Anthelmintic activity of trans-cinnamaldehyde and A- and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum)

Abstract

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Figure 1. Examples of an A-type and a B-type procyanidin dimer.

Figure 2. Anthelmintic effects of cinnamon bark extract against Ascaris suum.

(a) Inhibition of A. suum third-stage larvae migration after incubation in cinnamon bark extract. Inhibition is expressed relative to larvae incubated only in culture media. Results are the means of three independent experiments, each performed in triplicate. (b) Inhibition of A. suum fourth-stage larvae motility after incubation in the cinnamon bark extract. Results are from a single experiment performed in triplicate.

Figure 3. HPLC chromatograms of cinnamon bark extract.

(a) HPLC chromatogram of cinnamon bark extract: Top panel shows detection of trans-cinnamaldehyde. Bottom panel shows larger version of the area indicated by the red arrow in top panel, showing 2, A-type procyanidin (PC) dimer; 3, A-type PC trimer; 5, A-type PC trimer; 6, internal standard; 7, cis—Cinnamic acid; 9, trans—Cinnamic acid. (b) HPLC chromatogram of thiolysed cinnamon bark extract (BM = benzylmercaptan adduct): 1, Catechin; 2, A-type PC dimer; 3, A-type PC trimer; 4, Epicatechin; 5, A-type PC trimer; 6, Internal standard; 7, cis—Cinnamic acid; 8, A-type PC—BM trimer; 9, trans—Cinnamic acid; 10, cis Catechin—BM; 11, Epicatechin—BM; 12, A-type PC—BM dimer.

Figure 4. Anthelmintic effects of isolated proanthocyanidin (PAC) fractions.

(a) Inhibition of Ascaris suum third-stage larvae (L3) migration after incubation in PAC fractions F1 and F2. Inhibition is expressed relative to larvae incubated only in culture media. Results are the means of two independent experiments, each performed in triplicate. Error bars indicate the inter-replicate SEM. (b) Inhibition of A. suum fourth-stage larvae (L4) motility after incubation in PAC fraction F2. Results are from a single experiment performed in triplicate. Error bars indicate the inter-replicate SEM. (c) Incubation in PVPP does not remove anthelmintic effects of cinnamon bark extract. Results are the means of two independent experiments, each performed in triplicate.

Figure 5. Anthelmintic effects of pure trans-cinnamaldehyde (CA).

(a) Anthelmintic effects of CA against Ascaris suum third stage (L3) and fourth stage (L4) larvae. Mortality of A. suum was assessed after 12 hours incubation for L3 and 6 hours for L4. L3 results are the mean of three independent experiments, each performed in triplicate, and the L4 results are from a single experiment performed in triplicate. Error bars indicate the inter-replicate SEM. (b) Anthelmintic effects of CA against Oesophagostomum dentatum L3 and Trichuris suis first-stage larvae (L1). Mortality of O. dentatum was measured by agar-based migration inhibition assay after overnight incubation in CA, and T. suis mortality by observation of motility after 2 hours incubation. O. dentatum results are the mean of two independent experiments, each performed in triplicate, and T. suis results from a single experiment performed in triplicate. Error bars indicate the inter-replicate SEM.

Figure 6. Ultrastructural changes in Ascaris suum exposed to trans-cinnamaldehyde.

Transmission electron micrographs of A. suum fourth-stage larvae exposed to either culture media (Control) or 236 μM trans-cinnamaldehyde (CA) for 12 hours. For all panels scale bar indicates 2 μm. (a) Cuticle (cu) and underlying Muscular (mu) tissue—note the lesions in the muscle tissue underlying the cuticle and hypodermis in parasites exposed to CA (red arrows). (b) Digestive tissues showing the microvilli (mv) overlying the intestinal lumen—note the destruction of the villi (red circle) and the presence of large vacuoles (red arrow) in parasites exposed to CA.

Figure 7. Total Ascaris suum larval burdens and distribution of larvae in the intestine of pigs administered trans-cinnamaldehyde.

(a) Numbers of fourth-stage larvae (L4) in the small intestine (SI) at day 14 post-infection (p.i.) in pigs fed either trans-cinnamaldehyde (CA) in the diet daily (‘CA diet’), dosed with encapsulated CA at days 11 and 13 p.i. (‘CA capsules’), or not administered CA (‘Control’). Indicated is the mean and SEM. (b) Proportions of L4 recovered from the three groups in Segment 1 (proximal half of the SI), Segment 2 (third quarter of the SI) and Segment 3 (distal quarter of the SI). See materials and methods for further information.

Figure 8. Schematic experimental design.

Stepwise outline of the anthelmintic testing of cinnamon bark extract, derived proanthocyanidin fractions and pure trans-cinnamaldehyde.