Effect of papaya flower ethanolic extract ( Carica papaya ) on the time kill of tetracyclin against Escherichia coli

Papaya ( Carica papaya ) is found throughout Indonesia. Papaya flowers contain saponin, tannin, alkaloid, and flavonoid compounds which functions as antibacterial. Escherichia coli is a Gram-negative rod bacteria that could be found in the human large intestine as a cause of diarrhea. The study aimed to determine the effect of papaya flower extract and a combination of papaya flower extract with tetracycline antibiotics using the smallest concentration capable of inhibiting 50% of bacterial growth against Escherichia coli bacteria ATCC 25922 using the time-kill test method. This study used the method of Minimum Inhibitory Concentration (MIC) and time-kill . The MIC of the ethanol extract of papaya flower was 2 mg/mL. The time-kill test showed that the death phase was occurred at 4 - 24 hours. The bacteriostatic effect of the extract was obtained at 0 hours to 24 hours, while the antibiotic and its combination with the extracts had a bactericidal effect at 8 hours and 24 hours.


Methods
The use of concentration in inhibiting bacterial growth is based on the Minimum Inhibitory Concentration (MIC) value with the percentage of inhibitor formula. The bacterial inhibitor evaluation was based on the log10 CFU/mL Time Kill-Test data curve; which was seen on the growth phase of bacteria.

Raw material preparation
Criteria of the papaya flowers were green and white flowers, perfectly shaped into flowers, small and large circles, contains mucus, and do not contains harmful or toxic substances. Papaya flowers that have been collected as much as 2 kg and then washed with tap water until clean then dried in the sun and obtained a papaya flower weight of 306 gram, grinded then mashed by pounding it into powder simplicia, obtained weight of papaya flower powder simplicia of 275 gram. The simplicial was then stored in the closed and labeled container, at room temperature (Hamida et al., 2022).

Plant extraction
Papaya flower extract was made by maceration method. Weigh the simplicial powder as much as 200 gram with an analytical balance, then the simplicia is mixed with 600 mL of 96% ethanol in a dark container, then homogenized by stirring for 6 hours and let stand for 18 hours. The top layer obtained is a mixture of solvents and active compounds. The remainder from the first maceration is then remaceration twice. Every day the papaya flower extract is stirred evenly (Hamida et al., 2022).

Papaya flower powder phytochemical test
Papaya flower powder weighed as much as 5 gram. Add 50 mL 96% ethanol and let stand for 15 minutes. Samples were taken as much as 1 mL and put in each test tube for phytochemical tests (Hasibuan et al., 2020).

Identification of flavonoids
Papaya flower extract was taken as much as 1 mL then mixed with 3 mL of 70% ethanol after that homogenized and heated for 2-3 minutes. Then filtered and the filtrate obtained was added with 0.1 gram of Mg and 2 drops of concentrated HCl. The result is said to be positive if the color changes to red (Hasibuan et al., 2020).

Identification of saponins
Papaya flower extract was taken as much as 1 mL and added with 10 mL of distilled water later heated for 15 minutes after that homogenized waited for 5 minutes. The formation of stable foam indicates presence of saponin. (Hasibuan et al., 2020).

Identification of steroids
Papaya flower extract was taken as much 1 mL then added 3 mL of 70% ethanol, 2 mL of concentrated sulfuric acid, and 2 mL of anhydrous acetic acid. A positive result is if there is a color change from purple to blue or green (Hasibuan et al., 2020).

Identification of tannins
A total of 1 mL of papaya flower extract was taken and heated for 2 minutes after that added 2-3 drops of 1% FeCl3. The occurrence of a color change with the formation of brown or blackish blue indicates the presence of tannins (Hasibuan et al., 2020).

Identification of phenolic
A samples of papaya flower extract that have been allowed to stand can then be taken in amounts of up to 1 mL. After that, add 1% NaCl solution and 10% gelatin solution, each in an amount of 1 mL. Homogenize, then let stand. Observe the presence of white precipitate in the sample (Hasibuan et al., 2020).

Preparation of 0.5 mac farland standard solution
The composition of the McFarland 0.5 solution was 9.95 mL of 1% H2SO4 and 0.05 mL of 1% BaCl2 solution. Making 1 mL of 1% H2SO4 and then dissolving it with 10 mL of distilled water, while making 1% BaCl2 solution by weighing 0.1 BaCl2 then dissolved in 10 mL of distilled water. The McFarland solution was made by taking 9.95 mL of H2SO4 and then mixed with 0.05 mL of BaCl2 solution (Sari et al., 2021).

Media preparation Preparation of Mueller Hinton Agar (MHA) Media
MHA media weighed as much 38 grams and dissolved with 1 liter of aquadest, the mouth of the erlenmeyer covered with a cotton swab, and then heated until completely dissolved. The MHA medium was sterilized in an autoclave at 121°C for 15 minutes and cooled to 45°C. The cooled media was put into a sterile Petri dish and allowed to solidify (Lintang et al., 2020).

Inoculation of the Test Bacterial
Escherichia coli were inoculated on nutrient agar slant tubes (NAS) and nutrient agar plates (NA) by scratching 1 ose containing E.coli aseptically, by which the needle was to bring the mouth of the test tube and the edge of the petri dish closer to the Bunsen flame when the needle was scratched. The test tube was closed again with cotton and the petri dish was closed again and then incubated in an incubator at 37°C for 24 hours (Suhaillah & Santoso, 2018).

MIC Determination
MHB medium which had contained a bacterial culture by equalizing it with 0,5 Mcfarland's medium. The papaya flower extract were dissolved by using 1 ml of 100% DMSO and the antibiotic solubles by adding sterile aquadest. The second series of concentrations were diluted using a 10-fold dilution. The 24 cuvettes added as much as 110 µL of MHB medium contained E.coli bacteria culture with 90 µL of series of antibiotic concentrations (8,4,2, and 1 µg/mL) and extracts (2.1,0.5, and 0.25 mg/mL) placed in 4 cuvettes sequentially. The volume of each cuvette was 200 µL, each treatment was added to the cuvette and repeated 3 times with the volume according to the concentration (Natasya, 2019).
The sample in the cuvette was measured using a UV-Vis Spectrophotometer with a wavelength of 595 nm absorbance. Sample calculations were performed before incubation and after incubation for 24 hours. The formula for determining percentage of inhibition can use : (1) In this research, the determination of MIC was based on the lowest concentration that could inhibit the growth of the test bacteria, namely > 50% (Nurkanto, 2012).

Time-kill Test Method Treatment of time-kill test
Bacterial culture on MHB medium was prepared and then equilibrated to 10 6 CFU/mL (1:150 dilution of 0.5 McFarland). Each test tube contains 9 mL of MHB which has been added with papaya flower extract, tetracycline antibiotics along with a combination of papaya flower extract and tetracycline antibiotics (1/4 MIC : 1/4 MIC) then added with 1 mL of E.coli bacteria (Figure 1). Incubation for 24 hours at 37°C. There are 3 controls used in this test, namely growth, media and NaCl sterility. Each sample was repeated 3 times (April et al., 2016). Information : • T.1 : 9 mL of MHB and Extract + 1 mL of Bacterial Culture • T.2 : 9 mL of MHB and Antibiotics + 1 mL of Bacterial Culture • T.3 : 9 mL of MHB and a combination of extracts and antibiotics + 1 mL of bacterial culture • T.4 : 9 mL MHB + 1 mL Bacterial Culture (Growth Control/Negative Control)

Calculation of bacterial colonies
Each sample at 0, 4, 8 and 24 hours was taken as much as 100 µL and diluted 10-fold with 0.9% NaCl. 100 µL sample was taken then spread inoculated in a cup containing MHA media and incubated at 37°C for 24 hours. Calculation of bacterial colonies using Colony Counter (Natasya, 2019).

Figure 1. Time-Kill test treatment in each test tube
In each petri dish test that contained bacterial colonies, it was carried out 3 times and counted between 30-300 CFU/mL ( Figure 2) (Basri & Khairon, 2012). The number of bacterial colonies can be calculated using the formula: CFU mL = bacterial colonies ∶ 1 concentration × volume sample inoculation (2) (Natasya, 2019).

Time-kill Curve
Time-kill curve are constructed with "X" as the sample time and "Y" for the number of bacterial colonies in log10 CFU/mL. Curve data contains growth control data, papaya flower extract, tetracycline antibiotics and a combination of papaya flower extract and tetracycline antibiotics. The purpose of the time-kill curve is also to determine the growth phase of bacteria, including the lag phase/bacterial adaptation phase, the log phase, the stationary phase (experiencing a fixed amount of bacterial growth) and the Death/death phase (Sharah et al., 2015). According to (Silva et al., 2011), the effect on bacteristatic had a decrease of < 3 log10 CFU/mL and bactericidal had a decrease of > 3 log10 CFU/mL.

Data Analysis
The data obtained were analyzed statistically with the SPSS version 26.0 program and then the normality of the data was seen using the Shapiro-Wilk test and the variance of the data using the Levene test. If the data distribution is normal, then the analysis is continued by using the One Way ANOVA and Post Hoc Tukey parametric statistical tests. If the data is not normal, then a non-parametric test is performed using Kruskal-Wallis. From this test, it can be identified whether there is a significant difference in the results of the time-kill test.

RESULT AND DISCUSSION
Carica papaya flower based on the results of phytochemical tests, the data in Table 1 showed the characters of the extract. Carica papaya flower the results of phytochemical tests, it was found that the of papaya flowers (Carica papaya) contained alkaloids, flavonoids, saponins, triterpenoids, phenolic compounds and tannins (Table 1 and Figure 3). The phytochemical characters was affected by environmental growth factors, by which the physiological and biochemical properties of papaya flowers were dependence (Venkatachalam, 2019). Minimum Inhibitory Concentration is a dilution method in which the growth of test microorganism is inhibited by a minimum concentration based on the time before and after incubation. The MIC value was determined based on the percentage value of inhibitors of papaya flower extract and tetracycline, which was the lowest concentration obtained a percentage of inhibitors > 50% of the bacterial growth (Nurkanto, 2012).

Figure 3. Result of phytochemical screening: (a) flavonoid; (b) phenolic; (c) triterpenoid; (d) steroid; (e) tannins; (f) saponins; (g) alkaloid; (h) dragendorf; (i) wagner
Based on data on the percentage of bacterial inhibition ability in Table 2 using MIC50, which is the concentration used to inhibit 50% of bacterial growth (Solanki et al., 2015), it was found that papaya flower extract and tetracycline antibiotics have inhibitory activity on E. coli bacteria. The concentrations used for testing using the time-kill test method were MIC concentrations of 2 mg/mL in extracts and 1 g/mL in antibiotics. Time-kill test is the most appropriate method in determining bactericidal or fungicidal effect. This method is a powerful means of obtaining dynamic information between microbial strains and antimicrobial agents. The time kill test proves antimicrobial effect is concentration or time dependent (Rodríguez-Hernández, 2017). Maintenance time-kill was determined by sampling control and tube media containing antimicrobial agents in the incubation intervals (0, 4, 8, 10, 12 and 24 hours) and growth of colonies that spread on plate media. Interpretation of time-kill data was carried out on a timekill curve to measure decrease or increase in number of bacteria between combinations compared to the most active single antimicrobial agent (Pfaller et al., 2004).
Bacteristatic and bactericidal effects, as well as the growth phase of papaya flower extract and tetracycline antibiotics; the combination of papaya flower extract and tetracycline antibiotics with E.coli bacteria were tested by using the time-kill test method. The concentration would be used was seen from the lowest concentration of MIC which inhibits papaya flowers by 2 mg/mL, tetracycline antibiotics by 1 µg/mL while the concentration for the combination used was 1/4 : 1/4 MIC value. Based on the research of (Shi et al., 2017), 1/4 MIC was used to determine the inhibitory mechanism between extracts and antibiotics in E.coli bacteria. So that the concentration of the combination used by testing the timekill test method for papaya flower extract was 0.5 mg/mL and for tetracycline antibiotics was 0.25 µg/mL. Based on the results of the log time-kill test data contained in (Figure 4), the growth phase of the bacteria could be determined. In the KK treatment, from 0 to 8 hours there was a lag phase or adaptation phase, meanwhile at 24 hours there was a growth phase with a relatively short time. It was characterized by a gradual increase in the number of bacterial colonies. In the EBP treatment, there was a lag phase at 0 hour, and then there was at 4 hours to 24 hours there was a death phase which was marked by a decrease in the number of bacterial colonies. This should that papaya flower extract could inhibit the growth of E.coli bacteria. There was a lag phase at 0 hour, and then the death phase occurred at 4 hours to 24 hours which was marked by a decrease in the number of bacterial colonies every hour in ABT treatment. This shows that tetracycline antibiotics could inhibit the growth of E.coli bacteria. For EBP+ABT combination there was a lag phase at 0 hour, then at 4 to 24 hours the number of bacterial colonies showed a decrease so that it can be seen that the combination treatment occurred in. This is because the mechanism of action between papaya flower extract and tetracycline antibiotics inhibits the growth of E.coli bacteria and the extract has similarities with antibiotics.
In the research of (Prasetya et al., 2019), the growth curve of E.coli bacteria in time (t1), the growth phase of E.coli bacteria consists of lag phase, log phase and stationary phase. The first phase is lag phase which occurs at 0 to 8 hours. In the lag phase, the bacteria experience a process of adaptation to environmental conditions, namely pH, temperature, and nutrition, and an increase in the number of bacterial cells occurs slowly. In this phase, the bacterial isolates consisting of 4 isolates had rapid growth from the 9th to the 24th hour. Bacteria have not reached the stationary phase or equilibrium period. In this phase, metabolic activity slows down. Nutrient deficiencies, accumulation of waste products, and changes in pH that are toxic to cells are considered to be the causes of the cessation of cell exponential growth. In addition, the bacteria have not yet reached the death phase, so further incubation is needed for more than 24 hours.Papaya flower extract has antibacterial compounds including alkaloids, flavonoids, saponins, triterpenoids, phenolics, and tannins. Each compound has a different mechanism of action, including inhibition of the synthesis of cell walls, inhibition of protein synthesis, inhibition of nucleic acid (DNA or RNA) synthesis, or inhibition of the synthesis of essential metabolites (Bauman, 2012).
The mechanism of action of tetracyclines involves inhibiting protein synthesis in bacterial ribosomes. Tetracyclines enter bacteria through two processes, namely passive diffusion and active transport. Furthermore, tetracycline enters the ribosome and binds reversibly to the 30S ribosome and prevents the tRNA-aminoacyl binding to the ribosomal mRNA complex, which prevents the formation of new amino acids in the peptide from being formed. This can prevent elongation between new peptide chains and stop protein synthesis (Koto et al., 2010). In Figure 4, a bactericidal effect can be determined with a log value > 3 log10 CFU/mL and a log bacteristatic value of < 3 log10 CFU/mL after 24 hours of incubation (Silva et al., 2011). Antibacterial compounds are compounds that can interfere with the growth of bacteria. There are two properties of antibacterial toxicity, including bactericidal, which is antibacterial and can kill bacteria, and bacteriostatic, which is antibacterial and inhibits bacterial growth (Purnamaningsih et al., 2017). The log kill value is obtained from the difference between development control and each treatment. The bactericidal effect can be observed between 8 and 24 hours after the antibiotic treatment or combination treatment. While the extract treatment at 0, 4, 8, and 24 hours was included in the bacteristatic.

CONCLUSION
Based on this research, it can be concluded that papaya flower and a combination of papaya flower extract and tetracycline antibiotics can inhibit the growth of E. coli bacteria by using the time-kill test method, which is indicated by the curve results in the combination treatment and papaya flower extract experiencing a death phase at 4, 8, and 24 hours, which were marked by a decrease in E. coli bacterial colonies. The research of the log time-kill test data showed a bacteriostatic effect on papaya flower extract treatment at 0, 4, 8, and 24 hours, and a bactericidal effect at combined treatment at 8 and 24 hours.