**6. Antioxidant Activity (DPPH Radical Scavenging Activity)**

In vitro testing of the two dispersion dyes' antioxidant properties used their capacity to scavenge DPPH free radicals. The IC50 value of the dyes—the dose needed to suppress the production of DPPH radicals by 50%, was used to describe their antioxidant activity. Data from Table 4 show that disperse dye **22h** had a moderate antioxidant activity and outperformed ascorbic acid as the standard, which had an IC50 of 14.2, while disperse dye **22i** had a poor antioxidant activity with an IC50 of 191.6. (Figure 10).

**Figure 10.** Antioxidant activities of disperse dyes 22h and 22i.

#### **7. Antimicrobial Activities of Dyes 3, 5, 7, 9a–d, 22a–i and 26a,b**

The agar well diffusion method was used to investigate the antibacterial effects of the produced dyes **3**, **5**, **7** and **9a–d** against various bacteria and yeast. The information in Table 5 reveals encouragingly effective antibacterial activity. While the other disperse dyes exhibited moderate to poor antibacterial properties, disperse dyes **3** and **5** had strong antibacterial activity against Gram-positive bacteria.


**Table 5.** Inhibition zones of dyes 3, 5, 7, 9a-d, 22a–i and 26a,b.

(NI): No inhibition, (NC): Not checked.

All of the substances tested inhibited the development of *Candidia albicans* after six days of incubation. Additionally, the agar well diffusion method was used to test the dispersion dyes **22a–i** and **26a,b** for their antibacterial properties against a range of bacteria and yeast.

In addition, Figure 11 shows that *Candida albicans* re-grew in the formed zone surrounding the wells containing compound **3**. This may reflect the cytostatic effect of the

chemicals rather than their cytolytic effects. Note that the plate color changed with the increase in the incubation time indicates a complete diffusion of the chemical used in the agar as the incubation period increased.

**Figure 11.** *Candida albicans* treated with 10mg ml−<sup>1</sup> of compound **3** after one day (**A**), three days (**B**) and six days (**C**) of incubation. It is worth noting that Figure 10 shows the cytolyticeffects of dyes number 5, 7 and 9a on *Candida albican* where after one, three and six days of incubation, the inhibition zone did not change (Figure 12).

While, the cytostatic effect of the same dyes is clear on the plates inoculated with *Bacillus subtilus.* The growth of *B. subtilus* resumed in the inhibited area after six days of incubation, which may indicate that as the concentration/toxicity of dyes number **5**, **7** and **9a** reduce due to possible evaporation of these dyes or diffusion in the media, the effect of these dyes on *B. subtilus* decrease, and the organisms start growing again (Figure 13).

**Figure 12.** *Bacillus subtilus* treated with 10mg ml−<sup>1</sup> of dyes 5, 7, and 11a after one day (**A**) (1, 2, 3), three days(**B**) (4, 5, 6) and six days (**C**) (7, 8, 9) of incubation.

Based on data for the inhibition zone diameter for the dispersion dyes **22a–g**, Table 5 shows that all of the tested dyes demonstrated strong positive antibacterial activities against the studied pathogens. For all of the examined bacteria, disperse dye **22a** had a cytolytic effect, with no growth being seen in the inhibition zone.

**Figure 13.** *Candida albican* treated with 10mg ml−<sup>1</sup> of dyes 5, 7, and 9a after one day (**A**) (1, 2, 3), three days(**B**) (4, 5, 6) and six days (**C**) (7, 8, 9) of incubation.
