leucocytopenia, lymphopenia and MDA elevation than administration of diminazene aceturate alone. These potentiating activities of methanolic extract of A. fragrantissima against trypanosomiasis might be due to its phytochemical constituents of total fl avonoids and total polyphenolics a point needs further investigations.

Conflict of interest statement

The authors have declared that there is no confl ict of interest related to this paper.

Acknowledgments

This project (No. BCS06) was fi nancially supported by Promising Research Center in Biological Control and Agricultural Information (BCARC), Qassim University, Al Qassim, Kingdom of Saudi Arabia.

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Document heading 10.1016/j.apjtm.2016.01.032

IF: 1.062

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

15 December 2015

*

Ibrahim M. El-Ashmawy, Department of Veterinary Medicine, College of Agricultural and Veterinary Medicine, Qassim University, Kingdom of Saudi Arabia.

E-mail: i_elashmawy@yahoo.com

Tel: +966509601369

Fax: +966063801360

Foundation project: This project (No. BCS06) was financially supported by Promising Research Center in Biological Control and Agricultural Information (BCARC), Qassim University, Al Qassim, Kingdom of Saudi Arabia.