Sheep farmers believe from their experience and observation that there is a reduction in the load of intestinal parasites eggs, including Haemonchus contortus in animals feed with cassava. The researchers decided to investigate the hypothesis that this reduction is due to the presence of free cyanide in fresh cassava leaf extract that was established at the same time as sampling was done for the mobility test of L3 larvae of H. contortus.
Table 1 presents the results of the controlled bioassay, in which 100 isolated L3 stage larvae (in triplicate) were subjected to variations in the concentration of extract of fresh cassava leaves in water. The aqueous extract of fresh cassava leaves, as elaborated, showed a concentration range of 0.22 to 3.44 µg of CN- per ml.
Table 1 – Effect of fresh cassava extract and cyanide concentration on the motility of a hundred L3 larvae of H. contortus (Average of 3 repetitions).
Mass concentration of fresh cassava leaves in the water extract
|
CN-
Concentration
|
Number of mobile larvae per repetition
|
Mobility reduction
|
mg x ml-1
|
µg x ml-1
|
R1
|
R2
|
R3
|
Mean and standard deviation (*)
|
5,2
|
0,22
|
95
|
92
|
94
|
93,67
|
10,0
|
0,43
|
70
|
65
|
60
|
65,00
|
20,0
|
0,86
|
40
|
50
|
48
|
46,00 c
|
40,0
|
1,72
|
20
|
25
|
22
|
22,34 d
|
80,0
|
3,44
|
0
|
0
|
0
|
0,00 e
|
Ivomec® (FCL)
|
0,01%
|
04
|
03
|
0
|
2,34 e
|
Distilled water NCW)
|
0,00
|
98
|
99
|
96
|
97,66 24a
|
(*): Equal letters in the columns did not differ at the 0.5% level
The results confirmed the toxic effect over the sheathed larvae of H. contortus, an effect proportional to the concentration of extract of fresh cassava leaves. From the data presented in Table 1, a curve was fitted relating the mobility of L3 larvae with the concentration of the cassava leaves extract. The curve that best fit the results allowed was the logarithmic equation y = -33.39ln(x) + 40.517, with R2=0.996 (Figure 1), with a positive and significative correlation on the effect of increased immobility of larvae and the increasing free cyanide concentration in the cassava leaves extract. The equation highlights that the greatest effect was caused between 0.0 and 0.5 µg of CN x ml-1, when about 50% of L3 larvae lost mobility. With the increase in free cyanide content from this concentration, the curve becomes less accentuated, but it is not asymptotic before affecting the total of larvae, equating to the control of the commercial product based on ivermectin, which had an immediate impact on the mobility of the larvae.
The equation allowed us to calculate the dose that may cause total paralysis a hundred L3 larvae (DL100) as occurred with approximately 3.5 µg of CN- ml-1, corresponding to 80 mg of cassava leaves.ml-1 or 80g.l-1. This result agrees with the results from Sokerya, (2009) that the consumption of cassava leaves can reduce the number of intestinal parasites in sheep but may contradict the author's conclusion that a dose of 170 mg of HCN per kg can be innocuous as anthelmintic. Onwuka et al, (1992) and Sokerya, (2009) reported the need to use a 246 to 248 mg100 g of cyanide to achieve similar effects.
It is noteworthy that cassava leaves may vary in cyanide content depending on the cultivar or variety, age and time of year (Ravindran, 1995). The results obtained in this experiment partially agree with those of Suteky and Ji (2019) with 47.87% of the larvae immobilized with 12.5 mg by ml of cassava leaves extract, but this difference can be attributed to the fact that the authors were based on larval development and not on larval motility.
Al-Rofaai et al (2012) reported an efficiency of 57.33% on L3 larvae by an extract of 12.5 mg of cassava leaves.ml-1, which would be near to 22mg to achieve total larvae immobility. Although even less than the 80mg we founded to immobilize all of the L3 larvae, it is necessary to consider that the experiment did not eliminate the effect of the solvents used in the preparation of the cassava leaf extract. The author used solvents to evaluate, in addition to tannins, alkaloids, flavonoids, steroids and phenols. The solvents used were hexane, chloroform, ethyl acetate and methanol (80%), precisely the one that presented the best effect against the larvae, so it impossible to separate the cyanide effect from the effect of the solvents used.
The same use of solvents was founded in several other studies carried out to evaluate the anthelmintic action of cassava extract. Marie- Magdeleine et al (2010), before processing the extract, removed the cyanogen glycosides and then dehydrated the extract. The resulting powder was then diluted with three different extraction solvents, dichloromethane, methane and water, because the author focusses the action of tannins on the parasites.
In our experiment, we used only water, because the objective was to evaluate cassava leaves in its natural way, with minimal chemical interventions, so that we could have the closest scenario to the process of ingesting the plant leaves in the animal's rumen, guaranteeing the presence of all its compounds even those that are not soluble in water.
In several studies carried out to evaluate the anthelmintic action of cassava leaves extract, the authors use several solvents for the extraction, having in mind to study the effect of phenolic compounds. Al-Rofaai et al (2012) evaluated tannins and phenols, substances known for their antiparasitic effects, not only against H. contortus, but also on other nematode species. With the same purpose, alkaloids, flavonoids, steroids and phenols were also evaluated (Sokerya 2009; Marie-Magdeleine et al, 2010; Al-Rofaai et al, 2012; Suteky and Ji, 2019; Constant, 2020). Marie-Magdeleine et al (2010), for evaluating the effect of tannins on the parasites, took care to remove the cyanogen glycosides, and the resulting powder from the process was diluted with dichloromethane, methane and water.
Another experiment carried out in Malaysia by Sokerya (2009) explores the potential of cyanide present in cassava leaves, although focusing on goats and not sheep. The author evaluated the anthelmintic effects of cassava feed in animals contaminated with Haemonchus contortus, but used the cassava plant in different ways such as fresh, ensiled and dehydrated leaves. The count of eggs present in the feces of these animals was done after a period of time after ingestion and showed positive results in all groups in which the animals were fed with cassava leaves, but mainly in the consumption of fresh leaves. The authors justifies that they did not carry out the detoxification of cassava by drying, as carried out by Marie-Magdeleine et al (2010), precisely because the objective was to relate the forms of feeding the animals with cassava and the respective responses on the parasite load. However, the authors emphasizes that the risk of poisoning the animal by cyanide should not be neglected, which is why they measured the amount of HCN present in the plants used, which presented 585mg of CN- per kg in fresh leaves and 170mg of CN- per kg in silage made with the same leaves. The author also highlighted that the values found fall within the tolerance limits for ruminants, which is 2 to 6 mg/kg of the animal's live weight, according to Onwuka et al, (1992), reducing the risk of intoxication in these animals.
Although our bioassay makes it quite clear that the cyanide present in cassava leaves, and not only its phenolic compounds, has a direct action against H. contortus larvae, these results should be evaluated and adjusted by feed the sheep with fresh leaves.
The comparison of results with the literature highlighted the lack of literature that presents results of anthelmintic potential of cassava leaves evaluated by aqueous extract or that highlight the cyanogenic compounds of cassava, although the literature that points out the risks of cyanide consumption for human and animal health is very abundant.
The review also highlighted that the focus of the anthelmintic use of cassava leaves for feed sheep and goats has always been hypothesized by the effect of the phenolic compounds present, such as tannins, as they are reported in the literature for their antiparasitic effects, not just in H. contortus, as well as in other nematode species. Also in this aspect, it would be important to compare, in the same in vitro assay, the effect of cyanogenic and phenolic compounds, with and without the use of solvents.
On the other hand, the research started from the principle that, if the anthelmintic effect is proven, the use of leaves is easier and more feasible, since it is not necessary to use the roots, which have commercial value, or the branches, which are used as planting material. Leaves can be obtained without interruption during all year or all the summer in some south regions of South America, using pruning, as the literature has confirmed that up to 3 pruning’s over 12 months of cultivation does not affect root yields.
If cassava leaves contain compounds from the phenolic group, such as tannins and saponins, in addition to cyanogenic glycosides (Tao et al, 2019), it also has a high nutritional value (Pereira et al, 2018). The use of cassava leaves by feeding of sheep or goats is possible (Vilpoux et al. 2013) and may also be a powerful tool and can diversifying its antiparasitic power and avoid the occurrence of resistance to the present anthelmintic principles.
This type of local use could also add value to the production of cassava leaves, as assessed by Sagrilo et al. (2001) in weight of leaves harvested in commercial cassava plantations for starch extraction, with 12 and 24 months of cultivation with the 5 main cultivars planted. The authors found an average production of fresh leaves 2.4 ton. ha-1, ranging from 1.8 to 7.5 ton. ha-1, available throughout the year, with higher production in the beginning of cultivation and in the summer months in Brazil (September to May). Currently this volume of good quality protein material is in the field, without use.