INTRODUCTION:

Periodontitis is an inflammatory disease of the tooth supporting tissue. Based on the Global Burden of Disease analysis in 2019, there were 1.1 billion cases of severe periodontitis in the worldwide and it is one of the 11 most prevalent conditions in the world¹. This is supported by 2018 Riskesdas data that as many as 67.8-79.1% of the population ≥15 years old suffer from periodontitis and it is the second most common dental and oral disease in Indonesia². The high prevalence is caused by the role of Porphyromonas gingivalis which is the main periodontopathic bacteria in periodontitis.

Porphyromonas gingivalis (P. gingivalis) is a 'keystone' bacteria in the subgingival biofilm that causes periodontitis³. It affects the structure of the surrounding tissue, including alveolar bone resorption (bone loss)^4,5. This bacteria causes a host inflammatory response through the lipopolysaccharide (LPS), gingipain, fimbriae, and capsule⁶. These virulence factors from P. gingivalis trigger inflammatory cells such as macrophages to produce pro-inflammatory cytokines that impact the osteoclastogenesis signaling pathway^6,7.

Pro-inflammatory cytokines such as Interleukin-1 (IL-1), Interleukin-6 (IL-6), and Tumor Necrosis Factor-α (TNF-α) will increase the synthesis of receptor-activated of nuclear factor Kappa-B Ligand (RANKL) and suppress the synthesis of osteoprotegerin (OPG) by osteoblasts then bind to its receptor, Receptor Activator Nuclear Factor Kappa-B (RANK)⁸. This binding causes increased activation, proliferation, and differentiation of osteoclasts^5,6. These cytokines can also stimulate the expression of Mitogen-Activated Protein (MAP) kinase to induce osteoblast apoptosis^9,10. Therefore, there is an increase in the number of osteoclasts and a decrease in the number of osteoblasts, which can trigger the process of alveolar bone resorption.

Metronidazole is a broad spectrum antibiotic commonly used in the treatment of periodontitis¹¹. This drug is effective against anaerobic bacteria with the lowest percentage of drug resistance compared to other antibiotics^12,13. This antibiotic works by damaging and destabilizing the bacterial DNA helix. However, long-term use of metronidazole can cause diarrhea, xerostomia, sinusitis and pharyngitis¹⁴. Therefore, it is necessary to use natural resources as an alternative treatment for periodontitis.

In an agro-industrial country like Indonesia, it is necessary to make maximum use of natural resources, especially as therapy and treatment. 90% of cultivated coffee plants in Indonesia are robusta coffee (Coffea canephora)¹⁵. However, robusta coffee husk has not been utilized optimally. On the other hand, the anthocyanin, tannin and caffeine content in robusta coffee husk is known to have antibacterial and anti-inflammatory potential^16,17. Anthocyanins in the total dry weight content 2.171mg/L can inhibit the expression of Nuclear Factor-Kappa Beta (NF-κB) and various pro-inflammatory cytokines¹⁸. Tannins contained 13.143% in robusta coffee husk can inhibit the expression of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α^17,19. On the other hand, the 2.15% caffeine content in robusta coffee husk also has an anti-inflammatory effect which is related to inhibiting NF-κB signaling and the production of anti-inflammatory cytokines such as IL-10 so it can suppress the increase in the ratio of the number of osteoclasts and osteoblasts^20,21.

Research on robusta coffee husk in vivo has not been carried out to date. Previous research conducted by Cahyani, et al.(2021)²² showed that Arabica coffee husk extract (Coffea Arabica L.) at doses of 125mg/kg BW, 250mg/kg BW, and 500 mg/kg BW could work on liver soft tissue to induce immune responses and cell regeneration. Research by Prasetya, et al.(2021)²³ stated that the number of inflammatory cells in periodontitis rats that were given robusta coffee was less than in periodontitis rats that were not given robusta coffee after being euthanized on the 22nd day. This study aims to analyze the effect of robusta coffee husk extract (C. canephora) on the number of osteoblasts and osteoclasts in the alveolar bone of Wistar rats induced by P. gingivalis.

MATERIALS AND METHODS:

Ethical approval:

This research was conducted following the approval from the research ethics committee of the Faculty of Dentistry Jember University No. 2146/UN25.8/KEPK/DL/2023. Our experimental procedure especially treated the rats already following Animal Research: Reporting of In Vivo Experiments (ARRIVE) and our animal ethical protocol institution.

Preparation of robusta coffee husk extract:

The robusta coffee used in this research came from the Banjarsengon coffee plantation, Jember. Robusta coffee husk extract (C. canephora) was made by grinding the exocarp part of the robusta coffee husk that has been dried, then weighing 320 grams of simplicia powder and macerating it in 1500ml of 96% ethanol solution for 72 hours to obtain a 100% thick extract.

Preparation of P. gingivalis:

P. gingivalis ATCC 33277 (MediMark, France) was grown in TSA (Oxoid, England) containing 10% sheep blood, 0.4μl/ml vitamin K1 and 5μl/ml hemin, then grown for 21 days at 37oC in an anaerobic incubator with 80% N₂, 10% H₂and 10% CO₂. To make a bacterial suspension, the bacteria were subcultured and incubated for 3 days then harvested with a sterile plastic tube and dissolved in saline solution. The concentration of P. gingivalis was 1.5 x 10⁸ CFU/ml.

Preparation of metronidazole:

The metronidazole preparation was obtained from the antibiotic metronidazole tablet (Novapharin, INA) at a dose 250mg/kg BW which was crushed, then weighed to 0.14mg/g BW and dissolved in 1ml of distilled water. The choice of metronidazole dose of 0.14 mg/g BW was based on research by Pang, et al (2015)¹⁸ which stated that this dose could significantly reduce the gingival index of rats experiencing periodontitis.

Experimental animal group allocation:

24 male Wistar rats aged 2-3 months with a body weight of 180-200grams. Rats were given standard turbo feed and mineral water. The rats were adapted for one week and then randomly divided into 6 groups (consisting of 4 rats each group):

1. The normal group (KN), which was not induced by P. gingivalis, was not treated;

2. The negative control group (K-), which was made chronic periodontitis by P. gingivalis induced once every three days for 21 days;

3. The positive control group (K+), group of rats induced by P. gingivalis once every three days for 21 days and given metronidazole per day for 21 days;

4. group of rats induced by P. gingivalis once every three days for 21 days and given 125 mg/kg BW robusta coffee husk extract via oral sondase per day for 21 days (KP 125);

5. group of rats induced by P. gingivalis once every three days for 21 days and given 250mg/kg BW robusta coffee husk extract via oral sondase per day for 21 days (KP 250);

6. group of rats induced by P. gingivalis once every three days for 21 days and given 500mg/kg BW robusta coffee husk extract via oral sondase per day for 21 days (KP 500).

Periodontitis model:

The animal was anaesthetised using a combination of ketamine and xylazine. P. gingivalis bacteria was carried out at 0.05ml with a concentration of 2 x 10⁹ CFU/ml once every 3 days for 21 days using a 1 ml tuberculin syringe and a 30 gauge needle in the gingival sulcus of the buccal part of the lower left first molar. The condition of periodontitis was confirmed by clinical and radiographic features.

Haematoxylin Eosin staining:

Experimental animals were euthanized on the 22^nd day and the lower left jaw was removed from the mesial of the first molar to the posterior of the third molar and immersed in 10% buffered formalin fixation solution before decalcification and fixed in 10% formic acid for 14 days with daily vibration then embedded in paraffin, and cut into 5 micron. The results of the incision were selected and transferred on a glass object and labeled according to the label of the tissue being cut. The tissue preparation was dried on a hot plate at 48◦C for 24hours and stained by haematoxylin eosin.

Calculation of the osteoblast and osteoclast cells:

The number of osteoblast and osteoclast cells was carried out by three observers using a light microscope with 400x magnification, then the average was looked for. The osteoblast cells observed were characterized as cuboid or flat cells, dark blue/purple nuclei, mononucleated cells, had cytoplasm that appeared basophilic, and were located on the surface of the alveolar bone. The osteoclast cells observed were characterized as giant cells that had irregular cell edges, had many dark blue/purple nuclei, and were located on the concave surface of the alveolar bone.

Statistical analysis:

Data are expressed as mean and standard deviation. Statistical significance was determined using the one-way Anova and continued with the Least Significance Different (LSD) test with p-values of <0.05 considered significant using SPSS version 22.

RESULT:

Periodontitis was induced by injection of P. gingivalis bacteria once every 3 days for 21 days and was confirmed by clinical features and supported by radiographic images. The clinical picture shows inflammation in the gingival margin area which is characterized by a change in color to pale red, the texture of the gingiva is smoother and stiffer (Figure 1). Periodontitis was confirmed by radiographic images of the lower jaw in the left M1 region of the buccolingual and mesiodistal sections, showing alveolar bone resorption (Figure 2)^24,25,26. Osteoblasts and osteoclasts were observed on preparations with Haematoxylin Eosin (HE) staining using a binocular microscope on the periosteum of the buccal alveolar bone which was visible at 400x magnification (Figures 5 and 6).

Figure 1. Clinical features of gingival inflammation (red arrow)

Figure 2. Radiographic features of alveolar bone resorption.

Marked with a yellow arrow. Radiographs of the jaws of normal group rats buccolingual (A) and mesiodistal (A'); and periodontitis group buccolingual (B) and mesiodistal (B').

Alveolar bone loss can be known from the decreased of alveolar crest in histological features of each group using imageJ software. The decrease in the alveolar crest was calculated from the distance between the base of the sulcus to the peak of the alveolar bone (red asterisk) in Figure 3. The results showed that there was alveolar bone loss in rats induced by P. gingivalis that can be seen in figure 4.

Figure 3. Histological features of the height of alveolar crest. Normal group (A) and negative control group (periodontitis) (B)

Figure 4. Graphic of distance between the base of sulcus to the peak of alveolar crest

Alveolar Bone (AB), Cementum (C), Periodontal Ligament (PL)

Figure 5. Histological Image of Alveolar Bone (400x Magnification). Osteoblasts (black arrows) and osteoclasts (yellow arrows) in KN (a), K- (b), K+ (c), KP 125 (d), KP 250 (e), KP 500 (f)

Data on the number of osteoblast cells were analyzed for normality and homogeneity using the Shapiro-Wilk and Levene Test. The results of the analysis showed that the calculation data for the number of osteoblast and osteoclast cells in each research group was normal and homogeneous (p>0,05). A One-Way ANOVA results showed that there were differences between treatment groups in the number of osteoblasts (p=0,000) and osteoclasts (p=0,000). A post-hoc LSD test results showed that there were significant differences in several treatment groups which can be seen in Figure 6 and 7.

* = mean difference in significance <0,05

Figure 6. Graphic of average number of alveolar bone osteoblasts

* = mean difference in significance <0,05

Figure 7. Graphic of Average Number of Alveolar Bone Osteoclasts

DISCUSSION:

The results showed that the group treated with robusta coffee husk extract at a dose of 250mg/kg BW was significantly different and had a higher average number of osteoblasts than the negative group, which means that this dose could increase the number of osteoblasts in the alveolar bone of periodontitis rats. In addition, the number of osteoclasts in the treatment group with robusta coffee husk extract doses of 125, 250, and 500 mg/kg BW was on average lower than the negative control group and was significantly different from the negative control group. It means that robusta coffee husk extract at doses of 125, 250, and 500mg/kg BW can significantly induce a decrease in the number of osteoclasts in the alveolar bone of periodontitis rats.

The increase in the number of osteoblasts and decrease in the number of osteoclasts is thought to occur due to inhibition of the activation of the immune response from anthocyanins, tannins and caffeine in robusta coffee husk. These compounds can suppress the process of pro-inflammatory cytokine formation so that they can trigger osteoblast differentiation and inhibit osteoclastogenesis by blocking NF-κB signals^19,21,27.

The results of this study are supported by Du, et al., (2018)²⁸ who stated that tannin as a type of polyphenol can reduce the expression of Cyclooxygenase-2 (COX-2) due to inhibiting NF-κB activation. COX-2 is an enzyme that stimulates the inflammatory response. Blocked NF-κB activation by tannin can prevent COX-2 transcription which leads to failure of pro-inflammatory cytokine production and RANKL stimulation which can inhibit osteoclastogenesis²⁹. Omar et al., (2021)³⁰ and Winiarska-Mieczan, et al., (2023)³¹ explained that caffeine can block Nuclear Factor-kappa B (NF-κB) signals and stimulate osteoblastogenesis by regulating the expression of osteoblastic genes such as Alkaline Phosphatase (ALP) and osteocalcin. ALP and osteocalcin function in the differentiation of osteoblast cells so that they can accelerate mineralization and formation of alveolar bone³². In addition, a literature review written by Widjaja and Syaify (2023)³³explains that apart from modulating genetic transcription which increases the production of pro-inflammatory cytokines, NF-κB activation can also trigger the formation of Reactive Oxygen Species (ROS). Excessive amounts of ROS in the body can cause destruction of alveolar bone tissue because it can trigger osteoclast differentiation. Anthocyanins and tannins can prevent an increase in the amount of ROS by acting as H atom donors or as single electron transfers and contribute to increasing anti-inflammatory activity³⁴, it allows redox reactions to occur to clear free radicals more easily³⁵.

The results of this study prove that the group treated with robusta coffee husk extract at a dose of 250mg/kg BW had a higher ability to increase the number of osteoblasts in periodontitis rats compared to a dose of 125mg/kg BW. Robusta coffee husk extract at a dose of 125mg/kg BW is thought to have low levels of active compounds so it has less effect in suppressing the production of pro-inflammatory cytokines and may result in failure to induce osteoblast differentiation. The decrease in the average number of osteoblasts in the 500 mg/kg BW dose group compared to the negative control group was thought to be because the high dose could have a toxic impact and affect the viability of osteoblast cells.

This result in line with research by Cahyani, et al. (2021)²² who stated that arabica coffee husk extract at a dose of 250mg/kg BW could induce regeneration of rat hepatocyte cells induced by 15% ethanol because the active compound content at this dose was optimal in reducing the amount of inflammatory cell infiltration. A dose of 125 cannot work optimally on hepatocytes because the active compound content at a dose of 125 mg/kg BW is too low so it is unable to stimulate cell regeneration. On the other hand, arabica coffee husk extract at a dose of 500mg/kg BW was not able to increase hepatocyte cell regeneration because the compound content at that dose was too high and could induce cell degeneration and necrosis.

A drug given orally will become a unit in plasma concentration and reach bioavailability³⁶. A drug dose that is too low cannot reach the Minimum Effective Concentration (MEC) so that the concentration of the active compound in plasma becomes inadequate and the resulting therapeutic effect becomes ineffective. Conversely, doses that are too high can exceed the Maximum Therapeutic Concentration or Minimum Toxic Concentration (MTC) so that the concentration of active compounds in plasma will increase³⁷. When high concentrations of active compounds reach the target tissue, there will be an imbalance in extracellular and intracellular fluids which will result in dysregulation of cell function, damage to cell walls, or disruption of cell maintenance which can trigger cell damage³⁸.

This study used metronidazole as a positive control because it is often used in the treatment of periodontitis. The results of this study showed that the number of osteoblasts from robusta coffee husk extract at a dose of 250mg/kg BW was not significantly different from the positive control group, which means that robusta coffee husk extract at a dose of 250mg/kg BW had almost the same ability as metronidazole in increasing the number of osteoblasts compared to doses of 125 and 500mg/kg BW. The number of osteoclasts from robusta coffee husk extract at doses of 125 and 250mg/kg BW was not significantly different from the positive control group. This means that robusta coffee husk extract at doses of 125 and 250mg/kg BW has almost the same ability as metronidazole in reducing the number of osteoclasts in the alveolar bone of periodontitis rats.

These results are in accordance with research by Fatimatuzzahro et al., (2024)³⁹ which states that robusta coffee husk extract has antibacterial activity against P. gingivalis. Robusta coffee husk contains anthocyanin compounds that can stimulate intracellular ion leakage and failure of bacterial cell metabolism⁴⁰. Caffeine as the one type of polyphenol can change the structure of the amino acids of bacterial cell walls and DNA so it will occur and cause lysis of the bacterial nucleus and at high consentrations it will cause denaturation if microbial enzymes and proteins^41,42. In addition, tannins work by inhibiting bacterial growth by disrupting protein transport and causing damage to bacterial cell wall⁴³.

Based on the results of the research, administering robusta coffee husk extract at a dose of 250mg/kg BW is optimal in increasing the number of osteoblast cells and reducing the number of osteoclast cells in the alveolar bone of Wistar rats induced by P. gingivalis for 21 days. Further research needs to be carried out to develop robusta coffee husk extract at a dose of 250mg/kg BW as an anti-inflammatory drug for periodontal disease.

CONCLUSION:

Robusta coffee husk extract (Coffea canephora) can increase the number of osteoblasts and reduce the number of osteoclasts in the alveolar bone of Wistar rats induced by P. gingivalis for 21 days with an optimal dose of 250mg/kg BW.

DECLARATION OF COMPETING INTEREST:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

AKNOWLEDGEMENT:

The authors would also like to thank to Laboratory of Basic Dentistry, University of Jember and Rumah Sakit Gigi dan Mulut (RSGM), University of Jember.

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Received on 29.07.2024 Revised on 25.01.2025

Accepted on 04.06.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5472-5478.

DOI: 10.52711/0974-360X.2025.00789

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