Open Veterinary Journal, (2025), Vol. 15(7): 3341-3351

Short communication

10.5455/OVJ.2025.v15.i7.46

Investigation of cisplatin-induced acute kidney injury using LC-HRMS and network pharmacology approaches in mixed Curcuma longa and Curcuma zedoaria eExtracts

Putri Reno Intan^1,2, Sukmayati Alegantina³, Ani Isnawati³, Lucie Widowati³, Dian Sundari⁴, Frans Dany², Agus Saputra⁵, Sela Septima Mariya², Lina Noviyanti Sutardi⁶, Agus Setiyono⁷ and Ekowati Handharyani^7*

¹Animal Biomedical Study Program, IPB Postgraduate School, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia

²Center for Biomedical Research, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Center, Bogor, Indonesia

³Research Center for Pharmaceutical Ingredients and Traditional Medicine, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Center, Bogor, Indonesia

⁴Research Center for Public Health and Nutrition, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Center, Bogor, Indonesia

⁵Department of Anatomy, Physiology, Pharmacology, and Biochemistry, Faculty of Medicine and Veterinary Medicine, Universitas Nusa Cendana, Kupang NTT, Indonesia

⁶Division of Pharmacy, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia

⁷Division of Pathology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia

*Corresponding Author: Ekowati Handharyani. Division of Pathology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia. Email: ekowatieko [at] apps.ipb.ac.id

Submitted: 07/03/2025 Revised: 23/06/2025 Accepted: 25/06/2025 Published: 31/07/2025

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ABSTRACT

Background: Cisplatin-induced acute kidney injury (AKI) is a serious complication of cancer therapy. Natural compounds, such as those found in Curcuma species, may exert protective effects through anti-inflammatory and antioxidant mechanisms.

Aim: This study investigated the therapeutic mechanisms of combined Curcuma longa and Curcuma zedoaria extracts against cisplatin-induced kidney damage using Ultra-High Performance Liquid Chromatography–High Resolution Mass Spectrometry (UHPLC-HRMS) and network pharmacology.

Methods: An ethanolic extract was prepared and analyzed using UHPLC-Q-Orbitrap HRMS to identify active compounds. The antioxidant capacity was assessed using the Diphenyl-2-picryl-hydrazyl assay. Network pharmacology was used to predict compound-target interactions and identify key proteins involved in AKI pathways.

Results: UHPLC-Q-Orbitrap HRMS analysis identified 40 bioactive compounds, among which 22 met the OB >30% threshold. Network analysis revealed 14 overlapping protein targets associated with AKI from 485 compound-related targets and 198 disease-related targets.

Conclusion: This study demonstrated that key phytochemicals in C. longa and C. zedoaria may act on multiple protein targets implicated in AKI, highlighting their potential as multitarget therapeutics for kidney protection and future drug development.

Keywords: Curcuma longa, Kidney damage, UHPLC-Q-Orbitrap HRMS, Network pharmacology, Zedoaria.

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Introduction

Curcuma longa (turmeric) and Curcuma zedoaria (zedoary) are widely used in traditional medicine across Asia, particularly in India, Indonesia, and China. These Curcuma species were selected for investigation based on their rich content of bioactive compounds, particularly curcumin, which possesses potent anti-inflammatory and antioxidant properties that are crucial for mitigating renal damage in acute kidney injury (AKI). The complementary phytochemical profiles of these species offer potential synergistic protection against cisplatin-induced nephrotoxicity (Kocaadam and Şanlier, 2017; Santana de Oliveira et al., 2020). These plants, which are members of the Zingiberaceae family, are rich in bioactive compounds and have shown therapeutic potential for various diseases. One such compound, curcumin, has gained attention in nephrology because of its potent antioxidant and anti-inflammatory activities, which are relevant in mitigating kidney injuries, such as AKI and chronic kidney disease (CKD) (Kocaadam and Şanlier, 2017; Santana de Oliveira et al., 2020; Gharge et al., 2021).

AKI is a sudden condition characterized by a reduced glomerular filtration rate, which may progress to CKD. CKD involves persistent inflammation, fibrosis, and irreversible kidney damage (Sato et al., 2020).

Inflammation and oxidative stress are key drivers of these pathological processes. Several studies have suggested that Curcuma-derived compounds alleviate nephrotoxicity by modulating key inflammatory mediators. The combination of C. longa and C. zedoaria has shown the potential to reduce cisplatin-induced kidney damage by suppressing the expression of TNF-α, kidney injury molecule (KIM)-1, and caspase-3 (Intan et al., 2025). Curcumin modulates multiple molecular targets, including cytokines, transcription factors, and signaling proteins. It inhibits proinflammatory cytokines, such as TNF-α and IL-1β, reduces macrophage infiltration, and interferes with fibrosis pathways by downregulating TGF-β (Jacob et al., 2013; Ghosh et al., 2014; Sadeghian et al., 2023).

The identification of specific biomarkers has significantly advanced the early detection and management of kidney disease. As shown in recent research, a single intraperitoneal dose of cisplatin (10 mg/kg body weight) was used to develop a specific and sensitive model for the early identification of renal damage in male albino rats (Jana et al., 2023). Among the various biomarkers, interleukin (IL)-18 has been identified as a key indicator of cisplatin-induced kidney injury, AKI, hemodialysis, inflammatory kidney disease, diabetic renal disease, and polycystic renal disease (Mitra et al., 2025). This finding aligns with the findings that urine KIM-1, IL-18, nephrin, neutrophil gelatinase-associated lipocalin (NGAL), and serum cystatin C (Cys C) levels were greatly increased on day 3 after cisplatin treatment, whereas blood urea nitrogen and serum creatinine levels remained normal. This study confirmed these findings by upregulating the mRNA expression of KIM-1, IL-18, Cys C, and NGAL and downregulating the expression of nephrin in kidney tissue at an initial stage, demonstrating that utilizing an array of kidney impairment indicators has emerged as an earlier, more effective, and more reliable technique to diagnose AKI when compared with the most sophisticated signs now available (Jana et al., 2023).

Other phytochemicals, such as demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), terpenoids, and flavonoids, may also contribute synergistically to kidney protection, although their roles remain poorly understood.

Despite the existing knowledge of individual bioactive components, the combined effect of C. longa and C. zedoaria and their specific mechanisms in kidney protection, particularly in the context of cisplatin-induced nephrotoxicity, are not well defined; thus, this study sought to fill this gap.

In this study, UHPLC-Q-Orbitrap HRMS and network pharmacology approaches were used to identify the bioactive compounds present in a mixed extract of C. longa and C. zedoaria and to explore their therapeutic mechanisms against cisplatin-induced kidney damage. Through UHPLC-HRMS, phytochemical profiles were obtained, and network pharmacology was used to predict target proteins and biological pathways, revealing key interactions underlying nephroprotective effects (Hopkins, 2008).

The novelty of this study lies in its integrative approach combining LC-HRMS and in silico network analysis to unravel how complex natural mixtures act at the molecular level to alleviate drug-induced kidney injury, thereby offering new insights for therapeutic development in nephrology.

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Materials and Methods

Preparation and extraction of samples

Curcuma longa and C. zedoaria rhizomes were provided and authenticated by the Research Center for Tropical Biopharmaca at IPB University, Indonesia. Taxonomic verification was conducted using reference number 306/IT3. LL. P13/TA.00.03/M/B/2023 and 305/IT3. LL. P13/TA.00.03/M/B/2023, respectively. The extraction of Simplicia used a maceration technique employing pharmaceutical-grade ethanol 96% solvent (Merck-Darmstadt, Germany) over three successive 24-hour periods. The sample-to-solvent ratio was 1:10 (Indonesia Health Ministry, 2017). The rotary evaporator was set to approximately 50 °C, and the filtrate was evaporated. Extracts from both Curcuma species were mixed in equal proportions to form a 1:1 combination.

Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity

This study followed the methodology described by Salazar-Aranda et al. (2011) with some adjustments. 1,1-DPPH was obtained from Sigma-Aldrich (USA). Ascorbic acid was obtained from Merck (Billerica, MA). The study was initiated by combining equivalent amounts (100 µl each) of DPPH solution (125 µM) with sample concentrations of 31.25, 62.5, 125, 250, and 500 µg/ml. The absorbance was measured after a half-hour incubation. In this study, ethanol was used as both the solvent and negative control, and ascorbic acid was used as the positive control. The percentage inhibition of DPPH antioxidant activity was calculated using the following formula (Anandika et al., 2024):

The IC50 value represents the test sample concentration (µg/ml) that resulted in a 50% reduction in DPPH activity, effectively inhibiting or decreasing the oxidation process by half. A 0% value indicates no antioxidant activity, whereas a 100% value suggests complete attenuation, necessitating further dilution of the test solution to determine the limit of its activity concentration.

UHPLC-Q-Orbitrap HRMS for metabolite separation and putative identification

An Accucore C18 (100 × 2.1 mm, 1.5 µm) column and a UHPLC Vanquish Tandem Q Exactive Plus Orbitrap HRMS instrument (Thermo Scientific) were used to extract compounds from a 1:1 blend of C. longa and C. zedoaria extracts. Using a gradient elution technique, the mobile phases included 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) for the following durations: 0.0–1.0 minute (5% B), 1.0–25.0 minutes (5%–95% B), 25.0–28.0 minutes (95% B), and 28.0–33.0 minutes (5% B). An injection volume of approximately 2 µl was used to maintain a consistent flow rate of 0.2 ml/min. The following parameters were used for the UHPLC-Q-Orbitrap HRMS analysis: ESI positive ionization source from a Q-Orbitrap mass analyzer with a range of 100–1,500 m/z, fragmentation collision energies of 18, 35, and 53 eV, capillary temperature of 320°C, spray voltage of approximately 3.8 kV, and flow rates of 15 and 3 ml/min for the sheath and auxiliary gases, respectively. A complete MS/dd MS2 scan was performed in the positive-ion mode. Metabolite identification was conducted using mass spectra processed using mzCloud and ChemSpider and an in-house database derived from extensive Curcuma genus literature. Spectrum selection, retention time alignment, unknown compound identification and classification, composition prediction, mass list searches, gap filling, area normalization, and background compound labeling are all steps of this procedure.

Network pharmacology

As described in the UHPLC-Q-Orbitrap HRMS section on metabolite separation, the chemicals found in the combined extracts of C. longa and C. zedoaria were identified. The “Canonical SMILES” chemicals were obtained from PubChem. The SwissTarget Prediction tool, available at https://swisstargetpredicti on.ch/) was used to determine the targets of these chemicals. A list of herbs with high oral bioavailability (OB) (OB ≥ 30%) for each constituent was entered into the Traditional Chinese Medicine Systems Pharmacology database (https://tcmsp-e.com/) to forecast potential targets. The list includes a combination of C. longa and C. zedoaria extracts. To produce a complete list of targets for the compounds in the combined extracts of C. longa and C. zedoaria, the targets associated with each compound were selected and combined with those identified using SwissTarget Prediction, removing duplicates.

Using the term “AKI,” a search was performed in the Online Mendelian Inheritance in Man (OMIM) database (https://www.omim.org/) to identify targets linked to AKI. To show the overlap between the targets linked to AKI and the targets found for the chemicals in the combined extract of C. longa and C. zedoaria, a Venn diagram was created using the Jvenn platform (https://jvenn.toulouse.inra.fr/app/example.html). The overlapping targets were seed nodes.

The selected targets were entered into the STRING database (https://string-db.org/) with the organism “Homo sapiens” to create a protein–protein interaction (PPI) network. Networks were established and PPI data were manipulated using Cytoscape 3.10.3. Degree centrality, betweenness centrality, and closeness centrality were among the selection criteria, and each had to have at least as high as the median value. The compounds associated with these effective targets were considered active compounds, and the nodes that satisfied these criteria were determined to be effective targets of the combined extract of C. longa and C. zedoaria in AKI.

Data analysis

The UHPLC-Q-Orbitrap HRMS mass spectra were examined using a Compound Discoverer 3.2 (Thermo Fisher, Waltham, USA). The proposed software employs a custom database from extensive scientific literature on the Curcuma genus, specifically C. longa and C. zedoaria. Compounds were identified by matching the MS2 spectra with those in the database at https://cfmid.wishartlab.com (Wang et al., 2021).

Ethical approval

This study was approved by the Ethics Committee of the National Research and Innovation Agency (BRIN), Indonesia, with approval number 087/KE.02/SK/05/2023.

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Result and Discussion

Extraction yield

Curcuma longa and C. zedoaria were extracted using a 96% ethanol maceration technique. The yields from the extraction procedures were 14.465% and 5.414%, respectively. Several factors can influence the extraction yield, including the particle size of the Simplicia being extracted, extraction technique, solvent, material-to-solvent ratio, extraction time, temperature, evaporation temperature, and time. If the extraction process is too long or exceeds the appropriate limit, the phytochemical compounds in the dissolved substance are degraded. However, they are no longer effective because the solvent of the dissolved substance is saturated. However, maceration times that are too short may result in incomplete dissolution of all compounds in the solvent (Kim et al., 2007; Chan et al., 2009; Rafiee et al., 2011).

The maceration technique using 96% ethanol was selected on the basis of the target bioactive compounds and their physicochemical properties. Ethanol (96%) was chosen because of its optimal polarity profile for extracting a range of compounds of interest from C. longa and C. zedoaria. The polar protic nature of ethanol allows efficient extraction of polyphenolic compounds, particularly curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which are primary targets for their documented anti-inflammatory and antioxidant properties in attenuating kidney damage (Kocaadam and Şanlier, 2017; Santana de Oliveira et al., 2020). Comparative studies have demonstrated that ethanol extracts of Curcuma species yield significantly higher concentrations of polyphenolic compounds than other solvents, with 96% ethanol showing superior extraction efficiency for curcuminoids due to polarity matching (Wahyuningtyas et al., 2017). This aligns with the well-established principles of “like dissolves like” in phytochemical extraction science, in which flavonoids and related polyphenols with moderate polarity are optimally extracted in ethanol systems (Harborne, 1987).

Among the organic solvents, ethanol demonstrated superior maceration efficacy, producing the most significant extract. Methanol is not used because of its harmful nature (Drosou et al. 2015).

Evaluation of the DPPH radical scavenging capability

One of the most commonly used techniques for assessing the antioxidant capability of different substances, such as chemicals, extracts, and biological samples, is the DPPH free radical-scavenging assay. Diphenylpicrylhydrazine was formed in this experiment when the sample changed the DPPH solution’s color from violet to yellow. Changes in absorbance were measured at predetermined time points (Rafi et al., 2021). This technique is commonly used to assess the capacity of a substance to donate hydrogen or neutralize free radicals because of its rapidity, simplicity, and economic efficiency (Kedare and Singh, 2011).

The absorbance of DPPH was evaluated at 517 nm, the maximum wavelength identified following the introduction of a combined extract of C. longa and C. zedoaria at concentrations of 31.25, 62.5, 125, 250, and 500 µg/ml. This technique is based on the concept that DPPH compounds that have not reacted in ethanol produce absorbance readings at 517 nm, which can be observed as a color change from purple to light purple or pale yellow. Table 1 shows the DPPH absorbance values after the addition of different amounts of C. longa and C. zedoaria extracts. The mixed extract had an IC50 of 145.46 mg/L, whereas the reference compound, L-ascorbic acid, had an IC50 of 5.19 mg/LL.

When measuring antioxidant activity using the DPPH assay, the IC50 value of the extract indicated the concentration at which 50% DPPH was triggered a response. The ethanol extract derived from C. longa and C. zedoaria mixtures exhibited weaker antioxidant properties than thatose of ascorbic acid, which served as a positive control. This finding implies that ascorbic acid, a recognized standard in antioxidant assessment, has a stronger antioxidant capacity than the mixture of C. longa and C. zedoaria.

Several factors may have contributed to the reduced antioxidant capacity of the combined extracts. One possibility is that the mixture contains antioxidant compounds that are less effective or exhibit activities that are different from those of ascorbic acid. Ascorbic acid is well known for its strong ability to neutralize DPPH free radicals because of its chemical structure, which efficiently supports the suppression of free radicals (Ghasemzadeh et al., 2018). The low antioxidant capacity of the extract may also be influenced by the interactions between the compounds found in C. longa and C. zedoaria. When extracts are combined, antagonistic interactions can occur between their components, potentially diminishing their overall antioxidant effects. This interaction resulted in a higher IC50 value than for ascorbic acid, which contains a single active molecule without any efficacy-reducing interactions (Kedare and Singh, 2011). The limited antioxidant capacity observed in this study may also be attributed to variations in chemical makeup. In contrast to plant extracts that typically contain multiple components, ascorbic acid is a pure compound with potent antioxidant properties. The complex composition of plant extracts may reduce their antioxidant efficacy or lead to a more diffuse effect than a concentrated, pure, and active substance, such as ascorbic acid (Kedare and Singh, 2011).

However, the higher IC50 value (145.46 mg/L) compared with that of ascorbic acid (5.19 mg/L) does not necessarily indicate reduced therapeutic efficacy. Several factors contribute to this, including the multitarget activity of the compounds within the extract. These bioactive compounds may act on multiple biological pathways simultaneously, which is advantageous for addressing complex diseases, such as cancer (Herranz-López et al., 2018). Furthermore, although some flavonoids have low bioavailability, their interaction with gut microbiota may produce active metabolites that enhance their biological activity (Hu et al., 2025). These mechanisms support the therapeutic potential of the extract beyond that reflected by the IC50 value alone.

Additionally, antioxidant effectiveness was not solely determined by the IC50 values. Other factors, such as synergistic interactions and compound delivery methods, can enhance overall efficacy. For instance, curcumin combined with grape seed oil in a microemulsion system exerted a synergistic effect, significantly increasing antioxidant activity compared with the effects of either compound alone (Scomoroscenco et al., 2022). In addition, formulation strategies that improve compound bioavailability may further enhance antioxidant performance. Functionalized mesoporous silica nanoparticles improved antioxidant activity by enhancing compound stability, release behavior, and absorption. These findings support the notion that the observed IC50 is only one aspect of a compound’s functional potential, and improvements in formulation or synergistic use may further optimize its therapeutic value (Budiman et al., 2024).

Table 1. Efficiency of ethanol extract reduction and ascorbic acid.

Fig. 1. UHPLC-Q-Orbitrap HRMS chromatogram of C. longa and C. zedoaria ethanol extracts.

Putative identification of the compounds

The chromatogram profile in Figure 1 indicates that the mixed ethanol extracts of C. longa and C. zedoaria contained several high concentrations of compounds. Most compounds dissociated at retention times of 8–24 minutes. This study identified 40 compounds by examining an internal database and conducting literature reviews of C. longa and C. zedoaria species, the Curcuma genus, and the Zingiberaceae family. They compared the MS2 spectra for identification (Table 2). The most prevalent metabolite, with a mass of 303.2919 and retention duration of 18.779 minutes, was not identified. These substances belong to the sesquiterpenoid, alkaloid, and sesquiterpene categories.

UHPLC-Q-Orbitrap HRMS analysis identified 40 metabolites extracted using 96% ethanol from a combination of C. longa and C. zedoaria. Among the 40 detected metabolites, one was an unidentified compound with the molecular formula C₂₁H₃₇N, a molecular weight of 303.29192, and MS2 fragmentation patterns at 240, 136, 91, and 58. This compound is likely a novel substance, a metabolite derivative, or a minor product of metabolism. It is presumed to belong to a class of long-chain aliphatic amines or light alkaloid derivatives with a benzyl aromatic group and has not yet been documented in common metabolite databases (Klau et al., 2023; Wang et al., 2024).

Curcuma zedoaria, a member of the Curcuma genus, has been used to treat various ailments (Fuloria et al., 2022). Analysis of UHPLC-Q-Orbitrap HRMS data tentatively identified compounds in C. zedoaria extract, primarily consisting of sesquiterpenoids (24 compounds), alkaloids (4), and sesquiterpenes (3). Previous studies have identified various sesquiterpenoids with diverse biological effects. These compounds exhibit diverse biological activities, including anti-inflammatory, anticancer, antimicrobial, antioxidant, and immunomodulatory effects. Furthermore, studies have demonstrated their capacity to inhibit angiogenesis and suppress TNF-α release from macrophages (Matsuda et al., 2001; Cox-Singh et al., 2008; Yuandani et al., 2021; Fuloria et al., 2022).

Recent studies have shown that not all known C. zedoaria compounds are present in the extract. The untargeted metabolomics approach detected various sesquiterpenoid compounds, such as Curzeone, Zingiberene, Zedoalactone A and B, and Zedoarol. However, several compounds, including curcumin, curcumin, and neocurdione, have not yet been identified (Matsuda et al., 2001; Yuandani et al., 2021). In addition to sesquiterpenoids, only two known sesquiterpenes have been detected in C. zedoaria: curcumene and alpha-farnesene. The previously unreported sesquiterpene 3,4-Dihydrocadalene was also discovered. Flavonoids are powerful antioxidants that are regarded as secondary plant compounds (Jabbar et al., 2022). However, the flavonoid naringenin has not yet been identified. The cultivation location of a plant influences the presence and concentration of compounds in its extract. Numerous studies have consistently identified phenolic compounds, which are secondary compounds, as substances with antioxidant properties that can neutralize various free radicals (Taqi, 2014).

Table 2. Putative identification of compounds in C. longa and C. zedoaria ethanol extracts.

Several alkaloid compounds with relatively high concentrations were identified, including choline, betaine, L (-)-pipecolinic acid, and lycopsamine, which were not previously reported. Lycopsamine, a pyrrolizidine alkaloid detected at a retention time of 6.197 minutes, exhibits cytotoxic activity against human hepatoma HepG2 and Huh6 cells (Hadi et al., 2021).

Studies have shown that C. zedoaria rhizome contains over 10 sesquiterpenes, including ethyl p-methoxycinnamate, furanodiene, α-phellandrene, 1,8-cineole, β-elements, curcumin, β-turmerone, β-endemol, zingiberene, and dihydro curcumin (Lobo et al., 2009; Gharge et al., 2021). According to previous studies, the polarity of the organic solvent employed during extraction or fractionation affects the presence of several components in the crude extract of C. zedoaria rhizome, including alkaloids, saponins, terpenoids, flavonoids, and tannins (Pujimulyani and Yulianto, 2020; Setyani et al., 2020). Active chemicals found in almost all C. zedoaria species have pharmacological and antioxidant properties, indicating their potential use in therapeutic settings (Dhal et al., 2012).

Compounds with the highest concentrations (molecular weight, m/z 214) could not be identified. Previous studies have not yielded any molecules with this molecular weight, suggesting that thesre isare novel metabolites or fragments of larger molecules.

Network pharmacology

The application of OB >30% as a screening threshold in network pharmacology studies of Curcuma species for renal conditions follows established methodological standards that are widely implemented in similar research (Dong et al., 2021; Li et al., 2023). Following OB >30% screening, 22 bioactive chemicals were identified in the C. longa and C. zedoaria extracts (Table 3). The active drug targets were obtained from SwissTargetPrediction, and the target genes linked to these compounds were gathered from the OMIM database, resulting in 1,654 recognized therapeutic targets. The duplicate targets were eliminated, leaving 485 targets associated with the combined extract.

Similarly, 256 therapeutic targets for AKI were retrieved from the OMIM database, and 198 AKI-related targets remained after duplicates were removed. A Venn diagram of the overlapping targets of AKI and the combined extract is shown in Figure 2. Fourteen of the 198 AKI-related targets and 485 extract-related objectives overlapped and were designated as hub targets for further research.

Table 3. Bioactive compounds of combined C. longa and C. zedoaria extracts.

Fig. 2. Intersection of target genes between active compounds and AKIs. The blue circle represents AKI-related targets, whereas the red circle indicates active compound-associated targets. This overlapping section highlights the anti-AKI targets that interact with the active compounds in the combined extract.

The blue circle represents AKI-related targets, whereas the red circle indicates active compound-associated targets. This overlapping section highlights the anti-AKI targets that interact with the active compounds in the combined extract.

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Conclusion

A total of 40 compounds identified in C. longa and C. zedoaria extracts mixed with 96% ethanol were classified as sesquiterpenoids, alkaloids, and sesquiterpenes. Among them, 22 bioactive compounds were linked to 14 overlapping therapeutic targets for AKI, suggesting their potential role in AKI treatment or prevention and providing a foundation for further drug development.

Further studies involving experimental validation, such as in vivo and in vitro assays, are necessary to strengthen the findings on the therapeutic mechanisms of the combined C. longa and C. zedoaria extracts. Additionally, more detailed investigations of metabolite stability during extraction and the impact of interindividual variability on the composition of bioactive compounds are recommended to support future clinical applications.

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Acknowledgments

The authors thank Indonesia’s National Research and Innovation Agency for funding this study.

Conflict of interest

The authors declare that they have no conflicts of interest.

Funding

This study was funded by the National Research and Innovation Agency (BRIN) of Indonesia as part of its Research Project Program for the 2024 Fiscal Year.

Authors’ contribution

PRI, SSM, LNS, AS, and EH conceived and designed the study. PRI, SA, AI, LW, FD, ASA, SSM, LNS, AS, and EH contributed to data analysis and interpretation. PRI, SA, AI, LW, DS, FD, ASA, SSM, LNS, AS, and EH drafted the manuscript. PRI, SA, AI, LW, DS, FD, ASS, SSM, LNS, AS, and EH critically revised the manuscript. PRI, SSM, LNS, AS, and EH supervised this study. PRI, SA, AI, LW, DS, FD, ASA, SSM, LNS, AS, and EH contributed to the final approval of the manuscript.

Data availability

This manuscript contains all the necessary information to corroborate the results of this study.

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