Anthelmintic activity of Aloe vera and fractions against Onchocerca ochengi
The anthelmintic activity of leaves of A. vera on O. ochengi adult males was evaluated in terms of mortality after 48 h and 72 h of incubation. The plant extract induced mortality of O. ochengi adult male in a concentration-dependent (Fig. 1). The leave extract killed, with a LC50 = 20.71 ± 3.14 and 11.75 ± 0.73 µg/mL respectively after 48 h and 72 h (Table 1). Positive controls were active against O. ochengi with a respectives LC50 of 2.23 ± 1.96 µg/mL, 3.62 ± 1.88 µg/mL, and 4.34 ± 0.71 µg/mL for ivermectin, levamisole and albendazole after 72 h (Table 1).
Table 1
LC50 of A. vera crude extracts (MeOH-CH2Cl2) against Ochengi ochengi after 48 h and 72 h exposure
LC50 µg/mL after 72 h O. ochengi (after 48 h) |
Drugs | Ivermectin | Levamisole | Albendazole |
| 2.23 ± 1.96ns (5.27 ± 0.01)*** | 3.62 ± 1.88ns (6.9 ± 0.03)*** | 4.34 ± 0.71ns (8.0 ± 0.0)*** |
Leaves | 20.71 ± 3.14ns (11.75 ± 0.73)*** | 20.71 ± 3.14ns 11.75 ± 0.73*** | 20.71 ± 3.14ns (11.75 ± 0.73)*** |
Ns = non-significant (P > 0,05); significant with the threshold of 1‰ (P < 0,001;***= highly significant). |
The methanolic-methylene chloride extracts of A. vera have shown an anthelmintic activity similar to ivermectin, levamisole and albendazole after 48 h post incubation (P < 0.05). The LC50 values obtained are similar to the finding of Dikti et al. [46] and Kalmobé et al. [47] who evaluated the effect of Acacia nilotica on O. ochengi males (11.5 ± 0.1 µg/mL), and leave of Lophira lanceolata on O. ochengi males (11,6 ± 0.4µg/mL).
The HexAcOEt and AcOEt fractions were inactive on adult worms with LC50 of 50.52 µg/mL. As shown in Fig. 3, the highest activity in adults was observed with the AcOEtMeOH and MeOH (100: 0) fractions with a LC50 of 15.50 and 12.82 µg/mL respectively.
Anthelmintic activity of Aloe vera and its fractions on Caenorhabditis elegans wild-type and mutants
The anthelmintic activity of A. vera was assessed in vitro against wild-type (WT). The leaves of A. vera exhibited moderate activity on C. elegans WT. Figure 2 shows that worm motility decreased with increasing extract concentrations. The lowest concentrations of the extract required to cause 50% of mortality (LC50) was 1 937 µg/mL after 72 h (Table 2). The anthelmintic activity of ivermectin, levamisole and albendazole on the WT strain displayed LC50 of 2.17, 4.12 and 4.26 µg/mL respectively after 72 h incubation (Table 2). Statistical higher LC50 was observed between the MeOH-CH2Cl2 activity on C. elegans WT strain compared to ivermectin, levamisole and albendazole after 72 h incubation (Table 2) (P < 0.001).
Table 2
LC50 of A. vera crude extracts against Caenorhabditis elegans after 48 h and 72 h exposure
LC50 µg/mL after 72 h (after 48 h) |
Strains mutant | Leaves | Ivermectin | Levamisole | Albenazole |
WT | 1937 ± 1.58*** (2747 ± 0.78)* | 2.17 ± 0.66*** (2.41 ± 0.33)*** | 4.12 ± 0.31*** (4.15 ± 0.68)** | 4.26 ± 0.0*** (4.35 ± 0.57)*** |
CB3474 | 1950 ± 0.45*** (3170 ± 0.59)* | - | - | > 100 |
CB211 | 4360 ± 0.53*** (9650 ± 2.43) ns | - | > 100 | - |
VC722 | 3920 ± 0.84* (6790 ± 1.37) ns | > 100 | - | - |
The anthelmintic activity was assessed in vitro against three resistant strains of the free-living nematode namely CB211 resistant to levamisole, CB3474 resistant to albendazole and VC722 resistant to ivermectine. A tenfold increase of the LC50 value of C. elegans albendazole-resistant mutant CB3474, VC722 and CB211, was observed at 1 950, 3 920 and 4 360 µg/mL for 72 h respectively for the extract studied (Table 2).
Table 2 shows that there is a higher significant difference between mean LC50 values of albendazole resistant strain CB3474 (1 950 ± 0.45 µg/mL) to levamisole: CB211 (4 360 ± 0.53 µg/mL) and ivermectin: VC722 (3 920 ± 0.84 µg/mL) at 5% threshold after 72 h incubation (P < 0.001). From this comparison between the WT strain and the mutants, the methanolic-methylene chloride extract of A. vera had more effect on the WT strain with low lethal concentrations, followed by the albenazole mutant strain. This could mean that, the extract could act in the same way on the nematode. The plant extract slowed growth and induced an uncoordinated motion similar to that caused by albendazole.
The anthelmintic activity of plant fractions was assessed in vitro against WT and mutants strains of the free-living nematode C. elegans namely ivermectine. A tenfold increase of the LC50 value of C. elegans albendazole-resistant mutant CB3474, VC722 and CB211 respectively, was observed (Table 2). It appears from Table 2 that the WT strain has been very sensitive to different extract compared to the mutant strains (CB3474, VC722 and CB211). The LC50 obtained after 48 h is recorded in Fig. 4, except for Hex and HexAcOEt extracts, for which the WT strain was not very sensitive with LC50 value of 6 180 ± 012 µg/mL. The WT strain was very sensitive to the other fractions with LC50 ranging between 250 ± 0.1 and 790 ± 0.1 µg/mL. The anthelmintic activity can be at the origin of the bioactive molecules which could be found in these active fractions and this activity is due to the structure of the non-mutant gene of the WT which will favor the action of these fractions.
Some mutants were highly sensitive to some fractions, with values affecting half of the mortality of CB4374 varying between 4 590 and 6 548 µg/mL, VC722 (4 960 and 6 565 µg/mL), and CB211 (4 590 and 6 565 µg/mL) for all fractions (Figs. 5, 6 and 7). In Table 2, except for the fraction AcOEtMeOH (LC50 = 6 565 µg/mL), all the other fractions have an effect on the mutant and resistant strains of ivermectin VC722 (LC50 = 4 960, LC50 = 6 180 µg/mL). While for CB3474 strain (Fig. 6), with the exception of the fraction HexAcOEt with LC50 = 6 548 µg/mL, all other fractions were susceptible with LC50 varying between 4 960 and 7 700 µg/mL. For the levamisole-resistant strain CB211, all fractions were sensitive (LC50 = 4 590 and 1 364 µg/mL) with the exception of the fraction AcOEtMeOH (LC50 = 6 565 mg/mL). After comparing the activity of the fractions on the different mutant strains, it came out that the fraction which acted on the different mutant strains had a LC50 that varied between 4 590 and 6 100 µg/mL.
Secondary metabolites of methanolic-methylene chloride extracts and fractions of Aloe vera
The phytochemical composition of methanolic/methylene chloride extract was assessed to evaluate the chemical families present in this plant extract and which might be involved in its anthelmintic activity. Thus, tannins, polyphenols, flavonoids and saponins were quantified by spectrophotometer using a UV-biowave DNA Cambridge. The tannins, polyphenols, flavonoids and saponins were quantified and the results are shown in Table 3. It appears that polyphenol and tannins contents are the highest compared to flavonoids and saponins.
Table 3
Phytochemical composition of MeOH-CH2Cl2 extract and fraction of leaves
of Aloe vera
Parts used | Polyphenols | Tannins | Flavonoids | Saponins |
| | (mg/g) | | |
Crude of extract (mg/g ± ESD) | ++++ | +++ | + | ++ |
Crude of extract | 1015.01 ± 0.05 | 401.37 ± 0.05 | 25.35 ± 0.01 | 1.20 ± 0.05 |
Hex | 880.01 ± 0.01 | 788.89 ± 0.05 | 181.34 ± 0.003 | 0.51 ± 0.01 |
HexAcOEt | 14.51 ± 0.003 | 12.67 ± 0.02 | 25.76 ± 0.004 | 0.8 ± 0.1 |
HexAcOEt | 96.88 ± 0.004 | 74.29 ± 0.03 | 195.19 ± 0.002 | 0.08 ± 0.01 |
AcOEt | 252.80 ± 0.005 | 157.12 ± 0.03 | 195.19 ± 0.005 | 0.51 ± 0.01 |
AcOEtMeOH | 298.92 ± 0.004 | 240.10 ± 0.01 | 181.34 ± 0.05 | 0.94 ± 0.01 |
AcOEtMeOH | 893.60 ± 0.01 | 480.19 ± 0.09 | 181.34 ± 0.003 | 1.37 ± 0.01 |
MeOH | 641.60 ± 0.01 | 329.12 ± 0.02 | 225.76 ± 0.01 | 1.37 ± 0.01 |
Hexane (Hex) (1:0 v/v), hexane : acetate (HexAcOEt) (8:2 v/v), hexane : acetate (HexAcOEt) (6:4 v/v) acetate (AcOEt) (1:0 v/v), acetate: methanol (AcOEtMeOH) (8:2 v/v), acetate: methanol (AcOEtMeOH) (7:3 v/v) and methanol (MeOH) (1:0 v/v) |
+ = present; ++ = very present; +++ = highly present |
The results of these assays are shown in Table 3. It appears that the MeOH-CH2Cl2 extract of A. vera content higher amount/proportion of polyphenols (1015.01 ± 0.05 mg/g), tannins (401.37 ± 0.05 mg/g), flavonoids (25.35 ± 0.01 mg/g) and saponins (1.20 ± 0.05 mg/g).
The phytochemical study of MeOH-CH2Cl2 fractions revealed the presence of unevenly distributed bioactive elements (Table 3). The polyphenol and tannin content of the Hex, AcOEtMeOH and MeOH leave extracts reflects its higher anthelmintic activity (Table 3). Other fractions, although containing these chemical families, appeared to have no anthelmintic activity.
Antioxidant power assay
The investigation of the antioxidant activity of MeOH-CH2Cl2 extract of A. vera was carried out by the free radical scavenging method DPPH. The more the value of IC50 is low, the more the extract has strong antioxidant activity. The MeOH-CH2Cl2 extract of A. vera has an IC50 = 15 ± 0.12 µg/mL against standard which is not too far of that of the ascorbic acid used as standard (IC50 = 9 ± 0.22 µg/mL).
Assessment of acute toxicity of methanolic-chloride methylene extract of Aloe vera
In the study of acute toxicity test, oral administration of the methanol- chloride methylene extract of leaves of A. vera to the Witar rats showed no mortality to the dose up to 3 000 mg/kg and no signs of toxicity after oral administration. The suggested acute toxicity results of this methanol- chloride methylene extract from A. vera leaves are non-toxic up to this dose (3 000 mg/kg). These results are similar to those observed by Michayewicz [69], testing of ethanolic activity of A. vera leaves and obtaining no mortality up to 3000 mg/kg.