INTRODUCTION:

The rhizosphere is a complex habitat rich in microbial diversity, especially bacteria capable of producing various secondary metabolitesy¹. Pigment is one of these metabolites and has a wide range of possible uses in the manufacturing of paints, plastics, inks, cosmetics, food, and medicines. As a result, the demand for pigments has increased significantly. As an alternative to natural pigments, those produced by microbes, especially bacteria, are increasingly preferred by researchers due to their stability and abundant availability. Bacterially produced pigments exist in various forms, including the red pigment known as prodigiosin²^,³.

The chemical structure of the red pigment known as prodigiosin includes an alkaloid tripyrrole ring⁴. Several bacterial genera, including Streptomyces, Vibrio, and Serratia produced prodigiosin, with Serratia being the most widely reported genus⁵. Within the proteobacterial group, Serratia is a Gram-negative bacteria isolated from various environments, including soil, water, animals, plants, and the air. There are currently over ten species of Serratia in existence⁶. To date, the studies have focused on Serratia species capable of producing prodigiosin, including S. marcescens, S. rubidaea, S. plymuthica⁶, and S. nematodiphila⁷. Prodigiosin synthesis in Serratia is controlled by several gene clusters, including the Pig gene, which is ±20 kb in size⁸. The prodigiosin synthesis pathway is a complex process with prodigiosin synthetases (PigC) as condensing enzymes in the terminal pathway, 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amyl-pyrrole (MAP). Prodigiosin exhibits a broad range of bioactivities, including antibacterial, antifungal, immunosuppressive, algicidal, insecticidal, antimalarial, anticancer, antiprotozoal, and antioxidant activities⁹^,¹⁰^,¹¹.

Free radicals have received considerable attention from researchers because of their dual nature. Under normal conditions, they function as beneficial compounds when balanced by antioxidants. Nevertheless, they become toxic and may cause the build-up of reactive oxygen species (ROS) when their levels in the body become excessive. Oxidative stress, which is brought on by ROS, can harm biomolecules like proteins, DNA, and lipids and contribute to various degenerative diseases including, cancer, cardiovascular disease, atherosclerosis, metabolic disorders, and diabetes¹²^,¹³^,¹⁴^,¹⁵. Therefore, antioxidants are essential for preventing the production of free radicals, lowering oxidative stress, and enhancing immune function¹⁶. Several studies have reported that prodigiosin from S. marcescens SEM has antioxidant activity¹⁷. Similarly, Sajjad et al. (2018)¹⁸ also reported that the red pigment from Streptomyces sp. strain WMA-LM31 has antioxidant activities against DPPH radicals.

One of the strategies to meet current antioxidant requirements is to use synthetic antioxidants that are currently widely available, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and n-propyl gallate (PG). However, it is essential to highlight that these products have serious implications and pose a significant health risk when consumed in the long term. These negative effects include endocrine disruption, induction of oxidative stress, cytotoxicity, carcinogenicity, and tumor development. Therefore, natural antioxidants are an alternative that can be used as antioxidant agents with minimal negative effects¹⁹^,²⁰. The potency of red pigments as antioxidants has been extensively documented using various in vitro methods. However, no such evidence has been reported at the cellular level. In the present study, the model organism S. pombe was used to perform an assay at the cellular level.

Previous studies have successfully isolated red-colored bacteria from the rhizosphere soil of Rafflesia plants. The aim of this study was to investigate the potential role of these bacterial pigments as antioxidants by evaluating their activities both in vitro and at the cellular level. Our findings demonstrated that the antioxidant properties extend to the cellular level, suggesting the potential utility of S. nematodiphila RR16 prodigiosin pigments for further development in the health and pharmaceutical fields.

MATERIALS AND METHODS:

Materials:

The bacterial strain RR16 was previously isolated from the soil rhizosphere of Rafflesia (R. arnoldii) in Kepahiang, Bengkulu, Indonesia (3°41'24.6"S 102°31'48. 3"E) and routinely cultivated in Luria Berthani (LB) medium (Tripton 10g, NaCl 10g, and yeast extract 5g, and 1000 mL of distilled water). The yeast S. pombe ARC039 (h-leu1-32 ura4-294) was provided by Dr. Rika Indri Astuti (IPB University-Indonesia) and maintained on Yeast Extract Supplement (YES) medium (5g/L yeast extract, 20g/L agar (Himedia, India), 30g/L glucose, 0.128g/L histidine, 0.128g/L leucine, 0.128g/L adenine, 0.01g/L uracil, and 0.128g/L arginine).

Methods of Characterization of red pigment-producing bacteria:

The red pigment-producing isolate RR16 was characterized by morphological observations and hemolytic activity analysis. RR16 isolate was cultivated on LB agar medium. The morphological characteristics of the isolates included the shape, elevation, surface, edges, and color of the colonies. The hemolytic activity of the RR16 isolate was tested using blood agar. Staphylococcus aureus was used as a positive control, while Escherichia coli DH5α was used as a negative control⁸.

Molecular identification:

The isolate RR16 was molecularly identified based on the 16S rRNA gene. The Zymo Research Quick-DNATM Fungal/Bacterial Miniprep Kit (CA, US) was used to extract genomic DNA in accordance with the manufacturer's instructions. Amplification of the 16S rRNA gene was performed using primers 63F (5'- CAGGCCTAACAC ATGCAAGTC-3') and 1387R (5'-GGGCGGWGTGTACAAGGC-3') with an amplicon size of ±1300 bp²¹. The reaction mixture (50 µL) consisted of 25µL My Taq HS Red Mastermix 2x, 4 µL of each F/R primer (10pmol), 4µL DNA template (100ng) and 13µL nuclease-free water. The PCR conditions were a pre-denaturation step at 94°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1min and 30 s, and a final elongation at 72°C for 10 min. The PCR products were visualized on a 1% (w/v) agarose gel and sent to First Base Sequencing Service (Selangor, Malaysia) for further analysis. The nucleotide sequences were aligned to the National Center for Biotechnology Information (NCBI) GenBank database using BLASTn, and phylogenetic trees were constructed using MEGA 11 with the neighbor-joining method and a bootstrap value of 1000 times.

Optimization and extraction of red pigment from pigmented bacteria:

Isolate RR16 was cultured in LB medium for five days in the dark. The culture was then centrifuged at 4000 rpm for 30 min. The cell biomass was then extracted with organic solvents, including methanol, acetone, chloroform, and ethyl acetate while the supernatant was extracted with chloroform, ethyl acetate, and n-hexane in a 1:1(v/v) ratio. The best solvent selection was determined based on the OD value at a wavelength of 530nm²². Selected extracts from both cell biomass and supernatant were evaporated at 45°C using a rotary evaporator for further analysis²³.

Antioxidant potential of crude extract pigments:

The antioxidant activity test was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method²⁴. Each crude extract was dissolved in methanol at different concentrations. Briefly, 100μL of each extract was combined with 100μL of DPPH (125 μM in methanol) in a 96-well plate. The plate was then incubated for 30 minutes in the dark and the absorbance was measured at 517nm. Ascorbic acid was used as the positive control²⁵. Percent inhibition was calculated using the following formula:

^{Percent inhibition (%) = [1 - (A}sample ^{- A}control^{) / (A}blank ^{- A}control^{)] x 100%.}

^{Where, A}sample^{: Absorbance of the sample; A}control^{:
Absorbance of ascorbic acid; and A}blank^{: absorbance of the}DPPH and methanol. The percent inhibition obtained was then substituted into the linear equation to determine ^{the Inhibitory Concentration 50(IC}50^)
24.

Screening and quantification of the crude red pigment extracts:

The screening for red pigment was analyzed by presumptive test prodigiosin²³. The crude extract was divided into two test tubes. The first tube adds a drop of concentrated HCl, while the second adds a drop of concentrated ammonia.

The prodigiosin concentration was determined at 499 nm and 620 nm wavelengths. The OD values were ^{substituted
into the following formula}²⁶^{: Prodigiosin unit/cell = ([OD}499 ^{- (1.381 x OD}620^{)]) x
1000/ OD}620^{. Where, OD}499^{: Absorbance of pigment in
culture; OD}620^{: Absorbance of bacterial culture; 1.381: Constanta.}

Thin-layer chromatography (TLC) fractionation:

Fractionation of red pigments was performed according to the TLC method described by Prastya et al. (2020)²⁴ with modifications. Aluminium TLC plates coated with silica gel 60F-254 were used as the stationary phase²⁷^,²⁸. A total volume of 10μL of the crude extract of RR16 (7% (w/v) in chloroform) was applied to the TLC plate using the CAMAG Linomat 5. The mobile phase used for fractionation was dichloromethane and ethyl acetate in a ratio of 8.5:0.5 (v/v). Observations were made under UV light at a wavelength of 254nm, as done by Housheh et al. (2017)²⁹ and 366nm. Bioautographic TLC analysis was performed by spraying 5mM DPPH onto a TLC plate and incubating for 30minutes in the dark. The active fraction was then isolated by preparative TLC and the DPPH assay was performed as previously described.

Yeast oxidative stress tolerance assay (spot test):

S. pombe ARC09 was used as the model organism in this oxidative stress assay. Yeast colonies were cultivated ^{on YES medium and incubated
for 24 hours until the culture reached an optical density (OD}600^{) of
0.05. The}culture was then transferred to fresh YES medium containing different concentrations of crude extract and biopigment fractions (in DMSO) to a final volume of 3mL. The two positive controls used were cultures in YES medium supplemented with 5 µg/ml ascorbic acid and cultures in YES medium with a reduced glucose concentration (0.3% w/v). The negative control was culture in YES medium supplemented with DMSO (v/v). ^{The cultures were incubated at
120rpm for 24h, and OD}600 ^{values
were measured to obtain a value of 1. They}were then serially diluted to 10^-1, 10^-2, 10^-3, and 10^-4. The results of these dilutions were spotted on YES solid ^{medium
containing H}2^O2 ^{(0.5mM,
1, and 2 mM) in triplicate}³⁰^.

Mitochondrial activity assay:

For the mitochondrial activity assay, treatment and control conditions were performed as for the oxidative stress tolerance assay, followed by incubation for 18 h at 30°C. The culture was then centrifuged at 4000rpm for 3 min and washed with 0.1 M phosphate buffer (pH 7), followed by the addition of 200 nM rhodamine B. Mitochondrial activity was observed using an Olympus BX51 fluorescence microscope³¹.

Structural characterization of the red pigment fraction:

The crude extract and fraction of the red pigment were characterized by UV-Vis spectroscopy at wavelengths in the range of 200-700nm⁸. The functional groups of the fractionated red pigments were analyzed using Fourier Transform Infrared (FTIR) spectroscopy (Bruker-Tensor II) with Attenuated Total Reflectance (ATR) accessories, covering the frequency range of 4000-500 cm^-1 with a resolution of 2.0 cm³²^,³³. ¹H-NMR spectra of ^{the red pigment fraction reconstituted with CDCl}3 ^{were recorded using a Bruker Advance
700 MHz}spectrometer (Bruker Co., Billerica, MA, USA) as done by Balkrushna et al. (2018)³⁴ with modifications.

Detection of the PigC gene, domain prediction and three-dimensional structure modeling:

The PigC gene was amplified using primers SMf (5'-CGCTGGGCATTCTCAGCCTGGTGGAGACGG-3') and SMr (5'-GGCCGGGTCGCTTCGCGGCGTTCGGCC-3')³⁵. The PCR reaction used is the same as that used in the molecular identification method. The PCR conditions were 35 cycles, including pre-denaturation (94°C for 5 min), denaturation (95°C for 30 s), annealing (62°C for 30 s), elongation (72°C for 1 min), and post- elongation (72°C for 10min). The sequencing results were translated into amino acid sequences and aligned with the GenBank database of the National Center for Biotechnology Information (NCBI) database using BLASTx. A phenetic tree was constructed with MEGA 11 using the Jones-Taylor-Thornton (JTT) method with a bootstrap value of 1000. The amino acid sequence was aligned to the reference sequence using ClustalW. The prediction of the three-dimensional structural model of the partial PigC protein and the superposition analysis were performed using the SWISS-MODEL program, and the protein model structure was validated using the PROCHECK program. The partial PigC protein of RR16 isolate was aligned with the intact PigC protein of S. marcescens jx-1³⁶ to obtain the domain prediction of the partial PigC protein of the RR16 isolate.

Data analysis:

^{The observed parameter in this study was
the IC}50 ^{value.
The value analyzed using ANOVA
followed by}Duncan's significant difference test at the 5% level with a completely randomized design model.

RESULT:

Characterization of the red pigment-producing bacteria:

The morphological characteristics of RR16 colonies grown on LB agar medium showed a circular shape with convex elevation, smooth surface, entire edges, and red colony color (figure 1A). The isolate RR16 showed no hemolytic activity which did not form a zone on the medium as in the positive control (figure 1B).

Molecular identification:

The isolate RR16 based on 16S rRNA gene revealed a high level of similarity with S. nematodiphila DZ0503SBS1 (NR_044385.1) with a similarity of 98.06% (E-value 0.0, Query Cover 100%). The construction of a phylogenetic tree indicated a close relationship with S. nematodiphila DZ0503SBS1 (figure 1C). Based on these results, it was determined that isolate RR16 belonged to the S. nematodiphila species with RR16 serving as the strain code. The 16S rRNA gene sequence of S. nematodiphila isolate RR16 has been deposited in GenBank under the accession number OR098552.1.

Figure 1. Characterization and identification of the RR16 isolate. A) Colony morphology on LB agar medium (48h incubation) B) Hemolytic assay: a) S. aureus, b) RR16 isolate, and c) E. coli DH5α, C) Phylogenetic tree of S. nematodiphila RR16 and its related species constructed using neighbor-joining (1000x bootstraps).

Pigment extraction:

The red pigment which extracted from the cell biomass and supernatant of S. nematodiphila RR16 resulted absorbance values ranged 0.07-1.45 and 0.59-1.20, respectively (figure 2). The highest absorbance values were obtained using methanol for the cell biomass (RR16M) and chloroform for the supernatant (RR16K). The results suggest that the two solvents are the most effective in extracting red pigments and used for further test.

Figure 2. Solvent's effect on the extraction of red pigment. A) Red pigment extraction from the cell biomass and B) the supernatant.

Antioxidant assay:

The antioxidant activity of red pigments extracted from methanol (RR16M) and chloroform (RR16K) crude ^{extracts of S.
nematodiphila RR16 was assayed against DPPH radicals (Table 1). The IC}50 ^{as the measured}potential antioxidant activity indicates the extract concentration required to reduce 50% of the DPPH radicals. ^{The lower IC}50 ^{value indicates the higher level of
antioxidant activity. The result showed that the RR16K extract}exhibited superior activity compared to the RR16M extract (Table 1). Therefore, the RR16K extract was used for further analysis.

Table 1. Antioxidant activities of the crude red pigment extract of S. nematodiphila RR16 against DPPH radical

Extract code	^IC₅₀ ^{(µg/ mL)}
RR16M	255.94 + 2.64^c
RR16K	111.18 + 5.70^b
Ascorbic acid	2.5 + 0.038 ^a

Numbers in the same column followed by the same letter are not significantly different from the results of the Duncan's test (α = 0.05). The data represent the mean values from n = 3.

Fractionation of the red pigment extract:

The RR16K extract was fractionated by TLC and 19 fractions were visible in the UV light at 366 nm and 254 nm. TLC bioautography revealed six yellow fractions (F1, F2, F3, F4, F5, and F6) that indicate antioxidant activity, as shown in figure 3. Six fractions were isolated and extracted by preparative TLC for DPPH analysis.

Figure 3. Fractionation of RR16K extract by TLC. A) Bioautography with yellow band indicated as antioxidant active fractions, B) TLC profile at 366 nm, C) 254 nm, and D) the selected fraction on the TLC plate.

Antioxidant activities of the active fraction of red pigment extract:

The active fraction of the red pigment S. nematodiphila RR16 in reducing DPPH radicals in vitro showed that fraction four (F4, Rf 0.29) had the highest antioxidant activity (Table 2). The antioxidant potency of fraction F4 and its characteristic pink color (figure 3) on the TLC plate were the reasons for selecting fraction F4 for further testing and characterization.

Table 2. Antioxidant activities of the active fractions of S. nematodiphila RR16 against DPPH radical

Fraction code	^IC₅₀ ^{(µg/ mL)}
F1	385.04 + 3.67^d
F2	194.47 + 7.09^c
F3	523.54 + 2.21^e
F4	140.31 + 4.68^b
F5	1004.78 + 2.21^f
F6	2757.06 + 9.13^g
Ascorbic acid	2.5 + 0.038 ^a

Numbers in the same column followed by the same letter are not significantly different from the results of the Duncan's test (α = 0.05). The data represent the

mean values from n = 3.

Oxidative stress tolerance assay:

The ability of the RR16K extract and the F4 fraction to maintain S. pombe cells' viability under oxidative stress ^{conditions was evaluated using H}2^O2 ^{radicals. Colony-forming units were
used to measure each extract and}fraction's effect on cell viability under H2O2 stress conditions at each concentration. The results showed that both the crude extracts and active fractions preserved S. pombe viability better than the negative control. When ^{subjected to H}2^O2 ^{stress conditions (0.5-2mM), the negative
control could only maintain
cell viability up to 10-2}, whereas the extract at dilution 10^-3 to 10^-4 and the fraction up to 10^-4. The extracts and fractions of the red ^{pigment S. nematodiphila RR16
maintained the best cell viability at concentrations of 100µg/mL (1mM H}2^O2^{)
and 140µg/mL (2mM H}2^O2^{), respectively (figure 4). In
general, viability in the crude extract and active}fraction was similar to the positive control.

Figure 4. Oxidative stress response assay of red pigment from S. nematodiphila RR16 using S. pombe. A) Crude extract and B) active fraction, compared to the positive control ascorbic acid (5 µg/mL) and Calorie Restriction 0.3% (w/v) (CR), and negative control DMSO (C-).

Mitochondrial activity assay:

The optimal concentration for maintaining cell viability in the oxidative stress tolerance test for crude extract (100µg/mL) and active fraction (140µg/mL) was tested for their mitochondrial activity assay. The test conditions that can stimulate mitochondrial activity are indicated by the luminescence in yeast cells. The results showed that the extract and fraction of S. nematodiphila RR16 red pigment induced mitochondrial activity (figure 5). A similar effect was observed for the positive control, which included ascorbic acid and calorie restriction. In contrast, the negative control showed no luminescence in yeast cells, indicating that DMSO could not stimulate mitochondrial activity (figure 5). These results suggest that oxidative stress tolerance is induced by S. nematodiphila RR16 red pigment extracts and fractions through mitochondrial adaptive reactive oxygen species (ROS) signaling, similar to the positive control.

Figure 5. Mitochondrial activity assay with Differential Interference Contrast (DIC) and Ultraviolet Fluorescence (WU) observations.

Screening, quantification, and UV-Vis spectroscopy characterization of red pigment:

In this study, a presumptive prodigiosin test was performed as an initial screening for the type of red pigment in the RR16K extract. The results were positive as the extract showed a pink color under acidic conditions and a yellow color under alkaline conditions. The amount of prodigiosin produced by S. nematodiphila RR16 was approximately 862.499 units/cell when quantified under these culture conditions. Ultraviolet-visible (UV-Vis) spectral analysis of the crude red pigment and the fraction obtained from S. nematodiphila RR16 showed a peak absorption at 539 and 537nm (figure 6), respectively. This finding suggested that prodigiosin is likely to be the dominant content in the red pigment extracts and fractions.

Figure 6. Absorbance profile of the red pigment. A) crude extract and B) fraction red pigment peak absorbances.

Structural characterization of the red pigment fraction:

As shown in figure 7, the red pigment fraction of S. nematodiphila RR16 produced major vibrations that showed several functional groups characteristic of prodigiosin. Specifically, the vibrations at 2955, 2916, 2869, and 2848 cm^-1 corresponded to the asymmetric and symmetric stretching of the methyl and methylene groups. Moreover, the vibrations at 1658.28 and 1458.63 cm^-1 indicated the presence of C=N and methyl groups, while the vibrations at 1375.37 cm^-1 indicated the presence of C-O groups in prodigiosin. In addition, the vibrations at ^{1081.24
and 963 cm-1 correspond to the C-O-C single covalent bonds and –CH}2 ^{groups, respectively. The}analytical results indicate that the red pigment fraction of S. nematodiphila RR16 contains prodigiosin, as evidenced by the presence of characteristic functional groups.

The FTIR data were also supported by the results of NMR characterization performed on ¹H one-dimensional ^{with
a frequency of 700 MHz. The 12 δ}H ^{values showed characteristics of prodigiosin compounds. At δ}H ^{0.88 ppm (s), it was expressed as CH}3^{,
which was expected to be connected to OCH}3 ^{at the quaternary carbon (C-7) as well as to C-13 and
C-17. At δ}H ^{1.25,
1.28, 1.33, and 1.37 ppm (s) was stated as CH}2 ^{and was predicted to be connected to C-14 - C-17. The
position of the alkaloid shift was found to be generally around δ}H ^{7.07 - 7.77 ppm, which included the shift predicted at δ}H ^{7.07, 7.12, 7.17,
7.22, and 7.53 ppm (d) for
the H atoms connected to C-1, C-2, and C-3. In addition, δ}H ^{7.77 and 7.35 ppm (s) indicated the
position of the H atoms connected to}C-6, C-9, and C-11 (figure 7).

Figure 7. Structural characterization of the red pigment fraction. A) FTIR spectrum, B) Prodigiosin structure³² and C) ¹H-NMR spectrum

PigC gene detection, domain prediction and three-dimensional structure modelling:

PigC is a gene encoding the enzyme responsible for prodigiosin condensation. In this study, a partial PigC gene from the S. nematodiphila RR16 isolate was successfully amplified with 417 bp amplicons and subsequently deduced into 139 amino acids. The partial amino acids of PigC from S. nematodiphila RR16 are homologous to the prodigiosin biosynthetic protein from S. marcescens (ABL61530.1). This finding is supported by the results of the phenetic tree construction (figure 8A), which showed that the prodigiosin biosynthesis protein from S. nematodiphila RR16 belongs to the same clade as the prodigiosin biosynthesis protein from S. marcescens (ABL61530.1). The amino acid sequence of PigC from S. nematodiphila isolate RR16 has been added to GenBank with accession number PP747172.1.

The partial amino acid sequence of PigC S. nematodiphila RR16 aligned with the reference sequence revealed 19 conserved amino acid residues, including leucine (L), arginine (R), phenylalanine (F), and glutamate (E), as the dominant amino acids. The alignment between the partial amino acid sequence of PigC S. nematodiphila RR16 and the complete amino acid reference of prodigiosin synthase S. marcescens (ADV58253.1) demonstrated the domain prediction of the partial amino acid PigC S. nematodiphila RR16, which is located in the central domain (figure 8E).

Figure 8. Analysis PigC gene. A) Phenetic tree of PigC. The domain prediction of PigC protein based on S. marcescens (ADV58253.1): B) N-terminal ATP-binding domain, C) central domain, D) C-terminal domain and E) partial amino acids of PigC S. nematodiphila RR16.

The 3D structural model of the partial protein sequence for PigC S. nematodiphila RR16 (figure 9) exhibited an extremely high degree of similarity (97.84%) to the reference protein model for the S. marcescens PigC- synthesizing transferase (Q5W252). The partial protein model of PigC from S. nematodiphila RR16 showed excellent quality and topological similarity to the database template, with GMQE and RMSD values of 0.90 and 0.044 Å, respectively. Validation of the partial protein structure model for PigC S. nematodiphila RR16 using PROCHECK and the data in the form of Ramachandran plots revealed excellent model quality results (figure 9C), with the most favored regions consisting of 122 residues (97.6%), allowed regions consisting of three residues (2.4%), and G-factor values exhibiting a positive value of 0.14. The results of the superposition analysis indicated a strong overlapping structure between the partial sequence model of the PigC protein of S. nematodiphila RR16 and the reference protein model of PigC-synthesizing transferase of S. marcescens (Q5W252) (figure 9B).

Figure 9. Analysis of three-dimensional structural model. A) Three-dimensional model of PigC S. nematodiphila RR16 partial protein, B) superposition analysis of three-dimensional model of PigC S. nematodiphila RR16 partial protein against reference protein PigC Synthethizing transferase S. marcescens (Q5W252), and C) Ramachandran plot of PigC S. nematodiphila RR16 partial protein model.

DISCUSSION:

The red pigment of S. nematodiphila RR16 can be produced intracellularly (cell biomass) and extracellularly (supernatant)³⁷. Thus, it is necessary to compare different solvents to obtain large amounts of pigment. Similar studies have confirmed the efficacy of chloroform in extracting prodigiosin pigment from supernatant³⁸ and methanol in extracting pigment from cell biomass²² as this study (figure 2). The biological activity assay showed that the crude red pigment extract (table 1) had a higher level of antioxidant activity than its fractions (table 2). However, it is essential to note that the antioxidant activity of the crude extract and red pigment fraction was still higher than that of the prodigiosin pigment from S. marcescens TNU01, which had an IC50 value of 235 µg/mL³⁹. At the cellular level, the crude extract and red pigment fraction of S. nematodiphila ^{RR16 could maintain yeast cell viability under different
H}2^O2 ^{stress
levels. These results also indicated that the}viability of cells treated with the red pigment extract was higher than that of cells treated with its fractions (figure 4). The results of the in vitro antioxidant assay support the notion that the difference in cell density is due to synergistic interactions between compounds present in the unseparated crude extract, which was not subjected to TLC fractionation⁴⁰. This could potentially increase the ability of yeast cells to withstand oxidative ^{stress under H}2^O2 ^{stress conditions. A previous study reported that yeast
viability could be preserved by using a}100 µg/mL crude extract of the yellow pigment derived from Bacillus haikouensis AGS112⁴¹.

The extract and red pigment fraction of S. nematodiphila RR16 and ascorbic acid and calorie restriction (CR) treatment showed strong luminescence in the mitochondrial activity of S. pombe cells (figure 5). Masoro (2005)⁴² states that calorie restriction (CR) treatment reduces cell damage. It slows the aging process caused by reactive oxygen species (ROS) by increasing mitochondrial activity, activating ROS adaptive mitochondrial signaling. Calorie restriction (CR) treatment of S. pombe cells can increase cellular respiration in the log phase, which can produce intracellular ROS and trigger adaptive response mechanisms to oxidative stress⁴³. Based on these findings, it is believed that the extract and red pigment fraction of S. nematodiphila RR16 mimics the mechanism of CR treatment under oxidative stress conditions, showing an increase in mitochondrial membrane ^{potential similar to that of CR
treatment. These outcomes are consistent with the H}2^O2 ^{stress tolerance test}results, which show that the crude extract and its fractions are able to maintain cell viability under oxidative stress conditions (figure 4), which shows that the crude extract and its fractions are able to maintain cell viability under oxidative stress conditions (figure 4). Previous research has reported that Bacillus sp. SAB E-41 extract induces mitochondrial activity³⁰. The mechanism by which S. pombe withstands oxidative stress brought on by the extract and red pigment fraction of S. nematodiphila RR16, however, requires more investigation.

Initial characterization was performed using a presumptive test for prodigiosin, which revealed changes in the color of the extract at different pH levels. The research results are consistent with those reported by Darshan and Manonmani (2016)⁴⁴ that observed similar color changes in prodigiosin pigments produced by S. nematodiphila darsh1. The red pigment fraction (F4, Rf 0.29) showed a pink color on the TLC plate (figure 3), similar to the prodigiosin pigment from S. marcescens ATCC 9986 reported by Guryanov et al. (2013)⁴⁵. The UV-Vis characterization (figure 6), which is believed to be prodigiosin, was supported by the previous research from S. marcescens 11E at 539 nm³² and Serratia sp. BRL 41 at 537nm⁴⁶. The FTIR analysis showed the ^{presence of
characteristic peaks such as the C-O groups, C=N, methyl, and -CH}2 ^group^{(figure 7)}⁴⁷^,⁴⁸^,⁴⁹^,⁵⁰^,⁵¹^,⁵² ^thatrefer to prodigiosin. These data were further supported by the ¹H-NMR data, which exhibited the characteristics of prodigiosin pigments (figure 7) and displayed similar results in chemical shifts to previous research reports⁵³^,³². Finally, the characterization data were further supported by the molecular detection of red pigments, presumably prodigiosin. The partial PigC amino acid deduced from S. nematodiphila RR16 was homologous to the prodigiosin biosynthesis protein of S. marcescens (ABL61530.1) (figure 8A). The partial amino acid of S. nematodiphila RR16 aligned with the complete amino acid of the prodigiosin synthase S. marcescens jx-1 (ADV58253.1) shows that the partial amino acid of S. nematodiphila RR16 is located in the central domain (figure 8E), which is related to the condensation of MBC and MAP to the final product prodigiosin³⁶. According to You et al. (2018)³⁶, the position of this central domain in the complete amino acid sequence of the prodigiosin synthase S. marcescens jx-1 is in the amino acid sequence Thr299 - Ser779. The 3D structure of the partial PigC protein of S. nematodiphila RR16, constructed using a homology model, also supports these data (figure 9). The partial PigC protein model of S. nematodiphila RR16 has good quality and topology, similar to the template sequence⁵⁴^,⁵⁵.

CONCLUSION:

This study presents the first of the prodigiosin pigment from S. nematodiphila as an antioxidant agent. This data indicated that prodigiosin from S. nematodiphila RR16 could be a suitable candidate for antioxidant applications, as it effectively reduces radicals in vitro (DPPH), maintains S. pombe cell viability under oxidative stress, and increases mitochondrial activity, resulting in adaptive mitochondrial ROS signaling. However, further investigations are needed to explore the role of prodigiosin compounds from S. nematodiphila RR16 in activating genes involved in cellular defense mechanisms under oxidative stress.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This work was supported by the Ministry of Education, Culture, Research, and Technology, of the Republic of Indonesia, through Master’s Thesis Research (Penelitian Tesis Magister/PTM) [grant numbers 102/E5/PG.02.00PL/2023]. Therefore, we appreciate and thank to all supports given to this research.

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Received on 24.05.2024 Revised on 26.09.2024

Accepted on 18.12.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2305-2314.

DOI: 10.52711/0974-360X.2025.00330

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