Therefore, this study was conducted to determine the effect of different types of surfactant used in the formulation of transfersomes on the entrapment capacity of the transfersomes and the effectiveness of binahong leaf extract to heal wounds. The Pharmacy Department is one of the 6 departments of the i3L School of Life Sciences that focus on medicine. Therefore, wounds can be defined as the presence of damage and/or disruption of the skin integrity, mucous membrane, and also organ tissue.
Wounds that occur on the skin can disrupt the function of the epidermis to protect the body. One of the most common ways to treat wounds is by applying a topical antibiotic to prevent wound infections. One of the ways to solve this problem is by encapsulating the active ingredients within a carrier such as liposomes that are able to encapsulate hydrophilic molecules within its aqueous phase as well as hydrophobic molecules within its lipid bilayer (Lammari et al. , 2021).
One of the main components of the transfersome is a surfactant, which has the ability to destabilize the bilayer of the vesicles, resulting in a deformation, so that it can increase the penetration of the transfersome into the skin, which will be related to the effectiveness of the active ingredients ( Khan et al., 2021).
Scope of the project
Objective / Aim
Problem formulation and Proposed Solutions
The presence of flavonoid was checked by reacting BLE with 2N NaOH solution while the appearance of yellow colored solution followed by loss of color to colorless after adding a 37% HCl solution confirms the presence of flavonoid (Khalid et al., 2018). Finally, the presence of saponin was checked by diluting BLE with 5 mL of hot water in which the solution was shaken vigorously for 10 seconds. TPC for the extract was measured by reacting BLE with 10% Folin Ciocalteu which was then incubated for 5 minutes.
Then 7.5% NaOH solution was added and further incubated for 2 hours before the measurement using a UV-Vis spectrophotometer (UV-Vis Spectrophotometer Shimadzu 1280) with a wavelength of 750 nm. Making transfersome was started by dissolving the soy lecithin and surfactant in ethanol 96% which was heated and mixed using a homogenizer until all the soy lecithin and surfactant were dissolved. Then the temperature of the solution was brought to 60oC to stabilize the weight of the solution before the addition of BLE while mixing with a homogenizer.
The solution was then sonicated for 1 h before the solvent was evaporated at 60 oC using a rotary evaporator (Buchi, R-100 Rotavapor, B-100 heating bath, V-100 vacuum pump, F-105 recirculating cooler) until a thin film formed. The solution was then incubated for 2 hours at room temperature and finally sonicated for 2 hours. In vitro testing was performed for each formulation as well as for the extract using a scratch assay against human keratinocytes (HaCaT) and mouse embryonic fibroblast (3T3) cells.
To initiate treatment, a linear scratch was made in each well using a sterile 10 µL micropipette tip. The cells were then washed using a Dulbecco's Modified Eagle Medium (DMEM) to eliminate any existing dead cells. The wound area was then measured using Image J Java software and the percentage of wound closure was calculated using the equation below (Allaw et al., 2021):
FINDINGS 3
Result
- Qualitative Phytochemical Screening of Binahong Ethanolic Extract
- Quantitative Phytochemical Screening of BLE
- Entrapment Efficiency of Transfersome
- In Vitro Efficacy Testing Using Scratch Assay
The results for %EE for each formulation were calculated using a formula stated earlier in the method, which can be seen in Table 4. The calculation was based on the amount of trapped and free flavonoid as one of the main bioactive compounds with wound healing activity . From the figure, it can be seen that F2 has the highest %EE, which indicates that F1 has the highest ability to encapsulate the active ingredients, followed by F3 and F1.
Subsequently, the obtained data were analyzed using one-way ANOVA and Tukey HSD post hoc test to analyze the significance of the difference between each formulation, the result of which can be seen in Figure 1. However, the p-value is >0 .05 between F2 and F3 meaning there is no significant difference between those 2 formulations. The area of the wound represented by the cells was measured using Image J software, in which the rate of wound closure was calculated using a previously reported formula.
Wound closure for each treatment solution can be seen in Figure 2 and Figure 2B towards human keratinocyte cells (HaCaT) and rat fetal fibroblasts (3T3), respectively. After all data were collected, data analysis was performed using two-way ANOVA in which repeated measures were used after the same wells were observed repeatedly during the 48 hours of observation and followed by Tukey HSD post hoc test to analyze the significance. of the difference between each formulation. From the figures below, it can be seen that among all the formulations for both cells, none of them has a significant difference (p > 0.05) from each other, including the extract that acted as a control.
Looking at the value alone, it can be seen that F2 shows the highest % wound closure compared to the other formulations as well as the extract, especially on the HaCaT cells (Figure 2A) which shows a higher closure than the extract starting at 24 hour observation after the initial treatment. While for the treatment against 3T3 cells (Figure 2B), each treatment solution shows similar results starting from the beginning at 0 hours until the last observation 48 hours after the initial treatment, with F2 showing the highest wound closure rate at the 48 hour time point. compared to the other treatment solutions.
Discussion
Of the 3 formulations tested in this experiment, F2 shows the highest %EE, followed by F3 and F1 in last place. Mixing two surfactants together can make the HLB value of the mixture higher than tween 80, but still lower than tween 80, which can result in a better %EE than that expressed by tween 80. Another possible reason for a lower %EE in the transfersome that was generated using the 80 spacer is its effect on transfersome fluidity.
According to El Maghraby et al. 2004), the addition of tween 80 to a phospholipid can reduce the melting temperature (Tm) by 0.84oC, while the addition of span 80 reduces it by 7.3oC, which will be related to the fluidity of the transfersome, since the lower the Tm , the higher the fluid structure. Knowing that all the transfer reformulations were able to encapsulate the extract, specifically the flavonoid in the extract, an in vitro efficacy against HaCaT and 3T3 cells was performed using scratch assay. In line with the entrapment efficiency test results, F2 with the highest amount of flavonoid entrapped in the transfersome showed the highest % wound closure 48 hours after the initial treatment compared to the other formulations as well as the extract solution used as a control in both cells used.
In addition to the amount of bioactive ingredient trapped in the transfersome, a drug release property of the transfersome for each formulation has also become another major factor affecting the efficacy of the active ingredient to effect wound healing. Since there was one study that found a difference in the drug release of each surfactant formulation, these insignificant data may be obtained due to some errors during the work, as well as possible cytotoxic reactions caused by the contents of the transfersome. , especially of the surfactant. himself. It may be caused by the difference in the types of surfactants used in the manufacture of the drug.
This may also be related to the result of treatment with F3, which shows a similar but slightly higher % of wound closure than F1, which uses span 80 and tween 80 with the same ratio of surfactant and soy lecithin as the F1 formula. Another cell used was 3T3, as the results are shown in Figure 2B. Slightly different from the results of HaCaT cells, as treatments F1, F2 and F3 were all higher in % wound closure compared to BLE, despite the non-significant difference between all of them. However, the results of the previous study were obtained for the reaction on fibroblast cells isolated from the lung due to the limited research articles related to the cytotoxicity study of span 80.
CONCLUSION AND RECOMMENDATION
SELF REFLECTION
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