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Research Article
Anti-inflammatory potential of Curcuma heyneana: An in vitro and in silico investigation
expand article infoMelanny Ika Sulistyowaty‡, Fifteen Aprila Fajrin§, Mohammad Rizki Fadhil Pratama‡|, Dwi Setyawan‡, Anastasia Wheni Indrianingsih, Galih Satrio Putra#, Sabry A. H. Zidan¤, Takayasu Yamauchi«, Katsuyoshi Matsunami»
‡ Universitas Airlangga, Surabaya, Indonesia
§ Universitas Jember, Jember, Indonesia
| Universitas Muhammadiyah Palangkaraya, Palangka Raya, Indonesia
¶ Research Center for Food Technology and Processing, National Research Innovation and Agency (BRIN), Yogyakarta, Indonesia
# State University of Malang, Malang, Indonesia
¤ Al-Azhar University, Assiut, Egypt
« Hoshi University, Tokyo, Japan
» Hiroshima University, Hiroshima, Japan

Corresponding author: Melanny Ika Sulistyowaty ( melanny-i-s@ff.unair.ac.id )

Academic editor: Emilio Mateev
© 2024 Melanny Ika Sulistyowaty, Fifteen Aprila Fajrin, Mohammad Rizki Fadhil Pratama, Dwi Setyawan, Anastasia Wheni Indrianingsih, Galih Satrio Putra, Sabry A. H. Zidan, Takayasu Yamauchi, Katsuyoshi Matsunami.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Sulistyowaty MI, Fajrin FA, Pratama MRF, Setyawan D, Indrianingsih AW, Putra GS, Zidan SAH, Yamauchi T, Matsunami K (2024) Anti-inflammatory potential of Curcuma heyneana: An in vitro and in silico investigation. Pharmacia 71: 1-11. https://doi.org/10.3897/pharmacia.71.e120886
Open Access

Abstract

Rheumatoid arthritis (RA) is a progressive and chronic systemic autoimmune disease. However, currently treatment is often carried out using NSAIDs and corticosteroids. Although it reduces symptoms, this treatment has a high rate of side effects. This study aims to determine the anti-inflammatory property of Curcuma heyneana so that it can be used as an alternative treatment for RA. Based on author knowledge, this study on C. heyneana from the Indonesian region for the treatment of RA is still limited. C. heyneana’s rhizome was extracted by maceration with ethanol followed by fractionation process using n-hexane, ethyl acetate, and n-butanol. The anti-inflammatory invitro activities were determined by heat-induced hemolysis, effect on protein denaturation, and cyclooxygenases (COX) inhibition assay. Molecular docking of major predictive compounds was performed by AutoDock Vina. The results showed that some fractions of C. heyneana contained terpenoids, flavonoids, and alkaloids. The ethyl acetate fraction exhibited the highest anti-inflammatory activity, which was attributed to the presence of curcuminoids. Molecular docking studies further confirmed the potential of demethoxycurcumin, a curcuminoid identified in the ethyl acetate fraction, to inhibit COX-2. These findings suggest that C. heyneana, particularly its previously unreported curcuminoid-rich fractions, holds promise as a natural anti-inflammatory agent. Further research is warranted to explore the therapeutic potential of this plant in the management of RA and other inflammatory disorders.

Keywords

Rheumatoid arthritis, Curcuma heyneana, anti-inflammatory, curcuminoids, COX-2

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation of the joints. This inflammatory process leads to joint damage, pain, and functional impairment (Guo et al. 2018). The global prevalence of RA ranges from 0.5% to 1%, with women disproportionately affected compared to men (Costenbader et al. 2008).

The pathogenesis of RA involves a complex interplay of genetic and environmental factors that trigger an abnormal immune response. The immune system mistakenly attacks the body’s own tissues, leading to chronic inflammation. Key cellular and molecular mechanisms implicated in RA include the activation of T and B cells, the production of pro-inflammatory cytokines (such as interleukin-1, tumor necrosis factor-alpha, and interleukin-6), and the dysregulation of matrix metalloproteinases (MMPs). These factors contribute to the destruction of cartilage and bone within the affected joints (Yap et al. 2018; Kondo et al. 2021).

The immune system’s primary function is to protect the body against infection and disease. However, in autoimmune diseases like RA, this protective response becomes dysregulated. The innate immune system, composed of cells such as macrophages, mast cells, and dendritic cells, plays a crucial role in initiating the inflammatory response. The adaptive immune system, comprising T and B cells, provides a more specific and targeted response to foreign antigens. In RA, these immune responses become dysregulated, leading to chronic inflammation and tissue damage (Artis and Spits 2015; Marshall et al. 2018).

Current RA treatment strategies, often involving anti-inflammatory drugs like corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs), aim to inhibit key inflammatory mediators such as TNF-α, T lymphocytes, and cyclooxygenase (COX) enzymes to regulate and reduce the level of damage to articular tissue (Ahmed et al. 2021; Huang et al. 2021). However, long-term use of these medications can lead to significant side effects, including peptic ulcers, osteoporosis, and cardiovascular complications (Wongrakpanich et al. 2018; Bindu et al. 2020). The search for safer and more effective anti-inflammatory agents has led to increased interest in natural products.

Herbal-based drugs, such as those derived from plants, have demonstrated promising anti-inflammatory properties with fewer side effects compared to synthetic drugs (Yatoo et al. 2018). Natural substances have a long history of use in traditional medicine and continue to be a valuable source for drug discovery and development (Arozal et al. 2020; Atanasov et al. 2021). Indonesia, with its rich biodiversity, offers a wealth of potential medicinal plants for drug discovery and development. One such plant is Curcuma heyneana Val. & V. Zijp, a native Indonesian species belonging to the Zingiberaceae family (Widyowati and Agil 2009).

Curcuma heyneana, commonly known as temu giring, is renowned for its potential health benefits. The plant’s rhizomes, characterized by a pale-yellow color and bitter taste, are rich in various bioactive compounds, including flavonoids, essential oils, phenols, curcumin, and saponins. These compounds have been associated with antioxidant and anti-inflammatory properties (Firman et al. 1988; Jalil 2019).

Traditionally, C. heyneana has been used in Indonesian folk medicine for diverse applications, including as an anthelmintic and as a skincare agent. Notably, Javanese women have employed this plant for its skin-brightening and anti-aging properties (Firman et al. 1988; Kusumawati et al. 2018). Previous studies have reported the presence of bioactive compounds such as curcuminoids, sesquiterpenes, and diterpenes in this plant. For instance, Bos et al. (2007) identified curcuminoid content ranging from 0.98% to 3.21% in C. heyneana. Furthermore, Firman et al. (1988) isolated several sesquiterpenoid and diterpene compounds from the rhizomes of this plant. Saifudin et al. (2013) expanded on this research, isolating various compounds belonging to the germacrane, guaiane, spironolactone, sesquiterpene, and labdane groups. While previous studies have explored the phytochemical composition and bioactivity of C. heyneana, research on its potential as an anti-inflammatory agent, particularly in relation to RA, remains limited. This study aims to investigate the in vitro anti-inflammatory properties of C. heyneana and to elucidate its potential mechanism of action through molecular docking studies with the COX-2 enzyme.

Methods and materials

Analytical grade chemicals and solvents were purchased from Sigma-Aldrich, unless otherwise specified. Rhizomes of Curcuma heyneana were collected from Purwodadi, Indonesia, in March 2021. A voucher specimen (No. 754/PKN/FFUA) was deposited at the Herbarium of the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Indonesia.

All computations were performed on a Toshiba Portege Z30-C series Ultrabook equipped with an Intel Core i7-6600U processor (2.6 GHz) and Windows 10 Pro operating system. The following software tools were utilized for the study: OpenBabel 3.1.1: For ligand and receptor format conversion; AutoDockTools 1.5.7: For protocol configuration; AutoDock Vina 1.1.2: For docking calculations; UCSF Chimera 1.15: For preparation and analysis of docking results; and Discovery Studio Visualizer 20.1.0.19295: For visualization of docking results. All other software used in this study was freely available. Authors use non-generative AI functions from Grammarly and Google Gemini to proofread and improve the readability of manuscripts, without creating new content or changing pre-existing content.

Extraction and fractionation of C. heyneana

Dried rhizomes of C. heyneana were subjected to maceration extraction using ethanol. The resulting crude extract was further fractionated using a liquid-liquid partitioning technique with solvents such as n-hexane, ethyl acetate, and n-butanol. The obtained fractions were qualitatively screened for their chemical constituents using standard phytochemical tests. Subsequently, these fractions were evaluated in vitro for their anti-inflammatory potential using the following assays: heat-induced hemolysis, protein denaturation, as well as COX-1 and COX-2 inhibition assays. Fractions exhibiting significant anti-inflammatory activity were further purified using open column chromatography. The isolated compounds from the most potent fractions were subjected to structural elucidation using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

Phytochemical screening of C. heyneana

Phytochemical screening was conducted to identify the presence of alkaloids, flavonoids, and terpenoids in the crude extract and its fractions obtained from C. heyneana. Alkaloid screening was performed using Dragendorff’s reagent, which produces a reddish-brown precipitate, and Mayer’s reagent, which yields a white precipitate. Terpenoid content was assessed using the Salkowski test, which results in a golden yellow color. Flavonoid content was determined using the AlCl3 method (Farnsworth 1966).

In vitro anti-inflammatory assays

Preparation of erythrocyte suspension

This study was approved by the Ethical Committee of Medical Research, Universitas Jember, Indonesia (approval number: 1219/UN25.8/KEPK/DL/2021). Fresh human blood (5 mL) was obtained from the Indonesian Red Cross, Surabaya, and collected in an EDTA tube. The blood was centrifuged at 3000 rpm for 10 minutes to separate the plasma from the red blood cells (RBCs). The RBC pellet was washed three times with 5 mL of 0.9% normal saline and centrifuged at 2500 rpm for 5 minutes. Finally, a 10% (v/v) RBC suspension was prepared by diluting the RBC pellet with phosphate-buffered saline (PBS, pH 7.4), as described by Chandrappa et al. (2013).

Heat-induced hemolysis

Varying concentrations of diclofenac sodium (DS; 60, 70, 80, 100, and 120 µg/mL) and the extract and fractions of C. heyneana (60, 70, 80, and 100 µg/mL) were added to 0.1 mL of RBC suspension. The mixtures were incubated at 56 °C for 30 minutes in a water bath. Subsequently, the samples were centrifuged at 3000 rpm for 5 minutes to separate the clear supernatant from the cell debris. The absorbance of the supernatant was measured at 560 nm using a UV-Vis spectrophotometer, with each measurement performed in triplicate (Chanda and Juvekar 2019). The hemolysis rate was calculated according to the method described by Okoli and Akah (2004) using the following Equation 1, in which A1 is control absorption and A2 is test sample mixture absorption.

Inhibition of hemolysis (%)=(1-A2A1)×100% (1)

Effect on protein denaturation

Protein anti-denaturation activity was assessed using a modified method described by Williams et al. (2008) and Bailey-Shaw et al. (2017). Briefly, 50 mg of the positive control (DS) or the C. heyneana extract/fraction was added to 5 mL of 0.2% bovine serum albumin (BSA) solution. The mixture was incubated at 25 °C for 30 minutes, followed by heat treatment at 72 °C for 5 minutes. After cooling to room temperature, the absorbance was measured at 660 nm using a UV-Vis spectrophotometer. The percentage inhibition of protein denaturation was calculated using the following Equation 2, in which A1 is control uptake and A2 is uptake of the test sample mixture:

Inhibition of denaturation (%)=(1-A2A1)×100% (2)

COX-1 and COX-2 inhibition assay

The inhibitory effects of the C. heyneana extract and its fractions on COX-1 and COX-2 enzymes were assessed using a commercial colorimetric COX inhibitor screening assay kit (COX-2 human recombinant; COX Colorimetric Inhibitor Screening Assay Kit 701050; Cayman Chemical Company, US). Briefly, 150 µL of assay buffer, 10 µL of hemin, 10 µL of COX-2 enzyme, and 10 µL of the sample were added to each well of a 96-well plate and incubated at 25 °C for 5 minutes. The reaction was initiated by adding 20 µL of a mixture containing colorimetric substrate and arachidonic acid. The mixture was further incubated at 25 °C for 2 minutes. The absorbance of each well was measured at 590 nm using a microplate reader. A similar procedure was followed for the COX-1 inhibition assay.

Statistical analysis

Data from three replicates were analyzed using a one-way ANOVA followed by a Tukey’s HSD test using the Minitab 18.1 software to determine statistical significance at a significance level of p < 0.05. The fraction with the highest anti-inflammatory activity was selected for further analysis. Curcumin content was quantified using a validated LC-HRMS method. Additionally, in silico studies were conducted to predict potential bioactive compounds and their mechanisms of action.

Determination of curcuminoids

Standard curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) and selected fractions from C. heyneana were analyzed using thin-layer chromatography (TLC)-densitometry. The TLC plates were developed in a mobile phase consisting of chloroform and methanol (9.6:0.4 v/v). After development, the plates were air-dried and visualized under UV light at 365 nm. Densitometric analysis of the visualized bands was performed at a wavelength of 424 nm to quantify curcuminoid levels.

Detection with LC-HRMS

Non-targeted screening analysis of the main compounds in selected fractions of C. heyneana was performed using a Q-exactive LC-HRMS system (Thermo Fisher Scientific). Standard compounds were not utilized for identification. Instead, tentative identification of compounds was based on their accurate mass and mass spectral similarity to the mzCloud online library (https://www.mzcloud.org/). This full-scan data acquisition method is described in detail by Susilo et al. (2022).

Molecular docking study

Receptor preparation

The target receptor for this study was prostaglandin-endoperoxide synthase 2 (COX-2), a key enzyme involved in inflammation and pain. Previous in vitro studies have demonstrated the efficacy of COX-2 inhibition in reducing inflammation by blocking the conversion of arachidonic acid to prostaglandins (Jarapula et al. 2016; Gunaydin and Bilge 2018). The crystal structure of COX-2 (PDB ID: 3LN1) was utilized for molecular docking simulations. This structure, resolved at 2.40 Å resolution, features celecoxib, a selective COX-2 inhibitor, bound to chain A (Wang et al. 2010).

Ligands preparation

The study utilized a set of predictive compounds derived from selected fractions of C. heyneana as test ligands. For comparative analysis, 15 known anti-inflammatory compounds with potential COX-2 inhibitory activity were included (Pratama et al. 2021). Celecoxib, a selective COX-2 inhibitor and a co-crystal ligand of the receptor, served as a reference ligand. Prior to docking simulations, all ligands, excluding the reference ligand (which was directly extracted from the receptor), were sketched and energy-minimized using the MMFF94 force field. The optimized ligands were then saved in the PDBQT format for subsequent docking calculations.

The validation process for the docking protocol and molecular docking

This study employed a computational approach to evaluate the potential binding affinities of three test ligands to the target protein. A previously validated molecular docking protocol, as described in Pratama et al. (2021), was utilized for this purpose. The docking process was replicated three times for each ligand to ensure reproducibility. The docking results were visualized using a two-dimensional graph. The x-axis represented the difference in binding free energy (ΔG) between the test ligand and the reference ligand (in kcal/mol), while the y-axis represented the percentage of interactions formed by the test ligand compared to the reference ligand. The binding affinities of the three test ligands were compared to a reference ligand and other known anti-inflammatory ligands to assess their potential anti-inflammatory activity.

Results

Extraction and phytochemical screening of C. heyneana

A total of 3.0 kg of C. heyneana rhizomes was subjected to a three-day maceration process using ethanol as the solvent. This resulted in the extraction of 265.0 g of crude ethanol extract (EE). The EE was further fractionated using a solvent-solvent partitioning method to yield four fractions: n-hexane (HF, 130.9 g), ethyl acetate (EAF, 73.7 g), n-butanol (BF, 9.2 g), and water (25.7 g). Phytochemical screening of all extracts and fractions revealed the presence of terpenoids in all samples. Notably, flavonoids and alkaloids were detected only in the HF and EAF fractions, suggesting the potential presence of curcuminoids (Table 1). These findings indicate that the HF and EAF fractions are likely to possess anti-inflammatory activity.

Table 1.
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Phytochemical screening of C. heyneana.

Phytochemical EE HF EAF BF
Terpenoids + + + +
Flavonoids + + + -
Alkaloids + + + -

Note: EE: ethanol extract, HF: n-hexane fraction, EAF: ethyl acetate fraction, BF: n-butanol fraction, (+) color spots detected, (-) no color spots detected.

In vitro anti-inflammatory assays

Heat-induced hemolysis

The results of the antioxidant activity assay (Table 2) demonstrated that all C. heyneana extracts and fractions exhibited lower IC50 values than the standard drug (DS), indicating enhanced antioxidant potential. Notably, EAF demonstrated the highest antioxidant activity. In the membrane stabilization assay, a positive correlation was observed between the concentration of the extracts and the percentage of hemolysis inhibition. A statistically significant difference (p < 0.05) was noted between the membrane stabilization effects of EAF and DS, with EAF exhibiting superior activity. No significant differences were observed among the three fractions or between EE and the fractions. These findings suggest that C. heyneana extracts, particularly EAF, possess significant antioxidant and membrane-stabilizing properties, surpassing the effects of the standard drug.

Table 2.
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Heat-induced hemolysis inhibition of C. heyneana.

Component IC50 ± SEM (ppm)
DS 167.8 ± 8.57a
EE 96.77 ± 1.61b
HF 85.97 ± 0.59bc
EAF 68.46 ± 0.58bc
BF 83.84 ± 0.37bc

Data were analyzed using One-Way ANOVA followed by LSD Post Hoc test (p < 0.05). Means with the same superscript letter are not significantly different (Tukey’s HSD, n = 3). DS: diclofenac sodium; EE: ethanol extract; HF: n-hexane fraction; EAF: ethyl acetate fraction; BF: n-butanol fraction.

Effect on protein denaturation

Previous studies demonstrated that EE exhibited superior anti-inflammatory activity compared to DS. Therefore, this study focused on further investigating the anti-inflammatory properties of EE and EAF. Both EE and EAF demonstrated significant inhibitory activity against protein denaturation, as evidenced by a dose-dependent decrease in absorbance values. This suggests that these extracts can protect proteins from heat-induced damage. However, the IC50 values for both EE and EAF were higher than that of DS (Table 3), indicating that DS may possess stronger protein-stabilizing properties.

Table 3.
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Effect on protein denaturation of C. heyneana.

Component IC50 ± SEM (ppm)
DS 21.18 ± 0.45a
EE 234.91 ± 18.55b
EAF 228.1 ± 0.97b

Data were analyzed using One-Way ANOVA followed by LSD Post Hoc test (p < 0.05). Means with the same superscript letter are not significantly different (Tukey’s HSD, n = 3). DS: diclofenac sodium; EE: ethanol extract; EAF: ethyl acetate fraction.

COX-1 and COX-2 inhibition assay

Our results (Table 4) demonstrate that DS exhibited inhibitory activity against both COX-1 and COX-2 enzymes. This activity was significantly higher (p < 0.05) compared to EE for COX-2 inhibition. Interestingly, EAF displayed an IC50 value for COX-1 inhibition that was not statistically different from DS. However, EAF exhibited a lower IC50 value for COX-2 inhibition compared to both DS and EE. This suggests a higher selectivity of EAF towards COX-2 inhibition compared to the other fractions. Notably, EAF displayed the lowest IC50 values for both COX-1 and COX-2, indicating a potentially broader inhibitory effect compared to the other fractions.

Table 4.
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COX-1 and COX-2 inhibition assay of C. heyneana.

Component IC50 ± SEM (ppm)
COX-1 COX-2
DS 21.34 ± 10.04a 4.27 ± 0.56a
EE 860.08 ± 71.96b 243.21 ± 41.38b
EAF 187.56 ± 43.38a 162.67 ± 23.39c

Data were analyzed using One-Way ANOVA followed by LSD Post Hoc test (p < 0.05). Means with the same superscript letter are not significantly different (Tukey’s HSD, n = 3). DS: diclofenac sodium; EE: ethanol extract; EAF: ethyl acetate fraction.

Determination of curcuminoids

Based on the results of in vitro anti-inflammatory assays and phytochemical screening, EAF emerged as the most promising candidate for anti-inflammatory activity due to its curcuminoid content. To further investigate the specific compounds responsible for this activity, EAF was subjected to subfractionation using an open column chromatography technique with a solvent gradient ranging from 100% chloroform to 100% methanol. The resulting subfractions were coded and weighed, as detailed in Table 5.

Table 5.
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Subfraction of EAF from C. heyneana.

Subfraction Solvent Weight (g)
1 CHCl3 16.07
2 CHCl3 : MeOH (20 : 1) 18.47
3 CHCl3 : MeOH (10 : 1) 3.64
4 CHCl3 : MeOH (7 : 1) 1.73
5 CHCl3 : MeOH (5 : 1) 1.33
6 CHCl3 : MeOH (2 : 1) 1.74
7 CHCl3 : MeOH (1 : 1) 2.16
8 CHCl3 : MeOH (1 : 2) 1.46
9 MeOH 0.94

Nine subfractions were obtained from the EAF extract, with subfraction 2 exhibiting the highest weight, followed by subfraction 1. Given the potential correlation between weight and activity, curcuminoids were detected in subfractions 1 to 6 using a standard mixture of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. The eluent system employed for chromatographic separation consisted of a specific ratio of chloroform and methanol. Fig. 1 illustrates that all subfractions exhibited similar visual spots to those of the curcuminoid standard, confirming the presence of the three constituent curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in each subfraction. Notably, demethoxycurcumin appeared to be the most prominent component in all subfractions, suggesting its potential contribution to the overall anti-inflammatory activity of EAF.

Figure 1. 

TLC analysis of standard curcuminoids and EAF subfractions under UV light (365 nm). Lanes (a–e) represent curcuminoid standards at concentrations of 25, 50, 100, 150, and 250 ppm, respectively. Lanes (f–k) correspond to subfractions 1–6 of the ethanolic extract of C. heyneana. The standard curcuminoids include curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3).

Detection with LC-HRMS

TLC analysis revealed similar profiles for Subfractions 1–6 (Fig. 1). To identify the most promising subfraction, we assessed their in vitro anti-inflammatory activity using heat-induced hemolysis and COX-2 inhibition assays (Table 6). Subfraction 5 consistently exhibited the lowest IC50 values across both assays, significantly outperforming the other subfractions (except for a similar COX-2 inhibition by Subfraction 6). Based on these results, Subfraction 5 was selected for further analysis using LC-HRMS (Fig. 2).

Table 6.
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Heat-induced hemolysis inhibition and COX-2 inhibition assay of subfractions of EAF from C. heyneana.

Subfraction IC50 ± SEM (ppm)
Heat-induced hemolysis COX-2
1 >250 >250
2 92.63 ± 1.44a >250
3 112.76 ± 1.02a 186.12 ± 14.57ab
4 87.36 ± 0.87a 201.57 ± 32.5ab
5 77.55 ± 6.01b 71.99 ± 15.92b
6 88.02 ± 0.23a 104.18 ± 10.36b

Data were analyzed using One-Way ANOVA followed by LSD Post Hoc test (p < 0.05). Means with the same superscript letter are not significantly different (Tukey’s HSD, n = 3).
Figure 2. 

LC-HRMS spectrum of subfraction 5 from EAF, with peaks of (a) curcumin, (b) demethoxycurcumin, and (c) bisdemethoxycurcumin.

Subfraction 5 of the EAF was subjected to LC-HRMS analysis to identify and quantify its curcuminoid constituents. The obtained LC-HRMS spectra (Fig. 2) revealed three compounds with retention times between 14 and 18 minutes and m/z values ranging from 300 to 370, all in the [M+H]+1 ionic form or [M+H] (Table 7). These compounds were confirmed to be curcumin, demethoxycurcumin, and bisdemethoxycurcumin by comparing their mass spectra to the mzCloud database. Demethoxycurcumin was the most abundant curcuminoid in subfraction 5, accounting for approximately 57% of the total peak area. Curcumin and bisdemethoxycurcumin represented approximately 26% and 23%, respectively. These findings align with previous TLC analysis, which also indicated demethoxycurcumin as the predominant curcuminoid in this subfraction.

Table 7.
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Curcuminoid compounds in subfraction 5 from ethyl acetate fraction (EAF) of C. heyneana.

Compounds RT (minutes) m/z Ion
Bisdemethoxycurcumin 14.997 309.1110 [M+H]+1
Curcumin 15.319 369.1320 [M+H]+1
Demethoxycurcumin 17.583 339.2879 [M+H]

Molecular docking

The RMSD value obtained for the redocked celecoxib, a reference ligand, was 0.949 Å, well below the 2.0 Å threshold for acceptable docking results. As illustrated in Fig. 3, the overlay of the redocked and crystallographic poses demonstrates excellent agreement. Analysis of the celecoxib binding interactions revealed a predominance of weak forces like van der Waals and alkyl/π-alkyl interactions (Fig. 4). However, the significant contribution of five hydrogen bonds, primarily involving the sulfonamide group, explains the favorable binding energy (-12.5 kcal/mol). Notably, the trifluoromethylpyrazole group, another key pharmacophore, exhibited predominantly hydrophobic interactions without any halogen bonds, despite the presence of three fluorine atoms. These findings underscore the crucial role of the sulfonamide group in celecoxib’s anti-inflammatory activity, as previously reported (Ottonello et al. 1995; Pavase et al. 2018). Table 8 presents the detailed docking protocol and validation results.

Table 8.
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Validation results for the docking protocol and process.

Parameters Values
PDB ID 3LN1
Co-crystal ligand Celecoxib
Grid box dimensions (Å) 32 × 20 × 26
Grid box position x = 30.092
y = ‐22.559
Z = ‐15.758
RMSD (Å) 0.949
ΔG ± standard deviation (kcal/mol) ‐12.5 ± 0
Residues of amino acids 75-Hisa – 102-Valb – 106-Argb – 178-Glna – 335-Valc – 338-Leub – 339-Sera – 340-Glyb – 341-Tyrd – 345-Leud – 367-Pheb – 370-Leud – 371-Tyrd – 373-Trpd – 499-Arga – 502-Alab – 503-Ileb – 504-Phea – 508-Metb – 509-Valc – 512-Glye – 513-Alad – 516-Serb – 517- Leub
Figure 3. 

The redocking ligands (blue) are shown alongside reference ligands from crystallography data (green) at receptors 3LN1 with RMSD 0.949 Å.

Figure 4. 

Two-dimensional interaction between celecoxib and the 3LN1 receptor binding site. Bright green: hydrogen bonds; light green: Van der Waals interactions; violet: Pi-sigma interactions; pink: alkyl/Pi- alkyl interactions; maroon: amide-Pi stacked interactions.

Molecular docking simulations revealed significant differences in the binding affinities of the three tested ligands to the COX-2 protein. Demethoxycurcumin exhibited the lowest binding energy (ΔG = -9.37 kcal/mol) and the highest ligand-receptor interaction similarity (62.5%) compared to the other ligands, including curcumin (Fig. 5). While the binding energy of demethoxycurcumin is comparable to non-selective NSAIDs like ketoprofen and piroxicam, its higher interaction similarity aligns more closely with selective COX-2 inhibitors such as etoricoxib. Demethoxycurcumin formed 23 interactions with the receptor, the highest among the tested ligands. Notably, it exhibited a key pi-sigma interaction with Val509, a feature shared with celecoxib and other selective COX-2 inhibitors (Fig. 6). This observation suggests that demethoxycurcumin may possess selective COX-2 inhibitory properties. Furthermore, the binding energy of demethoxycurcumin was lower than those reported for various curcumin analogs in a previous study by Sohilait et al. (2017), indicating its potential as a promising lead compound for the development of novel COX-2 inhibitors.

Figure 5. 

Graph between the difference in the value of ΔG and % similarity of ligand- receptor interactions of the test (blue) and comparison (red) ligands compared to reference ligand on the 3LN1 receptor. Demethoxycurcumin shows better docking results than other test ligands.

Figure 6. 

Two-dimensional interaction between (a) demethoxycurcumin, (b) bisdemethoxycurcumin, and (c) curcumin and the 3LN1 receptor binding site. Bright or pale green: hydrogen bonds; light green: Van der Waals interactions; violet: Pi-sigma interactions; pink: alkyl/Pi-alkyl interactions; maroon: amide-Pi stacked interactions; red: Unfavorable bump/donor-donor.

Discussion

Numerous Indonesian plants have been reported to possess compounds with anti-inflammatory properties, including the ability to inhibit pro-inflammatory cytokines (Thieme et al. 2019; Hamsidi et al. 2021; Deyulita et al. 2022; Ong et al. 2022; Sandhiutami et al. 2022; Triastuti et al. 2022). Curcuma species, particularly Curcuma heyneana, have garnered attention due to their potential anti-inflammatory effects. Previous research has identified compounds like zedoarondiol and curcumin in Curcuma spp. as potent inhibitors of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, which play a crucial role in chronic inflammatory diseases like RA (Kusumawati et al. 2018). C. heyneana is known to contain essential oils rich in sesquiterpenes, diterpenes, and monoterpenes (Widyowati and Agil 2009). Kusumawati et al. (2018) further demonstrated the presence of curcuminoids, including curcumin, in ethanol extracts of C. heyneana through TLC.

Curcuminoids, particularly curcumin, have been extensively studied for their ability to modulate various inflammatory pathways. By inhibiting the activation of NF-κB, a key transcription factor involved in inflammatory responses, curcumin can effectively reduce inflammation (Panahi et al. 2016). The results of the heat-induced hemolysis inhibition assay demonstrated that both the crude extracts and fractions derived from C. heyneana exhibited significantly higher inhibitory activity compared to the positive control, DS. This suggests that the plant extracts possess potent antioxidant properties capable of neutralizing free radicals and protecting red blood cells from oxidative damage. The observed anti-inflammatory activity of C. heyneana extracts and fractions can be explained by the interaction of their bioactive compounds with cellular membranes. Flavonoids can interact with the polar head groups of phospholipids, leading to increased membrane stiffness and reduced fluidity, thereby stabilizing the membrane structure (Tarahovsky et al. 2014). Additionally, curcuminoids can form aggregates within the lipid bilayer, further enhancing membrane stability (Loverde 2014).

Previous research has highlighted the presence of flavonoids and curcuminoids in C. heyneana, compounds known for their antioxidant and anti-inflammatory properties (Lestari and Indrayanto 2014). Curcumin, a polyphenol derived from turmeric, has been shown to inhibit heat-induced protein denaturation with an IC50 value of 1.93 to 32.75 ppm (Hayun et al. 2019). Similarly, flavonoids from other Curcuma species, such as C. longa, have demonstrated protein denaturation inhibitory activity with an IC50 value of 106.21 ± 0.53 ppm (Altir et al. 2021).

The synergistic effect observed between the different extracts suggests that the combination of these bioactive compounds may enhance their overall therapeutic potential. This finding aligns with previous research demonstrating the synergistic effects of curcumin and other natural compounds in various health conditions (Sulthana et al. 2018). The immunomodulatory properties of C. heyneana rhizome, as reported by Fajrin et al. (2023), further support its potential as a therapeutic agent. By modulating the immune response, this plant may contribute to the prevention and treatment of various inflammatory diseases.

Among the three compounds with the most favorable docking scores (demethoxycurcumin, bisdemethoxycurcumin, and curcumin), hydrogen bonding emerged as a consistent interaction motif. Notably, all compounds formed hydrogen bonds with the amino acid residue ARG106, primarily involving their keto groups. This interaction appears to be crucial for binding affinity, as evidenced by the significantly higher ΔG values and lower % interaction similarity observed in the absence of this interaction. This finding aligns with previous research by Pratama et al. (2015), who reported similar hydrogen bonding interactions between curcuminoids and COX-2.

Demethoxycurcumin exhibited a unique interaction involving hydrogen bonds with two hydroxyl groups at its terminal ends, which was not observed in the other two compounds. This additional interaction likely contributes to the lower ΔG value of demethoxycurcumin compared to bisdemethoxycurcumin and curcumin. The importance of hydrogen bonding in molecular interactions is well-established, with a greater number of hydrogen bonds generally correlating with stronger binding affinity and stability (Zhao and Huang 2011).

Conclusion

The ethyl acetate fraction of C. heyneana exhibited the most potent anti-inflammatory activity among the tested fractions. Further investigation revealed that demethoxycurcumin, a curcuminoid identified in this fraction, demonstrated the strongest potential for inhibiting COX-2 activity.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

Experiments on animals: This research obtained research ethics approval from the Ethical Committee of Medical Research, Universitas Jember, Indonesia, with document number 1219/UN25.8/KEPK/DL/2021.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Funding

The authors gratefully acknowledge the financial support provided by the Directorate of Research and Community Service, Ministry of Research and Technology/National Research and Innovation Agency of the Republic of Indonesia through the Basic Research scheme (Grant Numbers 397/UN3.15/PT/2021 and 754/UN3.15/PT/2022).

Author contributions

Melanny Ika Sulistyowaty: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Validation; Roles/Writing - original draft. Fifteen Aprilia Fajrin: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Visualization; Roles/Writing - original draft. Mohammad Rizki Fadhil Pratama: Software; Validation; Visualization; Roles/Writing - original draft. Dwi Setyawan: Project administration; Supervision; Writing - review & editing. Anastasia Wheni Indrianingsih: Analysis; Writing - review & editing. Galih Satrio Putra: Validation; Writing - review & editing. Sabry A. H. Zidan: Supervision; Writing - review & editing. Takayasu Yamauchi: Supervision; Writing - review & editing. Katsuyoshi Matsunami: Supervision; Writing - review & editing. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Author ORCIDs

Melanny Ika Sulistyowaty https://orcid.org/0000-0002-9510-6822

Fifteen Aprila Fajrin https://orcid.org/0000-0001-5374-5389

Mohammad Rizki Fadhil Pratama https://orcid.org/0000-0002-0727-4392

Dwi Setyawan https://orcid.org/0000-0001-8009-6054

Anastasia Wheni Indrianingsih https://orcid.org/0000-0002-7073-2421

Galih Satrio Putra https://orcid.org/0000-0003-4192-3621

Sabry A. H. Zidan https://orcid.org/0000-0003-4975-5933

Takayasu Yamauchi https://orcid.org/0009-0002-1948-9726

Katsuyoshi Matsunami https://orcid.org/0000-0002-8034-0253

Data availability

All of the data that support the findings of this study are available in the main text.

References

  • Ahmed EA, Ahmed OM, Fahim HI, Ali TM, Elesawy BH, Ashour MB (2021) Potency of bone marrow-derived mesenchymal stem cells and indomethacin in complete Freund’s adjuvant-induced arthritic rats: roles of TNF- α; IL-10, iNOS, MMP-9, and TGF- β 1. Stem Cells International 2021: 6665601. https://doi.org/10.1155/2021/6665601
  • Altir NKM, Ali AMA, Gaafar ARZ, Qahtan AA, Abdel-Salam EM, Alshameri A, Hodhod MS, Almunqedhi B (2021) Phytochemical profile, in vitro antioxidant, and anti-protein denaturation activities of Curcuma longa L. rhizome and leaves. Open Chemistry 19: 945–952. https://doi.org/10.1515/chem-2021-0086
  • Arozal W, Louisa M, Soetikno V (2020) Selected Indonesian medicinal plants for the management of metabolic syndrome: molecular basis and recent studies. Frontiers in cardiovascular medicine 7: 82. https://doi.org/10.3389/fcvm.2020.00082
  • Bailey-Shaw YA, Williams LAD, Green CE, Rodney S, Smith AM (2017) In-vitro evaluation of the anti-inflammatory potential of selected Jamaican plant extracts using the bovine serum albumin protein denaturation assay. International journal of pharmaceutical sciences review and research 47: 145–153. https://globalresearchonline.net/journalcontents/v47-1/27.pdf
  • Bindu S, Mazumder S, Bandyopadhyay U (2020) Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochemical Pharmacology 180: 114147. https://doi.org/10.1016/j.bcp.2020.114147
  • Bos R, Windono T, Woerdenbag HJ, Boersma YL, Koulman A, Kayser O (2007) HPLC-photodiode array detection analysis of curcuminoids in Curcuma species indigenous to Indonesia. Phytochemical Analysis 18: 118–122. https://doi.org/10.1002/pca.959
  • Costenbader KH, Chang SC, Laden F, Puett R, Karlson EW (2008) Geographic variation in rheumatoid arthritis incidence among women in the United States. Archives of Internal Medicine 168: 1664–1670. https://doi.org/10.1001/archinte.168.15.1664
  • Deyulita S, Ilmi H, Nisa HK, Tumewu L, Widyawaruyanti A, Hafid AF (2022) Anti-inflammatory activity of water extract of Luvunga sarmentosa (BI.) Kurz stem in the animal models. Borneo Journal of Pharmacy 5: 56–62. https://doi.org/10.33084/bjop.v5i1.2968
  • Fajrin FA, Sulistyowaty MI, Ghiffary ML, Zuhra SA, Panggalih WR, Pratoko DK, Christianity FM, Matsunami K, Indrianingsih AW (2023) Immunomodulatory effect from ethanol extract and ethyl acetate fraction of Curcuma heyneana Valeton and Zijp: Transient receptor vanilloid protein approach. Heliyon 9: e15582. https://doi.org/10.1016/j.heliyon.2023.e15582
  • Hamsidi R, Wahyuni , Sahidin I, Apriyani E, Harsono , Azizah NA, Malik F, Yodha AWM, Purnama LOMJ, Fristiohady A (2021) Suppression of proinflammatory cytokines by Etlingera alba (A.D.) Poulsen rhizome extract and its antibacterial properties. Advances in Pharmacological and Pharmaceutical Sciences 2021: 5570073. https://doi.org/10.1155/2021/5570073
  • Hayun , Maggadani BP, Kurnia A, Hanifah A, Yuliandi M, Fitriyani I, Hadrianti SP (2019) Anti-inflammatory and antioxidant activity of synthesized mannich base derivatives of (2e,6e)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-(phenyl methylidene)cyclohexan-1-one. International Journal of Applied Pharmaceutics 11: 246–250. https://doi.org/10.22159/ijap.2019.v11s1.19448
  • Jalil M (2019) Temu giring (Curcuma heyneana Val.): a review of morphology; phytochemistry, and pharmacology. Journal of Biological Education 2: 104–116. https://doi.org/10.21043/jbe.v2i2.6296
  • Jarapula R, Garangapu K, Manda S, Rekulapally S (2016) Synthesis, in vivo anti-inflammatory activity, and molecular docking studies of new isatin derivatives. International Journal of Medicinal Chemistry 2016: 2181027. https://doi.org/10.1155/2016/2181027
  • Kondo N, Kuroda T, Kobayashi D (2021) Cytokine networks in the pathogenesis of rheumatoid arthritis. International Journal of Molecular Sciences 22: 10922. https://doi.org/10.3390/ijms222010922
  • Kusumawati I, Kurniawan KO, Rullyansyah S, Prijo TA, Widyowati R, Ekowati J, Hestianah EP, Maat S, Matsunami K (2018) Anti-aging properties of Curcuma heyneana Valeton & Zipj: A scientific approach to its use in Javanese tradition. Journal of Ethnopharmacology 225: 64–70. https://doi.org/10.1016/j.jep.2018.06.038
  • Loverde SM (2014) Molecular simulation of the transport of drugs across model membranes. Journal of Physical Chemistry Letters 5: 1659–1665. https://doi.org/10.1021/jz500321d
  • Okoli CO, Akah PA (2004) Mechanisms of the anti-inflammatory activity of the leaf extracts of Culcasia scandens P. Beauv (Araceae). Pharmacology Biochemistry & Behavior 79: 473–481. https://doi.org/10.1016/j.pbb.2004.08.012
  • Ottonello L, Dapino P, Scirocco MC, Balbi A, Bevilacqua M, Dallegri F (1995) Sulphonamides as anti-inflammatory agents: old drugs for new therapeutic strategies in neutrophilic inflammation? Clinical Science 88: 331–336. https://doi.org/10.1042/cs0880331
  • Panahi Y, Hosseini MS, Khalili N, Naimi E, Simental-Mendía LE, Majeed M, Sahebkar A (2016) Effects of curcumin on serum cytokine concentrations in subjects with metabolic syndrome: A post- hoc analysis of a randomized controlled trial. Biomedicine & Pharmacotherapy 82: 578–582. https://doi.org/10.1016/j.biopha.2016.05.037
  • Pavase LS, Mane DV, Baheti KG (2018) Anti-inflammatory exploration of sulfonamide containing diaryl pyrazoles with promising COX-2 selectivity and enhanced gastric safety profile. Journal of Heterocyclic Chemistry 55: 913–922. https://doi.org/10.1002/jhet.3118
  • Pratama MRF, Poerwono H, Siswodihardjo S (2021) Introducing a two‐dimensional graph of docking score difference vs. similarity of ligand‐receptor interactions. Indonesian Journal of Biotechnology 26: 54–60. https://doi.org/10.22146/ijbiotech.62194
  • Pratama R, Ambarsari L, Sumaryada TI (2015) Molecular interaction analysis of COX-2 against curcuminoid and xanthorizol ligand as anti-breast cancer using molecular docking. Current Biochemical Engineering 5: 139–149. https://doi.org/10.29244/cb.11.1.2
  • Saifudin A, Tanaka K, Kadota S, Tezuka Y (2013) Sesquiterpenes from the rhizomes of Curcuma heyneana. Journal of Natural Products 76(2): 223–229. https://doi.org/10.1021/np300694a
  • Sandhiutami NMD, Khairani S, Dewi RS, Hakim ZR, Pradani AR (2022) Anti-inflammatory and analgesic activity of Musa balbisiana peels in vivo. Borneo Journal of Pharmacy 5: 81–92. https://doi.org/10.33084/bjop.v5i2.3169
  • Sulthana N, Kuchana V, Bodupalli BM, Potnuri AG (2018) Evaluation of in-vitro anti-arthritic and anti-angiogenic activity of combination of curcumin and diclofenac sodium. Indian journal of medical research and pharmaceutical sciences 5: 27–33. https://doi.org/10.5281/zenodo.1214978
  • Susilo B, Rohim A, Wahyu ML (2022) Serial extraction technique of rich antibacterial compounds in sargassum cristaefolium using different solvents and testing their activity. Current Bioactive Compounds 18: e100921196341. https://doi.org/10.2174/1573407217666210910095732
  • Tarahovsky YS, Kim YA, Yagolnik EA, Muzafarov EN (2014) Flavonoid-membrane interactions: involvement of flavonoid-metal complexes in raft signaling. Biochimica et Biophysica Acta 1838: 1235–1246. https://doi.org/10.1016/j.bbamem.2014.01.021
  • Thieme C, Westphal A, Malarski A, Böhm V (2019) Polyphenols, vitamin C, in vitro antioxidant capacity, α-amylase and COX-2 inhibitory activities of Citrus samples from Aceh, Indonesia. International Journal for Vitamin and Nutrition Research 89: 337–347. https://doi.org/10.1024/0300-9831/a000481
  • Triastuti A, Pradana DA, Setiawan ID, Fakhrudin N, Himmi SK (2022) In vivo anti-inflammatory activities of Plantago major extract and fractions and analysis of their phytochemical components using a high-resolution mass spectrometry. Research in Pharmaceutical Sciences 17: 665–676. https://doi.org/10.4103%2F1735-5362.359433
  • Wang JL, Limburg D, Graneto MJ, Springer J, Hamper JRB, Liao S, Pawlitz JL, Kurumbail RG, Maziasz T, Talley JJ, Kiefer JR, Carter J (2010) The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: the second clinical candidate having a shorter and favorable human half-life. Bioorganic & Medicinal Chemistry Letters 20: 7159–7163. https://doi.org/10.1016/j.bmcl.2010.07.054
  • Widyowati R, Agil M (2009) Chemical constituents and bioactivities of several indonesian plants typically used in jamu. Chemical and Pharmaceutical Bulletin 66(5): 506–518. https://doi.org/10.1248/cpb.c17-00983
  • Williams LAD, O’Connar A, Latore L, Dennis O, Ringer S, Whittaker JA, Conrad J, Vogler B, Rosner H, Kraus W (2008) The in vitro anti-denaturation effects induced by natural products and non- steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds, without the use of animals, in the early stages of the drug discovery process. West Indian Medical Journal 57: 327–331. https://pubmed.ncbi.nlm.nih.gov/19566010/
  • Wongrakpanich S, Wongrakpanich A, Melhado K, Rangaswami JA (2018) Comprehensive review of non-steroidal anti-inflammatory drug use in the elderly. Aging and Disease 9: 143–150. https://doi.org/10.14336/AD.2017.0306
  • Yap HY, Tee SZY., Wong MMT, Chow SK, Peh SC, Teow SY (2018) Pathogenic role of immune cells in rheumatoid arthritis: implications in clinical treatment and biomarker development. Cells 7: 161. https://doi.org/10.3390%2Fcells7100161
  • Yatoo MI, Gopalakhrisnan A, Saxena A, Parray OR, Tufani NA, Chakraborty S, Tiwari R, Dhama K, Iqbal HMN (2018) Anti-inflammatory drugs and herbs with special emphasis on herbal medicines for countering inflammatory diseases and disorders - a review. Recent Patents on Inflammation & Allergy Drug Discovery 12: 39–58. https://doi.org/10.2174/1872213x12666180115153635
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