Background

African Animal Trypanosomiasis (AAT) is a neglected tropical disease caused by Trypanosoma brucei brucei, T. vivax, and T. congolense while human Trypanosomiasis is caused by two subspecies of Trypanosoma brucei: T. brucei gambiense, and T. brucei rhodesiense [1, 2]. The disease has an enormous economic impact in Africa since it affects the settlement patterns of people including land use and farming [3, 4]. In Ethiopia, AAT is one of the most significant and costly diseases because it hinders the effort made by the government to attain food sufficiency and affect the greater river basins of Abay, Omo, Ghibe, and Baro that have a high potential for agricultural development [5–8].

Trypanocides are used for the control of the disease in 37 African countries where animal trypanosomiasis is endemic but the available drugs are old, expensive, less effective, and face the problem of drug resistance [9–11]. The continued use of the same Trypanocides for years has resulted in drug resistance that has been largely responsible for the current chemotherapeutic failures in Ethiopia [9, 12]. Therefore, there is a need to develop alternative and efficacious drugs, either synthetically or from plant origins.

Herbal remedies are known to have been used for the treatment of this disease such as. Khaya senegalensis, Piliostigma reticulatum, Securidaca longepedunculata, ** was used to make smears on the slides and to monitor parasitaemia every other day microscopically at 400× total magnification. The degree of parasitaemia was determined using the “Rapid Matching” method of Herbert and Lumsden [38]. The wet smear was prepared in triplicates from each animal, and the mean value of slide counts was taken per sample examined microscopically. Logarithm values of these counts were obtained by matching with the table given by Herbert and Lumsden [38].

$$ \%\ \mathrm{change}\ \mathrm{in}\ \mathrm{parasitemia}\kern0.5em = \frac{\mathrm{Mean}\ \mathrm{parasitemia}\ \mathrm{on}\kern0.5em \mathrm{DAY}\kern0.5em 14-\mathrm{Mean}\ \mathrm{Parasitemia}\ \mathrm{on}\ \mathrm{DAY}\kern0.5em 0}{\mathrm{Mean}\ \mathrm{Parasitemia}\ \mathrm{on}\ \mathrm{DAY}\kern0.5em 0} \times 100 $$

Determination of packed cell volume (PCV)

Packed cell volume was measured using Wintrobe and Landsberg [42], and Wernery et al. [43] methods to predict the effectiveness of the test extracts in preventing hemolysis resulting from increasing parasitaemia associated with trypanosomiasis. It was monitored for infection three times until the 14th day (on day 0, 7, 14). 12 DPI was an important day to monitor since the treatment of mice with the extracts in each group began on the 12th day of post-infection (day 0 of treatment) when the infected mice showed peak parasitaemia (~108 trypanosomes/ml). The effect of extracts in improving the PCV of treated animals was compared with the controls.

$$ \%\ \mathrm{change}\ \mathrm{in}\ \mathrm{P}\mathrm{C}\mathrm{V} = \frac{\mathrm{Mean}\ \mathrm{P}\mathrm{C}\mathrm{V}\ \mathrm{on}\ \mathrm{DAY}\kern0.5em 7-\mathrm{Mean}\ \mathrm{P}\mathrm{C}\mathrm{V}\ \mathrm{on}\ \mathrm{DAY}\kern0.5em 0}{\mathrm{Mean}\ \mathrm{P}\mathrm{C}\mathrm{V}\ \mathrm{on}\ \mathrm{DAY}\kern0.5em 0} \times 100\kern0.5em \mathrm{and}\ \mathrm{on}\ 14\mathrm{D}\mathrm{P}\mathrm{I}? $$

Determination of body weight

The body weight (in gram) of each mouse in all groups was measured before infection, on the day treatment commenced (day 0) and every other day (on Day 2, Day 4, Day 6, Day 8, Day 10, Day 12 and Day 14) up to day 14.

$$ \%\ \mathrm{change}\ \mathrm{in}\ \mathrm{Body}\ \mathrm{weight} = \frac{\mathrm{Mean}\ \mathrm{body}\ \mathrm{weight}\ \mathrm{on}\ \mathrm{Day}\kern0.5em 14-\mathrm{Mean}\ \mathrm{Body}\ \mathrm{weight}\ \mathrm{Day}\kern0.5em 0\ }{\mathrm{Mean}\ \mathrm{Body}\ \mathrm{weight}\ \mathrm{on}\ \mathrm{Day}\kern0.5em 0} \times 100 $$

Determination of mean survival time

Mortality was monitored daily and the number of days from the date of inoculation of the parasite to death was recorded for each mouse in the treatment and control groups throughout the follow-up period for six weeks. The Mean Survival Time (MST) for each group was calculated as follows;

$$ \mathrm{Mean}\ \mathrm{survival}\ \mathrm{time} = \frac{\mathrm{Sum}\ \mathrm{of}\ \mathrm{survival}\ \mathrm{time}\ \mathrm{of}\ \mathrm{a}\mathrm{ll}\ \mathrm{mice}\ \mathrm{in}\ \mathrm{a}\ \mathrm{group}\ \left(\mathrm{days}\right)}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{mice}\ \mathrm{in}\ \mathrm{that}\ \mathrm{group}} $$

Data analysis

The data obtained from the study were summarized and expressed as mean ± standard error of mean (SEM). Data analysis was performed using Statistical Package for Social Science (SPSS), version 17.0. One-way ANOVA followed by Tukey’s multiple comparison tests were carried out to compare the results obtained from different groups and to determine statistical significance. P values less than 0.05 were considered significant.

Results

Yield for plant extract and phytochemical screening

The aqueous and methanol leaf extracts of V. sinaiticum gave 13.09%w/w and 18.13%w/w respectively. The phytochemical screening results are shown in Table 1. The methanol extract had more phytochemicals compared to the aqueous extract; however, anthraquinones and terpenes were absent in both extracts.

Table 1 Phytochemicals screened from the aqueous and methanol leaf extracts of Verbascum sinaiticum
Full size table

Acute toxicity test

The acute toxicity bioassay showed that the Lethal Dosage (LD50) of the aqueous (LD50 = 3807.9 mg/kg) and methanol (LD50 = 2154.1 mg/kg) leaf extracts of V. sinaiticum was above 2000 mg/kg and there was no evidence of an acute toxicity at the doses tested indicating good safety margin.

In vivo antitrypanosomal activity of aqueous and methanol crude extracts

Effect on parasitaemia

The reduction of parasitaemia showed variation among the administered doses of aqueous and methanol extracts of V. sinaiticum. The animals treated with 400 mg/kg dose of the aqueous extract had significantly (p < 0.001) low level of parasitaemia on days 8 and 10 when compared with other doses of the aqueous extract treated mice. The methanol leaf extract of V. sinaiticum at 100 mg/kg, 200 mg/kg and 400 mg/kg had kept parasitaemia at a significantly low level on day 6, 8, 10, 12 and 14 (p < 0.001) as compared with the negative control (Table 2).

Table 2 The effect of aqueous and methanol leaf extracts of Verbascum sinaiticum on parasitaemia level of Trypanosoma congolense infected mice
Full size table

Effect on packed cell volume

Animals treated with a higher dose (400 mg/kg) of the aqueous extract of V. sinaiticum had a statistically significant (p < 0.001) higher PCV value (47.14 ± 0.25) as compared to the negative control group (40.58 ± 0.28) on day 14 of treatment. Analysis of change in the percentage of PCV from day 7 to day 14 of treatment also showed that the aqueous extract at 200 and 400 mg/kg dose had prevented a drop in PCV associated with trypanosomes as compared to the negative control group (Table 3). In consistence with the results seen in PVC (Fig. 1), animals treated with the methanol extract of V. sinaiticum had higher PCV value (p < 0.001) as compared to the negative control groups at the end of the observation period (Table 3).

Table 3 Effect of the aqueous and methanol leaf extracts of Verbascum sinaiticum on packed cell volume of Trypanosoma congolense infected mice
Full size table
Fig. 1
figure 1

Comparison of the effect of aqueous and methanol leaf extracts of Verbascum sinaiticum on packed cell volume of Trypanosoma congolense infected mice

Full size image

Effect on body weight

The aqueous and methanol extracts of V. sinaiticum were capable of improving body weight of treated animals on days 8–14 as compared to the negative control group (p < 0.001). Animals treated with 400 mg/kg dose of both extracts of V. sinaiticum had a significantly (p < 0.001) higher body weight as compared to the negative control group (19.09 ± 0.34) on day 14 of treatment (Fig. 2).

Fig. 2
figure 2

Comparison of the effect of aqueous and methanol leaf extracts of Verbascum sinaiticum on body weight of Trypanosoma congolense infected mice

Full size image

Effect on mean survival time

Animals treated with 400 mg/kg of the methanol extract of V. sinaiticum had the highest mean survival time (40.20 ± 0.31 days) as compared to the negative control group (25.40 ± 0.43) while animals that received the positive control diminazine aceturate had a mean survival time of 44.00 ± 0.63 days (Fig. 3).

Fig. 3
figure 3

Mean survival time of Trypanosoma congolense infected mice treated with aqueous and methanol crude leaf extracts of Verbascum sinaiticum

Full size image

The overall activities of the extracts are shown in Fig. 4. The extracts had shown an increased values in the indices used for studying the potential of Verbascum sinaiticum against Trypanosoma congolense infected mice. The PVC and survival time had higher values as compared to negative control.

Fig. 4
figure 4

Comparison of the effects of aqueous and methanol leaf extracts of Verbascum sinaiticum on parasitaemia, packed cell volume, body weight, and survival time of Trypanosoma congolense infected mice at the end of the experimental study

Full size image

Discussion

The Verbascum sinaiticum extracts showed an LD50 above 2000 mg/kg indicating there was no lethal effect. The results validate why the plant has been used by several traditional healers for treatment of various disease including animal trypanosomiasis [21–24, 26], and toxicity and antidotes have not been reported. The experimental determination of this safety margin would justify that the plant is safe at the dose levels used in this study, which is an additional proof of the medicinal value of the plant in folk medicine. Though in previous studies, phytochemicals flavonolignans, hydrocarpin, sinaiticin, as well as two flavones, chrysoeriol and luteolin from the leaf of V. sinaiticum exhibited dose-dependent cytotoxicity when tested against cultured P-388 cells [44].

The trypanocidal and trypanostatic efficacy of V. sinaiticum aqueous and methanol extracts are associated with the presence of one or more biological active principals. This was shown to be true by the positive test for the presence of alkaloids, flavonoids, phenols, glycosides, saponins, steroids, and tannins. Tatli et al. [45] reported that Verbascum L. species has considerable saponins, iridoid and phenylethanoid glycosides, monoterpene glucoside, neolignan glucosides, flavonoids, steroids and spermine alkaloids that are responsible for biological activities thus their use in folk medicines. Previous in vitro and in vivo studies conducted on the antitrypanosomal activities of these phytochemicals have reported the trypanocidal and trypanostatic potential of these compound in human and animal trypanosomes [14, 15, 46–48]. Therefore, the observed in vivo antitrypanosomal activity of V. sinaiticum might be attributed either to the individual class of compounds antitrypanosomal activity or to the synergistic effect of each class of compounds in the extracts [49].

The reduction of parasitaemia (6.36 + 0.17) and prolonging of the lifespan (40.20 ± 0.31 days) of infected mice by a higher dose of methanol extract can be associated to the trypanosuppression of the phytochemical mainly the flavonoids that have been shown to exhibit potential to inhibit the growth of African Trypanosomes [14, 50]. These biologically active phytochemicals act at a single or multiple target sites associated with a physiological process [14] and interference with the redox balance of the parasites acting on the respiratory chain or on the cellular defenses against oxidative stress that partially eliminates the trypanosomes [51, 52]. Moreover, the flavonoids compounds have demonstrated promising antitrypanosomal activities on the trypomastigote forms, which are usually found in the bloodstream of mammalians [53, 54]. Alkaloids, flavonoids, phenolics, and terpenes have shown trypanocidal activity in an in vitro investigation, and the alkaloids have been shown to reduce the growth of trypanosomes by intercalating in the deoxyribonucleic acid (DNA) of trypanosomes [13] and inhibiting protein synthesis [53].

The trypanosomes are not eliminated from the blood stream of infected mice though animals treated with 400 mg/kg methanol extract had significantly (p < 0.001) lower mean parasitaemia (7.20 + 0.16) as compared to the negative control group (8.82 + 0.12) on day 14 of treatment. This result is similar to other studies conducted on Khaya senegalensis [55], Artemisia abyssinica [56], Adansonia digitata [18], Garcinia kola [57] and Carrisa edulis [58] with antitrypanosomal activities. The efficacy of crude extract might be masked with high parasite load in the host animal [55, 59], or it could be due to enzymatic inactivation of active compounds of the phytochemical in the host animal and impaired absorption from the site of administration [52, 60]. In addition, the concentration of the phytochemicals that reaches the target organs, the duration the phytochemicals take to effect a cure, and short half-life of the phytochemicals can reduce the efficacy of the crude extracts [61].

The effectiveness of diminazine aceturate was challenged in all mice approximately on days 12–14 of treatment and relapse of parasitaemia was observed [18]. T. congolense sequester in small vessels and capillaries of the heart, skeletal and other tissues, which often leads to a prolonged pre-patent period [62]. The relapse is also a clue to the existence of drug resistance trypanosomes in the South West and North West part of the country since the samples were acquired from the south-west part of Ethiopia. The test organism obtained from south-west part of Ethiopia can be a heterogeneous population of trypanosomes that are sensitive and resistant to diminazine aceturate; hence, the treatment with diminazine aceturate could have eliminated the sensitive sub-population through its therapeutic effects, so that the relapse is the manifestation of resistant population [6, 7, 63].

The reduction in packed cell volume (PCV) observed in extracts treated mice compared to diminazine aceturate treated mice could be due to acute hemolysis induced by the growing infection and increased susceptibility of red blood cell membrane to oxidative damage [64]. A higher PCV exhibited in mice treated with the extracts compared with the negative control group might be as a result of the phytochemicals reducing the parasite load, neutralizing the toxic metabolites produced by trypanosomes; the aetiological factors involved in the haemolysis of red blood cells and scavenging the trypanosome associated free radicals [59, 65, 66]. It could also be attributed to the potential antioxidant activity of the flavonoids and glycosides present in the leaf, which was also confirmed by similar studies previously done on related plant species from the same family [67–69].

The loss of body weight is associated with progression of infection followed by appetite decreases, and the animal loses condition as a result, there is wasting. The decreased supply of oxygen because of the anemia is also an important factor [70, 71]. However, animals, which received 400 mg/kg aqueous and methanol extract of V. sinaiticum gained weight by 1.07 and 1.67 % respectively, which was statistically significant (p < 0.001) as compared to the negative control groups. This shows that because of reduction in parasitaemia and prevention of drop in PCV as a result of the trypanosuppressive effect of the extracts against trypanosome infection, physical status of the treated mice was improved. Similar observations have been made by other researchers [59, 68, 69, 72–74].

Conclusion

In vivo tests usually show reliable antitrypanosomal activities of traditional antitrypanosomal medicinal plants. V. sinaiticum trypanocidal activity analysis indicated its antitrypanosomal potential and without toxicity effect on the host organism. The phytochemicals identified are known to have antitrypanosomal activities. The crude extracts have partially eliminated trypanosomes in a dose-dependent manner. Though unintentional, this study has also shown the existence of drug-resistant trypanosomes in the field stock. The study has shown that V. sinaiticum has a potential to be used as trypanocidal though further analysis is required to identify potent biologically active chemicals.