Tracking of the Antibiofilm Activities of Lakum Leaf Extract (Causonis trifolia Linn.) Against Staphylococcus aureus

MATERIALS AND METHODS

Study area: This research was carried out in the Laboratory of the Universitas Muhammadiyah Kalimantan Timur, Faculty of Pharmacy and Department of Pharmacy, Islamic University of Indonesia from October, 2021 to May, 2022.

This research was carried out using an in vitro experimental study design using observations of the results of phytochemical screening tests to determine the content of secondary metabolites in Lakum leaf extract and to determine the effectiveness of Lakum leaf anti-biofilm (Causonis trifolia L.) on Staphylococcus aureus through microplate reader reading which measures the absorbance value of the bacteria. Plants based on their optical density values to measure the percent inhibition of biofilms on Lakum leaves²⁵.

Research tools and materials: In this study, the research equipment used was an oven (Bioevopeak), glass jar (Kedaung), wooden stirrer, measuring cup (Iwaki), filter paper, Erlenmeyer (Iwaki), funnel, porcelain cup, rotary evaporator (Buchi), water bath (Thermo), aluminum foil, plastic wrap, stainless basin, stir bar, microtube, brown paper, autoclave (All American, test tube (Iwaki), test tube rack, Petri dish (Iwaki), micropipette (Dragon Lab), micropipette tip, plate reader 96 wells (Iwaki), analytical balance (Fujitsu FS-AR210, tools glasses, incubators (Labnet), ose needles, Bunsen, micro readers (HiPo-Biosan and Lenovo), microscopes (Olympus), laminar air flow (LAF) (Bioevopeak).

While the materials used in this study were Lakum leaf extract (Causonis trifolia) S. aureus bacterial culture (ATCC 25923), sodium broth media (NB), sodium agar media (NA), sterile distilled water and crystal violet (1% in distilled water), 96% ethanol, Clindamycin, Mayer’s reagent, Bouchardat’s reagent, Dragendorff’s reagent, Magnesium (Mg), concentrated Hydrochloric Acid (concentrated HCl), amyl alcohol, Liebermann’s reagent and others.

Methods
Sampling: Lakum leaf samples (Causonis trifolia) were obtained from Tenggarong City, Kutai Kartanegara Regency, East Kalimantan Province, Indonesia as much as 3 kg with a total viscous extract obtained of 37.3 g and a yield of 9.18%.

Plant determination: Lakum plants, to be precise, Lakum leaves to be used in this study were determined based on the morphological characteristics of the plant. The plant determination process will be carried out at the Forestry Laboratory of Mulawarman University, Samarinda.

Making simplicia powder: The simplicia of the Lakum leaves that have been taken will be wet sorted to separate dirt or foreign materials and other unwanted plant parts from the simplicia. After carrying out wet sorting, proceed with washing, chopping and drying the simplicia. The simplicia drying process is carried out using the sun until the simplicia is completely dry (obtained moisture content <10%)^21,26,27.

Then after the simplicia drying process is complete, is continued by carrying out dry sorting to separate foreign materials and simplicia that have not dried properly so that simplicia is guaranteed to be completely free of foreign materials. After that, the simplicia pollination process was carried out using a blender^21,26,27.

Preparation of Lakum leaf extract (Causonis trifolia): Lakum leaves were extracted using the maceration method in which 200 g of powdered Lakum leaves were weighed and 2250 mL of 96% ethanol was added. Extraction was carried out for 3×24 hrs or for 3 days. After extraction, the maceration results were filtered to separate the soluble extract and filtrate. Then the extract dissolves in the rotary evaporator until the extract thickens slightly or the solvent and extract have separated sufficiently. After that, the results of the rotary evaporator extract were in a water bath until the extract thickened.

Phytochemical analysis testing
Alkaloid compounds:

Flavonoid compounds:

After that, 5 mL of the test filtrate was added and 50 mg, 1 mL concentrated HCl and 2 mL of amyl alcohol were added to the test tube. After that, it was shaken and allowed to separate. If the solution produces a red or orange color in the amyl alcohol layer, then the positive extract contains flavonoids.

Phenolic compounds: As much as 50 mg of Lakum leaf extract was dissolved in 5 mL of distilled water. Then add a few drops of 5% neutral ferric chloride. If the solution produces a dark green color, then the positive extract contains phenolic.

Polyphenol compounds: Add 10% FeCl solution in distilled water. Then put it in the isolate that has been put in a test tube. If the solution produces a strong green, red, purple, blue, or black color, then the positive extract contains polyphenols.

Terpenoid compounds, sterols and steroids: Evaporate as much as 2 mL of the extract then the residue obtained from the evaporation process is dissolved again in 0.5 mL of chloroform, then added with 0.5 mL of anhydrous acetic acid. Furthermore, the solution was dripped with concentrated sulfuric acid as much as 2 mL through the wall of the test tube. If the solution produces a bluish-green color, then the positive extract contains terpenoids, sterols and steroids.

Another method for identifying these three compounds is by placing 1 mL of the extract solution in a test tube, then adding a few drops of Liebermann-Burchard’s reagent. If the solution produces a green or blue color, then the positive extract contains steroids. If the solution produces a purple or red color, then the positive extract contains terpenoids.

Tannin compounds: Take enough thick extract and add 10 mL of distilled water. Then the solution was heated for 3 min and cooled. After cooling, the filtrate was filtered and diluted again. After filtering, put it in a test tube and add 1-2 drops of FeCl₃ reagent. If the solution produces a black-green color, then the positive extract contains tannins.

Another method for identifying these tannin compounds is by inserting ±2 mL of the filtrate into a test tube. Then 2-3 drops of 1% FeCl solution were added. If the solution produces a green or bluish-black color, then the positive extract contains tannins.

Saponin compounds: Take enough thick extract and add 10 mL of hot distilled water (hot water). Then the filtrate was cooled and shaken vigorously. If the solution produces foam after shaking and does not disappear for >5 min, then the positive extract contains saponins.

Preparation of bacteria for assay: Staphylococcus aureus bacteria were cultured in sodium broth (SB) medium and incubated for 24 hrs at 37°C. The optical density of 600 of the bacterial culture is adjusted to the McFarland 0.1 standard, namely 0.5-1.5×10⁸ CFU mL^–1 by looking at the level of bacterial density using the help of a spectrophotometer. If the density of Staphylococcus aureus bacteria cells is not in accordance with the McFarland standard, then the bacterial media needs to be diluted using new growth media until the level of bacterial density meets the McFarland standard, which was OD₆₀₀ 0.01. If the cell density was in accordance with the McFarland standard, then the biofilm test bacteria can be used for testing^28-31.

Assay of biofilm inhibitory activity: A total of 5 μL of microbial suspension (10⁷ CFU mL^–1) and 75 μL of media containing test extracts with concentration series (0.125, 0.25, 0.5 and 1% w/v) were added to each well of the microtiter plate. In addition, 20 μL of aquadest control, 75 μL of media control, ethanol control and drug control in other wells were given as a comparison of the test results. Once ready, the plates were incubated for 24 and 48 hrs^28-31.

After completion of incubation for a predetermined time, the plate is washed using sterile aquadest three times to remove cells that are not attached. Then add 100 μL of 1% crystal violet to each well filled with the test sample and let it stand for 15 min. After that, the crystal violet solution was removed from the plate and then washed again using sterile aquadest three times. After washing, 100 μL of 96% ethanol was given to each well filled with the test sample and then tested the sample in a microreader^28-31.

As a control media, media without microbial growth was used and microbial suspensions were used as growth controls. As a drug control, a microbial suspension was used which was given Clindamycin at a rate of 1% w/v. The plates were then incubated at 37°C for 24 and 48 hrs. After that, the plate was washed using aquadest three times. To remove excess water after washing, the plates were dried for 5 min at room temperature^28-31.

Then, 125 μL of 1% crystal violet was added to each well to give color to the biofilm that formed. In this case, the building blocks of biofilms are dead cells and living cells. After that, the plate was incubated for 15 min at room temperature. After incubation, the microplates were washed again with running water to clean the remaining crystal violet and to dissolve the biofilm that formed, 200 μL of 96% ethanol was added to each well^28-31.

Optical density (OD) readings were carried out with a microplate reader at a wavelength of 620 nm. The test was carried out with three replications. The data obtained from the analysis of biofilm inhibition was in the form of OD values for each concentration of the test compound and the control without the test compound (growth control) obtained from readings with a microplate reader^28-31.

To determine the percentage of bacterial inhibition, the calculation used the OD value from the research analysis using the following equation^28-31:

The concentration of the sample which can inhibit at least 50% of biofilm formation is considered MBIC₅₀ (Minimal Biofilm Inhibition Concentration)^28-31.

Statistical analysis: The research data obtained were subjected to statistical testing in the form of the One Way ANOVA test. Before carrying out the test, a normality test will be carried out with a value of p>0.05 to ensure that the data is normally distributed and a variance test is carried out, so that the data must be homogeneous.

RESULTS AND DISCUSSION

Phytochemical screening of Lakum (Causonis trifolia) leaves: Phytochemical screening of Lakum leaf extract was carried out to find out which groups of compounds it contains. The compounds tested were alkaloids, flavonoids, phenolics, polyphenols, triterpenoids, sterols, steroids, tannins and saponins. The results of the phytochemical screening of Lakum leaves can be seen in Table 1.

Based on the results of the research conducted, Lakum leaves (Causonis trifolia) taken from Tenggarong District, Loa Ipuh Village, Tenggarong City, which grows wild in the plantations and settlements of the Tenggarong people, tested positive for containing alkaloids, flavonoids, phenolic compounds, polyphenols, tannins and saponins. This was in accordance with the statements of some researchers^21,32-34.

However, the leaves of Lakum (Causonis trifolia) are negative for triterpenoids, sterols, steroids and steroids. In this case, there are several factors that affect the absence of these compounds even though in some areas these compounds are contained in the same plant.

Factors that affect this are the age of the plants taken, the living environment of the plants²⁷ the location or altitude^35,36 and the nutrient content of the soil where the plants grow³⁵, the method of drying or the extraction process³⁷, the type of solvent used for the extraction process³⁷ and so on.

Results of antibiofilm activity against Staphylococcus aureus: In this study, the anti-biofilm potential of Causonis trifolia L. was tested in the inhibition phase of the biofilm against Staphylococcus aureus bacteria. The results obtained were that the Lakum leaf extract had an inhibitory effect on the growth of biofilms in the 48 hrs or maturation phase. The results of Lakum leaf anti-biofilm against Staphylococcus aureus bacteria can be seen in Table 2 and Fig. 1.

The anti-biofilm potency test of Lakum leaves (Causonis trifolia L.) was carried out during the maturation phase, namely the monomicrobial biofilm inhibition test against S. aureus bacteria. Lakum leaf extract exhibited biofilm inhibitory activity in the mid-phase (24 hrs) and the maturation phase (48 hrs) at all extract concentrations ranging from 1-0.125% w/v.

The highest inhibitory activity of Lakum leaf extract (Causonis trifolia L.) was in the mid and maturation phase in this study at 1% extract concentration, namely 76.95±0.0007 and 72.85±0.0003%. Meanwhile, the lowest biofilm inhibition activity was at a concentration of 0. Extract 0.125%, namely 65.65±0.0001% in the mid-phase and 59.71±0.0003% in the maturation phase.

In the control drug, Clindamycin 1% alone gave biofilm inhibitory activity in the mid-phase of 80.9±0.0004% and the maturation phase was 77.3±0.0004%. Based on the results of this study, the MBIC₅₀ value of the ethanol extract of Lakum leaves (Causonis trifolia L.) was at a concentration of 0.125% w/v in the maturation phase, which was 59.71±0.0003%. Meanwhile, the MIC value of this extract was found at the lowest concentration, namely 0.125% with the inhibition in the mid-phase (24 hrs) and maturation (48 hrs) of 65.65±0.0001 and 59.71±0.0003%.

According to Gosal et al.³⁸ and Elisabeth³⁹, there is greater inhibition in the middle phase compared to the maturation phase. This was because the biofilm EPS (extracellular polymeric substances) matrix produced in the middle phase is not as complex and structured as in the maturation phase so the ethanol extract of Lakum leaves inhibits the growth of biofilms more than in the maturation phase.

According to Elisabeth³⁹, during the maturation phase (48 hrs) the formation of biofilm cells has produced EPS which adheres to the surface of the bacterial cells and attaches to a collection of Staphylococcus aureus bacteria to form microcolonies. According to Purbowati⁴⁰, Kannappan et al.⁴¹ and Hamzah et al.⁴², during the maturation phase (48 hrs) biofilms will decrease anti-biofilm activity compared to the mid-phase (24 hrs). This was because the growth time of biofilms in the maturation phase is longer and the biofilm defense system will form a stronger defense system and biofilm cells will continue to grow for several hours^43,44.

Biofilm growth during the maturation phase results in a thicker and more complex cell structure when compared to the mid-phase. The mucus layer produced in the maturation phase is a difference in cell structure that can be seen because in this phase it can be seen that the mucus layer is more numerous and thick and is attached to the wells. This is what makes it difficult for some antibiotics to penetrate the EPS biofilm layer. In addition, according to Achinas et al.⁴⁴ and Muhammad et al.⁴⁵, the increase in biofilm EPS production occurs due to slow bacterial growth. This was due to the hydration of biofilm EPS which prevents dryness in some natural biofilms. This EPS also contributes to the antimicrobial resistance of the biofilm which causes obstructs the mass transport of antibiotics through the biofilm^46,47.

Even so, the inhibitory activity of the ethanol extract of Lakum leaves (Causonis trifolia L.) gave a good biofilm inhibitory activity in the mid-phase of 76.95±0.0007% and this result was almost equivalent to the control drug Clindamycin 1%, which was 80.9+0.0004%. In addition, in the maturation phase, Lakum leaf extract still provided good biofilm inhibition activity, although not as large as in the middle phase, which was 72.85±0.0003% and these results were close to the control of clindamycin 1% which was 77.3±0.0004%. Thus it was stated that the ethanol extract of the leaves of Lakum (Causonis trifolia L.) had biofilm inhibitor activity until the maturation phase.

Fig. 1:

Antibiofilm activity of Lakum leaf extract (Causonis trifolia L.) on Staphylococcus aureus bacteria Blue: Middle phase (24 hrs), Red: Maturation phase (48 hrs), K-: Negative control, K+: Positive control, K.E: Ethanol control, K.A: Aquadest control, K.M: Media control, E. 1%: Extract 1%, E. 0.5%: Extract 0.5%, E. 0.25%: Extract 0.25% and E. 0.125%: Extract 0.125%

Fig. 2:

Graph of antibiofilm activity results of Lakum leaves (Causonis trifolia L.) against Staphylococcus aureus bacteria Blue: Middle phase (24 hrs) and Red: Maturation phase (48 hrs)

According to Sari et al.³³, the ethanol extract of Lakum leaves has a very strong inhibitory effect on the growth of Staphylococcus aureus bacteria. At a concentration of 25%, Lakum leaf extract had an inhibition of 25.65 mm and at the lowest concentration of 10% it was 21.08±1.50 mm. Meanwhile, according to Opeña and Sotto²¹, Lakum leaf extract has Staphylococcus aureus antibacterial activity with an inhibition zone of 12 mm. In addition, according to Princeton et al.⁴⁷ Lakum leaf extract has antibacterial activity against Staphylococcus aureus with the highest inhibition zone of 25.33 mm and the lowest inhibition zone produced is 18.33 mm.

However, in this study, previous research as research literature regarding the biofilm inhibition test of Lakum leaf extract did not yet exist and this research is the first study regarding testing the anti-biofilm activity of Lakum leaf extract against Staphylococcus aureus bacteria.

However, in this study, there were several secondary metabolites that played the most active role in inhibiting monomicrobial biofilms on Staphylococcus aureus bacteria. In general, the mechanism for inhibiting biofilm growth is by means of active compounds interfering with communication signals (Quorum sensing) between bacteria that work to form biofilms by penetrating the bacterial cell wall resulting in the inactivation of the genes in the bacteria that form EPS synthesis so that the EPS layer cannot be formed^48,49.

Whereas specifically, according to Ma et al.⁴⁹ flavonoids and tannins play an important role in inhibiting biofilm formation. In the formation of Staphylococcus aureus biofilm, regulation occurs through ica-dependent biofilm production. In this regulation, flavonoid and tannin compounds directly inhibit the expression of the icaA and icaD genes^49,50 where these genes regulate the formation of polysaccharide intercellular adhesin (PIA) by activating sigma factor σ^B which can activate staphylococcal accessory regulator A (sarA). If sarA is activated, then the ica promoter will also be activated^51,52. Apart from being able to inhibit the formation of biofilms, flavonoids and tannins also play an active role as antibacterials in which these two compounds work by inhibiting bacterial cell adhesion which is the main factor in the formation of biofilms in the process of attaching bacteria to substrate surfaces and between bacteria⁵⁰.

In addition, Jagani et al.⁵² and Yamanaka-Okada et al.⁵³ stated that flavonoids and tannins are a class of polyphenolic compounds that play an active role in the formation of biofilms where these compounds work directly on an important factor, namely the process of bacterial cell adhesion to the substrate where this compound will reduce the hydrophobic nature of the bacteria so that the formation of biofilms becomes inhibited.

During the research, there were some research limitations occurred. The first limitation of this research is the lack of sources of previous research regarding the anti-biofilm activity of Lakum leaf extract against Staphylococcus aureus bacteria, so there is no source for comparison of test results. Apart from that, due to the COVID-19 pandemic, the duration of the laboratory’s working hours was cut so that the research process could not run long and freely in one day. In addition, due to the limited tools and materials used in the laboratory. So that when purchasing the necessary tools and materials, you have to wait some time until the research can be continued with the appropriate tools and materials.

CONCLUSION

Lakum leaves (Causonis trifolia L.) originating from Tenggarong Sub-District, East Kalimantan is known to contain positive alkaloids, flavonoids, phenolics, polyphenols, tannins and saponins. However, the Lakum leaves were declared to have no triterpenoid, sterol or steroid compounds using 2 phytochemical tests. Lakum leaves (Causonis trifolia L.) originating from the Tenggarong Sub-District, East Kalimantan was stated to be able to provide biofilm inhibition activity in the mid-phase with a large percentage of inhibition produced at 0.125-1% w/v of 65.65±0.0001, 68.5±0.0009, 73.6±0.0004 and 76.95±0.0007%. While in the maturation phase with the same concentration, it was 59.71±0.0003, 63.18±0.0006, 68.89±0.0003 and 72.85±0.0003%. The percentage of inhibition produced by the Lakum leaf extract was almost equivalent to the control drug clindamycin 1% which provided percentage of inhibition of the biofilm against Staphylococcus aureus bacteria by 80.9±0.0004% in the middle phase and by 77.3±0.0004% in the maturation phase. The active compounds that work as biofilm inhibitors are flavonoids, tannins and polyphenols.

SIGNIFICANCE STATEMENT

This research was conducted to obtain the results of the anti-biofilm activity in Lakum leaf extract against Staphylococcus aureus bacteria and active compounds that play an important role in inhibiting monomicrobial biofilms. Lakum leaves have biofilm inhibitory activity against Staphylococcus aureus with flavonoids, tannins and polyphenols which work as active substances in inhibiting the formation of biofilms where biofilms become one of the nosocomial infections in the urinary tract system called Catheter-Associated Urinary Tract Infection (CAUTI). It is known that approximately 80% of urinary tract infections due to catheters are related to biofilm formation.

ACKNOWLEDGMENT

Thank you very much to the Department of Pharmacy, Indonesian Islamic University and the Faculty of Pharmacy, Muhammadiyah University of East Kalimantan which has provided facilities in carrying out this research.