Dzun Haryadi Ittiqo1*, Sri Mulyani Pranmayanti1, Melati Permata Hati1, Yuli Fitriana2,
Alvi Kusuma Wardani2
1Department of Technology Pharmacy, Faculty of Health Sciences,
University of Muhammadiyah Mataram, Indonesia, 83127.
2Department of Phytochemical and Pharmacognosy, Faculty of Health Sciences,
University of Muhammadiyah Mataram, Indonesia, 83127.
*Corresponding Author E-mail: dzun.haryadi@gmail.com
ABSTRACT:
High glucose level conditions cause many fatal complications, such as the increased risk of developing gangrene. The incidences of amputation worldwide have happened of 25% and 14.3% mortality after amputation. Patients with diabetic gangrene are often treated with a broad-spectrum antibiotic. However, nowadays antibiotic-resistant bacteria are emerging problems to cure the infection in diabetic gangrene. Scientists are looking for natural compounds to develop new medication as an alternative antibacterial. Kadara (Caesalpinia bonduc) is a plant from Dompu, West Nusa Tenggara. Flavonoids, the primary constituent of Caesalpinia bonduc seeds, are responsible for broad-spectrum antibacterial action. Flavonoids are poorly soluble in water, so as the solution for increasing the stability of flavonoids, it needs development of dosage form in the self-nanoemulsion drug delivery system (SNEDDS). This study aims to determine the effectiveness of the Snedds ethyl acetate fraction of the extract of the seeds of the fruit (Caesalpinia bonduc) against diabetic gangrene bacteria. This research begins with maceration to obtain a thick extract followed by fractionation, then make the SNEDDS formula using the trial-error method. Characterization is carried out, and the antibacterial activity test is carried out. The results showed that the SNEDDS formula that met the criteria was obtained from a comparison of tween 80 and PEG 400 (7:2) with an emulsification time of 29.67±0.58 seconds, a transparent emulsion formation medium, and a particle size of 16.8nm. The results of the bacterial inhibition test showed that the SNEEDS formula was not significantly different from the tetracycline positive control (*p>0.05), with the inhibitory diameter values of 15.67±2.31mm and 15.67±1.15mm.
KEYWORDS: SNEDDS, Kadara, Gangrene, Antibacterial, Etil Acetate.
INTRODUCTION:
Treatment of diabetes mellitus (DM) is a chronic treatment even for life. High blood glucose levels can cause fatal complications, one of which is gangrene1. Epidemiological studies report more than one million amputations in people with diabetes each year with an amputation rate of 25%, and as many as 14.3% will die within a year after amputation2.
According to the research results conducted by Media Yutika et al. in 2015, after a qualitative test was carried out on pus from gangrene wounds with the mannitol test medium, the results were positive for staphylococcus Epidermidis bacteria and with Urea Agar media containing E. coli bacteria3. One of the holistic management of diabetic gangrene is the administration of broad-spectrum antibiotics, including gram-negative and gram-positive bacteria2. Based on several studies, diabetic gangrene patients experience resistance to several antibiotics, so alternative traditional drug therapies have begun to be developed to suppress the occurrence of resistance1.
Indonesia has the second-largest tropical forest globally and a high diversity of plants. In addition, it is known as one of the seven "megabio-diversity" countries, with a wealth of ecosystem biodiversity, genetic resources, abundant species, rich ethnic and cultural diversity. Each ethnic has its local wisdom, including using plants as traditional medicine4. West Nusa Tenggara Province is one of the provinces rich in medicinal plants. It is proved by the results of the Research on Medicinal Plants and Herbs (RISTOJA) in 2017 on the Bima ethnic, Korean ethnicity, Donggo ethnicity, and Dompu ethnicity, produced 290 medicinal plants and 102 phytochemical samples, one of which is Kaddara seeds (Caesalpinia bonduc) in Dompu Regency4.
The compounds in the flesh of the seed from the fractionation screening with aqua dest, N-Hexane, and ethyl acetate were all positive for flavonoid compounds5. Flavonoid compounds have several therapeutic properties such as antipyretic, antidiuretic, anthelmintic and antibacterial, anti-anaphylactic, antidiarrheal, and antiviral6,7. Although it has various pharmacological properties, it exhibits low oral bioavailability due to poor water solubility. There are many promising strategies to overcome this problem, such as product development through pharmaceutical technology. For example, nanotechnology combines active ingredients that are difficult to dissolve in water, namely self-nanoemulsion drug delivery system (SNEDDS)8.
The SNEEDS formulation can increase the penetration of the active compound into the body to increase the activity of the active compound, increase the effectiveness of treatment and avoid the occurrence of resistance1. Based on this, it is essential to research the Effectiveness of the SNEDDS Formula ethyl acetate fraction of Kadara Dompu Fruit Seed Extract (Caesalpinia bonduc) Against Diabetic Gangrene Bacteria.
MATERIALS AND METHODS:
Materials:
The material used in this study was Kadara fruit flesh obtained from Dompu Regency, NTB, Media Muller Hinton Agar (MHA), ethyl acetate (Merck), Tween 80 (Brataco), PEG 400 (Brataco), 70% ethanol (Merck), N-hexane (Merck), aqua dest (Brataco), VCO (Virgin Coconut Oil) (Brataco). Epidermidis staphylococcus bacteria, and E. Coli.
Research Methods:
Manufacture of the extract of the flesh of the seeds of the fruit of Kadara:
A total of 120grams of simplicia powder of fruit flesh sorted dry is soaked in 70% ethanol solvent for 3x24 hours, then strained then evaporated with a water bath to obtain a thick extract9,10.
Ethyl acetate fraction of Kadara Fruit Seed Flesh Extract:
Weighed 5grams of thick extract of the fruit's flesh and dissolved in 70% ethanol as a solvent, then partitioned with n-hexane and ethyl acetate in a separating funnel. The partition results were separated between ethanol, ethyl acetate, and n-hexane, then each partitioned filtrate was evaporated in a water bath at 50°C9.
Snedds Formula Making and Evaluation:
The composition of oil, surfactant, and co-surfactant was compared using the Trial and Error method, which was then evaluated:
a. Clarity Test:
An amount of 100.0L of the optimum formula was added with distilled water to a final volume of 5.0mL. The mixture was homogenized with the help of a vortex for 30 seconds. The results of homogeneous mixing and providing a clear visual appearance are an early sign of the success of making SNEDDS11,12.
b. Emulsification time:
The emulsification time was calculated for the SNEDDS formula in two media, namely distilled water and artificial gastric fluid without pepsin. The 250 mL medium was conditioned on a magnetic stirrer at a speed of 100rpm. A total of 1mL of SNEDDS Formula was dripped into the medium rapidly. Observations were made on time required by the formula containing the Ethyl Acetate Extract Fraction to form a nanoemulsion characterized by being thoroughly mixed in the media13,14,15.
c. Micelle Diameter Measurement:
One hundred microliter SNEDDS of tapioca fruit was chosen as the best formula from the clarity and emulsification time evaluation results. The diameter of the micelles formed was measured by adding aqua dest until the emulsion volume was 25mL. The size of this emulsion was observed for micelles (droplets) and their size distribution using the Particle Size Analyzer (PSA). The zeta potential of nanoparticles was measured using the NocompTm 380 ZLS Submicron Particle Size, which was carried out using a Flow cell in automatic mode16,17,18.
Test the effectiveness of SNEDDS Ethyl Acetate Fraction of Kadara Fruit Seed Extract against Diabetic Gangrene Bacteria:
a. Preparation of bacterial: suspension is by inserting 3ml of sterile aqua dest into each test tube. The inoculated colonies of staphylococcus epidermidis and E. coli were taken using sterile nose wire and then inserted into each test tube containing sterile aqua dest, then homogenized, or shaken until it forms the same turbidity as 0.5 McFarland solution19,20.
b. Test: Prepare 8 petri dishes containing MHA media, each marked using a marker with the words replication I, II, III, and control for each bacterium, then take the bacterial suspension that has been made using a sterile cotton swab by wetting the cotton swab sterile and then scratched all sides of the MHA media. After all, the media were scratched using a suspension of Staphylococcus epidermidis bacteria and E. coli, then allowed to stand for 15 minutes. The bacterial suspension was absorbed in the MHA media. After 15 minutes, each MHA media that replicated I, II, and III was made a hole in the middle of the MHA media. Then a 100 l SNEEDS sample of the ethyl acetate fraction of the seed flesh of the fruit (Caesalpinia Bonduc) was inserted into the hole in the MHA media. Then, for each bacterium’s positive and negative control, a hole was made in the negative control section, then 100 l of sterile distilled water was added as a negative control. Moreover, a tetracycline disc antibiotic was used for positive control, placed in the positive control section, and incubated the petri dish at 37°C. C for 24 hours then observed the inhibition zone formed, indicating no bacterial growth21,22,23.
RESULT:
Fractionation of Kadara Fruit Seed Flesh Extract:
The Simplisia powder of the content of the seeds of the fruit, which was obtained as much as 120grams, produced a thick extract of 69.82grams. This extract was purified by fractionation technique using the partition method (liquid-liquid), where the fractionation process aims to make the results obtained from the extract simpler because the initial extract is very difficult to separate with a single separation technique. Therefore, the initial extract needs to be separated into fractions with the same polarity and molecular size.
Fractionation using ethyl acetate and n-Hexane solvent (6:4).
The choice of this solvent was based on the difference in polarity of the compounds present in the extract so that the compounds in the extract can separate according to the level of polarity. The amount of Ethyl acetate fraction produced after the separation and evaporation process was 17.60mg. Flavonoids are secondary metabolites primarily found in plants with antimicrobial activity. Most flavonoids have good solubility in ethanol solvents and are very slightly soluble in ethyl acetate and n-hexane solvents. So that in the process of fractionation, ethyl acetate and n-hexane can attract compounds other than flavonoids in ethanol24.
SNEDDS Formula and Evaluation:
The SNEDDS formula was made with 3 components, namely VCO as the oil phase, tween 80, and PEG 400 as surfactant and cosurfactant, with the ratio shown in Table 1 with a dosage strength of 200mg/mL.
All formulas were tested for emulsification time and clarity in AGF medium to determine which formulas met the criteria. The results of the emulsification time observation showed that all formulas except formulas I and VI had emulsification times under 60 seconds. The formula that meets the emulsification time standard with the smallest standard error value is formula VII with a value of 29.67±0.58 seconds with a ratio of t between 80 and PEG 400 7:2. The value of emulsification time in the SNEDDS formula is significant because it determines when the preparation emulsifies spontaneously in the gastrointestinal tract with mild agitation in less than 60 seconds. The faster the emulsification time, the faster the dispersion of the drug in the gastrointestinal tract, and the shorter the onset of the drug to effect due to the absorption process immediately after emulsification25.
The clarity of a SNEDDS formula is crucial because it can represent the size of the droplets formed by looking at the clarity of the nanoemulsion. This study obtained that the clarity of formulas I to VI was still lacking or looked cloudy. Formula VII forms a clear solution in the medium; formula VII can form micelles with tiny sizes when in the aqueous phase. In contrast, the formula that produces a cloudy medium still forms giant micelles.
Table 1. Evaluation Results of SNEDDS Formula Ethyl Acetate Fraction of Kadara Fruit Seed Flesh Extract.
|
Formula |
Comparison of composition |
SNEDDS Characteristics |
||||
|
VCO |
Tween 80 |
PEG 400 |
Emulsification time (detik) |
Clarity |
Particle size (nm) |
|
|
I |
1 |
5 |
1 |
140,67±21,01 |
a little clear |
|
|
II |
1 |
5 |
2 |
30,67±0,58 |
a little clear |
|
|
III |
1 |
5 |
3 |
25,33±7,51 |
a little clear |
|
|
IV |
1 |
6 |
2 |
40,33±7,09 |
a little clear |
|
|
V |
1 |
6 |
3 |
46,33±4,93 |
a little clear |
|
|
VI |
1 |
7 |
1 |
82,67±4,51 |
cloudy |
|
|
VII |
1 |
7 |
2 |
29,67±0,58 |
clear |
16,8 |
Table 3. Test of Differences Between Treatment Groups
|
|
(I) Sample |
(J) Sample |
Mean Difference (I-J) |
Std. Error |
Sig. |
|
Tukey HSD |
Sample |
Positive Control |
.16667 |
1.30323 |
.991 |
|
Negative Control |
15.66667* |
1.30323 |
.000 |
||
|
Positive Control |
Sample |
-.16667 |
1.30323 |
.991 |
|
|
Negative Control |
15.50000* |
1.59613 |
.000 |
||
|
Negative Control |
Sample |
-15.66667* |
1.30323 |
.000 |
|
|
Positive Control |
-15.50000* |
1.59613 |
.000 |
The result of observing the characteristics of the SNEDDS ethyl acetate fraction of the seed extract of the fruit of the seeds can be concluded that the formula with a ratio of tween 80 and PEG as a surfactant and cosurfactant which is capable of producing fast and small micelle formation is formula VII (7:2). This formula was chosen to be the best formula that satisfies the characterization; then, the micelle size was tested using a Particle Size Analyzer.
The size of the emulsion micelles is a characteristic of the emulsion that can determine the rate of drug release and absorption. The maximum micelle size formed in the SNEDDS optimal formula for the ethyl acetate fraction of the seed flesh of this fruit, shown in Figure 1, is 16.8 nm with a PI (Polydispersity) value of 0.113. The droplet size value is in the nanometer range, and the PI value is below 0.5. A PI value less than 0.5 indicates that the droplet size distribution is even. Meanwhile, a PI value of more than 0.5 indicates a heterogeneous dispersion26.
Pigure 1. Results of Observation of Micelle Size Formula SNEDDS
Observation of Antibacterial Activity SNEDDS Ethyl Acetate Fraction of Kadara Fruit Seed Flesh:
The ethyl acetate fraction of the fruit flesh of the seeds in the SNEDDS formula was tested for antimicrobial activity against bacteria that generally cause gangrene in people with diabetes mellitus, namely Escherichia coli and Staphylococcus epidermidis. The positive control used was tetracycline and negative control Basis or SNEDDS component without ethyl acetate fraction. Escherichia coli and Staphylococcus epidermidis bacteria are normal flora bacteria on the skin's surface. However, if there is damage to the skin's barrier, these bacteria will cause disease.
This effectiveness measured the diameter of the inhibition of bacterial growth after being given the SNEDDS of the Kadara fruit fraction as shown in Table 2. The results showed that the effectiveness of the SNEDDS of the Kadara fruit fraction against E.coli was 15.67±1.15 mm with positive control of 17 mm and against S.epidermidis was 15.67±2.31 mm with positive control of 14 mm.
Table 2. SNEDDS Formula Effectiveness Test Results
|
Barrier Diameter(mm) |
|||
|
SNEDDS Formulas |
Positive Control |
Negative Control |
|
|
Staphylococcus epidermidis |
15,67±2,31 |
14 |
0 |
|
Escherichia coli |
15,67±1,15* |
17 |
0 |
Based on the statistical test using ANOVA with post hoc test technique to see the differences in the variables that have differences, the results show the effectiveness of the inhibitory power of the SNEDDS formula against S. epidermidis and E.coli bacteria is not significantly different from the positive control (p> 0.05). While the negative control, there was a significant difference both with the positive control and with the SNEDDS formula (p<0.05). So it can be concluded that the SNEDDS preparation has the same effectiveness as tetracycline antibiotics as a positive control against S. epidermidis and E. coli bacteria that cause diabetic gangrene. The results of statistical tests are as in table 3.
CONCLUSION:
1. The SNEDDS formula was obtained from the ratio of tween 80 and PEG 400 (7:2) with an emulsification time of 29.67±0.58 seconds, a transparent emulsion formation medium, and a particle size of 16.8 nm.
2. The activity of SNEDDS against S. epidermidis and E. coli was not significantly different from the positive control of tetracycline (p>0.05). While for the negative control, there is a significant difference (p <0.05).
CONFLICT OF INTEREST:
The author(s) declare(s) that there are no conflicts of interest regarding the publication of this article.
ACKNOWLEDGMENTS:
The author would like to express gratitude to:
1. LPPM (Institute for Research and Community Service) Muhammadiyah Mataram University for funding support so that this research can be completed;
2. Program and Implementation of Muhammadiyah Research Grants Batch V 2021 for funding support so that this research can be completed;
3. The Dean of the Faculty of Health Sciences, Muhammadiyah Mataram University for the support so that this research can be completed on time;
4. Drug, Food, and Cosmetic Testing Laboratory, Islamic University of Indonesia, Yogyakarta.
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Received on 14.02.2022 Modified on 29.08.2022
Accepted on 15.12.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(4):1823-1827.
DOI: 10.52711/0974-360X.2023.00299