INTRODUCTION:

Diabetic ulcer infections pose a significant challenge in wound management, as they are often associated with persistent bacterial biofilms that are resistant to conventional antibiotic treatments¹. Biofilms are intricate and ever-changing microbial colonies that flourish at the boundary layer between a solid surface and a liquid medium². The complex formations consist of microbial cells that are tightly integrated with one another and with the underlying surface, enclosed inside a self-generated matrix of extracellular polymeric compounds³. Biofilm formation is a fundamental survival strategy used by many microorganisms to acclimatise to their habitat, especially under unfavourable circumstances⁴. Because of the rise of bacteria and viruses that are resistant to antibiotics, choosing the right empirical antibiotic coverage for diabetic ulcer infection therapy has become more challenging in recent years⁵.

To successfully address severe diseases caused by biofilms, new and innovative therapeutic techniques are required⁶. Utilising nanogel formulations of naturally occurring substances with intrinsic antibiofilm characteristics⁷, like kratom extract, is one approach that shows promise. Mitragynine and 7-hydroxymitragynine are two of the active components found in kratom. These molecules target different types of bacteria, including those that cause infections in diabetic ulcers. Regardless, more in vitro and in vivo investigations are needed⁸. There has been a lot of buzz in the ever-evolving world of antimicrobial strategies about targeted and effective approaches to combat biofilm-related diseases⁹. Biofilms are naturally resistant to conventional methods, such as antibiotic use. As a result, researchers are actively seeking novel approaches to this persistent issue, and they have turned their focus to the fascinating realm of nanotechnology¹⁰.

In their battle against bacterial infections, researchers have shown that nanomaterials and carriers on the nanoscale may kill bacterial cells, slow down quorum sensing, and prevent or eliminate biofilms¹¹. Nanomaterials are a game-changer in the fight against biofilm-associated illnesses and antibiotic resistance. In instance, there are unique benefits to delivering natural chemicals with antibiofilm action via nanogel formulations¹². There are a number of benefits to using nanogels, which are hydrogel nanoparticles, to reduce biofilms¹³. It is easy for the hydrophilic nanocarriers to penetrate bacterial biofilms and disrupt their extracellular matrix. Because of this, antibiofilm medications may be delivered directly to bacterial cells¹⁴. Nanogels' unique properties, such as their scalability, surface charge, and sensitivity to environmental stimuli, might potentially enhance the efficacy of the encapsulated kratom extract¹⁵.

Studies have demonstrated that nanogel formulations may prevent biofilm production, break existing biofilms, and improve the efficacy of conventional antibiotics in killing bacteria trapped in biofilms¹⁶. A very promising treatment approach for diabetic ulcer infections, these nanogel formulations effectively target and dissolve bacterial biofilms¹⁷. In addition to increasing local antibiotic concentrations, nanogels' ability to transport active drugs straight to the infection site aids in the fight against antibiotic resistance by penetrating the protective biofilm matrix¹⁸. This focused strategy may help improve patient outcomes, lessen the likelihood of adverse effects, and lower the requirement for systemic antibiotics, which is particularly important in the treatment of diabetic foot ulcers caused by biofilm-associated infections¹⁹. The objective of this investigation is to ascertain the antibiofilm properties of the nanogel derived from kratom extract in the context of diabetic ulcer infections.

MATERIALS AND METHODS:

Sampling Place:

We collected samples of kratom leaves (Mitragyna speciosa) in the forest of Kuranji District, Tanah Bumbu Regency, South Kalimantan, Indonesia. The process of identifying plants was carried out at Universitas Lambung Mangkurat's Basic Laboratory of the Faculty of Mathematics and Natural Sciences, Banjarmasin, Indonesia.

Materials:

Materials used were ethanol 95% (Merck, Germany), ethyl acetate (Merck, Germany), chloramphenicol (Sigma-Aldrich, Germany), phosphate-buffered saline (PBS; Sigma—Aldrich), McFarland (HiMedia, India), CMC-Na (Merck, Germany), Oxoid (Brain Heart infusion) (Merck, Germany), crystal violet (Merck, Germany), Carbopol (Merck, Germany), Trietanolamine (Merck, Germany), Gliserol (Merck, Germany), Propilenglikol (Merck, Germany).

Equipment:

Micropipette (Gilson, France), microtiter plate reader (OIS 2100, Spain), tube (Socorex, Swiss), LAF (Sakura, Japan), incubator 2B (Sakura, Japan), Microplate flat-bottom polystyrene 96 well (Iwaki, Japan), spektro genesys (Thermo Scientific Spectronic, USA), autoclave (Sakura, Japan), analytical balances (Ohaus, USA).

Bacterial strain:

ATCC 25923 strains of standard Staphylococcus aureus were cultivated in tryptic soy broth (TSB) medium and subjected to a 72-hour incubation period at 37°C.

Extraction and identification:

The Kratom leaf samples were cleansed by the process of rinsing with flowing water and meticulously inspecting for any contaminants. After washing, we sliced and desiccated the plant samples in an oven at 40°C for 3 hours. Sample standardisation is conducted using precise criteria. We accomplished the Simplicia extraction by fully immersing 300g of powder in 96% ethanol. We agitate the mixture every 6hours and replace the solvent every 24hours, resulting in a total of 5 repetitions or changes of solvent. We subjected the liquid extract to evaporation in a water bath maintained at a temperature of 50°C until the weight reached a steady state. The levels of quinones, terpenoids, phenolics, saponins, steroids, alkaloids, and flavonoids were assessed using the tube technique.

Antibacterial effect of kratom extract against staphylococcus aureus:

The antibacterial efficacy was assessed by the use of microdilution and well techniques. The study was carried out on a 96-well flat-bottom polystyrene microtiter plate using various doses of test chemicals (0,125%, 0.25%, 0.5%, 0.1% w/v) to identify their effects on S. aureus. The positive control used was chloramphenicol. In order to get a concentration of 5x106 colony forming units per millilitre (CFU/mL), the bacterial solution in BHI was first adjusted to 0.5 McFarland units and then diluted. The microplate was filled with a solution of ethanol with a concentration of 96% and subjected to incubation for a duration of 15 minutes. An ELISA Reader was employed to conduct optical density measurements at a wavelength of 570 nm.

Formulation of nanogel:

Carbopol and CMC were the two nanogel ingredients used as a standard gel base.

Table 1. Formulation of nanogel extract

No	Ingredients	F1 (g)	F2 (g)	F3 (g)
1	Carbopol 940	0,8	0,8	0,8
2	CMC-Na	0,6	0,6	0,6
3	Gliserol	1,25	1,25	1,25
4	Propilenglikol	1,75	1,75	1,75
5	Trietanolamin	1,5	1,5	1,5
6	Nanoemulsion extract	0,75	0,75	0,75
7	Aquadest ad	20	20	20
	Ingredient nanoemulsion
1	Ekstract	10%	20%	30%
2	Etanol 96%	9 mL	9 mL	9 mL
3	Tween 80	27,5 mL	27,5 mL	27,5 mL

Inhibitory activity of kratom extract against staphylococcus aureus biofilms:

Biofilm formation was assessed during 24hours (intermediate phase), 48hours (maturation phase), and 72hours (eradication) using the Microbroth Dilution Technique (MDT). The microtiter plates were rinsed with distilled water to remove detachable cells, and then allowed to desiccate in the surrounding air at room temperature for a period of 5minutes. Each well was thereafter stained with a 125µL portion of a 1% solution of crystal violet to identify living and non-viable cells, as well as any other components of the biofilm. Following a 15-minute incubation period at ambient temperature, we proceeded to rinse the plate with flowing water in order to eliminate the purple hue. The optical density (OD) was determined by adding 200µL of 96% ethanol to each well and using a microplate reader with a 595nm wavelength. The bacterial test culture, was deposited onto a microtiter plate including coverslips. In order to promote the biofilm development process, Subsequently, the plate was placed in an incubator that was maintained at a temperature of 37°C for a duration of 24-48hours.

In vivo biofilm study:

Adult rats (Rattus norvegicus) weighing 150–200g were employed to conduct in vivo antibiofilm investigations. The animals were given unfettered access to water and nutritious food and were kept inside a controlled setting. In order to induce type II diabetes, a combination of streptozocin and nicotinamide was administered. The rats were administered a 240mg/kg intraperitoneal dose of nicotinamide after a 12hour fasting period. 15 minutes later, I.P. administration of streptozocin at a dosage of 100mg/kg occurred. The animals were subjected to a 12hour fast following the streptozocin treatment, and their fasting blood glucose levels were assessed 72hours later. The investigation categorised animals as diabetic only if their fasting blood glucose level exceeded 150mg/dL²⁰. Positive, negative, and the maximum concentration of gel were the three categories into which the mice were divided. To prevent infection, the researchers made an open incision on the rodent with a diameter of 1cm and administered bacteria. To serve as a positive control, chloramphenicol was administered daily to the lesion until it had fully healed.

SEM study:

Following a thorough washing with sterile distilled water, the coverslip was then subjected to glutaraldehyde during fixation. Methanol was used as a means of decreasing the water content, while the examination was conducted utilising a SEM (scanning electron microscope) functioned at a voltage of 10 kilovolts (kV).

RESULT:

Phytochemical screening

The extraction yielded a filtrate, which was further evaporated using a water bath at 50^oC to give a concentrated extract with a consistent weight. The leaf yield was 18.8% with some phytochemical compounds in Table 2.

Table 2: Phytochemical compound of kratom extract.

S. No.	Secondary metabolites	Result
1	Alkaloids (Dragendorff)	-
2	Alkaloids (Mayer)	-
3	Alkaloids (Wagner)	-
4	Steroids	-
5	Terpenoids	+
6	Tannin	-
7	Phenolic	+
8	Saponin	-
9	Flavonoids	+
10	Quinones	+

Antibacterial effect of kratom leave extract against Staphylococcus aureus:

Figure 1: Kraton extract's antibacterial efficacy against Staphylococcus aureus

The results demonstrated that the concentration of lollipop flower extract affected its efficacy. At a 1% w/v concentration, the extract successfully suppressed SA growth, showing a substantial (p<0.05) inhibition percentage of 74.226%±0.002, notably different from the drug control's 84.097% ±0.001.

KDNE's inhibitory action against Staphylococcus aureus mid-phase (24-hour) biofilms:

Figure 2. Percentage of inhibitory action against Staphylococcus aureus mid-phase (24-hour) biofilms

Kratom-derived nanogel extract (KDNE) at a concentration of 30% w/v showed strong antibiofilm activity against Staphylococcus aureus (SA) during the mid-phase biofilm, with an inhibition percentage of 79.21% ± 0.08. Notably, this inhibition was not significantly different (p > 0.05) from the control drug at a concentration of 1% w/v, which showed an inhibition percentage of 83.58% ± 0.042, as depicted in Figure 2.

KDNE's inhibitory action against Staphylococcus aureus maturation phase (48-hour) biofilms:

Figure 3. Percentage of KDNE's inhibitory action against Staphylococcus aureus maturation phase (48-hour) biofilms

During the biofilm maturation phase, KDNE showed an inhibitory activity of 74.12% ± 0,101 at a concentration of 30% w/v. Interestingly, this result did not differ significantly (p>0.05) from the inhibitory effectiveness of the pharmacological control, which showed a higher inhibitory effectiveness of 77.79% ± 0.083. These data provide support for the idea that as the biofilm development duration increases, the arrangement of the matrix rises, resulting in a stronger and more complex biofilm structure. The intricate nature of this structure reduces the efficacy of both the test substance and pharmacological control in preventing the growth of biofilms²¹.

KDNE's inhibitory action against Staphylococcus aureus eradication phase (72-hour) biofilms:

Figure 4. Percentage of KDNE's inhibitory action against Staphylococcus aureus eradication phase (72-hour) biofilms

The eradication-phase biofilm of Staphylococcus aureus (SA) was effectively inhibited by KDNE at a concentration of 30% w/v, exhibiting an inhibition percentage of 67.25% ± 0.046. Figure 4 shows that at a dose of 1% w/v, the control medication exhibited an inhibition percentage of 72.912% ± 0.023, and this inhibition was not substantially different (p > 0.05).

In vivo biofilm study:

(a) (b) (c)

Figure 5. 5a. untreated group; 5b. Occurrence of Ulcers; 5c. the wound begins to close.

Diabetes is known to hinder the host's immune response and create an environment that encourages the growth of microorganisms. Numerous studies have highlighted the elevated risk of infections in diabetics, including rhinocerebral mucormycosis, malignant external otitis, and foot infections. There is evidence that diabetes-induced alterations in the microcirculatory system and disruption of wound healing processes contribute to this increased susceptibility²². This study shows that nanogel treatment can improve wound healing in diabetic mice by increasing angiogenesis and reducing biofilm formation, which is not significantly different from the control group. The ulcers treated with nanogel closed and healed within 11 days, and the healing diameter was almost the same as the control group.

SEM Study:

(a) (b)

Figure 6: Figure 6a Result of SEM Biofilm with No Treatment, Figure 6b Result of SEM Biofilm with Administration of Kratom Extract 30% b/v.

Figure 6b shows that the SEM test showed that the KDNE effectively stopped and removed the Staphylococcus aureus (SA) biofilm. Figure 6a shows the results of the scanning electron microscopy investigation into the untreated SA biofilm, which reveals a compact cellular structure inside the extracellular polymeric substance (EPS) matrix and hence a safe habitat for the organism. Limitation, cell death, and EPS matrix damage were the outcomes of treating biofilm with a 30% w/v concentration of KDNE.

DISCUSSION:

Polyphenols also quinones, natural plant-derived compounds, have garnered significant attention for their remarkable antibiofilm properties. These molecules work in different ways to stop different stages of biofilm formation²³. For example, they stop cells from attaching at the start, stop the production of extracellular matrix, and mess up the signalling pathways for quorum sensing²⁴. Flavonoids, tannins, and other phenolic compounds have been shown to be able to get into the biofilm matrix, cause oxidative stress, and damage the integrity of the microbiome cell membrane, which makes it harder for cells that are embedded in biofilm to stay alive²⁵. Similarly, studies have shown that quinones, a class of aromatic compounds, interfere with bacterial quorum sensing systems, crucial for coordinating biofilm development and maturation²⁶. Because of their strong antibacterial and antibiofilm properties, flavonoids and quinones have attracted a lot of interest from antimicrobial researchers. Flavonoids have antibacterial properties because they disrupt bacterial membranes, inhibit bacterial nucleic acid replication, reduce bacterial efflux pump function, and block bacterial adenosine triphosphate synthase²⁷. Offlavonoids are effective against bacteria because they may bind to extracellular proteins to create complex molecules that compromise bacterial cell membrane integrity²⁸. Quinones have antibacterial properties because they can create amino acid compounds that negatively impact protein function. When terpenoids bind to carbs and lipids in bacteria, they make their cell membranes less permeable²⁹.

Recent research has shown that kratom extracts may effectively hinder the development and disturb the structure of biofilms created by several harmful bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus³⁰. Kratom has the potential to be used in the development of antibiofilm drugs that specifically target diabetic ulcer infections such as its ability to impede bacterial quorum sensing and disrupt biofilm formation³¹. By incorporating these bioactive molecules into nanogel formulations, their stability, solubility, and targeted delivery to the infection site may be improved³². Attachment of microbial cells to a surface is the first step in the multi-stage process that culminates in the detachment of cells from the biofilm after microcolonies have developed and the biofilm structure has matured³³. This data implies that it is more problematic to suppress bacteria during biofilm development, since the inhibition percentage is lower compared to bacteria in planktonic form³⁴. For bacteria in planktonic form, KDNE had a substantial inhibitory effect of 83.58% ±0.41; for bacteria that had developed a biofilm during the mid-phase, the inhibitory effect was somewhat lower, at 79.21% ±0.8. These data support the idea that as the duration of biofilm development increases, there is a rise in the arrangement of the matrix, leading to a stronger and more complex biofilm structure. The intricate nature of this structure reduces the efficacy of both the test substance and pharmacological control in preventing the growth of biofilms³⁵.

The physicochemical properties of nanomaterials, including hydrophobicity and pore size, can be regulated to optimise their antimicrobial properties. Researchers have reported that increasing the nanoroughness of the surface reduces bacterial adhesion to medical devices, implying the development of nanogel formulations to curb biofilm formation and bacterial attachment³⁶. Nanogel formulations can also work as nanovehicles, making it easier to deliver naturally occurring compounds that can break down biofilms. Nanogel forms of kratom extract have been shown in new research to effectively stop the growth of biofilms made by many bacteria that are clinically important, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. As a result, the active chemicals might be more effectively administered locally at the site of infection, which could lessen the likelihood of systemic bad effects associated with traditional antibiotic treatments³⁷.

CONCLUSION:

The results indicate that the activity of KDNE in inhibiting the growth of Staphylococcus aureus biofilms varied with its concentration. At a concentration of 30%, the extract exhibited equivalent antibacterial, mid-phase, maturation, and eradication efficacy to the control. Plant-derived compounds have shown efficacy against Staphylococcus aureus, suggesting their potential as natural antibacterial agents.

CONFLICT OF INTEREST:

Regarding this study, the writers do not have any competing interests.

ACKNOWLEDGMENTS:

Universitas Lambung Mangkurat's competitive grant program in 2024 (contract number 1374.28/UN8.2/ PG/2024) is much appreciated. The researchers would like to express their gratitude to the Universitas Muhammadiyah East Kalimantan and the Universitas Lambung Mangkurat for their support in carrying out this study.

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Received on 22.09.2024 Revised on 21.01.2025

Accepted on 01.04.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5216-5222.

DOI: 10.52711/0974-360X.2025.00752

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.