INTRODUCTION:

Regenerative endodontic treatment has emerged as a biological alternative for managing immature permanent teeth with necrotic pulp and/or apical periodontitis¹. The success of this treatment encompasses clinical resolution, healing of apical lesions, continued root development, and neurogenesis as evidenced by responses to pulp sensitivity tests². The concept of tissue regeneration within root canals was modified in the clinical protocol in 2004, emphasizing minimal instrumentation, intensive irrigation, and the use of intracanal medicaments to support regeneration³.

Regenerative endodontics relies on three essential components: stem cells, scaffolds, and growth factors. Transforming growth factor-β1 (TGF-β1) is a key growth factor involved in cell proliferation, differentiation, and recruitment of progenitor cells necessary for tissue regeneration ⁴. The dentin matrix serves as a natural reservoir of growth factors, which can be released through demineralization using irrigation solutions such as NaOCl or EDTA⁴. However, high concentrations of NaOCl reduce stem cell viability, making lower concentrations preferable⁵.

Although EDTA 17% effectively releases various growth factors, including TGF-β1, its side effects on stem cells, such as early apoptosis, are concerning⁶. In this context, natural materials like Sapindus rarak DC, which contains saponins, flavonoids, and polyphenols, have shown potential as alternative irrigation solutions⁷. Ethanolic extracts of Sapindus rarak DC fruit have demonstrated antibacterial and anti-inflammatory effects and efficient smear layer removal⁸.

This study highlights the importance of developing irrigation solutions derived from natural resources, particularly Sapindus rarak DC⁹. By utilizing Carbon Quantum Dots (CQDs) synthesized from Sapindus rarak DC extract, this research provides an eco-friendly and biocompatible alternative to conventional solutions in regenerative endodontics. The results of this study are expected to contribute significantly to the success of regenerative treatments by optimizing the release of TGF-β1 from the dentin matrix.¹⁰ Furthermore, this innovation opens new opportunities for using nanoparticles derived from natural resources as safe, effective, and cost-efficient irrigation solutions¹¹.

The unique properties of CQDs, including nanocrystal sizes of 2–10 nm, superior optical characteristics, and electronic capabilities, enable enhanced release of TGF-β1 from the dentin matrix. This study evaluates the effectiveness of 4% CQDs from Sapindus rarak DC extract as an alternative irrigation solution, intending to advance regenerative endodontic treatment by integrating nanotechnology and modern dental practices.

Material and Methods:

This laboratory experimental study, conducted from January to May 2024 at Universitas Sumatera Utara, utilized 27 mandibular premolars approved by the Health Research Ethics Committee (352/KEPK/USU/2024). The research involved sample treatment at the Faculty of Dentistry Laboratory, with additional analyses conducted at various university facilities, including the Analytical Chemistry, Physical Chemistry, and Integrated Laboratories, and histological assessments at the Anatomical Pathology Laboratory. Samples were divided into three groups: Group 1 used 1.5% NaOCl + 12.5% Sapindus rarak DC extract, Group 2 used 1.5% NaOCl + 4% CQDs Sapindus rarak DC extract, and Group 3 used 1.5% NaOCl + 17% EDTA, to evaluate the efficacy of each irrigation solution in regenerative endodontic treatments.

Preparation of Ethanolic Extract of Sapindus rarak DC:

The ethanolic extract of Sapindus rarak DC fruit was prepared using the maceration method based on the Indonesian Herbal Pharmacopeia (2017). Fresh fruits (1 kg) collected from Situbondo, East Java, were cleaned, sliced into 3 mm pieces, and homogenized with 1 liter of 70% ethanol. The mixture was stirred for 6 hours, rested for 18 hours, and filtered to obtain the first macerate. The residue underwent a second maceration with 500 ml of 70% ethanol. Both macerates were combined and concentrated using a rotary evaporator at 40°C to produce a viscous extract stored in glass bottles. To prepare a 12.5% Sapindus rarak DC extract, the concentrated extract (100%) was mixed with CMC-Na dissolved in distilled water until uniform. The 4% Carbon Quantum Dots (CQDs) Sapindus rarak DC extract was prepared via a hydrothermal method. Two grams of concentrated extract were dissolved in 25 ml of distilled water, placed in a vacuum autoclave, and heated at 180°C for 8 hours. After cooling to room temperature over 24 hours, the solution was filtered using Whatman No. 3 paper, centrifuged at 1500 rpm, and passed through a 0.22 µm syringe filter to ensure purity. The final filtrate was stored for subsequent characterization and analysis⁸.

Carbon Quantum Dots Preparation:

Carbon Quantum Dots (CQDs) from Sapindus rarak DC extract were prepared using a simple yet effective hydrothermal method. Two grams of concentrated extract (100%) were dissolved in 25 ml of distilled water and placed in a vacuum autoclave. The autoclave was then heated in an oven at 180°C for 8 hours to initiate the synthesis process. After heating, the autoclave was allowed to cool to room temperature over 24 hours. The synthesized product was filtered using Whatman No. 3 filter paper to remove large particles. The resulting solution was centrifuged at 1500 rpm for 10 minutes to separate finer residues. The clear filtrate was further filtered using a syringe filter with a pore size of 0.22 microns to ensure the purity of the CQDs solution. The final product was stored in sterile containers for further characterization and analysis¹².

Characterization of CQDs Sapindus rarak DC:

The characterization of CQDs Sapindus rarak DC involved measuring density, viscosity, and flow rate using a pycnometer, Oswald viscometer, and flow meter. Fluorescence properties were assessed by irradiating 4% CQDs with UV light at 365 nm, while UV-Vis spectrophotometry (200–800 nm) and photoluminescence analysis were used to evaluate absorbance and fluorescence. High-Resolution Transmission Electron Microscopy (HR-TEM) was utilized to determine particle size and distribution, with samples diluted in distilled water and analyzed using Image J and Origin (Pro8 software). These analyses confirmed the physical, optical, and structural properties of the CQDs¹³.

Sample Preparation and Treatment:

Twenty-seven single-rooted premolars extracted for orthodontic purposes were selected as the study samples. Residual soft tissues were removed with a periodontal curette, and the teeth were cleaned with 70% ethanol. The root canals were prepared using the crown-down technique to #25/.08. The canals were irrigated using three solutions: 1.5% NaOCl + 17% EDTA, 1.5% NaOCl + 4% CQDs Sapindus rarak DC extract, and 1.5% NaOCl + 12.5% Sapindus rarak DC extract, applied with a three cc syringe fitted with a 30G side-vented irrigation needle¹⁴.

ELISA Assay:

TGF-β1 levels released from dentin were quantified using an enzyme-linked immunosorbent assay (ELISA) kit. The sandwich ELISA technique was employed following the manufacturer's protocol. Samples were incubated with detection antibodies, and absorbance readings were obtained using an ELISA reader at 450 nm. The TGF-β1 concentration was calculated by plotting a linear regression curve from the optical density values¹⁵.

Data Analysis:

Data collected from the ELISA results were analyzed using SPSS software. Normality and homogeneity tests were conducted, revealing normal distribution (p>0.05) but non-homogeneous data (p<0.05). Consequently, statistical analysis was performed using the Kruskal-Wallis test and the Mann-Whitney test for pairwise comparisons.

RESULTS:

Using a laboratory experimental design, the study evaluated the effectiveness of different irrigation solutions in releasing TGF-β1, a key growth factor in regenerative endodontic treatment. The effectiveness of the solutions was assessed through various tests. The concentration of TGF-β1 released from dentin was quantified using an enzyme-linked immunosorbent assay (ELISA). The CQDs Sapindus rarak DC extract was also characterized for its density, viscosity, flow-rate, fluorescence properties, UV-Vis absorbance, and photoluminescence. Particle size distribution was analyzed using high-resolution transmission electron microscopy (HR-TEM). These analyses aimed to compare the performance of the irrigation solutions and their potential to support the success of regenerative endodontic treatment by optimizing TGF-β1 release.

Table 1 presents the physical properties of Sapindus rarak DC Carbon Quantum Dots (CQDs) solutions at concentrations of 2%, 4%, and 6% were analyzed and compared to distilled water (aquadest) as a reference. The results showed a slight increase in density with rising CQDs concentration, from 0.9862 g/mL at 2% to 0.9893 g/mL at 4%, followed by a slight decrease to 0.9866 g/mL at 6%. Viscosity increased consistently with concentration, from 0.0089 P at 2% to 0.0099 P at 6%. This trend was accompanied by longer flow times, from 3.47 seconds at 2% to 3.91 seconds at 6%, and a decrease in flow rate from 2.88 mL/s to 2.55 mL/s as the concentration increased.

These results indicate that higher CQDs concentrations result in thicker and less easily flowing solutions, which may influence their behavior as irrigation solutions in regenerative endodontic treatment. The moderate viscosity observed in the 4% CQDs solution provides an optimal balance, enabling adequate penetration and distribution within the root canal system without significantly impeding flow. This balance is critical for enhancing the release of TGF-β1 from dentin and supporting tissue regeneration. The findings suggest that the 4% CQDs Sapindus rarak DC solution offers favorable physical properties for effective irrigation, aligning with its potential role in regenerative endodontic applications.

Table 1. Flow Rate, Density, and Viscosity evalutaions of CQDs Sapindus rarak DC

CQDs Sapindus rarak DC	Density (g/mL)	Viscosity (P)	Flow Time (s)	Flow Rate (mL/s)
2%	0.9862	0.0089	3.47	2.88
4%	0.9893	0.0093	3.66	2.73
6%	0.9866	0.0099	3.91	2.55
Distilled Water	1	0.0089	3.47	2.88

Figure 1 illustrates the optical properties of CQDs Sapindus rarak DC through UV-Vis absorbance, photoluminescence (PL), and fluorescence under UV light. The UV-Vis spectrum (a) shows distinct absorbance peaks around 260 nm and 350 nm, indicating aromatic π-π* transitions and n-π* transitions from surface functional groups, characteristic of carbon quantum dots. The PL spectrum (b) reveals strong emission in the blue region (450–500 nm) across excitation wavelengths ranging from 220 nm to 400 nm, confirming the photoluminescent nature of the CQDs due to quantum confinement effects and surface state emissions. In image (c), the yellow arrow highlights the precursor solution (Sapindus rarak DC extract), which lacks fluorescence. In contrast, the red arrow points to the synthesized CQDs, exhibiting bright blue fluorescence under UV light (365 nm). This visual evidence confirms the successful synthesis of high-quality CQDs.

These findings demonstrate the excellent optical properties of CQDs Sapindus rarak DC, which are crucial for their application in regenerative endodontic treatments. The UV-Vis and PL results indicate strong surface passivation and structural integrity, while the blue fluorescence under UV light highlights the CQDs' functional capabilities. These properties suggest that the CQDs can interact effectively with dentin, releasing growth factors like TGF-β1 and enhancing the outcomes of regenerative endodontic treatments.

Figure 1. Profile of CQDs Sapindus rarak DC. (a) Ultraviolet-visible (UV-Vis) spectrum, (b) Photoluminescence (PL) spectrum, (c) Quality of CQDs: The yellow arrow indicates extract solution, and the red arrow indicates CQDs.

Figure 2 illustrates the High-Resolution Transmission Electron Microscopy (HR-TEM) analysis of Sapindus rarak DC Carbon Quantum Dots (CQDs). At 200,000x magnification (a), the CQDs are shown to be well-dispersed with a spherical morphology. At 300,000x magnification (b), the particles appear uniform in size with smooth surfaces. Using ImageJ software for particle size analysis (c), the CQDs were measured to have a size range of approximately 23.43 nm to 63.78 nm, with a mean particle size of ~40 nm. This consistent nanoscale size and shape indicate high-quality synthesis.

The nanoscale size of the CQDs (~40 nm) plays a crucial role in their functionality as an irrigation solution in regenerative endodontic treatment. Their small size and uniformity enhance the surface area-to-volume ratio, enabling efficient interaction with the dentin matrix and optimal release of growth factors like TGF-β1. Moreover, the spherical shape ensures consistent photoluminescence properties, as observed in previous optical characterizations. These characteristics allow the CQDs to penetrate dentinal tubules effectively, facilitating bioactivity and promoting tissue regeneration. Thus, the particle size and morphology of CQDs Sapindus rarak DC strongly support their potential as an innovative and functional agent for regenerative endodontic applications.

Figure 2. HR-TEM of CQDs Sapindus rarak DC. (a) Magnification at 200,000x, (b) Magnification at 300,000x, (c) Particle size distribution analyzed using Image J software

Table 2 presents the pH values of four irrigation solutions, including 12.5% Sapindus rarak DC extract, 4% CQDs Sapindus rarak DC, 1.5% NaOCl, and 17% EDTA, measured across five replications. The mean pH values indicate that the 12.5% Sapindus rarak DC extract has a mildly acidic pH of 4.17, while the 4% CQDs Sapindus rarak DC exhibits a more pronounced acidic pH of 3.38. In contrast, 1.5% NaOCl demonstrates a strongly alkaline pH of 12.15, and 17% EDTA has a mildly alkaline pH of 8.13.

The pH of these solutions plays a crucial role in their effectiveness during regenerative endodontic treatments. The acidic nature of the 12.5% Sapindus rarak DC extract and the 4% CQDs Sapindus rarak DC suggests their ability to demineralize dentin and release growth factors, such as TGF-β1, from the dentin matrix. Notably, the more pronounced acidity of 4% CQDs Sapindus rarak DC indicates its potential for enhanced TGF-β1 release, essential for promoting tissue regeneration. On the other hand, 1.5% NaOCl, with its strongly alkaline pH, primarily functions as a disinfectant and is less effective in releasing growth factors. Meanwhile, 17% EDTA, with a mildly alkaline pH, is recognized for its ability to chelate dentin and release TGF-β1, but it may have limitations compared to the acidic CQDs solution. These findings highlight the potential of 4% CQDs Sapindus rarak DC as a promising alternative irrigation solution. Its acidic pH supports effective demineralization and enhances the release of bioactive molecules, such as TGF-β1, making it a valuable addition to regenerative endodontic applications.

Table 2. pH Values of Irrigation Solutions

Replication	*12.5% Sapindus rarak DC* Extract**	4% CQDs Sapindus rarak DC	1.5% NaOCl	17% EDTA
1	4.19	3.38	12.14	8.13
2	4.17	3.38	12.15	8.12
3	4.17	3.38	12.16	8.11
4	4.16	3.38	12.17	8.10
5	4.15	3.38	12.14	8.19
Mean	4.17	3.38	12.15	8.13

Figure 3 illustrates the release of TGF-β1 from the dentin matrix after root canal treatment using three different irrigation solutions: 4% CQDs Sapindus rarak DC + 1.5% NaOCl, 12.5% Sapindus rarak DC extract + 1.5% NaOCl, and 17% EDTA + 1.5% NaOCl. The highest TGF-β1 release was observed in the group treated with 12.5% Sapindus rarak DC extract + 1.5% NaOCl (0.421 ng/mL), followed by 17% EDTA + 1.5% NaOCl (0.364 ng/mL) and 4% CQDs Sapindus rarak DC + 1.5% NaOCl (0.349 ng/mL). The TGF-β1 release is critical in promoting tissue regeneration by recruiting and stimulating progenitor cells during root canal treatment. While the 12.5% Sapindus rarak DC extract exhibited the highest release, the 4% CQDs Sapindus rarak DC solution demonstrated comparable efficacy with potential advantages. Its moderate release ensures an optimal balance for effective regeneration without the risk of excessive inflammatory responses. Additionally, the nanoparticle nature of CQDs enhances penetration into dentinal tubules and improves growth factor release efficiency compared to traditional solutions like EDTA. These findings highlight that moderate TGF-β1 release, as achieved by 4% CQDs Sapindus rarak DC, is the most suitable for regenerative endodontics, providing an ideal balance for clinical outcomes. Thus, moderate release becomes the primary target in achieving optimal results in regenerative endodontic treatments.

Figure 3. Release of TGF-β1 from the dentin matrix after root canal treatment with different irrigation solutions.

The Kruskal-Wallis test was conducted to evaluate differences in TGF-β1 release among the three treatment groups: NaOCl 1.5% + 4% CQDs Sapindus rarak DC, NaOCl 1.5% + 12.5% Sapindus rarak DC extract, and NaOCl 1.5% + 17% EDTA. The test revealed a Kruskal-Wallis value of 10.884 with a significance level of 0.004, indicating significant differences in TGF-β1 release among the groups. Pairwise comparisons using the Mann-Whitney test showed that the TGF-β1 release in the NaOCl 1.5% + 12.5% Sapindus rarak DC extract group was significantly higher compared to the 4% CQDs Sapindus rarak DC solution (p=0.004) and 17% EDTA (p=0.009). Meanwhile, the comparison between NaOCl 1.5% + 4% CQDs Sapindus rarak DC and NaOCl 1.5% + 17% EDTA showed no statistically significant difference (p=0.268).

These results suggest that the NaOCl 1.5% + 12.5% Sapindus rarak DC extract provides the highest TGF-β1 release, likely due to its strong demineralizing ability, making it the most effective solution in this study for promoting tissue regeneration. On the other hand, the 4% CQDs Sapindus rarak DC solution exhibited comparable TGF-β1 release to 17% EDTA, indicating its potential as an alternative irrigation solution with the added benefits of nanoparticle properties, such as enhanced penetration and improved biocompatibility. Overall, the findings highlight the effectiveness of Sapindus rarak DC-based irrigation solutions in supporting regenerative endodontic treatments, with the 12.5% extract being the most optimal and 4% CQDs Sapindus rarak DC offering a promising innovative approach.

DISCUSSION:

This study evaluates the effectiveness of various irrigation solutions—NaOCl 1.5% combined with 12.5% Sapindus rarak DC extract, 4% CQDs Sapindus rarak DC, and 17% EDTA—in releasing TGF-β1 from the dentin matrix, a key factor in tissue regeneration during endodontic treatment. The findings demonstrate significant differences in TGF-β1 release, highlighting the superior efficacy of 12.5% Sapindus rarak DC extract and the promising potential of 4% CQDs as an innovative, biocompatible alternative to EDTA. The discussion explores how the properties of these solutions, including pH and nanoparticle characteristics, influence TGF-β1 release, emphasizing their role in optimizing bioactivity and clinical outcomes in regenerative endodontic therapy.

Carbon Quantum Dots (CQDs) have emerged as a promising tool in regenerative endodontics due to their ability to regulate and optimize the release of growth factors like TGF-β1 from biological matrices ¹⁶. As nanoscale particles, CQDs possess unique properties such as a high surface area, biocompatibility, and functional surface groups¹⁷, which enable effective interaction with dentin and controlled modulation of bioactive molecule release. Their nanoscale size (3–10 nm) allows for efficient penetration into dentinal tubules, enabling deeper interaction with the dentin matrix. Functional groups like hydroxyl (-OH) and carboxyl (-COOH) on the surface of CQDs can bind to growth factors, facilitating gradual and controlled release¹⁸. This reduces the risk of excessive demineralization or cellular stress, ensuring a balanced biological response¹⁹.

The slightly acidic pH of CQDs (Sapindus rarak DC) at 3.38 supports their role in effectively demineralizing dentin and releasing TGF-β1 without causing structural damage²⁰. This aligns with studies demonstrating that acidic solutions enhance TGF-β1 release from dentin matrices²¹. Unlike strongly acidic agents, CQDs maintain moderate acidity, reducing the risk of over-demineralization while achieving efficient growth factor activation. Furthermore, their biocompatibility makes CQDs a safer alternative to traditional solutions like 6% NaOCl²², significantly reducing SCAP viability, weakening dentin, and leading to persistent microbial growth²³. Though effective in chelating dentin, EDTA has also been shown to impair SCAP proliferation and induce early apoptosis²⁴. In addition to their role in modulating TGF-β1, CQDs enhance bioavailability through chelation. The polyphenols and flavonoids in Sapindus rarak DC CQDs contribute to TGF-β1 mobilization, as observed in studies on natural extract-based irrigation solutions²⁵. This combination of chelation and nanoscale penetration ensures consistent release and distribution of TGF-β1, crucial for tissue regeneration²⁶.

UV light and spectrophotometric analyses characterized the 4% CQDs Sapindus rarak DC. Under UV light, the solution exhibited cyan fluorescence, indicating carbon ring structures²⁷. UV-Vis analysis revealed major absorption peaks at 223 nm and 285 nm, attributed to π-π* transitions of aromatic C=C bonds and n-π* transitions of surface functional groups like C=O. Photoluminescence testing showed the highest intensity at a 360 nm excitation wavelength, reflecting efficient energy absorption and emission²⁸. HR-TEM analysis confirmed the nanoparticle size, with an average diameter of 7 nm, placing the solution within the CQD category²⁹. Additionally, physical properties showed a density of 0.9893 g/mL, viscosity of 0.0093 P, and flow rate of 2.73 mL/s. While the flow rate exceeded the ISO standard (~0.25 mL/s), the low viscosity indicates improved penetration into dentinal tubules³⁰.

Statistical analysis revealed significant differences in TGF-β1 release among the tested irrigation solutions (p=0.004). The Kruskal-Wallis test showed that 12.5% Sapindus rarak DC extract released the highest amount of TGF-β1, followed by 17% EDTA and 4% CQDs Sapindus rarak DC. Pairwise comparisons using the Mann-Whitney test indicated no significant difference between 4% CQDs and EDTA (p=0.268), suggesting that CQDs can achieve comparable TGF-β1 release while offering additional nanoparticle benefits, such as better dentinal tubule penetration. The reduced TGF-β1 release in 4% CQDs compared to 12.5% extract may be attributed to lower saponin content. Saponins, classified as terpenoids, are known to stimulate TGF-β1 synthesis, secretion, and activation. The acidic pH of 4% CQDs and 12.5% extract further supports dentin demineralization, aligning with findings that weak organic acids like citric acid enhance TGF-β1 release³¹.

CONCLUSIONS:

CQDs of Sapindus rarak DC provide a balanced approach to releasing TGF-β1, ensuring controlled activation of regenerative pathways. Their biocompatibility, nanoscale size, and chelating properties make them a promising alternative to conventional irrigation solutions. While the 12.5% Sapindus rarak DC extract demonstrated the highest TGF-β1 release, 4% CQDs offer innovative advantages with comparable efficacy, making them suitable for regenerative endodontic applications.

ACKNOWLEDGMENTS:

The authors gratefully acknowledge the Oral Biology Laboratory, Faculty of Dentistry, Universitas Syiah Kuala, for the preparation of Carbon Quantum Dots from Sapindus rarak DC. This work was supported by the TALENTA Research Grant, Universitas Sumatera Utara, under contract number 137/UN5.4.10.S/PPM/KP-TALENTA/RBI/2024.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 17.01.2025 Revised on 10.05.2025

Accepted on 16.07.2025 Published on 03.04.2026

Available online from April 06, 2026

Research J. Pharmacy and Technology. 2026;19(4):1660-1666.

DOI: 10.52711/0974-360X.2026.00238

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.