INTRODUCTION:

Periodontitis is a chronic inflammatory disease that predominantly results from periodontal bacterial infection and an inflammatory host response. It destroys alveolar bone and connective tissue. The pathophysiology of periodontal breakdown is largely dependent on the particular periodontal bacteria found in the subgingival plaque¹.

In Indonesia, the prevalence of periodontitis, especially chronic periodontitis, increased by 42.8% (1995), 70% (2001), 96.58% (2004), and 60% (2012), according to statistics from Riskesdas 2018. In 2018, this prevalence reached at 67.8%². Damage to the tissues that support teeth, such as the cementum, gingiva, alveolar bone, and periodontal ligament, is a hallmark of periodontal disease³. A key contributing factor to tooth loss, which can affect mastication, appearance, self-confidence, and quality of life, is periodontal disease⁴. Since this disease rarely manifests as complaints or discomfort, many people are unaware of it until it becomes too late, at which point the disease has severely damaged periodontal tissue⁵. The pathophysiological mechanism of gingivitis is in response to the bacterial biofilm adhering to tooth surface. Gingivitis is the most prevalent form of periodontal disease epidemiologically. Gingivitis can lead to periodontitis, a severe form of periodontal disease, if treatment is not received. However, in certain situations, periodontitis may not develop from gingivitis⁶. Periodontal disease, which starts as gingivitis and progresses to periodontitis, is mostly caused by bacteria. A gram-negative bacteria known as Porhyromonas gingivalis (P. gingivalis) is a periodontitis pathogen. P. gingivalis is even present in the host epithelium without clinical periodontal damage⁷. Patients with chronic periodontitis had subgingival plaque with up to 85.75% P. gingivalis bacteria. As a result, P. gingivalis is the primary cause of periodontitis⁸. P. gingivalis bacterium's primary virulence agents include bacteria, lipopolysaccharides (LPS), and proteases, which affect periodontal tissue⁹. The oral biofilm is complex containing about 700 different species of bacteria, most of which are opportunistic in nature. When the conditions are right, they penetrate the periodontium and trigger an intense immuno-inflammatory reaction. As a result, periodontal tissue is rapidly destroyed¹⁰.

P. gingivalis infections can take the form of bacteria or lipopolysaccharide (LPS) products. They are identified by TLR-2 or TLR-4 receptors in the gingival epithelial tissue, where they activate the NF-κB pathway by releasing IκB (NF-κB Inhibitor) and allowing NF-κB to enter the nucleus. Pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 are examples of inflammatory mediators whose transcription and translation are started when NF-κB is activated into the nucleus¹¹. Proinflammatory mediators (IL-1β, IL-6, and TNF-α) produced in the gingiva as a result of P. gingivalis virulence factors can be inhibited by NF-κB. This implies that a major target for host modification therapy may be this cell signaling pathway¹².

Treating periodontal disease primarily aims to eradicate any harmful bacteria that may infect periodontal tissue. The most effective method of treating periodontal disease is scaling and root planing, which eradicates the germs that cause the illness¹³. The fundamental component of first periodontal therapy is the mechanical debridement of the dental biofilm and the removal of underlying irritant factors¹⁴. The bacteria known as periodontopathogenics can reside in pockets or periodontal tissue, impeding the process of scaling and root planing and preventing the growth of pathogenic bacteria. These bacteria are responsible for the development of periodontitis. The residual bacteria can harm the alveolar bone and its supporting connective tissue, as well as result in recurrent periodontitis. This indicates that further therapy is required to eradicate any periodontopathogenic bacteria¹³.

A viable approach to aiding in the local application of medicinal plant materials to periodontal tissue to facilitate the repair of chronic periodontitis with minimal adverse effects is to employ these materials as an alternative¹⁵. Since ancient times, medicinal plants have been employed in many different countries for the prevention and treatment of many diseases. Herbal ingredients might be chewable stems, seeds, leaves, powder, paste, or gum. In dentistry, medicinal plant extracts have been employed as an antiplaque agent, anti-inflammatory, antiseptic, antioxidant, antimicrobial, antifungal, antibacterial, antiviral, and analgesic in addition to preventing histamine release¹⁶.

Water hyacinth is one plant that has several uses. The Pontederiaceae family of flowering plants includes water hyacinth. This plant, a freshwater hydrophyte that grows in tropical and subtropical regions, is regarded as a weed¹⁷. Water hyacinth, however, has an antimicrobial effect on P. gingivalis survivability¹⁸. Therefore, turning water hyacinth—which is considered a nuisance—into a substitute material for periodontal tissue repair is a problem for humans. The water hyacinth, or Eichornia crassipes, is a plant with potential medical uses that include antibacterial, antioxidant, anticancer, and wound healing acceleration¹⁹. It also demonstrates antimicrobial action against gram-negative bacteria. The test results for the viability of water hyacinth against fibroblast cells at a concentration of 1.56% were 75.98%. In vitro with a Minimum Inhibition Value (MIC) at a concentration of 1.56%²⁰.

According to earlier studies, metabolites found in water hyacinth include tannins, saponins, flavonoids, terpenoids, phenols, anthraquinones, alkaloids, quinones, glycosides, reducing sugars, and steroids^21,
22. Microbes and the host are impacted by the contents of water hyacinth, particularly the flavonoid mechanism. Alkaloids, phenols, and flavonoids also prevent NF-κB from triggering cytokines²³. A 2% content is utilized based on the discussion of the components of water hyacinth and its application as a therapeutic element, as well as studies indicating that the MIC is 1.56% and the viability test for water hyacinth against fibroblast cells at a concentration of 1.56% is 75.98%. The aim of this study was to investigate if the administration of water hyacinth, a mediator that controls inflammation, especially in periodontal disease, reduced the amounts of IL-6 and NF-κB in the gingiva of mice exposed to P. gingivalis bacteria.

MATERIAL AND METHODS:

This research is a laboratory experimental study with a post-test-only control group design. The research was carried out in vivo on 35 male Wistar rats (Rattus novergicus), aged 2-3 months, weighing 200-250 grams, with healthy physical condition and no defects, kept in the same conditions and place. Ethical feasibility is submitted to the Health Research Ethics Committee (KKEPK) Faculty of Dentistry, Airlangga University, before conducting research. The research was carried out after obtaining an ethical certificate with number: 294/HRECC.FODM/III/2023.

Producing Extract from Water Hyacinth Leaf:

After the powdered water hyacinth leaf (530 grams) was weighed, 2.5 L of 70% ethanol was used for maceration. With the aid of a Buchner funnel and filter paper, the residue was removed from the 70% ethanol filtrate. After the residue was completely submerged in the jar, about 2.5 L of 70% ethanol was added. The jar was put on a digital shaker, and it was shaken for a full day at a speed of 50 revolutions per minute. An Erlenmeyer flask was used to collect the filtrate after it was passed through a sieve. Using a rotary evaporator, the first and second extract results were combined and evaporated. Four hours are needed for evaporation. After two hours in a water bath, the extracted material evaporated. 40 milliliters of water hyacinth leaf extract were made from 600g of leaves and 5 liters of 70% ethanol. CMC Na, a suspending agent, is added after the extract is diluted with distilled water to produce 2% extract.

Treatment of Experimental Animals:

Before treatment, Wistar rats were adapted for 7 days in cages measuring 41x32x11cm which were divided into 7 groups, namely normal group (N), control group (K1, K2, K3), and treatment group (P1, P2, P3) with each Each cage consisted of 5 experimental animals. P. gingivalis bacteria for control (K) and treatment (P) groups were prepared. In group K, Wistar rats were tested for P. gingivalis bacteria in the gingival sulcus of the mandibular incisors using a micropipette with a volume of 2x106 CFU of 0.03 ml 1x48 hours for 7 days. Bacteria are given every morning at 7 am. This treatment aims to cause the gingiva to become inflamed²⁴. In group P, Wistar rats were also tested for P. gingivalis bacteria in the gingival sulcus of the mandibular incisors using a micropipette with a volume of 2x106 CFU of 0.03ml 1x48 hours every morning at 07.00 WIB for 7 days. Then Wistar rats that had been exposed to P. gingivalis were given 0.03 ml of water hyacinth leaf extract 2 times a day for 7 days in the gingival sulcus of the lower incisors. Water hyacinth leaf extract was dropped using a micropipette and left for 1 minute by retracting the mouth cavity of the Wistar rat so that it would not be swallowed during application^25,26. One advantage of local antimicrobial therapy delivery to periodontal pockets is that it maximizes drug concentration at the target site while reducing systemic exposure to the medication²⁷.

Immunohistochemistry Preparation Protocol:

Tissues were fixed with 10% NBF. The tissue was cut to a thickness of 2-4mm. The fixed tissue was then washed using 70% alcohol for about 15 minutes, 80% alcohol for 1 hour, 95% alcohol for 2hours, 95% alcohol for 1 hour, ethanol for 1 hour, and repeated 2x. Next, the tissue is cleared by placing it in xylol solution for 1 hour, then xylol for 2hours, and repeating once. The tissue was impregnated by soaking the tissue in paraffin (temperature 56-58ºC) for 2 hours, done twice. Paraffin melted to a boiling point of 56-60ºC is poured into a mold. The embedding cassette is placed into the mold with the surface of the tissue to be cut facing the bottom. Then the liquid paraffin is poured into the mold and waited until the liquid paraffin hardens.

A sliding microtome was used to cut paraffin blocks to 4 µm thickness. The results of the tissue incision in a paraffin block are taken with a brush and placed on the surface of the water in a water bath with a constant temperature of 56-58ºC until the incision extends. The tissue incision is then placed on a poly-L-lysine microscope slide (preparation). Preparations containing socket tissue are placed on a hot plate at a temperature of 30ºC-35ºC for a minimum of 12hours. Cut the periodontal tissue of the mandibular central incisor in a 4μm paraffin block. Expression of IL-6 was carried out by counting the number of epithelial cells in gingival tissue that expressed IL-6 using IHC staining at 400x magnification in 6 fields of view.

Data analysis:

IL-6 calculation data were analyzed using statistical tests, namely: the normality test using the Shapiro-Wilk Test. Because the data did not have a normal distribution, a nonparametric test was carried out using Kruskal-Wallis. To determine the differences between groups, the Mann-Whitney test was continued.

RESULTS:

IL-6 Expression Data:

IL-6 expression in Wistar rat gingiva in the second region of the mandibular central incisors was calculated from epithelial cells that expressed IL-6.

Figure 1. Comparison graph of the average IL-6 expression in each control and treatment group on days 1, 3, and 7.

Day 1, Day 3, and Day 7 findings from the Kruskal Wallis difference test showed that all treatment and control time groups had Sig values less than 0.05. This indicates that on days 1, 3, and 7, IL-6 expression significantly decreased in all groups.

Figure 2. IL-6 expression (arrow) in gingival epithelial cells of Wistar rats (A) normal group, (B) control group on day 1, (C) control group on day 3, (D) control group on day 7, (E) treatment group on day 1, (F) treatment group on day 3, (G) treatment group on day 7.

The Mann-Whitney test comparison of IL-6 expression showed a significant increase between the normal group and the control group from day 3 to day 7 (sig.0.05). There was a significant increase between the control group on day 1 and day 7 (sig.<0.005). The treatment group did not show significant differences on day 1 to day 3, as well as day 3 and day 7 (sig.>0.05). Analysis of IL-6 expression between the control group and treatment on day 1 did not show a significant difference (sig.>0.05), however, between the control group and treatment on day 3 and day 7 there was a significant difference (sig<0.005). Thus, it can be said that administering water hyacinth leaf extract for 7 days gave good results in inhibiting the increase in IL-6 expression. This can be seen from the comparison of the averages of the control and treatment groups on days 3 and 7, which shows that the difference in average IL-6 on day 7 is greater than on day 3.

DISCUSSION:

When gum disease, or gingivitis, is left untreated, it can lead to periodontitis. The ligaments and bone that support the teeth are affected by infection and inflammation that originates in the gingiva²⁸. Plaque bacterial collection in the gingival sulcus triggers an inflammatory response, which is the first step towards the development of periodontal disease. Evidence points to a hereditary component to the aetiology of the disease, in addition to the involvement of pathogenic microflora and other environmental risk factors in its pathogenesis. The localized immunopathology of periodontal disease appears to be significantly influenced by cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)²⁹.

Following the administration of P. gingivalis, wistar rats showed clinical signs of gingivitis from the first day. Gingivitis is clinically characterized by bleeding readily and redness of the gingival papillae and borders of the lower central incisors. From days 1 to 7, there seems to be an increase in these clinical indicators. According to study, giving P. gingivalis at this concentration and duration will worsen gingival inflammation, which will result in periodontitis on days 14²⁵.

According to immunohistochemistry, there was an increase in IL-6 expression on the first day when P. gingivalis was administered. The control group experienced a greater rise in IL-6 expression than the treatment group. From day one to day seven, the control group's pattern of rising IL-6 persisted. On the other hand, IL-6 inhibition happened in the therapy group from day 1 to day 7. After being exposed to inflammation during the first 24hours, IL-6 rises^30,31,32. This upregulation of IL-6 expression suggests that P. gingivalis is causing inflammation in the epithelium. On the gingival epithelium, exposure to P. gingivalis activates NF-κB, which in turn causes the induction of pro-inflammatory cytokines such IL-6¹¹.

Because P. gingivalis possesses virulence factors that alter immune system function, gingivitis is caused by P. gingivalis. The virulence factors that support P. gingivalis include adhesin domains, gingipain, fimbriae, LPS, and capsule. These elements of virulence are also P. gingivalis Pathogen-Associated Molecular Patterns (PAMPs). According to research, the three most significant P. gingivalis virulence factors in the development of periodontitis are LPS, gingipains, and fimbriae⁸. Through TLRs, or pattern recognition receptors (PRR), these virulence factors elicit an immunological response³³. P. gingivalis's pathogenicity towards gingival epithelial tissue is identified by the TLR-2 or TLR-4 receptor¹¹.

IL-6 used as a marker in this study. Epithelial cells release IL-6, which is crucial in controlling the acute phase and the body's reaction to damage and infection³⁴. Because IL-6 is a warning signal for tissue injury, it was used in this study. TLR can trigger the production of IL-6 through the NF-Kb pathway upon detecting the presence of LPS from P. gingivalis³⁵. The primary proinflammatory cytokines in periodontal tissue injury are IL-1, IL-6, and TNF-α³⁶. The study's findings showed that there was a significant difference in IL-6 expression between the treatment and control groups from day 1 to day 7. IL-6 expression increased in both the treatment and control groups. This is because P. gingivalis has the ability to alter the immune response by causing the gingival epithelium to produce more of the cytokine IL-6³⁷. In addition to activating IL-6, gingipain in P. gingivalis aids in the degradation of TLRs in cell membranes³⁸. Dysregulation and increased severity are also brought on by autocrine IL-6, particularly in periodontal tissues³⁹. This demonstrates that the study's findings support the hypothesis that IL-6 will rise during the first 24 hours of exposure and then fall for the next three weeks⁴⁰.

Based on the study's findings, there were no appreciable changes between the treatment group on days 1 through 3 or between days 3 and 7. There was no significant difference in the analysis of IL-6 expression between the treatment group and the control group on day 1, however there was a significant difference between the treatment group and the control group on days 3 and 7. The treatment group that received 2% water hyacinth leaf extract showed a greater drop in IL-6 expression. This demonstrates that 2% extract of water hyacinth can lower the expression of IL-6 in epithelial cells. The presence of phenolic, alkaloid, and flavonoid components in 2% water hyacinth leaf extract is one mechanism that could explain the observed reduction in IL-6 following administration of the extract. The water hyacinth leaf extract contains phenol gallic acid, which prevents lipid peroxidation, promotes Nrf2 signaling pathway expression and activation, and decreases NF-κB and IκB signaling pathway expression and activation. When Nrf-2 is released into the nucleus, the HO-1 enzyme is expressed. By inhibiting phosphorylation of NF-κB/IκB, this enzyme decreases NF-κB activity and its translocation to the nucleus, which in turn reduces the inflammatory cytokine IL-6 production⁴¹. The water hyacinth leaf extract contains alkaloids that modulate the transcription factor NF-κB by controlling the activity of both NF-κB and IκB. This results in a reduction of IL-6 levels. Alkaloids inhibit by first blocking TLR, then IKK's catalytic activity and IκB phosphorylation. Alkaloids also prevent IκB and NF-κB from being released, which lowers the expression of genes that promote inflammation. The inhibition of NF-κB entry into the nucleus will result in a decrease in proinflammatory cytokine expression, particularly IL-6⁴². Quarcetin 7-methyl ether, which is included in water hyacinth leaf extract, also blocks signaling pathways linked to inflammatory processes. Quercetin 7-methyl ether decreases NF-κB translocation into the nucleus, suppresses IL-6 expression beginning at TLR on the cell membrane, and subsequently phosphorylates IκB and NF-Κb⁴³.

The study's findings demonstrated that 2% water hyacinth leaf extract might suppress IL-6 and NF-κB expression. Gingival epithelial inflammation can be decreased by 2% water hyacinth leaf extract inhibition of IL-6. An essential function of the NF-κB pathway is to promote inflammation. In order to prevent inflammation from spreading, particularly in periodontal tissue, a particular strategy is to suppress the NF-κB activity pathway. According to this study, 2% water hyacinth leaf extract may be utilized as a substitute medication to prevent periodontal disease from getting worse.

CONCLUSION:

Administration of 2% water hyacinth (Eichornia crassipes) leaf extract can reduce IL-6 expression in mice exposed to P.gingivalis bacteria.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 18.02.2024 Revised on 17.08.2024

Accepted on 21.11.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1696-1701.

DOI: 10.52711/0974-360X.2025.00243

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