Fig 1.
Scatter plot of tested compounds after primary screening at the single concentration of 1 μM.
The growth of C. parvum HNJ-1 strain after exposure to each compound at a single concentration of 1 μM for 45 h, as determined by means of fluorescence microscopy. The percent inhibition is presented as the percentage of inhibited parasites compared with the positive control (untreated wells) after subtraction of the negative control (uninfected HCT-8 cells). Compounds with a growth reduction of more than 60% (dot line) were considered to be compound that inhibit C. parvum parasite growth. Blue dots represent the compounds and the red dot represents the comparative drug nitazaxonide (10 μM). This is single experiment with the means of triplicate wells.
Fig 2.
Chemical structures of the hit compounds.
The nine compounds identified in the primary in vitro screen using 1 μM as the highest concentration. The most effective compounds are indicated by boldface letters. These compound structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov).
Table 1.
The nine lead anti-C. parvum compounds identified in the first screen at a concentration of 1 μM and tested against the C. parvum HNJ-1 strain.
Table 2.
Biological activity of hit compounds evaluated against the in vitro growth of C. parvum.
Fig 3.
Dose-response curves and growth inhibitory effects of four hit compounds on C. parvum.
The growth of C. parvum HNJ-1 strain after exposure to various concentrations of four hit compounds for 45 h, as determined by means of fluorescence microscopy. The EC50s were determined from dose response curves using non-linear regression (curve fit analyses). The values from triplicate experiments are shown. (A) Dose-response curves and the half maximum inhibition concentration (EC50) value of alisol-A for C. parvum (122.9± 6.7 nM), (B) Dose-response curves and the (EC50) value of alisol-B (79.58±13.8 nM), (C) Dose-response curves and the (EC50) value of atropine sulfate (253.5±30.3 nM), (D) Dose-response curves and the (EC50) value of bufotalin (63.43±18.7 nM).
Fig 4.
In vivo infected mice treated with compounds showed growth inhibition of C. parvum.
Two compounds (atropine sulfate and bufotalin) reduce the number of oocysts shedding in mice compared to control drugs. C. parvum oocysts were inoculated orally into SCID mice. Infected mice left untreated served as a positive control. The mice were treated with 200 μl of atropine sulfate or bufotalin 3 days after infection (i.e., from Day 3) until Day 13 at three different concentrations for each compound (atropine sulfate: 200, 100, and 50 mg/kg; bufotalin: 0.1, 0.05, 0.025 mg/kg). Nitazaxonide was administered orally at 100 mg/kg as a comparative drug. The number of oocysts in the feces was determined by using the sugar flotation method, and the total number of oocysts per gram (OPG) was calculated. Data shown are the mean and SEM (n = 3 mice in each group). Asterisks indicate levels of statistical significance as evaluated by the difference in parasitemia between the control and drug-treated groups by use of a one-way ANOVA with the post-hoc Tukey HSD test, *: p < 0.05, **: p<0.01.
Table 3.
Efficacies of TCM compounds with nitazoxanide for reducing oocyst shedding from the distal colon of C. parvum-infected neonatal SCID mice.
Fig 5.
Histological observation with immunohistochemistry detection of C. parvum infection in ileum tissues.
Histological sections of small intestine (ileum) of different animal groups (treated with nitazoxanide, atropine-sulfate, bufotalin, and control groups). Except for the negative control group, all mice were orally inoculated with Cryptosporidium parvum oocysts (1×105), then treated with each compound. The left column is the lower magnification for hematoxylin and eosin (HE), the middle column is the higher magnification for HE, and the right column is immunohistochemistry for C. parvum. The severity of C. parvum infection was scored from–to 3+. (A-C) Ileal sections of uninfected mouse (negative control) (Score: -). (D-F) Ileal sections of non-treated mouse (positive control) showing inflammatory cell infiltration of the mucosal layer. Numerous C. parvum oocysts are attached to the intestinal villi (Score: 3+) (arrowheads: C. parvum oocysts). Ileal sections of nitazoxanide-treated groups (G-I), Nitazoxanide-treated group (Score: 2+) showing a number of oocysts. (J-L) Atropine sulfate-treated group showing less reduction of oocysts (Score: 2+). (M-O) Bufotalin-treated group showing markedly reduced number of oocysts (Score: 1+). Bar = 100 μm.
Fig 6.
Scanning electron microscopic (SEM) findings the compound’s effects of ileum tissues.
The SEM images of intestinal ileum tissue of different groups of mice infected with C. parvum and treated with hit compounds, as well as the negative (A-C) and positive (D-F) control groups, and comparative drug (nitazaxonide)-treated group (G-I). Atropine sulfate- (J-L) and bufotalin- (M-O) treated mice showed no abnormalities or visible parasites in the intestine. The SEM shows the intracellular structures as well as the surface of C. parvum attached to the host cells. These compounds had no adverse effects on the ileum tissue.