Effects of ginger (Zingiber officinale) on gingival fibroblasts: An in vitro study

Abstract Objectives Ginger, the powdered rhizome of the herb Zingiber officinale, is commonly used as a traditional medicine in many areas around the world. Anti‐inflammatory actions of its extract have been previously reported. The aim of this study was to investigate the effect of ginger extract on matrix metalloproteinase (MMP) and interleukin (IL) expression from human gingival fibroblasts (HGFs) in vitro. Material and Methods HGFs were obtained from subcultures of biopsies from clinically healthy gingival tissues of 10 patients. Ginger extract was prepared from commercial powder of rhizome of Z. officinale (GZO) and its effect on cell viability was assessed using the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide cytotoxicity assay. Cells were then incubated and treated (except for the control samples) with either GZO, lipopolysaccharides (LPS), and GZO before or after LPS stimulation. Culture supernatants of all five samples were collected for the Milliplex analysis to measure MMP‐1, MMP‐2, MMP‐8, MMP‐9, IL‐1β, and IL‐8. One‐way analysis of variance and Duncan multiple range tests were used to compare the mean values of all groups. Results The gingerextract showed minimal cytotoxicity to HGFs even with the maximum tested concentration. Compared to the control group, GZO treatment alone caused little or no effect on the levels of expression of MMP‐1, MMP‐2, MMP‐8, MMP‐9, IL‐1β, and IL‐8. While GZO treatment after LPS stimulation significantly reduced the expression of MMP‐1, MMP‐2, MMP‐8, MMP‐9, and IL‐8 when compared to LPS alone. Comparing the control to LPS stimulation after GZO treatment, significant differences were detected for all tested MMPs and cytokines. Conclusions These findings suggest a potential role for ginger extract in inhibiting MMP and IL HGFs' expression in inflamed gingival tissues.


| INTRODUCTION
Periodontal diseases are infectious diseases characterized by periodontal attachment loss and bone destruction. They are considered one of the most common chronic debilitating diseases affecting a large number of people, causing considerable pain and discomfort, and are costly to control and treat. Their impact is, therefore, significant to both the individual and the wider community. The main etiological infectious agent involved in the pathogenesis of periodontitis is the Gram-negative bacteria, such as Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Tannerella forsythia. These microbial agents trigger a complex host immune-inflammatory reaction leading to the characteristic tissue destruction response (Kinane, 2001;Lovegrove, 2004;Popova et al., 2013;R. Saini et al., 2009).
Human gingival fibroblasts (HGFs) are the main cell type residing in gingival mesenchymal tissues and are responsible for the integrity of the extracellular matrix. They produce the essential gingival connective tissue proteins and glycoproteins as well as most of the immunoregulatory cytokines and chemical mediators including matrix metalloproteinases (MMPs). Their role is deemed to be crucial in periodontal disease pathogenesis and tissue destruction. Regulation of HGFs cellular reaction has been suggested as an essential pathway in preventing and controlling the progression of periodontal tissues' pathological alterations as well as healing processes (Dahan et al., 2001;Page, 1991).
Ginger (botanically known as Zingiber officinale) is a herbaceous plant that has been, for centuries, extensively used in many regions around the world as a spice. While India is considered the biggest producer of ginger in the world, the plant is also grown extensively in other countries, such as China, Nepal, the United States, Bangladesh, Taiwan, and Jamaica among other parts of the world. Z. officinale is also commonly used as a medicinal plant in Chinese, Indian Ayurvedic, and Muslim's Tibb-Unani herbal medicines for the treatment of many diseases, including asthma, stroke, digestive problems, rheumatoid arthritis, nervous diseases, and diabetes (Bhatt et al., 2013;Khalil et al., 2011;Moghaddasi & Kashani, 2012;Santo & Sankari, 2017).
Z. officinale has previously been shown to possess an anti-inflammatory effect in an in-vitro periodontal disease model as well as in an in vitro Candida albicans model (Eslami et al., 2015;Koontongkaew et al., 2013).
The purpose of this study was to assess the effect of Z. officinale on the HGFs' MMPs (MMP-1, MMP-2, MMP-8, and MMP-9) and inflammatory cytokines (interleukin [IL]-1β and IL-8) expression as an attempt to explore potential scientific bases for the claims of its anti-inflammatory role and the benefits of its use in several commercially available gingercontaining mouthwash. Effects of ginger extract were tested in vitro on cultured HGFs, in the presence or absence of lipopolysaccharide (LPS). Tissues were then cut into small fragments of approximately 1 mm 3 and placed in cell culture dishes, air dried, and incubated for 5-7 days at 37°C and 5% CO 2 in low-glucose (100 mg/L) Dulbecco's modified Eagles medium supplemented with 15% fetal bovine serum (Hyclone, Logan, Utah, United States), Two hundred millimoles of L-glutamine, 100 U/ml penicillin and 50 μg/ml gentamycin that was changed every 2 days. Explants were subcultured out of the cells that grew out and

| Preparation of ginger solution
The ginger solution was prepared from commercial powder of rhizome of the ginger plant (Z. officinale: GZO) (DRUERA; Dru Era Pty Ltd., Wilmington, DE, USA) The extraction was done by dissolving 40 g of powder in 200 ml of distilled deionized water and incubated at room temperature for 24 h with shaking at 120 rpm. After extraction, the solids were removed by centrifugation at 3000g at 25°C for 10 min and filtered with a 0.22 μm membrane filter, and then stored at −80°C.

| Cellular cytotoxicity assay
Cellular cytotoxicity was assessed using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based cytotoxicity assay. HGFs were seeded in 96-well plates at a density of 2 × 10 5 cells/well in a 100 µl optimized medium. The total number of cells was determined by the trypan blue exclusion test (0.4%) using a cell counter. Cells were allowed to settle for 24 h before treatment with individual serial concentrations of GZO (6.25,12.5,25,50 with LPS 5 µg/ml for 24 h before being exposed to GZO (50 µg/ml) while Sample 5 included cells pretreated with GZO 50 µg/ml for 24 h before being exposed to LPS (5 µg/ml). Culture supernatants of all five samples were collected for the Milliplex analysis. Internal controls and standards were included in every assay.

| Milliplex analysis
Following the addition of sample supernatants and beads, the resulting mixture was incubated overnight at 4°C. The plate was scanned using a Bio-Plex array reader with the Luminex 200 software system.

| Statistical analysis
Data were analyzed using SPSS 21.0 version (IBM Inc., Chicago, IL, USA) statistical software. One-way analysis of variance test and Duncan's multiple range tests were used to compare the mean values of the study groups. A p < .05% and 95% confidence intervals were used to report the statistical significance and precision of mean values.
The effect of GZO on HGF expression of MMPs, IL-1β, and IL-8 levels was determined by a comparison between all groups including the control group, as shown in Table 1

| DISCUSSION
One of the key destructive pathways in periodontitis is the loss of connective tissue attachment as a result of collagen fiber degradation. The extracellular proteolytic system that is involved in periodontal tissue breakdown and is responsible for this collagen degradation includes mainly MMPs. IL-1 and IL-8 are also important inflammatory mediators produced as a part of the host response and significantly contribute to tissue destruction. Studies have indicated that their levels directly correlate with the severity of periodontal disease.
Higher levels of inflammatory cytokines and metalloproteinases are significantly associated with the severity of the periodontal disease and the extent of tissue destruction (Gamonal et al., 2000;Silva et al., 2007;Yucel-Lindberg & Båge, 2013).
Ginger is the rhizome of the plant Z. officinale, widely used as a spice, delicacy, and traditional medicine around the world. Its name stems from the genus or family (Zingiberaceae). Other well-known members of this herbal family are turmeric, cardamom, and galangal.
Preliminary research indicated that active compounds found in ginger F I G U R E 1 Cell viability assay values (mean and standard deviation). Comparison between groups done using one-way analysis of variance test with Dunnett test as multiple comparisons to compare each group with the control. GZO, Zingiber officinale may possess antimicrobial, antioxidant, anti-inflammatory, and antinecrotic activities (Ali et al., 2008;Grzanna et al., 2005;Shahrajabian et al., 2019), which may explain its occasional use as an added ingredient in some of the commercially available mouthwashes as well as in some of the homemade mouthwash recipes. In this study, we investigated the effects of the ginger extract on MMPs and inflammatory cytokines expression from HGFs in vitro.
It is clear from the data of the current study that, compared to the control group, GZO treatment alone caused little or no effect on the levels of expression of MMPs, IL-1, or IL-8. Nevertheless, the data also indicated that when compared to the LPS-stimulated HGFs, GZO treatment resulted in a significant reduction in the levels of expression of MMP-1, MMP-2, MMP-8, MMP-9, and IL-8. Given the fact that the latter would more closely simulate the condition of periodontitis-affected inflamed tissues, these findings may partially confirm the claims of using GZO as an effective botanical antiinflammatory compound in mouthwashes. We might also hypothesize that, based on these findings, while GZO may not have any Several studies have evaluated ginger's effectiveness as an antiinflammatory agent. Previous in vitro studies suggested that ginger extracts block the formation of inflammatory compounds, such as prostaglandins and leukotrienes. In addition, animal data showed that in rat models of severe inflammatory arthritis, ginger oil effectively reduced inflammation and swelling (Flynn et al., 1986;Kiuchi et al., 1982;Sharma et al., 1994). Case series of patients with rheumatoid arthritis reported varying degrees of relief in pain and swelling and improved symptoms following supplemental ginger (Srivastava & Mustafa, 1989). In the current study, ginger extract  (Escalona et al., 2016;Finoti et al., 2017).
Similar to our findings, Lantz et al. (2007), studying the effects of extracts from ginger rhizome on inflammatory mediator production using an in vitro test system, have shown that compounds found in ginger have anti-inflammatory effects and are highly effective at inhibiting LPS-induced production of PGE2. Koontongkaew et al. (2013)  and erythema after a 20-day treatment period. They reported that patients were more satisfied with ginger mouthwash due to fewer side effects and it could possibly be recommended as an alternative to nystatin mouthwash in the treatment of denture stomatitis.
Targeting the MMPs has been traditionally suggested as an adjunctive therapeutic option in periodontal diseases since the progression of the disease is marked by increased collagen destruction, partly mediated by fibroblasts and their secretion of those factors. Our results showed that GZO significantly decreased MMP-2 and MMP-9, also referred to as gelatinases, as well as MMP-1 and MMP-8, the two main collagenases, expression in HGFs when compared with LPS-stimulated HGFs. Therefore, we hypothesize that GZO active ingredients may alleviate periodontal proteolysis and ease inflammation in the periodontitis-affected gingival tissues.
These suggestions, however, remain to be further confirmed in pharmacokinetic and clinical studies.
In conclusion, this study has shown that ginger extract would potentially be an effective natural botanical additive that may help alleviate inflammation and tissue destruction characteristics of periodontal diseases. In addition, it is expected to be safe and

AUTHOR CONTRIBUTIONS
Nouf Alshibani, Reem Alkattan, Lamees Alssum, and Eman Allam contributed to the design of the study, performed the experiments, and participated in the analysis of the results as well as writing and revising the manuscript.