Microwave-Assisted Extraction of Melastoma candidum D. Don Leaf and Chitosan–Tripolyphosphate Nanoparticles: Antioxidant and Antibacterial Activities

Muhammad Amin Nasution¹, Didi Nurhadi Illian^1,5, Hilda Maysarah¹, Dhea Nur Fadhilah¹,

Nur Irhamni Sabrina¹, Rena Meutia¹, Muhammad Andry², Muhammad Fauzan Lubis³*,

Nia Novranda Pertiwi⁴

¹Department of Pharmacy, Faculty of Mathematics and Natural Sciences,

Universitas Syiah Kuala, Aceh 23111, Indonesia.

²Department of Pharmacy, Faculty of Mathematics and Natural Sciences,

Universitas Sriwijaya, Sumatera Selatan 30662, Indonesia.

³Department of Pharmaceutical Biology, Faculty of Pharmacy,

Universitas Sumatera Utara, Sumatera Utara 20155, Indonesia.

⁴Departmen of Pharmacy, Faculty of Pharmacy, Universitas Mulawarman 75119, Samarinda, Indonesia.

⁵Doctoral Program of Pharmaceutical Sciences, Universitas Gadjah Mada, Yogyakarta, Indonesia.

*Corresponding Author E-mail: fauzan.lubis@usu.ac.id.

ABSTRACT:

Bacterial infections resulting from Gram-negative and Gram-positive bacteria are prevalent health issues. Due to increasing antibiotic resistance, alternative treatments are needed. The pharmacological activities of Melastoma candidum D. Don plant extracts are promising. Nanoparticle extracts enhance particle interaction, surface solubility, and antibacterial activity, making them attractive in health sciences. The study evaluated the in vitro antibacterial effectiveness of nanoparticles obtained from Melastoma candidum D. Don leaf extract against microorganisms using the disc diffusion technique, both gram-positive and gram-negative. The ethanol extract of Melastoma candidum D. Don leaf was obtained using Microwave-Assisted Extraction (MAE) with 96% ethanol. The total flavonoid quantity and antioxidant activity were assessed using the DPPH technique by UV-Visible spectrophotometry. Ionic gelation using 0.2% chitosan and 0.1% sodium tripolyphosphate (Na-TPP) produced nanoparticles. Nanoparticles were analyzed utilizing a Particle Size Analyzer (PSA) and Scanning Electron Microscopy (SEM). The antibacterial activity was evaluated at concentrations of 2.5%, 5%, 7.5%, and 10%. DMSO was the negative control and chloramphenicol the positive control. The ethanol extract contained 44.78± 0.18mgQE/g of total flavonoids. In the DPPH experiment, the IC₅₀ value was 12.51μg/mL, indicating strong antioxidant activity. PSA analysis determined an optimal nanoparticle size of 283.55nm, while SEM analysis revealed particle morphology at 500x and 1000x magnifications. The nanoparticles inhibited all tested bacteria, with inhibition zones for Escherichia coli ranging from 11.24 to 13.13mm across concentrations" to avoid implying higher concentrations were less active, which is the highest inhibitory power. The additional microorganisms examined comprised Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, and Cutibacterium acnes. Nanoparticles of the ethanol extract of Melastoma candidum D. Don leaf exhibited antibacterial activity. This study indicates that chitosan-based nanoparticles may function as an alternative antibacterial agent by suppressing the proliferation of gram-positive and gram-negative bacteria.

KEYWORDS: Nanoparticle, Melastoma candidum, MAE, Antioxidant, Antibacterial.

INTRODUCTION:

The increasing prevalence of bacterial infections and oxidative stress-related disorders has led to extensive research on natural sources of bioactive compounds. Medicinal plants, in particular, are valuable due to their rich phytochemical compositions and historical use in traditional medicine. Melastoma malabathricum is renowned for its anti-inflammatory, antibacterial, and wound-healing qualities. However, while Melastoma malabathricum has been extensively studied, its closely related species, Melastoma candidum, has received less attention despite showing similar medicinal properties and potential applications in antimicrobial therapies. In Melastoma candidum leaves, flavonoids, tannins, and phenolic substances have antibacterial and antioxidant effects. This study hypothesizes that the leaf extracts of Melastoma candidum can be effectively used to produce nanoparticles with enhanced antibacterial and antioxidant activities, offering a novel approach to combating bacterial infections and oxidative stress-related diseases^1-3.

An imbalance between free radicals and antioxidants causes oxidative stress, which is linked to cancer, cardiovascular, and neurological illnesses. Plant-derived natural antioxidants have garnered interest for their ability to neutralize free radicals and mitigate oxidative damage^4,5. Concurrently, the rise of antibiotic-resistant microorganisms has driven the search for alternative antimicrobial agents from natural sources^6,7. Nanotechnology provides a promising solution by augmenting the effectiveness of plant-derived bioactive chemicals. Nanoparticle formulations improve solubility, stability, and bioavailability, leading to enhanced antibacterial and antioxidant activities⁸. Prior research has shown that nanoparticles can enhance the medicinal efficacy of plant extracts by facilitating targeted administration and minimizing the necessary dosages⁹.

While Melastoma candidum is known for its medicinal properties, including its antimicrobial potential, conventional extraction treatments, such as maceration, are time-consuming and solvent-intensive.

This research gap points to the need for more efficient and sustainable methods of extraction, particularly for producing nanoparticles^10,11.

Microwave-Assisted Extraction (MAE) is developing as an advanced green extraction method that markedly decreases extraction time, solvent usage, and improves the effectiveness of compound separation. Despite the potential benefits of MAE, there is a lack of research on its application for the extraction of nanoparticles from Melastoma candidum leaves^12,13.

This study uses Microwave-Assisted Extraction (MAE) to create nanoparticles from Melastoma candidum leaf extracts to fill this gap. We hypothesize that MAE will improve the extraction efficiency of bioactive chemicals and facilitate the development of stable nanoparticles. The antibacterial efficacy of the produced nanoparticles will be assessed against relevant bacterial strains to determine their potential as innovative antimicrobial agents. This study intends to increase plant-derived nanoparticles' antibacterial and antioxidant properties through more efficient and environmentally friendly methods.

MATERIALS AND METHODS:

Materials:

This study employed materials including Melastoma candidum D.Don leaf, distilled water, 96% ethanol, acetic anhydride, nitric acid, sulfuric acid, amyl alcohol, iron(III) chloride, bismuth(III) nitrate, iodine, potassium iodide, magnesium powder, mercury(II) chloride, alpha-naphthol, lead(II) acetate, toluene, chloroform, n-hexane, hydrochloric acid, quercetin, aluminum chloride, sodium acetate, isopropanol, chitosan, sodium tripolyphosphate (Na-TPP), chloramphenicol, dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose (Na CMC), Mueller-Hinton Agar (MHA), Nutrient Agar (NA), and Nutrient Broth (NB).

Instrumentation:

The equipment used included Petri dishes, an autoclave, a centrifuge (Hitachi), an incubator (Memmert), a vernier caliper, an oven (Memmert), micropipettes, a vortex mixer, an analytical balance, a hotplate, a rotary evaporator, a Vasco Particle Size Analyzer (PSA), a Hitachi TM3000 Scanning Electron Microscope (SEM), a microwave-assisted extraction system, and a UV-Visible spectrophotometer.

Preparation of Ethanolic Leaf Extract of Melastoma candidum D.Don with Microwave-Assisted Extraction (MAE):

The leaf powder of Melastoma candidum D. Don (250 g) was added with a solvent of 96% ethanol (2500mL), thus forming a ratio of 1:10. Next, the extraction procedure was performed for 8 minutes with 450 watts of power. The extraction was performed at an internal temperature range of 40- 60^oC . The resulting mixture was subsequently filtered with Whatman No. 1filter paper. The filtrate was subsequently concentrated utilizing a rotary evaporator^14,15.

Examination of extract characterization:

As part of characterizing an extract, the amount of water present, the amount of water-soluble juice present, the amount of ethanol-soluble juice present, the amount of total ash present and the amount of acid-insoluble ash present are all measured¹⁵.

Phytochemical Screening of Ethanolic Leaf Extract of Melastoma candidum D.Don (EEMCL):

Phytochemical analyses were conducted on the ethanol extract of Melastoma candidum leaf (EEMCL) to detect flavonoids, phenolic compounds, triterpenoids, steroids, and alkaloids. Flavonoids were identified by adding hydrochloric acid and magnesium to the aqueous layer, resulting in a color change from orange to red. Phenolic compounds were detected by adding ferric chloride, causing a blue color change. Steroids and triterpenoids were identified by adding acetic anhydride and sulfuric acid to the chloroform layer, with green indicating steroids and red indicating triterpenoids. Alkaloids were detected by grinding the sample with sand and a chloroform-ammonia solution, followed by the addition of sulfuric acid and Mayer's reagent, which produced a white precipitate^16,17.

Determination of Total Flavonoid Content of Ethanolic Leaf Extract of Melastoma candidum D.Don (EEMCL):

We transferred the EEMCL into a 25mL volumetric flask and subsequently added methanol until the solution reached the 1000μg/mL level. Subsequently, 1mL of the liquid was introduced into a 10mL volumetric flask with a syringe. Subsequently, 1.5mL of methanol, 0.1mL of 10% aluminum chloride, 0.1mL of 1 M sodium acetate, and 2.8mL of distilled water were combined. After that, the methanol was added until the right amount was found. After being stirred until it was all the same, it was put away and left alone for 6 to 9 minutes. A 515nm lambda peak was then used to record the absorption. The value of absorption was shown as the mean±standard deviation of six separate tests^18,19.

DPPH (1,1diphenyl2 picryhidrazil) Method:

Sample Preparation:

A 25mg sample of Ethanol Leaf Extract of Melastoma candidum D.Don (EEMCL) was precisely measured and placed into a 25mL volumetric flask. The volume was then adjusted to the calibration mark using methanol to achieve a stock solution concentration of 1000μg/mL. Subsequently, 5mL of the stock solution was transferred into a second 25mL volumetric flask, and methanol was added to the calibration mark. The final concentration was established at 200μg/mL²⁰.

Preparation DPPH Solution:

The ethanol extract solution at a concentration of 200 μg/mL was pipetted in volumes of 0.3mL, 0.4mL, 0.5 mL, 0.6mL, and 0.7mL into separate 10mL volumetric flasks. Each flask was then supplemented with 1mL of DPPH solution at a concentration of 200μg/mL, and the volume was adjusted to the mark with methanol, resulting in final concentrations of 6ppm, 8ppm, 10 ppm, 12ppm, and 14ppm. The absorbance was measured at the peak wavelength of 515nm using a UV-Vis spectrophotometer²¹.

Preparation of Chitosan Solution:

The EEMCL solution (200μg/mL) was pipetted for 0.3, 0.4, 0.5, 0.6, and 0.7mL, respectively, and poured into a 10 mL-volumetric flask²².

Preparation of NaTPP Solution:

Sodium tripolyphosphate (NaTPP) was weighed to 0.1 g, then added to distilled water in a 1000mL beaker. The solution was stirred with a magnetic bar until completely dissolved. The NaTPP solution was adjusted to a final volume of 1000mL, resulting in a concentration of 0.01% (w/v)²³.

Preparation of Nanoparticles of Ethanolic Leaf Extract Melastoma candidum D.Don (EEMCL):

One gram of EEMCL was measured and dissolved in 35 mL of 96% ethanol and 15 mL of distilled water within a 1000mL beaker. Upon complete dissolution of the extract, 100mL of chitosan solution (1%w/v in 1% acetic acid) was included into the mixture. The pH of the chitosan–NaTPP mixture was carefully controlled to maintain a pH of around 5.5, as this is crucial for proper ionic gelation and particle size control. Next, 350 mL of NaTPP solution (0.01% w/v) was slowly poured into the mixture while stirring with a magnetic bar at a constant speed for 2hours. After stirring, the mixture was centrifuged to separate the chitosan from the NaTPP nanoparticle colloids of EEMCL. The nanoparticle solids were then refrigerated at approximately ±3°C to obtain a dry mass^24,25.

Preparation of nanoparticle test solution of Ethanolic Leaf Extract of Melastoma candidum D.Don (EEMCL):

0.1g of EEMCL nanoparticles was measured and dissolved in dimethyl sulfoxide (DMSO) to achieve a total volume of 2mL. The solution was agitated until fully dissolved, and the subsequent concentrations were formulated: 10%, 7.5%, 5%, and 2.5% (w/v)²⁶.

Antibacterial Activity Assay:

The antibacterial activity of EEMCL nanoparticles in various concentrations was investigated. Paper disc diffusion (Kirby–Bauer–type method) was used for this analysis²⁷.

Bacteria Assays:

This study utilized the following bacterial strains: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Streptococcus mutans (ATCC 25175). The inoculum (0.1mL) was produced to a density corresponding to the 0.5 McFarland standard. The inoculum was uniformly distributed across the surface of the Nutrient Agar (NA) medium, which had been previously placed onto sterile Petri dishes. The inoculation medium's temperature was sustained at 45 to 50°C for solidification. After solidification, paper discs were submerged in the EEMCL solution and EEMCL nanoparticles at different concentrations for 15minutes. The discs were subsequently positioned on the surface of the inoculated agar medium. The Petri plates were incubated at 35±2°C for a duration of 18 to 24hours. Following incubation, the diameter of the clear zone (inhibition zone) surrounding each paper disc was measured with a caliper. The mean diameter of the inhibitory zone from the three replicates was utilized for subsequent analysis²⁸.

RESULT:

Microwave Assisted Extraction (MAE) of Melastoma candidum D.Don Leaf:

Extraction on MAE was conducted at 450 watts for 8 minutes with 96% ethanol as the solvent. A rotary evaporator was employed to concentrate the extract through evaporation. The thick extract obtained in MAE extraction amounted to 32.590g (13.036%) with 250g of simplisia powder²⁹.

Results of Characterization Examination of Melastoma candidum D.Don Leaf Simplisia Powder:

Characterization of Melastoma candidum D.Don leaf extract yielded 4.6% water, 7.25% water-soluble essence, 0.43% extract that dissolves in ethanol, 58% total ash, and 19% acid-insoluble ash^30,31.

Phytochemical Screening Results:

Table 1. Phytochemical screening results of simplisia and Melastoma candidum D.Don leaf extracts

S. No.	Group of compounds	Simplisia	Extracts
1.	Alkaloids	+	+
2.	Flavonoids	+	+
3.	Tanins	+	+
4.	Glycosides	+	+
5.	Saponins	+	+
6.	Steroids/Triterpenoids	+	+

The analysis of secondary metabolites aims to provide a preliminary overview of the compound groups present within a sample. In this study, the ethanoli extract of Melastoma candidum D.Don leaf was analyzed to determine its phytochemical composition. Flavonoids, polyphenols, tannins, saponins, steroids, and alkaloids were some of the secondary metabolites that were found in the Melastoma candidum D.Don leaf samples Table 1³².

Results of Determination of Total Flavonoid Levels in Melastoma candidum D.Don Leaf:

The sample included milligrams of quercetin equivalents (QE) of flavonoids. Because it makes up 60–75% of plant flavonoids, quercetin was chosen as the standard. Additionally, quercetin's C-4 keto and C-3 or C-5 hydroxyl groups can form acidic compounds with aluminum chloride (AlCl₃). Six analyses quantified total flavonoids in Melastoma candidum D.Don leaf ethanol and ethyl acetate extracts. By comparing Melastoma candidum D.Don leaf ethanol extract absorption values, the calibration curve equation may calculate total flavonoid concentration. The study found 44.78 ± 0.18 mgQE/g in the ethanol extract of Melastoma candidum D.Don leaves. Table 2 indicates total flavonoids³³.

Results of Antioxidant Activity Analysis using the DPPH Method:

Melastoma candidum D.Don leaf ethanol extract antioxidant efficacy compared to ascorbic acid by % inhibition at different doses is shown in Table 3.

The IC₅₀ value for the ethanol extract of Melastoma candidum D.Don Leaf leaf is 12.51µg/mL. This would be classified as showing very strong antioxidant activity (IC₅₀ < 50µg/mL), as it falls well below the 50µg/mL threshold³⁴.

Particle size distribution results:

The Liquid nanoparticles were prepared by mixing chitosan and linking it with NaTPP. PSA measured particle size distribution for each recipe, which can be seen in (Figure 1)

Figure 1. Particle size distribution

The figure 1, we can observe a sharp peak near 0.1 µm on the x-axis, which indicates a large concentration of nanoparticles around this size. The distribution then drops off rapidly as the particle size increases. This suggests that most of the nanoparticles are within a narrow size range, with very few particles reaching larger sizes. The PSA test results for the nanoparticle extract from Melastoma candidum D.Don leaf were 283.55nm . This is a good result because it falls within the nanoparticle size range, which is below 1000nm³⁵.

Zeta potential:

Zeta potential evaluates the effective electrical charge on a nanoparticle's surface by quantifying its charge. When a nanoparticle possesses a surface charge, this charge is modulated by the concentration of counterions in proximity to the nanoparticle's surface. The layers of oppositely charged ions migrate with the nanoparticle and together with the layer. Zeta potential magnitude determines particle stability. Zeta potential results appear in Table 4.

Table 4. Zeta Potential Result

Replication	Zeta potential (mV)
1	−29.1
2	−29.2
3	−28.9
Mean	−29.07
SD	0.15

Particle surface charge is represented by zeta potential. Charge causes particles to agglomerate or repel. The zeta potential is -29.07V, which is less than -30mV and greater than +30mV, indicating better stability. The results obtained fail to meet the specified criteria, indicating that the zeta potential of Melastoma candidum D.Don leaf symplisia nanoparticles is less stable, suggesting a diminished repulsive force among the particles, which leads to a propensity for aggregation and dispersion³⁶.

Particle morphology results:

The dried solid particles produced from the Ethanolic Leaf Extract of Melastoma candidum D.Don were analyzed for shape and morphology using Scanning Electron Microscopy (SEM) at 500x and 1000x magnifications, as shown in (Figure 2).

Figure 2: SEM results of formula A with magnification of (a) 500 times magnification and (b) 1000 times magnification.

The SEM images reveal that the particles are predominantly irregular in shape, with some aggregates visible in both magnification levels (Figures 2a and 2b). These particles exhibit an uneven surface, characteristic of nanoparticles formed through the ionic gelation process. The irregular shape and aggregation suggest that the nanoparticles are not uniform and have a rough surface texture, which could be attributed to the nature of the ionic gelation process used in the synthesis. This process often results in particles that are prone to aggregation and variability in size and morphology, the particle size obtained was 283.55nm, determined from the SEM image, with a nanoparticle size range of 10-1000nm. In addition, the particle size distribution was observed to have a wide range, with a certain degree of aggregation, indicating that the particles were not uniform in size. The scale bars in the images represent 50µm (A) and 100µm (B), providing a reference for particle dimensions ³⁷.

Antibacterial Activity Test Results of Ethanol Leaf Extract Nanoparticles of Melastoma candidum D.Don

(a) (b)

(c) (d)

(e) (f)

Figure 6 Ethanol extract nanoparticles against (a) Staphylococcus epidermidis, (b) Cutibacterium acnes, (c) Pseudomonas aeruginosa, (d) Staphylococcus aureus, (e) Escherichia coli and (f) Streptococcus mutan

DISCUSSION:

The specific gravity of the Melastoma candidum leaf extract was determined to be 7.25% in water and 0.43% in ethanol, indicating a moderate solubility profile. These values are consistent with those found in similar plant extracts, such as Melastoma malabathricum, which also demonstrates solubility in both aqueous and ethanol mediums, as reported in previous studies. The total ash content of the simplisia was found to be 58%, which is in line with the range of 50-60% ash content typically reported for medicinal plants. This suggests that a significant proportion of the extract consists of inorganic components. The acid-insoluble ash content was 19%, suggesting the presence of non-physiological impurities such as soil or sand particles, which is a common characteristic observed in plant-based extracts, as noted in pharmacopoeial standards³⁸.

The antioxidant activity of Melastoma candidum D.Don leaf extract and ascorbic acid was tested after 60 minutes. At saturation levels of 6, 8, 10, 12, and 14 μg/ml, the test solution was added. Melastoma candidum D.Don leaf ethanol starch antioxidants donate electrons to DPPH radicals to fight them. This electron transfer changes the solution's colour from purple to yellow or reduces its intensity. Thus, this colour change reduces DPPH free radical absorption⁴³. Acidic Ascorbic Acid (Vitamin C) is used as a standard. The purpose of using ascorbic acid in the antioxidant activity test is to find out if Melastoma candidum D.Don. has the ability to be used to get antioxidants. When comparing it to ascorbic acid, its sources are very important. If the sample's IC50 value is very close to that of ascorbic acid, then it's likely that this sample is an important part of a strong antioxidant. The IC50 value for the ethanol extract of Melastoma candidum D.Don is 12.51µg/ml, as shown in Table 3. Besides that, it shows that the more concentrated the fluid or product is, the better it is at getting rid of free radicals. This is because more hydrogen atoms are added, and the DPPH level goes down. This makes the DPPH color lighter³⁹.

Table 5 shows that the inhibition zone diameter increases with higher concentrations of Melastoma candidum D.Don leaf nanoparticle extract, indicating that higher concentrations provide more active compounds. The absence of bacterial growth in the clear zone around the treatment suggests the antibacterial efficacy of the nanoparticles, while bacterial growth was observed in other areas. The largest inhibition zone was seen at a 2.5% concentration (11.24mm) against Escherichia coli. At higher concentrations (5%, 7.5%, and 10%), significant efficacy was observed against Staphylococcus epidermidis, Cutibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Streptococcus⁴⁰.

Interestingly, Escherichia coli demonstrated the largest inhibition zone, which could be attributed to its unique cell wall structure. E. coli, being a Gram-negative bacterium, has a thinner peptidoglycan layer and an outer membrane that may facilitate better penetration of the nanoparticles compared to other Gram-positive bacteria, which have thicker peptidoglycan layers that act as a stronger barrier. This difference in cell wall structure could explain the heightened sensitivity of E. coli to the nanoparticles. Furthermore, the nanoparticles' interaction with the bacterial membrane, and their ability to disrupt cell function, might be more effective against E. coli due to its membrane composition and permeability. Antibacterial activity was also demonstrated against Staphylococcus aureus and Pseudomonas aeruginosa at 5%, 7.5%, and 10% doses. Nanoparticle extract had narrower inhibitory zones than chloramphenicol. However, Melastoma candidum leaf nanoparticles' antibacterial activity against Gram-positive and Gram-negative bacteria suggests they may be effective antibacterial agents⁴¹.

CONCLUSION:

The ethanolic leaf extract of Melastoma candidum D. Don, obtained through Microwave-Assisted Extraction (MAE), showed promising phytochemical and functional properties. The extract yielded 13.036% and complied with standard quality parameters, though high ash levels (58% total ash, 19% acid-insoluble ash) suggest significant inorganic materials. Phytochemical screening identified alkaloids, flavonoids, tannins, glycosides, saponins, and steroids/triterpenoids, indicating pharmacological potential. It contained 44.78 ± 0.18 mgQE/g flavonoids and exhibited strong antioxidant activity (IC50 = 12.51 µg/ml). The extract formed nanoparticles using chitosan-NaTPP crosslinking, showing irregular morphology. These nanoparticles demonstrated strong antibacterial activity, especially against Escherichia coli (11.24 mm at 2.5%). This extract has potential as an antioxidant and antimicrobial agent, though nanoparticle optimization is needed for improved pharmaceutical or cosmeceutical applications.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest pertaining to this work.

ACKNOWLEDGMENTS:

The authors thank Universitas Syiah Kuala Research Laboratory for scientific and technical support. Universitas Sumatera Utara and Syiah Kuala supplied facilities and equipment for the research.

REFERENCES:

1. Addissouky TA. El Sayed IET. Al MM. Wang Y. El Baz A, Elarabany N, Khalil AA. Oxidative stress and inflammation: elucidating mechanisms of smoking-attributable pathology for therapeutic targeting. Bulletin of the National Research Centre. 2024; 48(1): 16. doi.org/10.1186/s42269-024-01174-6.

2. Qureshi KA. Parvez A. Khan MMU. Aspatwar A. Atiya A. Elhassan GO. Jaremko M. Exploring nature's hidden treasure: Unraveling the untapped phytochemical and pharmacological potentials of Clinopodium vulgare L.–A hidden gem in the Lamiaceae family. Heliyon. 2024; 10(2). doi.org/10.1016/j.heliyon.2024.e24781.

3. Zheng WJ. Ren YS. Wu ML. Yang YL. Fan Y. Piao XH. Wang SM. A review of the traditional uses, phytochemistry and biological activities of the Melastoma genus. Journal of Ethnopharmacology. 2021; 264: 113322. doi.org/10.1016/j.jep.2020.113322.

4. Leyane TS. Jere SW. Houreld. NN. Oxidative stress in ageing and chronic degenerative pathologies: molecular mechanisms involved in counteracting oxidative stress and chronic inflammation. International Journal of Molecular Sciences. 2022; 23(13): 7273. doi.org/10.3390/ijms23137273.

5. Fitri K. Andry M. Khairani TN. Nasution MA. Bu'ulolo AAC. Lubis MF. Antioxidant Potential of Ethanol Extracts Leaf and Stem of Nelumbo nucifera Gaertn. with DPPH, ABTS, and Frap Methods. Research Journal of Pharmacy and Technology. 2024; 17(12): 6084-6090. doi.org/10.52711/0974-360x.2024.00922.

6. Lubis LD. Prananda AT. Juwita NA. Nasution MA. Syahputra RA. Sumaiyah S. Atiqah JF. Unveiling antioxidant capacity of standardized chitosan-tripolyphosphate microcapsules containing polyphenol-rich extract of Portulaca oleraceae. Heliyon. 2024; 10(8). doi.org/10.1016/j.heliyon.2024.e29541.

7. Akbari B. Baghaei‐Yazdi N. Bahmaie M. Mahdavi AF. The role of plant‐derived natural antioxidants in reduction of oxidative stress. BioFactors. 2022; 48(3): 611-633. doi.org/10.1002/biof.1831.

8. Nasution MA. Andry M. Lubis MF. Illian DN. Rani Z. Fauzi ZPA. Phytochemical Analysis and Nanoparticle Formulations of Extracts Myristica fragrans Houtt Leaf as Antibacterial. Trends in Sciences. 2024; 21(10): 8335-8335. doi.org/10.48048/tis.2024.8335.

9. Elumalai K. Srinivasan S. Shanmugam A. Review of the efficacy of nanoparticle-based drug delivery systems for cancer treatment. Biomedical Technology. 2024; 5: 109-122. doi.org/10.1016/j.bmt.2023.09.001.

10. Peres Fabbri L. Cavallero A. Vidotto F. Abriele M. Bioactive Peptides from Fermented Foods: Production Approaches, Sources, and Potential Health Benefits. Foods. 2024; 13(21): 3369. doi.org/10.3390/foods13213369.

11. Gazizova A. Datkhayev U. Amirkhanova A. Ustenova G. Kozhanova K. Ikhsanov Y. Berdgaleyeva A. Phytochemical Profiling of Mentha asiatica Boriss. Leaf Extracts: Antioxidant and Antibacterial Activities. ES Food and Agroforestry. 2025; 19: 1355. doi.org/10.30919/esfaf1355.

12. Usman M. Nakagawa M. Cheng S. Emerging trends in green extraction techniques for bioactive natural products. Processes. 2023; 11(12): 3444. doi.org/10.3390/pr11123444.

13. Da Silva RF. Carneiro CN. de Sousa CBDC. Gomez FJ. Espino M. Boiteux J. Dias FDS. Sustainable extraction bioactive compounds procedures in medicinal plants based on the principles of green analytical chemistry: A review. Microchemical Journal. 2022; 175: 107184. doi.org/10.1016/j.microc.2022.107184.

14. Bitwell C. Indra SS. Luke C. Kakoma MK. A review of modern and conventional extraction techniques and their applications for extracting phytochemicals from plants. Scientific African. 2023; 19: e01585. doi.org/10.1016/j.sciaf.2023.e01585.

15. Boli E. Prinos N. Louli V. Pappa G. Stamatis H. Magoulas K. Voutsas E. Recovery of bioactive extracts from olive leaf using conventional and microwave-assisted extraction with classical and deep eutectic solvents. Separations. 2022; 9(9): 255. doi.org/10.3390/separations9090255.

16. Mokaizh AAB. Nour AH. Ukaegbu CI. Microwave-assisted extraction of phenolic compounds from Commiphora gileadensis leaf and their characterization. Results in Engineering. 2024; 24: 102892. doi.org/10.1016/j.rineng.2024.102892.

17. Anggraini L. Emriadi Y. Zulaiha S. Pardi H. Stainless 37 Steel Corrosion Inhibition in a Hydrochloric Acid Solution with Senggani (Melastoma Candidum D. Don) Leaf Extract. Portugaliae Electrochimica Acta. 2023; 41(3): 199-210. doi.org/10.4152/pea.2023410302.

18. Andry M. Ginting I. Nasution MA. Lubis MF. Impact of Solvent Type on Total Flavonoid Content and Sun Protection Factors in Centella asiatica (L.) Urban Leaf Extract. Malaysian Journal of Fundamental and Applied Sciences. 2025; 21(3): 1998-2007. doi.org/10.11113/mjfas.v21n3.3603.

19. Erenler R. Yaman C. Demirtas L. Hakki Alma M. Phytochemical investigation of Hypericum heterophyllum flowers: LC-ESI-MS/MS analysis, total phenolic and flavonoid contents, antioxidant activity. The Natural Products Journal. 2023; 13(7): 37-45. doi.org/10.2174/2210315513666230112165545.

20. Ihsanpuro SI. Gunawan S. Ibrahim R. Aparamarta HW. Extract with high 1, 1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory capability from pericarp and seed of mangosteen (Garcinia mangostana L.) using microwave-assisted extraction (MAE) two-phase solvent technique. Arabian Journal of Chemistry. 2022; 15(12): 104310. doi.org/10.1016/j.arabjc.2022.104310.

21. Baliyan S. Mukherjee R. Priyadarshini A. Vibhuti A. Gupta A. Pandey RP. Chang CM. Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of Ficus religiosa. Molecules. 2022; 27(4): 1326. doi.org/10.3390/molecules27041326.

22. Liu M. Chen H. Pan F. Wu X. Zhang Y. Fang X. Peng W. Propolis ethanol extract functionalized chitosan/Tenebrio molitor larvae protein film for sustainable active food packaging. Carbohydrate Polymers. 2024; 343: 122445. doi.org/10.1016/j.carbpol.2024.122445.

23. Ghadiri L. Naji L. Javanbakht M. Fabrication of high performance cation-exchange membrane based on sodium tripolyphosphate (NaTPP)-grafted graphene oxide for electrodialysis. Separation and Purification Technology. 2024; 330: 125402. doi.org/10.1016/j.seppur.2023.125402.

24. Lakikza I. Benguerba Y. Boublia A. Aouni SI. Lahbib H. Ferkous H. Ernst B. Comprehensive evaluation of Alysicarpus compactum extract as a natural corrosion inhibitor for St37 carbon steel in acidic media. Journal of Industrial and Engineering Chemistry. 2025; 147: 161-178. doi.org/10.1016/j.jiec.2024.12.010.

25. Fahmawati T. Akbar RIS. Kaswati NMN. Syakuran LA. Safithri M. The evaluation of antioxidant activity of cinnamon bark (Cinnamomum burmannii) nanopowder in comparison with extracts. Materials Today: Proceedings. 2024. doi.org/10.1016/j.matpr.2024.03.037.

26. Šukele R. Lauberte L. Kovalcuka L. Logviss K. Bārzdiņa A. Brangule A. Bandere D. Chemical Profiling and Antioxidant Activity of Tanacetum vulgare L. Wild-Growing in Latvia. Plants. 2023; 12(10): 1968. doi.org/10.3390/plants12101968

27. Mohammed MJ. Anand U. Altemimi AB. Tripathi V. Guo Y. Pratap-Singh A. Phenolic composition, antioxidant capacity and antibacterial activity of white wormwood (Artemisia herba-alba). Plants. 2021; 10(1): 164. doi.org/10.3390/plants10010164.

28. Quan H. Wang S. Xi X. Zhang Y. Ding Y. Li Y. Liu Y. Deep learning enhanced multiplex detection of viable foodborne pathogens in digital microfluidic chip. Biosensors and Bioelectronics. 2024; 245: 115837. doi.org/10.1016/j.bios.2023.115837.

29. Situmorang VC. Ramadhani S. Okselni T. Angelina M. Dewi RT. Rahmi EP. Septama AW. Microwave-assisted green synthesis of Cassia alata-mediated gold nanoparticles and evaluation of its antioxidant, anti-inflammatory, and antibacterial activities. Biomass Conversion and Biorefinery. 2025; 15(6): 8701-8714. doi.org/10.1007/s13399-024-05950-2.

30. Prasetya F. Salam S. Rijai HR. Kuncoro H. Rusli R. Rahmadani A. Rijai L. Pharmacognostic Profile of Simplicia and Ethanolic Leaf Extract from Indonesian Piper betle var. nigra. Pharmacognosy Journal. 2022; 14(5). doi.org/10.5530/pj.2022.14.143.

31. Febriyanti MK. Nurhasnawati H. The characterization of the simplies and ethanol extracts of limpasu leaf and fruit (Baccaurea lanceolata (Miq.) Müll. Arg.). Journal of Pharmacognosy and Phytochemistry. 2023; 12(2): 131-137. doi.org/10.20527/jps.v6i2.7345.

32. Dubale S. Kebebe D. Zeynudin A. Abdissa N. Suleman S. Phytochemical screening and antimicrobial activity evaluation of selected medicinal plants in Ethiopia. Journal of experimental pharmacology. 2023; 51-62. doi.org/10.2147/jep.s379805.

33. Vaou N. Stavropoulou E. Voidarou C. Tsigalou C. Bezirtzoglou E. Towards advances in medicinal plant antimicrobial activity: A review study on challenges and future perspectives. Microorganisms. 2021; 9(10): 2041. doi.org/10.3390/microorganisms9102041.

34. Jain A. Bhise K. Biogenic zinc oxide nanoparticles from Saraca asoca: cytotoxicity, antioxidant, antimicrobial evaluation, and topical gel development. Journal of Applied Pharmaceutical Research. 2025; 13(5): 147-164. doi.org/10.69857/joapr.v13i5.1300.

35. Zając M. Kotyńska J. Zambrowski G. Breczko J. Deptuła P. Cieśluk M. Naumowicz M.Exposure to polystyrene nanoparticles leads to changes in the zeta potential of bacterial cells. Scientific Reports. 2023; 13(1): 9552. doi.org/10.1038/s41598-023-36603-5.

36. Faisal S. Jan H. Shah SA. Shah S. Khan A. Akbar MT. Syed S. Green synthesis of zinc oxide (ZnO) nanoparticles using aqueous fruit extracts of Myristica fragrans: their characterizations and biological and environmental applications. ACS omega. 2021; 6(14): 9709-9722. doi.org/10.1021/acsomega.1c00310.

37. Tiji S. Benayad O. Berrabah M. El Mounsi I. Mimouni M. Phytochemical profile and antioxidant activity of Nigella sativa L growing in Morocco. The Scientific World Journal. 2021; 2021(1): 6623609. oi.org/10.1155/2021/6623609.

38. Vinci G. D’Ascenzo F. Maddaloni L. Prencipe SA. Tiradritti M. The influence of green and black tea infusion parameters on total polyphenol content and antioxidant activity by ABTS and DPPH assays. Beverages. 2022; 8(2): 18. doi.org/10.3390/beverages8020018.

39. Wołosiak R. Drużyńska B. Derewiaka D. Piecyk M. Majewska E. Ciecierska M. Pakosz P. Verification of the conditions for determination of antioxidant activity by ABTS and DPPH assays—A practical approach. Molecules, 2021; 27(1): 50. doi.org/10.3390/molecules27010050.

40. Yadav A. Kumar H. Kumar P. Rani G. Maken S. Syzygium cumini leaf extract mediated green synthesis of ZnO nanoparticles: A sustained release for anticancer, antimicrobial, antioxidant, and anti-corrosive applications. Journal of Molecular Structure. 2025; 1325: 141017. doi.org/10.1016/j.molstruc.2024.141017.

41. Hamid L. L. Hamed H. Al-Fahdawi A. M. Hamid S. L. Mutter T. Y. Ali H. H. Truffle mediated preparation of bacterial culture medium and mycosynthesis of titanium oxide nanoparticles loaded with polyvinyl alcohol/sodium alginate aerogel beads for antibacterial activity. Journal of Sol-Gel Science and Technology. 2024; 112(2): 512-523.

Received on 05.08.2025 Revised on 16.12.2025

Accepted on 21.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3273-3280.

DOI: 10.52711/0974-360X.2026.00466

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

Test Solution	No.	Sample Weight (g)	Absorbance	Concentration (mg/ml)	Total Flavonoid Content of Extract (mgQE/g)	Actual Level (mgQE/g Extract)
Ethanol Extract of Melastoma candidum D.Don Leaf	1.	0.025	0.342	4.4605	44.605	44.78 ± 0.18 mgQE/g extract
	2.	0.025	0.343	4.4737	44.737
	3.	0.025	0.343	4.4737	44.737
	4.	0.025	0.344	4.4868	44.868
	5.	0.025	0.345	4.4868	44.868
	6.	0.025	0.345	4.4868	44.868

Sample	Concentration	% Inhibition	IC₅₀ Values
Extract Ethananol Melastoma candidum D.Don leaf	6 μ/ml	16.73%	12.51 μ/ml
	8 μ/ml	29.64%
	10 μ/ml	38.01%
	12 μ/ml	49.27%
	14 μ/ml	58.88%

Concentration of Ethanol Extract of Melastoma candidum D.Don leaf (%)	Diameter of Area Inhibition zone (mm)
	Staphylococcus epidermidis	Cutibacterium acnes	Pseudomonas aeruginosa	Staphylococcus aureus	Escherichia coli	Streptococcus mutans
2.5	10.23±0.15^a	9.60±0.10^a	9.93±0.15^a	10.40±0.10^a	11.24±0.05^a	9.73±0.20^a
5	11.46±0.15^b	10.80±0.10^b	10.60±0.10^b	11.53±0.37^b	12.23±0,30^b	10.41±0.07^ab
7.5	13.30±0.20^c	11.51±0.10^c	10.73±0.20^b	12.50±0.20^c	13.13±0,41^b	10.86±0.25^bc
10	14.16±0.15^d	12.50±0.10^d	11.46±0.20^c	14±0.10^d	14.80±0.10^c	11.60±0.43^c
Control Positive (Chloramphenicol	33.80±0.30^e	32.83±0.35^e	33.4±0.20^d	35.10±0.26^e	31.10±0.26^d	29.86±0.30^d