INTRODUCTION:
Honey is a naturally occurring substance with a highly saturated or semi-solid texture. It is produced by Apis mellifera bees from various sources, including plant nectar, plant secretions, and excretions of insects that feed on live parts of plants1,2. Honey consists of an intricate mixture of more than 200 diverse organic and inorganic substances, which include water and sugar. Honey’s composition can vary depending on a variety of factors such as botanical origin, storage condition, and geographical source3,4. Throughout history, honey has been widely recognized as a valuable source of traditional remedies and therapeutics. Its efficacy in healing various ailments has been acknowledged since ancient times5.
Yemeni Sidr honey, which is exclusively produced in Yemen, is one of the most well-known varieties of monofloral honey.
The Sidr tree (Ziziphus spina-christi L.) is a significant honey-producing source in the region, and Yemeni Sidr honey is exclusively derived from its nectar6. The Z. spina-christi plant is indigenous to tropical and subtropical areas, particularly in the Middle East. Its extracts are of great importance in the development of drugs due to their notable pharmacological activities7.
Honey contains pharmacologically active phytochemicals that exhibit versatile biological actions, including antibacterial, antioxidant, antiviral, and antifungal effects8. In addition to its ability to promote the healing of wounds, it also has anti-inflammatory properties. Amongst the various types of honey worldwide, Yemeni Sidr honey is widely regarded as the most superior and highly valued honey in Yemen, which can provide numerous health benefits, including immune system enhancement and healing properties. Due to its abundance of essential minerals and nutrients, this honey is renowned for its potent anti-inflammatory, antipyretic, antimicrobial properties, and analgesic effects9,10.
For centuries, honey has been used as a form of complementary and alternative medicine, known as apitherapy, to treat many oral and dental health problems, including periodontal disease, stomatitis, oral candidiasis or thrush, halitosis, mouth ulcers, and healing of sockets after tooth extraction. Additionally, honey has been utilized for its preventative effects against dental plaque formation, which can lead to dental caries, gingivitis, and periodontitis. Honey's anti-inflammatory and anti-bacterial characteristics are recognized to stimulate new granulation tissue formation, which can aid in repairing damaged cells that have been impacted by oral infections11,12.
The primary objective of periodontal therapy is to manage inflammation of the periodontal tissue and facilitate reliable regeneration of the periodontium that was lost due to periodontal disease13,14. Several treatment methods have been suggested to repopulate the area of missing periodontal structure with desirable cells. Of all the cell groups that contribute to periodontal wound healing, fibroblasts and osteoblasts are some of the most vital for maintaining the periodontium. This is due to their ability to renew the essential elements of periodontal tissue, including gingiva, alveolar bone, periodontal ligament, and cementum15.
While there have been several studies conducted on the positive effects of honey, there is limited research available on the effect of Yemen Sidr honey on specific cells such as fibroblasts and osteoblasts. As a result, there is potential for honey to be used as an alternative approach to conventional treatments for periodontal diseases. However, more research is required in the area of regeneration-based treatments for periodontal diseases. Thus, the aim of this study is to explore the potential beneficial effects of Yemen Sidr honey for human gingival fibroblast and osteoblast cells, specifically in terms of cell viability and proliferation.
MATERIALS AND METHODS:
Study design:
A true experimental laboratory study with post-test only control group design. The protocol ofthe study was approved by ethical clearances No.386/HRECC.FODM/VIII/2020 (Date approval: 31 August 2020) from the Faculty of Dentistry Research Ethics Commission, Airlangga University, Surabaya, Indonesia.
Yemen Sidr Honey:
Yemen Sidr Honey was sourced from the city of Hadhramaut in Yemen and collected in sterile cups or culture bottles. The honey was then passed through a sterile mesh or gauze to remove any debris and stored at a temperature of 2-8°C until it was ready for use16.
Cell line:
GT1 Fibroblast cell culture, and MC3T3 Osteoblast cell culture were obtained from the reservoirs located in the Research Centre/Faculty of Dental medicine/ Universitas Airlangga/Surabaya/Indonesia.
Fibroblast and Osteoblast Cell Cultures:
Preparation is done in laminar flow. The fibroblast cell cultures GT1 are stored in a roux bottle containing Dulbecco’s Modified Eagle Medium (DMEM) and Fetal bovine serum 10% (FBS). Then it was incubated for 24 hrs at 37°C. Next, the DMEM media and FBS 10% as growth media were removed from the roux bottle. Furthermore, the fibroblast cell in a roux bottle was then washed with PBS. The cells that are attached to the bottom of the roux bottle are removed with trypsin 0.05% for 5 minutes. The released cell culture was then given a new DMEM media containing FBS 10%. Cell cultures replaced with the new media are divided into 96 wells of tissue cultures plate according to the number of samples and controls. Cells are inserted into the well plates for cell culture with growth media for the treatment groups, cell control group, and growth medium only. Each well contains 1x104 fibroblast cells and 100μl DMEM. The plates were then incubated in 5% CO2 at 37°C for 24hrs. Similar culture preparation is done for MC3T3 osteoblast cells17.
MTT Assay:
The viability and growth rate of cells were analyzed using the MTT assay, which utilized MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)18. The experimental procedure involved placing fibroblast cells into individual wells of a 96-well plate with a density of 1x104 cells per well in a medium of 100μL. After seeding the fibroblast cells, the plate was incubated in a CO2 incubator at a temperature of 37°C for 24hours to allow the cells to attach to the well surface. Following the attachment, the cells were subjected to different concentrations of honey, which were prepared through serial dilution. The culture media in each well was withdrawn following the 24-hour incubation period, and a new medium was added19.
The cells were treated with varying concentrations of honey ranging from (25%; 250μL to 0.50%; 0.2μL) for a duration of 24hrs inside a CO2 incubator kept at a temperature of 37°C. Each well was then filled with 10 μL of the MTT working solution, and the plate was incubated for 4hrs in a CO2 incubator at 37°C. Then, the medium was aspirated, and the formazan crystals produced were dissolved by adding 50μl of DMSO to each well and incubating for approximately 30 minutes in a CO2 incubator at 37°C. Finally, the degree of the dissolved formazan crystals (which were purple) was measured at 590nm using an ELISA plate reader. The amount of absorption (intensity of the color generated) correlated with the percentage of viable cells. The same procedure is done for osteoblast cells20.
The percentage of cell viability for both fibroblast and osteoblast cells treated with Yemen Sidr honey was determined using the following formula:
Treatment group-blank group
Cell viability % = ---------------------------------------------- x 100
Control group-blank group
Statistical Analysis:
The data was analyzed statistically using SPSS software, version 25. Statistical analysis was conducted with ANOVA(One-Way) test and Post hoc (HSD Tukey) test with significance level of 0.05 (α = 0.05).
RESULT:
The research findings of the cytotoxicity test of Yemen Sidr honey on cell culture of GT1 fibroblasts and MC3T3 osteoblast using the MTT assay method were read through an ELISA reader. The result obtained is the optical density values quantifying both the living cells and cell death percentages or the absorbance value of the sample color. The observation and reading results of the absorbance values of the toxicity tests on Yemen Sidr honey in fibroblasts and osteoblasts were divided into six treatment groups at different concentrations, each undergoing the treatment on eight occasions accompanied by the controls (cell control and media control).
Figure 1 displays the impact of honey on the growth rate of fibroblast in culture. The results demonstrate that the optimal concentration of honey for enhancing the viability of GT1 human fibroblasts was found to be 0.50% (5μl), as it resulted in 78% living cells compared to the control group. Meanwhile, the lowest number of fibroblast cells at a concentration of 12.5%, and 25% with the percentage of living cells was 10% and 19% respectively. Figure 2 revealed that the lower the concentration of honey, the higher the percentage of living cells. There was a significant difference in the number of fibroblasts from control cells against treatment groups including 25%, 12.5%, and 5%, with a p-value < 0.05.
Figure 1: GT1 Fibroblast cell under light microscope at 10× magnification (a)0.50%Yemen Sidr honey (b) 25% Yemen Sidr honey (c) Control cell
Figure 2: The percentage of GT1fibroblast cell proliferation for different concentration of Yemen Sidr honey
Regarding the significant difference in the number of fibroblasts between treatment groups, there is a significant difference in fibroblasts in the Yemen Sidr honey treatment group at 25%, 12.5%, 5% against fibroblast cells in the treatment groups 1%, 0.75%, 0.50with a p-value = 0.00.
Regarding MC3T3 osteoblast cells, Figure 3 represent that the highest number of osteoblast cells in Yemen Sidr honey is at a concentration of 0.50% (5μl), with the percentage of living cells was 72%. While the lowest number of osteoblast cells with a concentration of 25% with the percentage of living cells was 65%.
On the other hand, Figure 4reported that the percentage of the live cells in all groups of Yemen Sidr honey are slightly different, which indicates that there is slight increase in cell percentage as the concentration of the honey decreases. The comparison of the mean survival percentages of the osteoblast control group, i.e., 100%, demonstrates the rise in the number of osteoblast cells, which was not statistically significant.
Figure 3: Osteoblast cells under light microscope at 10× magnification (a) 0.50%Yemen Sidr honey (b) 25% Yemen Sidr honey (c) Control cell
Figure 4: The percentage of osteoblast cell proliferation for different concentration of Yemen Sidr honey
DISCUSSION:
Sidr honey's ability to inhibit and eliminate bacteria responsible for periodontal diseases makes it a promising treatment option. The primary objective of treating periodontitis is to control the inflammation of periodontal tissue and achieve predictable regeneration of the lost periodontium. To achieve this goal, various treatment approaches have been proposed to repopulate the area of the lost periodontal structure with desired cells21. Researchers have previously demonstrated that the periodontal ligament is a significant source of fibroblast and osteoblast cells. These cells play a crucial role in the regeneration and repair of periodontal tissue during wound healing and the formation of new bone. However, there is a limited amount of research that has investigated the impact of honey on human periodontal fibroblast and osteoblast cells. In general, there are only a few reports available that specifically focus on the proliferation and differentiation of periodontal ligament cells15,22.
Cell culture methods are often used for testing biological effects on the initial level of a material used in dentistry to know the effects of toxicity. The toxicity of materials used in dentistry related to the viability of living cells 23. This study utilized the MTT assay, a widely used colorimetric assay, to evaluate cell viability or cytotoxicity. This assay determines cell viability by assessing the mitochondrial function of cells by measuring the activity of mitochondrial enzymes. The results of this assay can be measured by the absorption of light at a specific wavelength. The amount of absorbent indicates the number of viable cells in the culture media. The advantages of this assay are accurate and sensitive measurements due to the use of a spectrophotometer that can obviously detect changes in metabolism, easy manipulation, commonly used equipment is available in the laboratory, saves time and effort, and uses no radioactive isotopes24.
According to the results of this study on fibroblast cells, the absorbance value of the treated group at a concentration of 0.50% was higher than other treatment groups. On the other hand, the groups treated with honey concentrations of 25% and 12.5% had significantly lower absorbance values compared to the control cells. These findings are in agreement with a previous study that evaluated the effect of Tualang honey on fibroblast cells, which demonstrated that low concentrations of honey stimulated cell proliferation. In contrast, high concentrations had an inhibitory effect12. Another study found that low concentrations of honey stimulate lymphocyte proliferation and activate phagocytes25. The enhanced proliferation observed with honey treatment may be due to a nutritional effect, as honey contains carbohydrates that serve as substrates for glycolysis26.
Regarding osteoblast cells, the highest average absorbance value was similar to fibroblast concentration i.e., 0.50% compared to the control cell. While 25% concentration of honey was the lowest. A previous study reported that the rate of cell proliferation decreased as the dose increased in a dose-dependent manner27. A study stated that a material is considered non-toxic if the living cells are more than 50%28. Therefore, based on the results for both the fibroblast and osteoblast cells, all treatment groups of honey concentrations had a percentage of living cells > 50% except for the higher concentrations of honey in fibroblast namely 25, 12.5%, and 5% have low cell percentage that is below 50%.
A limitation of the study is that several factors can influence absorbance readings, such as the type of solvent used, pH levels, temperature, the concentration of electrolytes, and the presence of interfering substances. However, further research is required to validate these findings, including longer study durations, in vivo experiments, and clinical trials. These future investigations could potentially contribute to the development of new treatments for periodontitis utilizing Yemen Sidr honey. Nevertheless, based on the cell percentage results for both fibroblast and osteoblast cells, it can be seen that the optimum dose of the treatment group of Yemen Sidr honey was 0.50%.
CONCLUSION:
The findings of this study suggest that Yemen Sidr honey has a viability effect on the fibroblast and osteoblast cells, suggesting its biocompatibility and non-toxic nature towards these cell types.
CONFLICT OF INTERESTS:
No conflict of interest.
ACKNOWLEDGMENTS:
This study supported by Directorate of Research, Technology and Community Service, Indonesian Ministry of Education, Culture, Research and Technology.
REFERENCE:
1. Khan SU, Anjum SI, Rahman K, et al. Honey: Single food stuff comprises many drugs. Saudi J Biol Sci. 2018; 25(2): 320-325. doi:10.1016/j.sjbs.2017.08.004
2. Roshan ARA, Gad HA, El-Ahmady SH, Khanbash MS, Abou-Shoer MI, Al-Azizi MM. Authentication of monofloralyemeni sidr honey using ultraviolet spectroscopy and chemometric analysis. J Agric Food Chem. 2013; 61(32): 7722-7729. doi:10.1021/jf402280y
3. Pasupuleti VR, Sammugam L, Ramesh N, Gan SH. Honey, Propolis, and Royal Jelly: A Comprehensive Review of Their Biological Actions and Health Benefits. Oxid Med Cell Longev. 2017; 2017: 1-21. doi:10.1155/2017/1259510
4. Ansari MJ, Al-Ghamdi A, Khan KA, et al. Validation of botanical origins and geographical sources of some Saudi honeys using ultraviolet spectroscopy and chemometric analysis. Saudi J Biol Sci. 2018; 25(2): 377-382. doi:10.1016/j.sjbs.2017.09.005
5. Purbafrani A, Ghazizade Hashemi SA, Bayyenat S, Moghaddam HT, Saeidi M. The benefits of honey in Holy Quran. Int J Pediatr. 2014; 2(3): 67-73. doi:10.22038/ijp.2014.3417
6. Masalha M, Abu-Lafi S, Abu-Farich B, et al. A New Approach for Indexing Honey for Its Heath/Medicinal Benefits: Visualization of the Concept by Indexing Based on Antioxidant and Antibacterial Activities. Medicines. 2018; 5(4): 135.
7. Asgarpanah J, Haghighat E. Phytochemistry and pharmacologic properties of Ziziphus spina christi (L.) Willd. Afr J Pharm Pharmacol. 2012; 6(31): 2332-2339. doi:10.5897/ajpp12.509
8. Sam Jebaraj A, Gopinath P. Antibacterial Activity of Honey Against Clinical Isolates of Pseudomonas aeruginosa. Res J Pharm Technol. 2016; 9(8): 1174-1176. doi:10.5958/0974-360X.2016.00224.9
9. Mohammed F, Abdulwali N, Guillaume D, Bchitou R. Element content of Yemeni honeys as a long-time marker to ascertain honey botanical origin and quality. LWT - Food Science and Technology. 2018; 88(October): 43-46. doi:10.1016/j.lwt.2017.09.040
10. Alzubier AA, Okechukwu PN. Investigation of anti-inflammatory, antipyretic and analgesic effect of Yemeni Sidr honey. World Acad Sci Eng Technol. 2011; 80(8): 47-52. doi:10.5281/zenodo.1330264
11. Pasupuleti VR, Sammugam L, Ramesh N, Gan SH. Honey, Propolis, and Royal Jelly: A Comprehensive Review of Their Biological Actions and Health Benefits. Oxid Med Cell Longev. 2017; 2017. doi:10.1155/2017/1259510
12. Ahmad S, Sharma S, Ahmad S. Honey: A Natural Remedy in Dental and Oral Diseases. Science Documents. 2018; 1(1): 3-7. Accessed April 3, 2023. https://www.academia.edu/89831205/Honey_A_Natural_Remedy_in_Dental_and_Oral_Diseases
13. Kumar KM, Varghese S. Effect of Nonsurgical Periodontal Therapy on Serum Lipid Profile- Prospective Study. Res J Pharm Technol. 2019; 12(8): 3664-3668. doi:10.5958/0974-360X.2019.00625.5
14. Madhavan S, Gajnedran PL. A Preliminary Study to compare The Pain Perception of Topical gel Versus Injected Local Infiltration/Block Anaesthesia during Non-Surgical Periodontal Therapy. Res J Pharm Technol. 2018; 11(10): 4257-4262. doi:10.5958/0974-360X.2018.00780.1
15. Yu LX, Taib H, Berahim Z, Ahmad A, Zainuddin SLA. The effect of Tualang honey on human periodontal ligament fibroblast proliferation and alkaline phosphatase level. Sains Malays. 2015; 44(7): 1021-1025. doi:10.17576/jsm-2015-4407-14
16. Mama M, Teshome T, Detamo J. Antibacterial Activity of Honey against Methicillin-Resistant Staphylococcus aureus: A Laboratory-Based Experimental Study. Int J Microbiol. 2019; 2019. doi:10.1155/2019/7686130
17. Saczko J, Dominiak M, Kulbacka J, Chwiłkowska A, Krawczykowska H. A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation. Folia Histochem Cytobiol. 2008; 46(1): 117-119. doi:10.2478/v10042-008-0017-4
18. Bahuguna A, Khan I, Bajpai VK, Kang SC. MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh J Pharmacol. 2017; 12(2): 115-118. doi:10.3329/BJP.V12I2.30892
19. Sasikala M, Sundaraganapathy R, Mohan S. MTT Assay on Anticancer Properties of Phytoconstituents from Ipomoea aquatica forsskal using MCF–7 cell lines for breast cancer in Women. Res J Pharm Technol. 2020; 13(3): 1356-1360. doi:10.5958/0974-360X.2020.00250.4
20. Rianti D, Kristanto W, Damayanti H, et al. The Characteristics and Potency of Limestone-based carbonate hydroxyapatite to Viability and Proliferation of Human Umbilical Cord Mesenchymal Stem Cell. Res J Pharm Technol. 2022; 15(5): 2285-2292. doi:10.52711/0974-360X.2022.00380
21. Siaili M, Chatzopoulou D, Gillam DG. An overview of periodontal regenerative procedures for the general dental practitioner. Saudi Dental Journal. 2017; 30(1): 26-37. doi:10.1016/j.sdentj.2017.11.001
22. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004; 364(9429): 55. doi:10.1016/S0140-6736(04)16627-0
23. Shahi S, Özcan M, MalekiDizaj S, et al. A review on potential toxicity of dental material and screening their biocompatibility. Toxicol Mech Methods. 2019; 29(5): 368-377. doi:10.1080/15376516.2019.1566424
24. Aslantürk ÖS. In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages. In: Genotoxicity - A Predictable Risk to Our Actual World. InTechopen; 2018: 64. doi:10.5772/intechopen.71923
25. Abuharfeil N, Al-Oran R, Abo-Shehada M. The effect of bee honey on the proliferative activity of human B- and T-lymphocytes and the activity of phagocytes. Food Agric Immunol. 1999; 11(2): 169-177. doi:10.1080/09540109999843
26. Martinotti S, Ranzato E. Honey, wound repair and regenerative medicine. J FunctBiomater. 2018; 9(2): 34. doi:10.3390/jfb9020034
27. Kannan TP, Ali AQ, Abdullah SF, Ahmad A. Evaluation of Tualang honey as a supplement to fetal bovine serum in cell culture. Food and Chemical Toxicology. 2009; 47(7): 1696-1702. doi:10.1016/j.fct.2009.04.020
28. Telli C, Serper A, Dogan AL, Guc D. Evaluation of the cytotoxicity of calcium phosphate root canal sealers by MTT assay. J Endod. 2000; 25(12): 811-813. doi:10.1016/S0099-2399(99)80303-3
Received on 07.11.2023 Modified on 14.01.2024
Accepted on 21.02.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(8):3764-3768.
DOI: 10.52711/0974-360X.2024.00585