Anthelmintic activity

H. contortus (HC) eggs and L3-stage larvae were used to evaluate anthelmintic activity. Parasites of the strain INIFAP were obtained from the National Institute for Forestry, Animal and Agronomic Research (INIFAP).

Egg hatching test

HC eggs were obtained following a protocol approved by the Bioethics Committee of the Institute of Agricultural Sciences of the Universidad Autonoma del Estado de Hidalgo. Briefly, a Hampshire lamb (3-months and 37 kg PV), clinically healthy and free of gastrointestinal nematodes, was infested with HC L3-stage larvae (350 larvae kg⁻¹ PV). Twenty-one days after infestation, fecal samples were collected and the number of eggs per gram of feces was determined by the McMaster method (^{Cordero-Miguel et al., 2000}). To recover HC eggs, the methodology described by ^{Olmedo et al., (2017)} was followed. Thirty grams of feces were washed with distilled water in 200-, 100-, 75-, and 37-µm sieves and concentrated in the 37-µm sieve. The material retained in the last sieve was washed with 6 mL of saturated saline solution and centrifuged at 3000 rpm for 3 min. The supernatant was discarded, and the sediment was washed three times with distilled water to obtain free eggs.

A 96-well ELISA plate was used for the assay. Each well was added with 150-200 eggs in 50 µL of distilled water and 50 µL of extract (200, 100, 50, 25, or 12.5 mg mL⁻¹). Each extract concentration was assayed with four replicates, using Ivermectin (5 mg mL⁻¹) and distilled water as positive and negative controls, respectively. The plates were incubated at 30 °C for 48 h in a constant-humidity chamber. After incubation, ten 10-μL aliquots were observed under the microscope to count the number of unhatched eggs and larvae (dead or alive) per well. Finally, the percentage of egg hatching inhibition was calculated using the equation 1.

Larval mortality test

Feces from infested lambs were mixed with distilled water and polyurethane foam (1.5 × 1.5 × 0.5 cm) and incubated for 10 days at room temperature (15-20 °C). After incubation, HC L3-stage larvae were recovered following the Baermann technique. L3-stage larvae were unsheathed with 3% sodium hypochlorite (NaClO) for one minute and washed three times with distilled water to remove residual NaClO.

The assay was performed in 96-well plates. Each well was added with 150-200 larvae suspended in 50 µL of water and 50 µL of K. daigremontiana extract (200, 100, 50, 25, or 12.5 mg mL⁻¹). Each extract concentration was assayed with four replicates, using Ivermectin (5 mg mL⁻¹) and distilled water as positive and negative controls, respectively.

The plates were incubated in a constant-humidity chamber at 30° C for 72 h. After incubation, ten 10-µL aliquots were observed under the microscope to determine the number of larvae alive or dead per well. Mortality rate for each extract concentration was determined using equation 2:

Larval motility test

For this assay, unsheathed L3-stage HC larvae were placed in 96-well plates. Each well was added with 150-200 larvae suspended in 50 µL of water and 50 µL of extract (200, 100, 50, 25, or 12.5 mg mL⁻¹). Each extract concentration was assayed with four replicates, using Ivermectin (5 mg mL⁻¹) and distilled water as positive and negative controls, respectively. The plates were incubated in a constant-humidity chamber at 30° C for 72 h. After incubation, 10-µL aliquots were observed under the microscope to determine the number of alive/dead, motile/immobile larvae per well. The rate of motility inhibition for each extract concentration was determined using equation 3:

Antibacterial activity

The microorganisms used to determine the antibacterial activity of the hydroalcoholic extract from K. daigremontiana were Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Salmonella typhimurium (ATCC 14028), Pseudomonas aeruginosa (ATCC 9027), Listeria monocytogenes (ATCC 19113), and Escherichia coli (ATCC 35218).

The broth microdilution method described by Kaewpiboo et al., (2012) was used with some modifications to determine the minimal inhibitory concentration (MIC) of the K. daigremontiana hydroalcoholic extract. Two-fold serial dilutions of K. daigremontiana hydroalcoholic extract (from 400 to 0.781 mg mL⁻¹) were prepared by duplicate (100 μL per well). A bacterial cell suspension was adjusted to 0.5 McFarland units (approximately 1.5 × 10⁶ colony forming units [CFU] mL⁻¹). A 10-μL aliquot was added to each well. Kanamycin (AppliChem 4K10421™) was used as a positive control (64-0.5 µg mL⁻¹) and nutritive broth was used as a negative control. Plates were incubated at 37 °C under agitation (70 rpm) for 24 h.

After incubation, 20 μL of 0.04% (w/v) p-iodonitrotetrazolium solution was added to each well and incubated for 30 min. A change in color from yellow to pink indicated the reduction of the dye due to bacterial growth. The MIC was determined for each extract concentration as the lowest concentration at which no microbial growth was observed, as determined by the absence of color change (Figure 1).

Figure 1 Lethal concentration (LC₅₀ and LC₉₀ ) of Kalanchoe daigremontiana hydroalcoholic extract to inhibit the hatching of Haemonchus contortus eggs 

Statistical analysis

Data were analyzed by one-way analysis of variance and the Tukey-Kramer post hoc test (α = 0.05). Concentrations that inhibited 50% (LC₅₀) and 90% (LC₉₀) of HC egg hatching and motility, as well as those that killed 50% and 90% of larvae, were calculated by a PROBIT analysis using the statistical package SAS 9.0.

Anthelmintic activity

The mean efficacy of the hydroalcoholic extract from K. daigremontiana against HC is shown in Table 2. A significantly different egg hatching inhibition rate (EHI%) with respect to positive and negative controls (P < 0.0001) was found in the range 100-200 mg mL⁻¹ (99.5%). On the other hand, mortality (MOR%) was also significantly different with respect to controls (P < 0.0001), being the highest MOR% values observed at 50 and 25 mg mL⁻¹ (16.4 and 14.5%, respectively). Finally, significant differences were found among treatments in terms of motility inhibition (IMOT%). The highest IMOT% value was observed at 200 and 100 mg mL⁻¹ (85.2 % and 73.1 %, respectively).

As shown in Figures 1 and 2, the LC₅₀ and LC₉₀ values for egg hatching were 66.5 and 87.3, and 1.5 and 240.9 for motility inhibition in HC L3-stage larvae.

Figure 2 Lethal concentration (LC₅₀ and LC₉₀) of Kalanchoe daigremontiana hydroalcoholic extract to inhibit motility in Haemonchus contortus L3-stage larvae  

Antibacterial activity

As shown in Table 3, K. daigremontiana hydroalcoholic extract showed antibacterial activity against both Gram-negative (P. aeruginosa) and Gram-positive (L. monocytogenes, B. subtilis, and S. aureus) bacteria. The MIC was 100 mg mL⁻¹ for P. aeruginosa and L. monocytogenes and 0.781 mg mL⁻¹ against B. subtilis and S. aureus.

Our results suggest that the K. daigremontiana hydroalcoholic extract has a significant activity against H. contortus egg hatching in vitro (Figure 1). The activity of the extract was as good as that of the positive control (Ivermectin 5 mg mL⁻¹), indicating the feasibility of using this plant species as an anthelmintic agent. Since no significant differences were found between 100 and 200 mg mL⁻¹ treatments, the lowest dose could be used to reduce the risk of nematode resistance to the drug. Interestingly, death was not the main effect of the extract, but motility inhibition.

K. daigremontiana hydroalcoholic extract is capable of inhibiting egg hatching and larval motility (Figure 2). Other plant-derived molecules, like flavonoids, flavones, saponins, alkaloids, xanthones (^{Rivero et al., 2016}), polyphenols (^{Akkari et al., 2016}), tannins (Desrues et al., 2016) and pyrazole-5-carboxamide derivatives (^{Jiao et al., 2017}), have also been reported to reduce larval motility. The presence in K. daigremontiana of flavonoids and polyphenols (^{Karwatzki et al., 1993}) has been reported, and it is feasible that these compounds play a role in larval motility inhibition.

Our results show that the K. daigremontiana hydroalcoholic extract inhibits the motility of HC L3-stage larvae but are not effective in killing them. This could be relevant for the proposed use of K. daigremontiana extracts as an anthelmintic, since (^{Moradpour et al., 2013}) have described the morphological changes in abomasal tissues due to parasite migration to different parts of sheep abomasa. The results of anthelmintic activity herein reported are similar to those observed by ^{Phatak (2013)}, who did not find any activity in methanolic and petroleum-ether extracts from Kalanchoe pinnata on larval survival; however, the extracts reduced larval motility Unlike that study, however, we also evaluated egg hatch inhibition, finding that 99.5% of eggs did not hatch when exposed to 100 and 200 mg mL⁻¹ of the extract, with an LC₅₀ of 66.5 mg mL⁻¹ and an LC₉₀ of 87.3 mg mL⁻¹.According to ^{Lunkad et al., (2016)}, extracts of species of the subgenus Bryophyllum, such as B. pinnatum, showed anthelmintic activity in various concentrations (30 and 50 mg/ml) against Indian earthworms Pheretima posthuman, counting as antihelmintic activity the paralisis and death of more than 50% of the organisms.

A possible action mechanism of plant secondary metabolites on the eggs and larvae of nematodes like H. contortus involves the inhibition or delay of parasite growth and maturation by the affinity of glycoproteins in the cuticle of the parasite to phenolic compounds (mediated by proline residues); polyphenols may bind these proteins, inhibiting parasite motility, feeding, and reproduction, eventually causing their death; additionally, saponins have membranolytic actions (^{Hernández et al., 2018}; ^{Irshad et al., 2010}).

With respect to antibacterial activity, K. daigremontiana hydroalcoholic extract showed a higher activity on gram-positive than on gram-negative bacteria; this could be explained considering that gram-negative bacteria have a phospholipidic outer membrane and porins; the phospholipidic membrane that covers structural lipopolysaccharide components make the cell wall impermeable to lipophilic solutes.

^{Mothana et al., (2009)} evaluated 25 plants with antibacterial activity, including Kalanchoe farinacea. The methanolic extract of this plant had inhibitory activity on S. aureus, B. subtilis, and multi-resistant Staphylococcus epidermidis and S. aureus at a concentration of 4 mg mL⁻¹, producing 15- and 16-mm inhibition zones on S. aureus and multi-resistant S. epidermidis and S. aureus cultures, respectively, and 10-mm inhibition zones on B. subtilis cultures, but had no effect against gram-negative bacteria.

Dichloromethane extracts from K. pinnata leaves produced 18-mm inhibition zones when assayed against E. coli, but had no effect on S. aureus nor P. aeruginosa. Those results are in partial agreement with those herein reported. K. daigremontiana hydroalcoholic extract had no effect on E. coli, but was effective against P. aeruginosa, with a MIC of 100 mg mL⁻¹. The same study reported that the methanolic extract contained saponins, cardiac glycosides, and steroids as secondary metabolites with possible antibacterial activity.

^{Richwagen et al., (2019)} reported antibacterial activity of extracts from two Kalanchoe species, K. mortagei and K. fedtschenkoi, against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter cloacae). Growth inhibition greater than 50% (IC₅₀) was generally observed at concentrations of 256 μg mL^-1 and 128 μg mL^-1.

^{De Souza Barboza et al., (2016)}, corroborated antimicrobial activity of aqueous extracts of leaves and flavonoids occurring in Kalanchoe pinnata (Lam.), concentrations of 100 μg.mL-1, showed a growth reduction higher than 50% for Pseudomonas aeruginosa and Citrobacter freundii.

^{Akinnibosun et al., (1994)} reported that an ethanolic extract from K. pinnata leaves was more effective against S. aureus, E. coli, and P. aeruginosa than aqueous and methanolic extracts of the same plant, showing inhibition zones of 17.3 ± 1.2, 12.7 ± 0.9, and 8.3 ± 0.9 mm for S. aureus, E. coli, and P. aeruginosa, respectively. A qualitative chemical analysis of the ethanolic extract detected flavonoids, steroids, alkaloids, tannins, cardiac glycosides, and reducing sugars, along with secondary metabolites with reported antimicrobial activity, mainly saponins and phenolic compounds like tannins.