A study of cytotoxicity and proliferation of Cosmos caudatus Kunth leaf extract in human gingival fibroblast culture

Background: Post-extraction dental sockets clinically resolve within a period of 3-4 weeks. However, complete healing and bundling of gingival fibers may require several months. Medication is therefore required to accelerate the healing process. Cosmos caudatus (C. caudatus), a local plant with antioxidant properties and high calcium content, has the potential to promote wound healing while also reportedly capable of strengthening bone. Previous studies have demonstrated the effectiveness of C. caudatus as an alternative treatment for post-menopausal osteoporosis by investigating the dynamic and cellular parameters of bone histomorphometry. Purpose: The study aimed to examine the citotoxicity and proliferation of human gingival fibroblast cells culture after the application of C. caudatus extract. Methods: Cultures of human gingival fibroblast cells with 5x10 cell density were divided into two groups and placed in a 30-well culture dish. The control group contained human gingival fibroblast cell culture without extract, while the experimental group consisted of human gingival fibroblast cells culture with extract. The concentrations of extract were 1200 μg/ml, 600 μg/ml, 300 μg/ml, 150 μg/ml, and 75 μg/ml. A toxicity test was conducted and the optimum concentration evaluated using an MTT assay, while fibroblast numbers on were calculated days 1 and 2 by means of a hemocytometer. Research data was analyzed using a one-way ANOVA test. Results: No toxicity was found. The optimum concentration was 600 μg/ml and fibroblast proliferation was significantly higher in the experimental group compared to the control group, p=0.002 (P<0.05). Conclusion: C. caudatus leaf extract is non-toxic and increases the proliferation of human gingival fibroblast culture at an optimum concentration of 600 μg/ml.


INTRODUCTION
A wound is considered healed if tissue has regenerated to its original anatomic structure, function and within a reasonable period.Most wounds result from minor injuries, but some do not heal fully in a timely fashion.Several local and systemic factors can impede wound repair by disrupting the process of balance improvement, resulting in chronic wounds failing to healing. 1 Fibroblasts, cells in the connective tissue that affect the wound healing process, enter the wound area three days after extraction and became dominant after 6-7 days. 2 Fibroblasts will experience certain changes to the phenotype and become myofibroblasts that serve to retract the wound.Fibroblasts produce extracellular matrix, collagen primer and fibronectin which promote cell migration and proliferation.Fibroblasts derived from the undifferentiated mesenchymal cells produce mucopolysaccharides, amino acid glycine and proline, a basic ingredient of collagen fibers, that binds wound edges.However, in cases of significant bone damage, the natural repair processes within the body cannot restore its function or promote clinical improvement. 3osmos caudatus (C.caudatus), known as kenikir in Indonesia, had been used in eastern regions of the country to reduce high blood pressure.As well as acting as an anti-

Research Report
Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740.Accredited No. 32a/E/KPT/2017.Open access under CC-BY-SA license.Available at http://e-journal.unair.ac.id/index.php/MKGDOI: 10.20473/j.djmkg.v51.i4.p179-184 hypertensive, it possesses anti-diabetic, anti-inflammatory, bone protective, anti-microbial and anti-fungal properties. 4he polyphenol content of C. caudatus is high, as is the level of vitamin E, vitamin C, folic acid, β-carotene and polyphenols in the C. caudatus lycopene. 5C. caudatus also promotes oral health, especially as an antibacterial, as it contains polyphenol derivates such as flavones, flavonols, flavonones, catechins and isoflavones.The catechin derivate has antioxidant and anti-inflammatory properties.Other substances contained in C. caudatus include phytochemical flavonoids.Previous studies have shown flavonoids to have anti-inflammatory and antioxidant properties, the capacity to accelerate tissue regeneration and to act as a natural antioxidant that enhances the wound healing process. 6Another study stated that C. caudatus leaves contain terpenoids (essential oils), alkaloids and saponin. 7C. caudatus was studied in this experiment because it is readily available, widely consumed and used in the manufacture of drugs to enhance the process of postextraction healing.
Tooth extraction socket healing occurs through a secondary healing process that involves the formation of granulated tissue and subsequent covering by epithelium.C. caudatus leaf properties have proved capable of balancing the production of reactive oxygen species (ROS) and promoting cell growth, such as fibroblasts. 8his research was conducted as a preliminary study of the development of drugs applied to promote fibroblast proliferation.It followed the successive stages of drug development, namely: in vitro examination, experimentation on live subjects (in vivo), clinical trials and a market launch. 9The initial step of this study prior to analyzing the proliferation of human gingival fibroblast (HGF) culture was that of conducting a citotoxicity assay/ study. 10he study aimed to quantify the citotoxicity and proliferation of human gingival fibroblast cell culture after the application of C. caudatus extract.C. caudatus leaf properties were able to balance the production of ROS and promote cell growth such as fibroblast so that tooth extraction socket healing occurred through a secondary healing process that increased the proliferation of granulated tissue and the epithelium.

MATERIALS AND METHODS
This was an experimental laboratory study using a post-test control group design.The experiment involved C. caudatus leaf extract application to primary cell cultures of human gingival fibroblasts.
Preliminary cytotoxicity and optimum concentration tests were conducted using a colorimetric assay that measured the reduction of yellow 3-(4.5-dimethythiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) assay by mitochondrial succinate dehydrogenase.Five concentrations of extract, consisting of 1200 μg/ml, 600 μg/ml, 300 μg/ml, 150 μg/ml, and 75 μg/ml based on the IC 50, were tested.The optimum concentration for cell growth was found to be approximately 504.840 μg/ml. 11he MTT results did not demonstrate toxicity after 24 hours with an ELISA reader used to calculate cells spectrophotometrically with a wavelength of 620-650 nm based on formazan crystal absorbance values.
Extract concentrations of 600 μg/ml were selected because, at this concentration, viable cell numbers of HGF were optimal with no bias in spectrophotometric absorbance.These concentrations correspond to the proliferation of fibroblasts compared to ones below 600 μg/ ml because lower extract concentrations will not provide maximum results.
Two groups, a control group and an experiment group each containing five samples placed in 30-well culture disk, were used in this study, together with a monolayer HGF culture of 5x10 4 cell density.A control group was HGFcultured without extract application, while the experiment group was HGF-cultured with the application of 600 μg/ml extract.C. caudatus leaf extract was applied by means of a 1 ml pipette with the fibroblast numbers being counted after 24 hours and 48 hours.
Fibroblast cell counts were conducted using a hemocytometer after trypan blue staining.Cells were taken at the ratio 1:1 and resuspended with trypan blue stain and observed under a light microscope at 100x magnification.Cells were considered viable if they were single, clear and round.An overlap between two or more cells was counted as two cells.Non-viable cells were defined as those absorbing the color of trypan blue.The measurement results of the observations were added together and calculation values obtained for the subjects of research using the formula: 12

Number of cells = (A+B+C+D) 4 x x 2 x sample dilution
Note: Count the number of cells -both viable (unstained) and nonviable (stained) in each of the four corner quadrants (A, B, C and D).
Data were then analised statistically using one-way ANOVA.A p-value less than 0.05 was considered statistically significant.

RESULTS
All concentrations increased the number of fibroblasts.There were differences in formazan crystal formation between each concentration.This was due to the difference in the number of living cells in each extract concentration as presented in Figure 1.The optimum concentration was 600 μg/ml which produced a high level of HGF proliferation as shown in Table 1.Extract concentrations of 1200 μg/ ml produced a false positive result due to the dark color as shown in Figure 1A.
The proliferation of HGF culture in both the control group and experiment group at 600 μg/ml extract applications showed a higher density under light microscope examination in the experiment group on both the first and second days, as shown in Figures 2 and 3.
The number of fibroblast in experiment groups were higher compared to control groups.Anova statistical analysis showed that there were significant differences in average fibroblast numbers between groups, p=0.002 (p<0.05) as presented in Table 2.

DISCUSSION
This was an experimental in vitro study with an application of C. caudatus leaf extract on HGF cell culture.The study was carried out following a drug development sequence which comprised of in vitro examination, animal experiments (in vivo), clinical trials and preparation for marketing. 9Cell culture was obtained from human gingival cells because these could be cultured into primary cell line culture demonstrating a similar pattern of behavior to in vivo reactions compared to cell lines. 13Cell lines have been used for decades to produce culture, resulting in stronger cells often genetically and phenotypically different from their original networks which underwent morphological changes.Unlike cell lines, primary cells are isolated directly from the network, have a limited lifespan and expansion capacity.On the positive side, primary cells demonstrate normal cell morphology and retain many important observable markers and functions, and the data obtained from primary cells is more relevant for interpretation and can be generalized into an in vivo setting. 14his finding was in line with that produced by the research conducted by Wong et al 15 indicating that primary cells are more sensitive to drugs than cell lines and tend to survive at higher concentrations and proliferation.In contrast, the lifespan of primary cells was shorter at 16-24 hours after the administering of drugs at lower concentrations.The administering of 20 μM (6 μg/ml) cisplatin to primary cells resulted in a viability range of 64.0% cells, while the cell line with 33 μM (6.6 μg/ ml) cisplatin concentration produced a viability level of 71.6% cells. 15tract concentrations were set at 1200 μg/ml, 600 μg/ ml, 300 μg/ml, 150 μg/ml and 75 μg/ml.Dose reduction was based on a geometric series of measurements to evaluate the dose-cell response relationship to the exposure of the extract. 11All concentrations of C. caudatus extract were non-toxic as shown in Table 1.The concentration of 600 μg/ml was selected because it showed the highest number of proliferated living cells.Moreover, the absorbance value of formazan crystals at a concentration of 600 μg/ml could be read spectrophotometrically without bias caused by the extremely dark color.This concentration was suitable for fibroblast cell proliferation compared to those below 600 μg/ml because, according to the dose-response relationship, weaker extract concentrations will produce a lower number of living fibroblasts or will fail to produce optimum results.The concentration of 1200 μg/ml was rejected because it was found that significantly elevated proliferation rates would induce dense cell growth resulting in greater competition for nutrients in the media.As a result, the cells did not mature rapidly and experienced high mortality rates culminating in a false negative result.The possibility of such a result occurring also existed because the concentrated color of dense cells caused the spectrophotometric reading to indicate a higher level of absorbance.
Fibroblast proliferation was significantly higher in the experiment group compared to that in the control group, p = 0.002 (p <0.05).This was due to the active ingredients in C. caudatus leaf extract containing catechins, polyphenols, flavonoids, vitamins C and E and saponins. 9Schuck et al 16 , citing Weisburg et al ( 2004), stated that catechins do not induce oxidative stress in normal human cells originating in the oral cavity because catechins in extracts can reduce ROS   Polyphenol content promotes both anti-microbial and antifungal activities that play a role in maintaining the growth of cell cultures.The catechins present in polyphenols can reduce ROS levels in normal cells, while flavonoids stimulate fibroblast synthesis and are able to balance the production of ROS with antioxidant capacity that promotes fibroblast growth. 8ther ingredients found in C. caudatus include high levels of vitamin C and E. Flavonoids increase the effectiveness of vitamin C conducive to the formation of fibroblasts. 14Ascorbic acid, also known as vitamin C, is necessary for normal responses to physiological stressors and its requirement increases during injury or stress.Studies have shown that physiological stress produces excessive ROS leading to vitamin C playing the role of activating intracellular signaling which regulates fibroblast proliferation. 17erpenoids or essential oils, as well as polyphenol, promote antibacterial and antifungal activity.Fibroblasts are able to proliferate effectively because of the eugenol content of essential oils that damage the cell walls of bacteria causing damage to the cells themselves for example those of gram-positive bacteria, especially Streptococcus aureus.Phenolic compounds present in essential oils cause protein denaturation and bacterial cell death. 8aponins, active substances that act as anti-microbes, increase cell membrane permeability and have antioxidant propoerties. 18Saponins can stimulate the formation of a cellextracellular matrix by stimulating fibronectin synthesis in fibroblasts capable of promoting the wound healing process. 19The saponin content stimulating transforming growth factor-beta 1 (TGF-β1) can affect fibroblast growth factor (FGF) in fibroblast cells thereby promoting the formation of collagen fibers.When FGF is stimulated by TGF-β1 fibroblast proliferation will be increased. 20lkaloids represent one of the other active components in C. caudatus leaves with toxic properties that significantly affect physiological activity.Most alkaloids that have been isolated from plants are crystalline solids with a certain melting point. 21The toxic content of alkaloids in C. caudatus leaf extract was at small levels, caused no effect on cell culture and does not increase ROS but induced oxidative stress in normal cells.In this study, cell death occurred but there were also fibroblast proliferation that did not effect the results. 22he wound healing process is evident from several indicators, including an increase in the number of fibroblasts that will stimulate the collagen forming extra-cellular matrices.The proliferation of fibroblasts promoted by transforming growth factor-β (TGF-β) will increase collagen and fibronectin synthesis and promote extracellular matrix deposition. 20 the next stage, there is a decrease in endothelial cell proliferation and the number of fibroblast cells, but fibroblasts become more progressive in synthesizing collagen and fibronectin involving changes in the composition of extracellular matrix (ECM). 19The ECM provides a sub-layer for cell attachment and effectively regulates cell growth, movement and differentiation propagated by growth factors and cytokines namely platelet-derived growth factor, FGF, TGF-β and interleukin-1, interleukin-4, immunoglobulin-g produced by leukocytes and lymphocytes during collagen synthesis.Remodeling will commence after an extracellular matrix is formed. 20n inherent weakness of this study was the fact that dense fibroblasts in the culture plate can perish because of an over-proliferation of their cells.Further research needs to be undertaken by reducing the number of cells to less than 5x10 4 .Further study will be conducted to develop a herbal drug which promotes post-extraction tooth socket healing.It can be concluded that C. caudatus leaf extract is non-toxic to human gingival fibroblast culture resulting in a significant increase in fibroblast numbers.The optimum concentration of C. caudatus leaf extract to increase fibroblast proliferation is 600 μg/ml.

Figure 2 .
Figure 2. Microscopic picture of fibroblasts on the first day (40x magnification).(A) Control fibroblasts on the first day.(B) Treatment of fibroblast cells.

Figure 3 .
Figure 3. Microscopic picture of fibroblasts on the second day (40x magnification).(A) Control fibroblasts on the second day.(B) Treatment of fibroblast cells.

Table 1 .
Toxicity test results of C. caudatus leaf extract

Table 2 .
Statistical analysis results of fibroblast numbers in normal cells, while causing intracellular oxidative stress in cancer cells.This indicates that the cathechin effect on normal and cancer cells worked in different biochemical pathways that induce normal cell proliferation.