The Difference in the Antimicrobial effect of Katuk Leaf Extract (Sauropus Androgynus (l. ) Merr. ) Concentration against Escherichia Coli
Abstract
Purpose – The aim of this study is to determine the differences in the antimicrobial activity of katuk leaf (Sauropus androgynus (L.) Merr) against Escherichia coli.
Design/Methodology/Approach – The method used in this study was experimental posttest using a control group design. Analysis of the effect of katuk leaf was performed in the dilution method with 20%, 40%, 60%, 80%, and 100% concentration. The data were analyzed using one-way ANOVA test (α = 0.05) and was then tested using the least significant difference (LSD) test.
Findings – Bacterial colony counting that used total plant count found the average of E. coli amount at 20% of concentration (526.820 CFU/ml), 40% of concentration (449.380 CFU/ml), concentration of 60% (255.710 CFU/ml), concentration of 80% (194.110 CFU/ml), and at concentration 100% (168.600 CFU/ml). This study concluded that the katuk leaf extract at 20%, 40%, 60%, 80%, and 100% of concentration had antimicroba effect with significant influence. The 100% of concentration had the most significant effect compared with the other concentrations.
Research Limitations/Implications – Katuk leaf could be used as one of the alternative herbal choices that has a compound antimicrobial effect.
Originality/Value – This study increases the theoretical understanding of the difference of antimicrobial effectivity of katuk leaf extract (S. Androgynus (L.) Merr.) concentration against E. coli
Keywords
Citation
Husna, C.A., Muqsith, A. and Hasibuan, S.L. (2018), "The Difference in the Antimicrobial effect of Katuk Leaf Extract (
Publisher
:Emerald Publishing Limited
Copyright © 2018, Cut Asmaul Husna, C. Al Muqsith, C. Soya Loviana Hasibuan.
License
Published in the Emerald Reach Proceedings Series. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode
1. Introduction
Infectious diseases are the leading cause of morbidity and mortality in the world. World Health Organization (WHO) in 2012 mentioned that one-third of the 25 million deaths worldwide are caused by infectious diseases (Depkes, 2012). Bacteria are one of the most common contributing microorganisms (Garg et al., 2016). The high incidence of infection requires treatment in killing the bacteria, one of them by using antimicrobials, but the high use of antimicrobials is the greatest trigger for the emergence of bacterial resistance (Sylvia and Wilson, 2006). Some bacteria can cause resistance to antimicrobials, one of which is the bacterium Escherichia coli, causing important problems for public health (Roland, 2013). E. coli bacterial infections in Asia have been reported to be resistant to antimicrobial use of about 60–79% (US National Library of Medicine National Institute of Health, 2015). The results of Antimicrobial Resistance in Indonesia (AMRIN-Study) study showed that from 2,494 individuals in Indonesia, 43% of E. coli samples from thousands of individuals are resistant to several antimicrobials, 34% ampicillin, 29% co-trimoxazole, and 25% chloramphenicol (Gabriela et al., 2015).
Indonesia is a biodiversity mega-country rich in medicinal plants and very potential to be developed, one of which is katuk leaf (Hariana, 2015). Katuk leaf (Sauropus androgynus (L). Merr.) is a plant that is utilized as a traditional medicinal ingredient due to its antimicrobial ability, improving and facilitating milk secretion, overcoming skin disorders, fever, and osteoporosis (Suprayogi et al., 2004; Handayani, 2013). Results of research conducted by the National Working Group of Indonesian Medicinal Plants showed that katuk leaves contain several chemical compounds, including flavonoids and tannins that function as antibacterial (Rukmana and Indra, 2003). Previous research conducted by Fatimah et al. (2014) explains that leaf extract of katuk with a concentration of 60–100% can inhibit the growth of Staphylococcus aureus bacteria in vitro, but does not affect the concentration of 20% and 40%. Research on the effectiveness of katuk leaves at various concentrations in inhibiting E. coli has not obtained the data.
2. Method
This research is a laboratory experimental study using true experimental post-test only control group design. The sample of this research is katuk leaf (S. androgynus (L.) Merr.) which is obtained from Uteunkot village, Lhokseumawe, and pure culture of E. coli bacteria is obtained from Microbiology Laboratory of Faculty of Pharmacy, University of Sumatera Utara. The size of the sample is calculated by Federer's formula, and the result is five repetitions. The experimental materials were divided into five groups (according to concentrations of 100%, 80%, 60%, 40%, and 20%) plus two control groups (Mc Farland control and negative control of katuk leaf extract without suspension).
The leaves of katuk were extracted by the maceration method, until the powder was obtained about 300 g, followed by dissolution with 96% ethanol for three days, then evaporated using vacuum rotary evaporator to produce 30 ml liquid extract. The results of pure extraction were diluted with aquades to reach concentrations of 20%, 40%, 60%, 80%, and 100%. Furthermore, the dilution results are homogenized with the vortex.
Test bacteria from pure cultures that have been identified, about 0.1 ml bacterial inoculum were fed to each extract tube for each concentration, then tested the effectiveness of antimicrobial by dilution technique, which is to be assessed for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Next is calculated total plate count (TPC) to get the average value of colony of E. coli on katuk leaf extract (S. androgynus (L.) Merr.) with a concentration of 20%, 40%, 60%, 80 %, and 100%.
This study used completely randomized design (RAL) with five treatments each with five repetitions. The research was analyzed using one-way ANOVA test with significance level (= 0.05).
3. Results
3.1. MIC and MBC extract of katuk leaf (s. androgynus (l.) merr.) against E. coli bacteria on dilution 10−2 and 10−3
The result of MIC and MBC of katuk leaf extract (S. androgynus (L.) Merr.) in E. coli bacteria showed the lowest colony count in 100% katuk leaf extract (see Table 1). While not found in MBC against E. coli bacteria on katuk leaf extract (S. androgynus (L.) Merr.) at concentrations of 20%, 40%, 60%, 80%, and 100% both on dilution 10−2 and 10−3.
The result of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Consentration (MBC) on dilution test of katuk leaf extract (S. androgynus (L.) Merr.) Against Escherichia coli (CFU/ml)
| Repetition | Dilution | Concentration | ||||
|---|---|---|---|---|---|---|
| 20% | 40% | 60% | 80% | 100% | ||
| I | 10−2 | 1,180 | 824 | 524 | 433 | 622 |
| 10−3 | 1,062 | 785 | 478 | 326 | 419 | |
| II | 10−2 | 971 | 793 | 569 | 396 | 432 |
| 10−3 | 960 | 766 | 392 | 281 | 254 | |
| III | 10−2 | 1,371 | 896 | 598 | 378 | 288 |
| 10−3 | 872 | 801 | 422 | 393 | 263 | |
| IV | 10−2 | 1,126 | 822 | 733 | 446 | 312 |
| 10−3 | 888 | 810 | 417 | 382 | 303 | |
| V | 10−2 | 1,124 | 1,023 | 827 | 598 | 276 |
| 10−3 | 897 | 896 | 523 | 334 | 254 | |
Next TPC is calculated in Table 2 to get the average value of colony of E. coli on katuk leaf extract of various concentrations.
The calculation of Average Total Plate Count (TPC) of Katuk Leaf Extract (Sauropus androgynus (L.) Merr.) Against Escherichia coli
| Concentration (%) | Average Number of Colony (CFU/ml) |
|---|---|
| 20 | 526.820 |
| 40 | 449.380 |
| 60 | 255.710 |
| 80 | 194.110 |
| 100 | 168.600 |
The result of calculation of total plate count (TPC) found that the average number of bacteria E. coli at a concentration of 20% had the highest amount of bacterial colony, while at 100% concentration, had the lowest amount of bacterial colony.
3.2. Efficacy of antimicrobial leaf extract katuk (s. androgynus (l.) merr.) against E. coli bacteria
Shapiro–Wilk Normality Test on Antimicrobial Effectiveness of Katuk Leaf Extract (S. androgynus (L.) Merr.) Against Escherichia coli Bacteria
| Group | Number of Sample | Average (%) | P value | α Score |
|---|---|---|---|---|
| Concentration 20% | 5 | 99.3 | 0.988 | |
| Concentration 40% | 5 | 84.8 | 0.188 | |
| Concentration 60% | 5 | 90.6 | 0.443 | 0.05 |
| Concentration 80% | 5 | 81.7 | 0.110 | |
| Concentration 100% | 5 | 82.4 | 0.126 |
Normality test results show that the average percentage data of E. coli bacteria in TPC are normal distribution (P > 0.05) so that further data can be used for one-way ANOVA test ( Table 3).
Furthermore, the homogeneity test is needed to determine the data variance used is the same or not. The test used is the Levene test ( Table 4).
Levene Homogeneity Test against Percentage of Total E. coli Bacteria
| Levene Statistic | df1 | df2 | P value | α Score |
|---|---|---|---|---|
| 0.684 | 4 | 20 | 0.611 | 0.05 |
Homogeneity test results indicate that data variance in this research data is homogenous (P> 0.05) so that further data can be used for one-way ANOVA test.
One-Way ANOVA Test of Percentage of TPC of Escherichia coli Bacteria
| Variable | Mean | SD | P value | α Score |
|---|---|---|---|---|
| Concentration 20% | 127,198 | 14,489.28 | ||
| Concentration 40% | 95,222 | 9,637.68 | ||
| Concentration 60% | 69,484 | 12,848.53 | 0.00 | 0.05 |
| Concentration 80% | 48,452 | 8,674.80 | ||
| Concentration 100% | 41,586 | 15,172.01 |
One-way ANOVA test results in Table 5 shows a significant difference in each concentration with P value (0.00). The above data were then tested again using post hoc least significant difference (LSD) to determine whether or not there was a significant effect between the concentration of katuk leaf extract (Sauropusandrogynus (L.) Merr.) At concentrations of 20%, 40%, 60%, 80%, and 100%.
Post-Hoc Test LSD on percentage of TPC of Escherichia coli.
The result of post-hoc LSD test in Table 6 shows significant difference among concentration 20%, concentration 40%, and concentration 60%
| (I) Group | (J) Group | Mean Difference (IJ) | Sig. |
|---|---|---|---|
| Concentration 20% | Concentration 40% | 31.976 | 0.001 |
| Concentration 60% | 57.714 | 0.000 | |
| Concentration 80% | 78.746 | 0.000 | |
| Concentration 100% | 85.612 | 0.000 | |
| Concentration 40% | Concentration 20% | −31.976 | 0.001 |
| Concentration 60% | 25.738 | 0.004 | |
| Concentration 80% | 46.770 | 0.000 | |
| Concentration 100% | 53.636 | 0.000 | |
| Concentration 60% | Concentration 20% | −57.714 | 0.000 |
| Concentration 40% | −25.738 | 0.004 | |
| Concentration 80% | 21.032 | 0.015 | |
| Concentration 100% | 27.898 | 0.002 | |
| Concentration 80% | Concentration 20% | −78.746 | 0.000 |
| Concentration 40% | −46.770 | 0.000 | |
| Concentration 60% | −21.032 | 0.015 | |
| Concentration 100% | 6.866 | 0.393 | |
| Concentration 100% | Concentration 20% | −85.612 | 0.000 |
| Concentration 40% | −53.636 | 0.000 | |
| Concentration 60% | −27.898 | 0.002 | |
| Concentration 80% | −6.866 | 0.393 |
4. Discussion
The results of this study showed that there was KHM in all leaf katuk extract concentration tested against E. coli. While no value of minimum inhibitory level (MIC) was obtained in katuk leaf extract at all concentrations. TPC calculations show that extract of katuk leaf (S. androgynus (L.) Merr.) at concentrations of 20% and 40% has a weak effectiveness in inhibiting E. coli while at concentrations of 60%, 80%, and 100% have strong activity. Concentration of 100% is the most effective concentration in inhibiting E. coli compared with other concentration variations.
The higher concentration of katuk leaf extract (Sauropusandrogynus (L.) Merr.) will decrease the number of bacterial colonies that survive. This suggests that with increasing concentrations the greater the levels of active ingredients that act as antimicrobials, so that their growth in inhibiting bacteria is also greater. This is in line with research (Fatimah et al., 2014) which assessed the effectiveness of katuk leaf extract in inhibiting the growth of S. aureus bacteria in vitro.
Different concentrations in variables’ treatment affect the linearity of the optical density (glass and air medium) of the bacteria. Thus, the higher the concentration, the smaller the optical density, which means fewer bacteria can survive. This shows that with the increasing concentration, the greater the level of active ingredients that function as antibacterial, so that its ability in inhibiting bacterial growth is also greater (Ajizah, 2004).
The ability of an antimicrobial agent in negating the micro-organism's survival depends on the concentration of the antimicrobial agent, meaning that the amount of antimicrobial agent in a bacterial environment is critical to the life of exposed bacteria. In addition to the concentration factor, the antimicrobial type also determines the ability to inhibit bacterial growth. In this study, suspected bacterial sensitivity of E. coli is because of the chemical content in katuk leaf extract which consists of antimicrobial nature (Schlegel, 1994).
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All papers within this proceedings volume have been peer reviewed by the scientific committee of the Malikussaleh International Conference on Multidisciplinary Studies (MICoMS 2017).
Corresponding author
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