Figure 1.
Chemical structures of the six isoflavones included in the study.
Table 1.
Composition of sublingual films of biochanin A.
Table 2.
IC50 values (µM) of isoflavones on C. pneumoniae and C. trachomatis inclusion counts.
Figure 2.
Effect of isoflavones on C. trachomatis inclusion size.
A) Immunofluorescence images of C. trachomatis infected HeLa cells (untreated and treated with 100 or 10 µM biochanin A). Chlamydia inclusions are stained in green (polyclonal rabbit antibody raised against formalin fixed C. trachomatis elementary bodies [30]) and host cell nuclei are stained in blue with DAPI. B) Quantitation of average inclusion sizes in C. trachomatis infections treated with the isoflavones. Inclusion sizes are expressed as relative units proportional to the untreated controls. In a pairwise comparison of different concentrations, the mean inclusion size in biochanin A, genistein and genistin treated samples were statistically significantly smaller than in formononetin, daidzein and daidzin treated samples, respectively (p<0.05, Student's t-test).
Figure 3.
Impact of biochanin A on C. pneumoniae replication.
A) Inhibition of C. pneumoniae inclusion counts and infectious progeny production by different concentrations of biochanin A. B) Effect of host cell or elementary body (EB) pretreatment with biochanin A on C. pneumoniae inclusion counts. C) Effect of delayed administration of biochanin A on the inhibitory capacity of biochanin A on C. pneumoniae inclusion counts (1st passage) and infectious progeny production (2nd passage). Biochanin A (50 µM) was added into infected cell cultures at 2, 6, 12 or 24 h post infection. In all experiments, the cultures were stained at 72 h as described in Materials and Methods. *** In B) and C) indicates statistically significant difference (p<0.05; unpaired t-test) compared to untreated control infection.
Figure 4.
Permeability of biochanin A across porcine buccal mucosa.
Cumulative amount of biochanin A was measured from the acceptor compartment of the diffusion cell.
Figure 5.
Optical microscope images of the film formulations.
Top view: blank films (A–F), bottom view: Biochanin A containing films (G–L). Formulations: Faa (A,G), Fbb (B,H), Fdd (C, I), Fee (D, J), Fii (E,K) and Fjj (F,L) at magnification 40x.
Table 3.
Properties of sublingual films of biochanin A. Data are presented as mean ± standard deviation (an = 12 and bn = 3–4).
Figure 6.
Dissolution profiles of biochanin A from buccal films of different composition, physical mixture (PM, corresponding to formulation Faa) and powder at pH 6.8. Data are presented as mean ± standard deviation (n = 3).
Figure 7.
X-ray diffraction and DSC measurements of film formulations.
A) X-ray diffraction patterns of (a) biochanin A powder, (b) genistein powder, (c) HPMC powder, (d) film Fii, (e) blank film Faa without biochanin A, (f) film Faa, (g) film Fjj and (h) physical mixture of formulation Faa. B) DSC thermograms of (a) biochanin A powder, (b) HPMC powder, (c) film Fii, (d) blank film Faa without biochanin A, (e) film Faa, (f) film Fjj and (g) physical mixture of formulation Faa.