## 4 Discussion  
Algae have been used within food and medicine for many centuries. Spirulina--as a blue-green alga--has antioxidant, anticancer, immunomodulation and antimicrobial effects. These effects can be attributed to some components such as phycocyanin [19]. Phycocyanin is a natural blue-colored protein complex that is water-soluble and has a peak absorption between 580-660 nm. This material showed some potential biologic activities that can be considered as a PS when irradiated using the appropriate wavelength [11]. The level of singlet oxygen production is not increased in the non-irradiated phycocyanin group, which confirms that the generation of singlet oxygen is dependent on laser irradiation [10]. Chlorophyll derivates composed of chlorophyllin metal complexes enjoy advantages such as good water-solubility and high photosensitivity and excitation with wavelength in the region of visible red (650-670 nm). This mixture can produce ROS--especially 1O2 in a higher amount and OH\({}^{\circ}\) in less amount--when irradiated with lasers of visible red range of wavelengths [20]. The potential of ROS production of this agent from the higher level to lower one is in this order: Fe-CHL > Mg-CHL > Cu-CHL. Hence, Fe-CHL shows the strongest photodynamic action [21]. In this study, we used Photoactive+ as a PS which includes phycocyanin and chlorophyll. Since chlorophyll is not water soluble, it is poorly absorbed by the body. The chlorophyll part in Photoactive+ has changed to possess the ability to dissolve in water in forms of sodium-magnesium-chlorophyllin (200 mg) and sodium-copper-chlorophyllin (100 mg). In addition, this mixture has 200 mg phycocyanin. Phycocyanin is water soluble and can be absorbed by the body. For highest bioavailability, in Photoactive+, the phycocyanin is embedded in a liposome to improve its absorbance in body. Lipsosomal structure of phycocyanin can incorporate hydrophilic, hydrophobic and amphiphilic substances to achieve improved infiltration of the PS into microorganisms. We prepared the final concentration of 0.01 g/mL for our experiment. The chlorophyllin can be activated by either red or blue wavelengths or a combination of both. The results of this study  
Figure 3: Cell cytotoxicity assessment. (**a**) MTT assay control group (1.4), Photoactive+ (1.19); Photomicrographs of HuGu cells under light inverted microscopy: (**b**) control group; (**c**) cells exposed to Photoactive+ at concentration of 0.01 g/mL. showed that aPDT with Photoactive+ and 635 nm diode laser showed the greatest reduction in the bacterial count, but when activated by LED (420-480 nm) did not significantly reduce the bacterial count. In addition, our results showed that R1-Blue was better than the Blue-R1 treatment. This can be related to the phenomenon that this complex was more activated by red wavelength rather than blue one. It seemed that that the chlorophyllin part of this mixture had no contribution as an antimicrobial photosensitizer when activated by LED. If a blue laser with a more accurate wavelength were used, it may have had an effect on bacterial reduction. In agreement with our results, [PERSON] et al. in assessing the efficacy of chlorophyllin-phycocyanin mixture activated by 635 nm diode laser against Streptococcus _mutans_ cultured on enamel slabs concluded that this mixture can reduce the number of living bacteria within the biofilms of _S. mutans_[22]. [PERSON] et al. evaluated the effect of chlorophyllin-Sodium-Copper activated by blue light (400 nm) with power density of 20 mW/cm2 on _Bacillus cereus_ (Gram-positive bacteria) which led to a significant reduction in bacteria [23]. In addition, [PERSON] et al. used chlorophyllin-Sodium-Copper using a homemade LED device at wavelength of 625 nm and power density of 8.3 mW/cm2 during 1 h for inactivation of _Staphylococcus aureus_ and _Escherichia coli_. The results of their studies showed efficacy for inactivation of Gram-positive bacteria. The results of our study were in line with above-mentioned studies, as it focused on _E.