|Year : 2019 | Volume
| Issue : 3 | Page : 90-94
Viability test of ethanol extract of beluntas (pluchea indica) leaves on In vitro fibroblast cells
Rani Wulan Sari1, Natallia Pranata2, Vinna Kurniawati Sugiaman2
1 Faculty of Dentistry, Maranatha Christian University, Bandung, Indonesia
2 Department of Oral Biology, Maranatha Christian University, Bandung, Indonesia
|Date of Submission||22-Apr-2019|
|Date of Decision||15-Aug-2019|
|Date of Acceptance||04-Sep-2019|
|Date of Web Publication||14-Oct-2019|
Dr. Vinna Kurniawati Sugiaman
Department of Oral Biology, Maranatha Christian University, Bandung
Source of Support: None, Conflict of Interest: None
Background: Tooth extraction is the most frequently conducted dental care procedure. In Indonesia, there is an extremely high utilization of dental and oral health services for tooth extraction, reaching 79.6%. Pain is a side effect of extraction. Pain due to extraction wounds can be treated with analgesic drugs, but alternative drugs with minimal or no side effects are still being researched. An herbal active beluntas leaf substance can reduce pain from extraction wounds. The beluntas plant not only aids in healing wounds but also exhibits anti-inflammatory and antipyretic effects. Objectives: In this study, the aims were to determine the 50% inhibitory concentration (IC50value) and examine the viability effect of an ethanol extract of beluntas leaves on fibroblast cell cultures in vitro. Methods: Laboratory experiments were carried out. Beluntas leaves were obtained; their leaf extracts were prepared using ethanol as the solvent; phytochemical tests were performed. Triplicate measurements for the viability of 3T3 BALB/c fibroblast cells were carried out using the Methylthiazol sulfophenyl (MTS Assay) method. The extract concentrations were 500, 250, 125, 62.50, 31.25, 15.63, and 7.81 μg/mL. Results: Data analysis was carried out by one-way analysis of variance statistical test. Analysis results revealed that extract concentrations of 500, 31.25, 15.63, and 7.81 μg/mL exhibit a significant difference in the effect of cytotoxicity (P < 0.05) on fibroblast cells, and the IC50value is 265.388 μg/mL. Conclusion: A significant difference in the cytotoxicity effect between the concentrations of the ethanol extract of beluntas (P. indica) leaves on the fibroblast cell cultures in vitro was observed. The beluntas leaf extract at an IC50value of 7.81 μg/mL did not affect cell viability; hence, it is considered safe.
Keywords: Beluntas leaves, cytotoxicity, ethanol extract, fibroblast cells, MTS assay, viability
|How to cite this article:|
Sari RW, Pranata N, Sugiaman VK. Viability test of ethanol extract of beluntas (pluchea indica) leaves on In vitro fibroblast cells. Sci Dent J 2019;3:90-4
|How to cite this URL:|
Sari RW, Pranata N, Sugiaman VK. Viability test of ethanol extract of beluntas (pluchea indica) leaves on In vitro fibroblast cells. Sci Dent J [serial online] 2019 [cited 2020 Jan 21];3:90-4. Available from: http://www.scidentj.com/text.asp?2019/3/3/90/268999
| Background|| |
Tooth extraction is common in dental practice. In Indonesia, there is extremely high utilization of dental and oral health services for tooth extraction, reaching 79.6%. Dental extraction is a surgical procedure that deals with soft and hard tissues in the oral cavity. Pain is a side effect of extraction. Pain starts to disappear during the proliferation phase in the proliferation process of fibroblast cells. The healing process of extraction occurs in several phases, viz., inflammatory phase, proliferation phase (complete wound closure and epithelial formation occur in this phase), and maturation. The wound-healing process is strongly affected by the migration and proliferation of fibroblasts in the wound area, and fibroblasts play a key role in the repair process.
Pain due to extraction wounds can be treated using analgesic drugs, but alternative drugs with minimal or no side effects are remained unclear. Flavonoids are herbal active substances that can reduce pain caused by extraction wounds. Besides flavonoids, beluntas leaves also contain other active compounds that can accelerate wound healing, including alkaloids, saponins, polyphenols, tannins, sterols, sodium, citrus oils, amino acids, fats, calcium, magnesium, phosphorus, Vitamin A, and Vitamin C. The beluntas plant can not only aid in wound healing but also exhibit anti-inflammatory and antipyretic effects.,
Flavonoids can stop the production of prostaglandin; therefore, it reduces pain, renders antibacterial effects, functions as an anti-inflammatory agent, and exhibits a working mechanism for the inhibition of lipid peroxidation, which serves to reduce reactive oxygen species; hence, it can slow tissue death, increase vascularity and collagen, prevent cell damage, and increase DNA synthesis.
Tannins also exhibit an effect that can stimulate not only the production of fibroblast cells but also the formation of collagen tissues in wound healing. Tannins exhibit antimicrobial and antioxidant effects that can help prevent infections and fight free radicals; therefore, tannins accelerate the wound-healing process.
Beluntas leaves can be used as therapeutic agents for oral mucous tissue damage. To serve as therapeutic agents, beluntas leaves must satisfy the requirements for biocompatibility, i.e., must not cause irritation and toxicity to the body. Therefore, it is imperative to conduct a standardized study that can determine the effect of the beluntas leaves extract on cell viability involved in tissue healing, including fibroblast cells.
In this study, the 50% inhibitory concentration (IC50) value was determined, as well as the cytotoxicity effect of the ethanol extract of beluntas (P. indica) leaves on fibroblast cell cultures in was determined.
| Materials and Methods|| |
Ethanol extract of beluntas
A ± 10-year-old beluntas leaf sample was obtained from the experimental garden in Manoko Spice and Medicinal Plant Research Institute (Balittro), which was determined in the laboratory of identification and determination of the Faculty of Life Sciences, Institut Teknologi Bandung (ITB).
A crude beluntas leaf extract was prepared by the maceration method using 70% ethanol, which was carried out at the Aretha Medika Utama, Biomolecular and Biomedical Research Center.
The dilution of the ethanol extract of the beluntas leaf started with the preparation of a stock solution, which had a concentration of 500 μg/mL, followed by the preparation of a series of working solutions with concentrations of 250, 125, 62.50, 31.25, 15.63, and 7.81 μg/mL.
Phytochemical analysis by the Farnsworth method was carried out to identify the chemical groups of alkaloids, saponins, tannins, flavonoids, terpenoids, phenols, and steroids/triterpenoids.
The MTS assay was performed to measure the viability of fibroblast cells, which was based on the conversion of tetrazolium salts to colored formazan by the mitochondrial activity of living cells. The amount of the produced formazan depends on the number of cells that are feasible in the culture, which is measured in triplicate by a spectrophotometer at 490 nm, with 24 h of incubation.
The viability test of the beluntas leaf extract was carried out using confluent 70%–80% 3T3 BALB/c fibroblast cells and planted with a density of 5000 cells/well in 96 wells/plate using the cell culture medium of the Dulbecco's modified Eagle medium, 10% fetal bovine serum, and 1% antibiotic-antimycotic solution, followed by incubation for 24 h in an incubator at a temperature of 37°C and a CO2 level of 5%.
The old medium was removed and replaced with 200 μL of a new medium, and 20 μL of the extract with various series of concentrations was added to each well, followed by incubation for 24 h with a temperature of 37°C and a CO2 content of 5%. After 24 h, 20 μL of the MTS reagent was added to each well and incubated for 3 h at the same temperature and CO2 levels, and the cells were calculated on the basis of their absorbance and curve integration. Based on the number of viable cells, the cytotoxicity of a material can be classified [Table 1].
IC50 was calculated from the plot of concentration as a function of the percentage of viability.
The obtained data were first processed by a normality test, i.e., the Kolmogorov–Smirnov test. Second, one-way analysis of variance with α = 0.05 and an advanced test, i.e., the post hoc test (Tukey), were carried out. Finally, probit analysis was performed to process the IC50 data.
| Results|| |
Phytochemical test results of the beluntas leaf extract revealed the presence of alkaloids, saponins, flavonoids, phenols, terpenoids, steroids, and tannins [Table 2]. These results are in agreement with those reported previously.
[Table 2] and [Figure 1] summarize the results obtained from the viability test. Based on the classification of cell viability, the table data revealed that concentrations of 500, 250, and 125 μg/mL are classified as extremely toxic. By contrast, lower concentrations of 62.5, 31.25, and 15.63 μg/mL are classified as slightly toxic. A concentration of 7.81 μg/mL is classified as nontoxic.
|Figure 1: Relationship between the concentrations of the beluntas leaf extract on the viability of 3T3 BALB/c fibroblast cell|
Click here to view
Then, the probit IC50 test was performed, and the IC50 value (safe concentration) of the ethanol extract of the beluntas was 265.388 μg/mL [Table 3] and [Figure 1].
| Discussion|| |
Healing occurs in several phases, viz., homeostasis, inflammation, proliferation, and maturation. The wound-healing process is strongly affected by the migration and proliferation of the fibroblasts in the wound area, and fibroblasts play a key role in the repair process.,
Flavonoids in the beluntas leaves play a role in the anti-inflammatory process because it can shorten the inflammation time; therefore, proliferation can occur immediately and inhibit bleeding. The flavonoid activity can accelerate the wound-healing process, and it is supported by antioxidant mechanisms for the inhibition of the free radical activity.
Antioxidants can block the initiation of free radical and trigger the proliferation of fibroblast cells. Besides flavonoids, beluntas leaves also contain saponins, which can not only stimulate collagen formation but also increase the fibroblast density via the activation of TGF-β.,,
Phenols, alkaloids, and tannins can serve as antibacterial agents, and terpenoids are active ingredients that help to accelerate the formation of collagen fiber produced by fibroblast cells.
Hence, to develop materials for the natural treatment of postextraction wounds, materials must be subjected first to biocompatibility tests in accordance with the material requirements in dentistry, especially those used in the mouth. One test to determine the various properties of a dental material is a cytotoxicity test on tissues. To determine the cytotoxicity of the ethanol extract of the beluntas leaves (P. indica), a fibroblast cell was tested using the MTS assay.
Parameters for the cytotoxicity test are based on the absorbance value, i.e., if the cell color becomes thicker (purple), the absorbance value is higher, implying that more cells are alive; however, if the cell color fades, the absorbance value is lower, implying that several cells die.
The cell viability decreased after the administration of the extracts to cells, and significant differences between concentrations were related to the differences in the cell response to the concentration, including the number of active ingredients at different concentrations, indicating that the extract exhibits cytotoxic properties on fibroblast cells. The mechanism and intensity of cell death depend on material content or cell contact. Beluntas leaves contain eugenol, which is a phenol derivative. Eugenol can cause cell cytotoxicity, which can damage the protein structure by a number of physical and chemical elements.
Cells exposed to materials or extracts exceeding the peak of exposure cause cell death. It can cause toxicity to cells via different mechanisms, such as the destruction of cell membranes; prevention of protein synthesis; irreversible binding to receptors; inhibition of the polydeoxynucleotide elongation; and other enzymatic reactions.,,
In a particular cell, cytotoxic agents can also be metabolized without any observable effect although most cells experience necrosis when confronted with toxic compounds. Necrosis can occur when cells are exposed to conditions that are extremely different from their physiological conditions or when the compound content of the extracts can damage cell membranes. Necrosis starts from the disruption of the cell's ability to maintain homeostasis because it can cause the entry of water and extracellular ions. Intracellular organelles, especially mitochondria and all other cells, can swell and rupture (cell lysis).
As a result of the disruption of the plasma membrane, the cytoplasmic contents, including the lysosome enzyme, are released into the extracellular fluid. The activity of these enzymes in extracellular media can be used to determine the level of necrosis. Besides these factors, the dose, exposure duration, and mechanism of cytotoxic agents are other factors that can cause cell death.,,
The IC50 value for the ethanol extract of beluntas leaves on fibroblast cells is 265.388 μg/mL, which is categorized as safe because it does not interfere with the viability of fibroblast cells; however, further research is required to investigate the effect of the beluntas leaves extract on the proliferation of fibroblast cells.
| Conclusion|| |
A significant difference in the cytotoxicity effect between the concentrations of the ethanol extract of beluntas (P. indica) leaves on fibroblast cell culturesin vitro was observed. The beluntas leaf extract at an IC50 concentration of 7.81 μg/mL did not affect cell viability; hence, it is considered safe.
We would like to acknowledge the Aretha Medika Utama, Biomolecular and Biomedical Research Center Laboratory.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Data Center and Information Ministry of Health of the Republic of Indonesia. [Dental and Oral Health Situation]. Jakarta; 2014.
Widiyastomo W, Andari WK, Permatasari I. Effect of starfruit fruit juice (Averrhoa carambola
Linn.) On increasing the number of fibroblasts in Wistar mouse socket after tooth socket. Prodenta J Dent 2013;1:62-70.
Kartikasari ID, Soelistiono, Prihartiningsih., Effect of Salvadora persica Stem Extract on the Growth of Streptococcus
Bacteria after Isolation of Mandibular Third Molar Tooth Extract (in vitro
study). Kedokt Gigi Univ Gajah Mada. 2008;1-6. FKG UGM 2008;1-6.
Hafsari AR, CahyantoT, Sujarwo T, Lestari RI. Antibacterial activity test of beluntas
(L.) Less.) extract against Propionibacterium acnes
cause of acne. J ISTEK 2015;9:141-61.
Ibrahim N', Wong SK, Mohamed IN, Mohamed N, Chin KY, Ima-Nirwana S, et al.
Wound healing properties of selected natural products. Int J Environ Res Public Health 2018;15. pii: E2360.
Suranto A. The Enormity of Propolis to Cure Disease. Jakarta: Agro Media Pustaka; 2010.
Ahmed KA, Abdulla MA, Mahmoud FM. Leaf extract in experimental rats. Middle East J Sci Res 2012;11:1614-8.
Widowati W, Mozef T, Risdian C, Yellianty Y. Anticancer and free radical scavenging potency of Catharanthus roseus
, Dendrophthoe petandra
, Piper betle
and Curcuma mangga
extracts in breast cancer cell lines. Oxid Antioxid Med Sci 2013;2:137-42.
Miret S, De Groene EM, Klaffke W. Comparison ofin vitro
assays of cellular toxicity in the human hepatic cell line HepG2. J Biomol Screen 2006;11:184-93.
Heravi F, Ramezani M, Poosti M, Hosseini M, Shajiei A, Ahrari F, et al
. In vitro
cytotoxicity assessment of an orthodontic composite containing titanium-dioxide nano-particles. J Dent Res Dent Clin Dent Prospects 2013;7:192-8.
Widyawati PS, Wijaya CH, Hardjosworo PS, Sajuthi D. Evaluation of antioxidant activity of beluntas
) leaf extract based on differences in leaf segments. Rekapangan J Teknol Pangan 2011;5:1-17.
Li B, Wang JH. Fibroblasts and myofibroblasts in wound healing: Force generation and measurement. J Tissue Viability 2011;20:108-20.
Gonzalez AC, Costa TF, Andrade ZA, Medrado AR. Wound healing – A literature review. An Bras Dermatol 2016;91:614-20.
Muslihah K, Sumono A, Fatmawati DW. Cytotoxicity effect of pectin extract from coffea robusta (Coffe acenephora
) fruit peels on human dental pulp fibroblast cell. Pustaka Kesehatan 2018;6:173-8.
Laksmitawati DR, Prasanti AP, Larasinta N, Syauta GA, Hilda R, Ramadaniati HU, et al
. Anti-Inflammatory potential of gandarusa (Gendarussa vulgaris
Nees) and soursoup (Annona muricata
L) extracts in LPS stimulated-macrophage cell (RAW264.7). J Nat Remedies 2016;16:73-81.
Bahuguna A, Khan I, Bajpai VK, Kang SC. MTT assay to evaluate the cytotoxic potential of drug. Bangladesh J Pharmacol 2017;12:115-8.
Gunasekaran S, Vinot KT, Lakshamana SS, Suganya P, Rincy Y, Amrutha C. Screening ofin vitro
cytotoxic activity of brown seaweeds against hepatocellular carcinoma. J Appl Pharm Sci 2017;7:51-60.
Nejad SM, Ozgunes H, Basaran N. Pharmacological and toxicological properties of eugenol. Turk J Pharm Sci 2017;14:201-6.
Susanty A, Rimayanti R, Sukmanadi M. Antibacterial activity of the ethanol extract Pluchea indica
less leaves against Escherichia coli
by in vitro
. Veterinaria Medika 2008;1:29-32.
Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al.
Molecular mechanisms of cell death: Recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 2018;25:486-541.
Aslantürk OS.In vitro
cytotoxicity and cell viability assay: Principles, advantages, and disadvantages. In: Larramendy ML, Soloneski S, editors. Genotoxicity-A Predictable Risk to Our Actual World. London: Intech Open; 2018. p. 1-18.
Van Tonder JJ. Development of anIn vitro
Mechanistic Toxicity Screening Model Using Cultured Hepatocytes [PhD Thesis]. Pretoria: University of Pretoria; 2011.
[Table 1], [Table 2], [Table 3]