Comparison of the antimicrobial reduction effect of photodynamic inactivation with the addition of chlorophyll and curcumin photosensitizer in Aggregatibacter actinomycetemcomitans and Enterococcus faecalis

Introduction

The oral cavity is one of the most important parts of the body that must be maintained. Infectious diseases of the teeth and mouth that are often found are periodontitis and endodontics. Periodontitis is a bacterial infection of the teeth that causes inflammation of the supporting tissues of the teeth, which include the gingiva, ligaments, cement, and alveolar bone.¹ Periodontitis is caused by pathogenic bacteria, predominantly gram-negative, anaerobic, or microaerophilic in the subgingival area.² Aggregatibacter actinomycetemcomitans bacteria are found in dental plaque, periodontal pockets, and buccal mucosa in up to 36% of the normal population.³ Aggregatibacter actinomycetemcomitans bacteria can infect patients when the human immune system decreases and inhibits other organisms’ growth in the oral mucosa, teeth, and nasopharynx.

In general, gram-positive bacteria, Enterococcus faecalis (E. faecalis), are found in the root canals of teeth. The bacterium Enterococcus faecalis is ovoid, with a diameter between 0.5 and 1 μm.⁴ These bacteria are facultative anaerobes and can survive in extreme environments such as highly alkaline pH and high salt concentration conditions. The number of these bacteria in the human body can be minimized by paying attention to the food consumed and environmental conditions such as humidity. Furthermore, E. faecalis bacteria resist calcium hydroxide and antibiotics such as tetracycline.⁵ Systemic treatment in the form of antibiotics has been widely used to treat periodontitis. However, several studies have reported cases of antimicrobial resistance to certain types of antibiotics.⁶ So alternative therapy is needed that is effective and does not cause antibiotic resistance.⁷ Therefore, the recommended alternative therapy in this study is photodynamic inactivation (PDI).

Photodynamic inactivation (PDI) is a method of inactivating microorganisms by utilizing light to activate a photosensitizer (PS) agent that produces reactive oxygen species (ROS), causing cell lysis.^8,9 The suitability of the light spectrum with the PS absorption spectrum is the key to photophysical reactions, namely the absorption of light energy by PS agents, which will trigger photochemical and photobiological reactions to produce antimicrobial effects^10,11 and biomodulation.¹² PS is a light-sensitive molecule that plays a role in absorbing light energy.¹³ PS is divided into two types, namely endogenous and exogenous photosensitizers. The addition of exogenous PS aims to increase the effectiveness of light energy absorption.¹⁴ Some natural ingredients that are exogenous PS include chlorophyll and curcumin. Chlorophyll is a green substance found in green plants that photosynthesizes.¹⁵ In photosynthesis, chlorophyll acts as a light catcher, energy transfer, and light conversion and can absorb a maximum wavelength range of 400-700 nm.¹⁶ Chlorophyll acts as a photosensitizer because it is naturally able to absorb light.¹⁷ The chemical structure of chlorophyll consists of a porphyrin ring that serves as the core framework and long hydrophobic side chains. The bioactivity of chlorophyll is due to its ability to act as an antioxidant, antimutagen, and anticarcinogen. Its unique chemical structure allows chlorophyll to capture harmful free radicals, reduce DNA damage, and modulate cellular processes involved in disease progression. In addition, its hydrophobic side chains facilitate interactions with biological membranes, affecting cellular uptake and signaling pathways.¹⁸

Curcumin is a curcuminoid compound with yellow pigment in turmeric rhizome, which is antitumor, antioxidant, anticarcinogenic, anti-inflammatory, antiviral, antifungal, antispasmodic, and hepatoprotective.¹⁹ The absorption spectrum of curcumin is in the wavelength range of 375-475 nm.²⁰ The advantage of curcumin as PS is that there are reactive functional groups, including ketone and phenol groups, that play a role in phototherapeutic activity. Phenol groups play an important role in the interaction of curcumin and the biological membranes of bacteria because the attachment of hydrogen groups plays an important role.²¹

Previous studies have reported the effectiveness of using PS chlorophyll in alfalfa leaves with a blue LED activator of 20.48 J/cm² for the inactivation of A. actinomycetemcomitans bacteria by 81%.²² The results of another study with the addition of PS curcumin and diode laser activator 403 nm 15.83 J/cm² in Staphylococcus aureus resulted in a mortality rate of 85.48%.²¹ Then, another study using curcumin and blue LEDs on S. aureus bacteria resulted in a mortality rate of 91.49%.²³ Continuing previous studies,^19,20,22 this study aims to compare the effectiveness of antimicrobial reduction from photodynamic inactivation (PDI) on bacteria A. actinomycetemcomitans and E. faecalis with PS curcumin and chlorophyll Medicago sativa L. using a 405 nm diode laser. Diode laser irradiation was carried out at various lengths of irradiation time, namely 30, 60, 90, 120, 150, and 180 seconds. In addition, this study aims to determine the effective laser irradiation time to reduce the bacteria A. actinomycetemcomitans and E. faecalis with PS curcumin and chlorophyll Medicago sativa L.

Methods

Results

The chlorophyll and curcumin extracts were tested using UV-Vis at a wavelength range of 325 nm to 705 nm to determine the absorption spectrum of light. Then, the results of characterizing the absorption spectrum of chlorophyll and curcumin to light are obtained, as shown in Figure 1. Based on Table 1, the characterization results show the peak wavelength of the diode laser at 405 nm with the stability of the output power at a distance of 1 cm (2.49 ±0.07) mW. The temperature characterization showed the optimum temperature stability (26.60±0.01) °C for bacterial growth. Thus, the irradiation energy density of the diode laser is 405 nm with an output power of 2.49 mW and a beam area of 0.28 at various exposure times (30, 60, 90, 120, 150, 180) seconds are 0.26, 0.53, 0.79, 1.06, 1.32, and 1.59 J/cm².

Figure 1. Graph of curcumin and chlorophyll absorption spectrum.

After that, antibacterial tests were carried out on Aggregatibacter actinomycetemcomitans and Enterococcus faecalis bacteria, which were exposed to a diode laser with and without a photosensitizer. So, the viability of the bacteria Aggregatibacter actinomycetemcomitans and Enterococcus faecalis is shown in Figures 2 and 3. Based on bacterial viability, the percentages of death of A. actinomycetemcomitans and E. faecalis bacteria by diode laser irradiation with the addition of curcumin photosensitizer and Medicago sativa L chlorophyll treatment are shown in Tables 2 and 3.

Figure 2. Graph of bacterial viability of A. actinomycetemcomitans in various treatments with and without the addition of curcumin and chlorophyll Medicago sativa L.

Figure 3. Graph of bacterial viability of E. faecalis in various treatments with and without the addition of curcumin and chlorophyll Medicago sativa L.

Based on the results of statistical tests, it was shown that diode laser irradiation with an energy density of 1.59 J/cm² gave a percentage of E. faecalis bacteria death of 36.7% without adding a photosensitizer. Then, the percentage of death of E. faecalis bacteria was 69.30% with the addition of PS chlorophyll Medicago sativa L. and 89.42% with the addition of PS curcumin. Meanwhile, the results of statistical tests on bacteria A. actinomycetemcomitans with diode laser irradiation at an energy density of 1.59 J/cm² gave the percentage of bacterial death of 35.81% without the addition of PS. Then, the death of A. actinomycetemcomitans was 64.39% with the addition of PS chlorophyll and 89.82% with the addition of PS curcumin.

Discussion

This research was conducted using the PDI technique using a blue diode laser as a light source, chlorophyll Medicago sativa L and curcumin as PS to reduce bacteria E. faecalis and A. actinomycetemcomitans. The wavelength of light is an important factor in the photoinactivation process. The diode laser used in this study has a wavelength of 405 nm and an output power of 2.49 mW. The results of the characterization of power against time and temperature show the stability of power and temperature so that the temperature factor does not cause the death of bacteria.

PS is a light-sensitive molecule. Exogenous PS is PS that is added to assist the photoinactivation process. This study used exogenous PS chlorophyll Medicago sativa L and curcumin. Medicago sativa L chlorophyll absorbance used for a laser wavelength of 405 nm was 85.1% and for curcumin was 80.64%.²³ The photoinactivation process occurs due to a photophysical mechanism initiated by the absorption of light by PS. The energy of the absorbed photon will cause the excitation of the electron to increase to a higher energy level. If the energy excitation state overlaps with the triplet excitation state, an intersystem crossing occurs, a spin reversal that places the electron in a triplet excited state and triggers a photochemical reaction.

The mechanism of photodynamic inactivation (PDI) is illustrated in Figure 4. During photodynamic therapy, chlorophyll and curcumin act as photosensitizers. A photosensitizer is a solution that is able to absorb laser light optimally through a photophysical process. The first process that occurs is that the photosensitizer absorbs photon energy from a 405 nm laser diode, causing excitation from a low state (¹PS) to an excited state (¹PS*). In the ¹PS* state, the photosensitizer can return to the ¹PS state by emitting fluorescence or internal conversion. In addition, the photosensitizer can also convert to a more stable T nearest excitation level before finally returning to the ¹PS state by emitting energy in the form of phosphorescence. This energy conversion is what requires oxygen through the photochemical process. Photochemical reactions are divided into two types. The first type is the transfer of electrons to a biological substrate in the form of a redox reaction and produces singlet oxygen. The second type is the transfer of energy to the triplet electrons to produce singlet oxygen. Singlet oxygen is radical. Reactive oxygen species (ROS) can damage the bacterial cell membrane and cause lysis. This is the earliest stage in bacterial cell death. Photochemical activity generates ROS and triplet oxygen radicals that oxidize unsaturated fatty acids, producing hydroperoxides. These radicals interfere with the synthesis of saturated fatty acids, causing harmful hydroperoxides known as lipid peroxidation. This disruption of the bacterial cell wall leads to cell lysis.²³ The photochemical reactions in PDI are generally of the second type.

Figure 4. Mechanism of bacterial inactivation.

The study’s results on E. faecalis bacteria showed a significant difference between treatments. Diode laser irradiation with an energy density of 1.59 J/cm² gave the percentage of bacterial death of E. faecalis 36.7% without the addition of PS, 69.30% with the addition of PS chlorophyll Medicago sativa L and 89.42% with the addition of PS curcumin. Meanwhile, in A. actinomycetemcomitans bacteria with energy density diode laser irradiation 1.59 J/cm², the percentage of bacterial death was 35.81% with the addition of PS, 64.39% with the addition of PS chlorophyll Medicago sativa L and 89.82% with the addition of PS curcumin.

The results showed that adding PS curcumin increased the effectiveness of reducing E. faecalis and A. actinomycetemcomitans bacteria. PDI with PS curcumin was effectively used to reduce bacteria because its absorption followed endogenous porphyrins.²⁴ The addition of energy density will increase the reduction effect without and with the addition of PS Medicago sativa L¹⁷ and curcumin.¹⁹ In addition, the results showed that the administration of Medicago chlorophyll PS was more effective in reducing E. faecalis bacteria than A. actinomycetemcomitans. This is because the wall structure of E. faecalis bacteria is thinner than A. actinomycetemcomitans.

A. actinomycetemcomitans is a Gram-negative bacterium measuring 0.4-0.5 μm × 1.0-1.5 μm.²⁵ Enterococcus faecalis bacteria are Gram-positive bacteria with a cell wall thickness of about 40 nm.²⁶ The cell wall structure of gram-negative bacteria consists of lipopolysaccharides. lipoproteins, lipopolysaccharides, and peptidoglycans. The cell wall of gram-negative bacteria is more complex, so it is more difficult to penetrate antibacterial compounds. The cell wall structure of gram-positive bacteria is relatively simpler so that antibacterial compounds easily enter the cell.²⁵

In this study, PS curcumin has a higher percentage of bacterial death than PS chlorophyll (Medicago sativa L). This is in accordance with research conducted by Avianti et al. (2020) that PS curcumin in photodynamic therapy has a greater bacterial death effect than PS chlorophyll on E. faecalis. In addition, the research by Avianti et al. (2020) was also conducted at 60 and 90 seconds of irradiation duration. Then, it was proven that the duration of 90 seconds on all PS proved effective in increasing the death of E. faecalis bacteria. This is related to the nature of curcumin and its molecular structure, which enhances its function in photodynamic therapy. It shows that the longer the duration of laser irradiation given, the greater the bacterial death caused.²¹

Research conducted by Balhaddad et al. (2020) showed that irradiation of S. mutans biofilm through 100 μg/mL TBO and an energy dose of ≈180 J/cm² resulted in a higher number of dead S. mutans colonies compared to the control (p < 0.001). Light energy dose and PS concentration optimize bacterial death.²⁷ Later, Pordel et al. (2023) reported that chlorhexidine and curcumin with a blue diode laser at 500 mW output power had the highest reduction in the number of S. mutans colonies (p < 0.001). The curcumin group was more effective than the riboflavin group. The results of the study by Pordel et al. (2023) showed that aPDT using curcumin as a photosensitizer plus a blue diode laser with an output power of 500 mW and a power density of 1.0 W/cm² at a wavelength of 445 nm can effectively reduce the colonies of S. mutans.²⁸ In addition, Ashtiani et al. (2024) also reported the results of their research that toluidine blue O (TBO) and phycocyanin (PC) activated with a 635 nm diode laser with a power density of 1592 W/cm² were more efficient in increasing the death of E. faecalis bacteria compared to a 636 W/cm² diode laser (p = 0.00). Light power density optimizes the reduction of E. faecalis bacteria.²⁹

In vivo studies are found in other journals.^30,31 The effect of bacterial photoinactivation on wounds in vivo due to photodynamic therapy in the red LED exposure group was 88%, the blue LED exposure group was 94%, and the red and blue LED combination exposure group was 95%.³¹ Blue and red lasers combined with ozone treatment effectively accelerated wound healing of incisions infected with MRSA bacteria.³² Thus, LED antimicrobial photodynamic therapy is effective for bacterial inactivation and accelerates wound healing in mice. Biofilm research is also found in other journals.^31,32 The difference in the reduction rate of PDT increased when ozone was added, thus helping biofilm reduction compared to PDT.³¹ The results showed that the Chlo+Ozon+Laser combination treatment at 20 seconds of ozone exposure with 4 minutes of irradiation time caused a decrease in biofilm activity by 80.26%, which was the highest efficacy of all treatment groups. The combination of laser, chlorophyll, and lower ozone concentration increased the effectiveness of photodynamic inactivation.³³

Conclusion

Based on statistical tests, the results of research on E. faecalis bacteria showed that laser irradiation with an energy density of 1.59 J/cm² gave a percentage of E. faecalis bacteria mortality of 36.7% without the addition of PS, 69.30% with the addition of PS chlorophyll Medicago sativa L and 89.42% with the addition of PS curcumin. Meanwhile, A. actinomycetemcomitans showed that the energy density diode laser irradiation of 1.59 J/cm² gave the percentage of bacterial death 35.81% without the addition of PS, 64.39% with the addition of PS chlorophyll Medicago sativa L and 89.82% with the addition of PS curcumin. So it can be concluded that the role of PS is significant for the success of PDI. The addition of PS curcumin increased the effectiveness of reducing bacteria E. faecalis and A. actinomycetemcomitans compared to chlorophyll Medicago sativa L.

Author contributions

DA contributes to the data curation, methodology, validation, original draft preparation of the work and editing of the work. SDA contributes to the conception, methodology, analysis, funding acquisitions, project administration, supervision, validation, review, original draft preparation of the work and editing of the work. SRM, SBL, DZIN, and DAH contribute to the conception, data curation, methodology, investigation, validation, original draft preparation of the work and editing of the work. S contributes to the conception, methodology, investigation, analysis, supervision, validation, review, original draft preparation of the work and editing of the work. YS contributes to the conception, data curation, methodology, investigation, validation, original draft preparation of the work and editing of the work. AS contributes to the conception, methodology, analysis, supervision, validation, review, original draft preparation of the work and editing of the work.