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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 4  |  Page : 389-394  

Action mode of ursolic acid as a natural antioxidant and inhibitor of superoxide dismutase: In vitro and in silico study


1 Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang, Indonesia
2 Department of Conservative Dentistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang, Indonesia
3 Department of Oral Biology, Faculty of Dentistry, Universitas Padjadjaran, Bandung, Indonesia

Date of Submission09-Apr-2021
Date of Decision26-Jun-2021
Date of Acceptance19-Jul-2021
Date of Web Publication20-Oct-2021

Correspondence Address:
Dr. Dikdik Kurnia
Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.japtr_90_21

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  Abstract 


Recently, the antioxidant is applied for the teeth bleaching treatment as an alternative of toxic material of hydrogen peroxide that is used in teeth bleaching. One of natural sources antioxidant is Uncaria gambir those containing active antioxidant agents. To be applied as a new bioactive constituent in teeth bleaching treatment, a preexperimental study is performed. The aim of the study is to identify the antioxidant constituent of U. gambir and predict their activity including action mode as an inhibitor of enzyme superoxide dismutase (SOD) through in vitro and in silico method. Combination of chromatography methods and spectroscopic analysis is used for isolated bioactive antioxidant constituent. The antioxidant activity was evaluated by in vitro assay against diphenylpicrylhydrazyl (DPPH) and SOD, respectively, while prediction of action mode of the active compounds as SOD-mutant enzyme inhibitor was conducted by in silico study using AutoDock 4.2 program. Antioxidant of ursolic acid was isolated from U. gambir with inhibitory concentration50 values 1721 ± 30.6 and 392 ± 53.57 μg/mL, respectively, against DPPH and SOD. By in silico study presented that ursolic acid inhibited SOD enzyme with a binding affinity of − 5.4 kcal/mol those higher than a quercetin as a positive control. The ursolic acid was identified as a potential natural antioxidant with potentially activity to inhibit SOD mutant.

Keywords: DPPH, superoxide dismutase enzyme, Uncaria gambir, ursolic acid


How to cite this article:
Somantri AD, Kurnia D, Zainuddin A, Dharsono HD, Satari MH. Action mode of ursolic acid as a natural antioxidant and inhibitor of superoxide dismutase: In vitro and in silico study. J Adv Pharm Technol Res 2021;12:389-94

How to cite this URL:
Somantri AD, Kurnia D, Zainuddin A, Dharsono HD, Satari MH. Action mode of ursolic acid as a natural antioxidant and inhibitor of superoxide dismutase: In vitro and in silico study. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 Nov 29];12:389-94. Available from: https://www.japtr.org/text.asp?2021/12/4/389/328636




  Introduction Top


The most popular dental care for the public is teeth whitening.[1] One of the methods used to whiten the teeth is bleaching.[2] The study showed that antioxidants play a role in the effect of tooth bond strength caused by bleaching method those can produce free radicals that are left in the tooth structure; therefore, antioxidants are needed as free radical scavengers to increase the strength of dental restoration materials bonding to the substrate.[3] An antioxidant is a substrate or molecule that can inhibit free radical reactions and is available in synthetic or natural forms since natural antioxidant is hardly safer than a synthetic antioxidant.[4],[5]

Natural antioxidant agents found in medicinal plants including Uncaria gambir Roxb. In Indonesia, those classified as herbal were originally used as a treatment for inflammation, oral problems, diarrhea, and as a component in betel chewing. The extracts of U. gambir were reported active as antioxidants against DPPH, as well as against superoxide anion radicals using the phenazine methosulfate nicotinamide adenine dinucleotide (PMS-NADH) method.[6],[7],[8] Some antibacterial, antidiabetic, and anti-inflammatory actions against damage produced by the process of mediating free radicals have been documented, but activity against the enzyme superoxide dismutase (SOD) has not yet been identified.[9]

The study focuses on the isolation and bioactivity assessment of antioxidant constituents from U. gambir using an in vitro technique against DPPH and superoxide radicals using nonenzymatic SOD. The mechanism of the antioxidant compound's molecular interaction with the SOD enzyme is then hypothesized using an in silico approach.


  Materials and Methods Top


Materials

The leaves of U. gambir were planted in Sumatra Barat, Indonesia, in May 2019. The voucher specimen (NP-0153) was identified and deposited in the Laboratory Taxonomy, Department of Biology, Universitas Padjadjaran, Bandung, Indonesia. Distilled organic solvents and water were utilized for isolation, whereas proanalyzed solvents from Sigma-Aldrich were used for spectroscopic analysis. The chromatography was used Silica G 60 from Merck and ODS RP-18 from Nacalai Tesque while for thin-layer chromatography (TLC) was used a plate of ODS RP-18 F254 and Silica G 60 F254 from Merck those visualized by spraying 10% of H2SO4 (v/v) in ethanol.

For antioxidant assay, DPPH from Wako was utilized, and for SOD assay, tetramethylethylenediamine (TEMED) 99% MB 026–100 ml and nitro blue tetrazolium (NBT) 98% MB 107–250 mg from HiMedia, riboflavin 98% from Sigma Aldrich, the solution of 1.0 M phosphate buffer pH 7.4 (VWR, E404–100 TABS), and water injection from Generic were employed.[10]

The AutoDock 4.2 software was used for molecular docking. [11] The protein data bank of the Research Collaboratory for Structural Bioinformatics (www.rcsb.org/) included the crystal structure of human SOD 1 complexed with naphthalene-catechol (protein data bank ID: 5YTO) and the chemical formulas of naphthalene-catechol (CID 134828057), quercetin (CID 5280343), and ursolic acid (CID 64945). The chemicals were found in the PubChem database (http://www.ncbi.nlm.nih.gov/pccompound). The Open Babel program was used to create the 3D structure in PDB format using SMILE notation.[12] Docking results were analyzed in Discovery Studio to establish the kind of interaction on residues.

Instruments

The compound structure was identified by ultraviolet (UV)-Vis 8452A Diode Array, infrared (IR) by FTIR Shimadzu 8400, 1D and 2D-NMR using JEOL type ECA (500 MHz), and mass spectrometry (MS) using water acquit ultra-performance liquid chromatography type triquadrupole. UV detector lamps with maximum wavelengths of 254 and 365 nm were used to examine the TLC plates. NEST flat-bottom 96-well microplates micropipettes from Eppendorf 1.5 mL microtube (GenFollower), incubator Memmert, and microplate reader EZ 400 (Biochrom, Germany) were utilized for the antioxidant activity test.

Methods

Isolation procedure of compound 1

The leaves of U. gambir (1 kg) were macerated in methanol and subsequently fractionated with n-hexane, ethyl acetate, and water, yielding crude extracts weighing 298.9, 17.4, 55.7, and 44.5 g, respectively. Extracts were tested for antioxidant activity against DPPH and SOD at various doses. The active ethyl acetate extract (30 g) was purified by chromatographed on Silica G 60 eluted with n-hexane-ethyl acetate in 5% gradient resulted fractions I–VI. The purification of II (0.1812 g) by re-chromatographed on ODS RP-18 eluted with methanol-H2O of 1% gradient to give five identical fractions of II 1–5 (0.0451 g) and then after washed with ethyl acetate resulted pure active compound 1 (0.0107 g).

Compound 1 structure determination

The chemical structure of 1 was established using spectroscopic data analysis of Uv-Vis, IR, NMR, and MS. The original spectra are available in the supplement material section [Figure S1], [Figure S2], [Figure S3], [Figure S4], [Figure S5],[Figure S6], [Figure S7], [Figure S8], [Figure S9], [Figure S10].



Antioxidant activity evaluation of the extracts and compound 1

The U. gambir extracts and ursolic acid were tested against DPPH assay. The assay concentrations of 5–50 μg/mL for extracts and 500–2000 μg/mL for ursolic acid were adjusted, and 60 μg/mL of DPPH solution was added in methanol in 96-well microplate and homogenized by diluting it using a micropipette, then left in a dark room for 30 min while the reaction takes place. The final reaction was measured at 517 nm by ELISA reader to determine the absorbance and inhibitory concentration (IC) 50 value, respectively.[13]

The SOD-like activity was determined according to published procedures.[10] The series concentration sample of extracts and compound 1 of 40 μL was added to 96 well microplate, and the solution was divided into two parts of solution A (aquadest, phosphate buffer pH 7.4, NBT, TEMED, and riboflavin) and solution B (blank mixture without riboflavin), and both were added to in the amount of 200 μL. The sample was diluted with a micropipette and irradiated for 10 min and their absorbance was measured at 560 nm for determine of IC50 values.

In silico study of the ursolic acid against superoxide dismutase

Canonical SMILES obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/64945) were used OPEN BABEL 2.4.1 software to transform the chemical structure of an ursolic acid compound into 3D in PDB format, and the RSCB (https://www.rcsb.org/structure/5YTO) was used to retrieve the 3D-structure of SOD.

AutoDock Vina software was used for docking and virtual screening of ligand-protein interaction. Naphthalene-catechol [Figure 1] was used as ligand control. Blind docking was undertaken using a box of size 40 x 40 x 40 points, covering the whole protein target, with coordinate X = 70.133, Y = 75.513, and Z = −12.173. BIOVIA/discovery studio was used to visualize the docking results. BIOVIA program showed the ligand-residue and docking position in a 3D molecule.
Figure 1: Structure of ursolic acid (1); quercetin (2); naphthalene-catechol (3)

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  Results Top


Compound 1 isolation procedures

The purity of compound 1 (10.7 mg) was evaluated by 2D-TLC analysis on ODS RP-18 eluted with methanol-water (1:9 v/v) with Rf = 0.47.

Compound 1 structure determination

Compound 1 was separated in the form of a white powder and dissolved in methanol. The IR spectrum of 1 showed absorptions at 3434, 2926, 1749, 1465, and 1059 cm − 1 those corresponding to hydroxyl, CH sp3, carboxyl, C = C and C-O functional group, respectively.

By NMR measurement, the 13C-NMR, and DEPT 135° spectra indicated that 1 to have thirty carbon signals including carbons for the seventh methyl, nine methylene sp3, six methine sp3, one carbon sp2, and seventh quaternary carbons, respectively, and were identified as six quaternary sp3 carbons at δc 39.8, 40.7, 43.2, 48.5, and 49.9 ppm, together with one quaternary sp2 carbon at 180.3 as carbon of carboxylate group. The 1H-NMR spectrum of 1 showed proton signals for two secondary methyl, one methine at δH 3.14 (1H, dd), one olefinic at 5.22 (1H, s), and some overlap methylene signals at 1.2–2.2 ppm, respectively.

Signals identification by 1H-1H-COSY of 1 presented correlations between H-11 with H-9, H-2 with H-1, H-6 with H-5, and H-19 with H-29 (δH 0.87), respectively. Another signals in heteronuclear Multiple Bond Correlation spectrum of 1 showed correlation of methine proton H3 at δH 3.14 to methane carbon C3 at 79.6, indicating a hydroxyl group attached at C3, and a carboxyl group (δc 180.3; C30) attached to quaternary carbon C28. For structural confirmation, the molecular mass of 1 was measured and showed of m/z 455.54 [M-H] corresponding to the molecular formula of C30H47O3 or m/z 456.54 for C30H48O3, respectively. Based on the spectral analysis together comparison data with published report, compound 1 was suggested to have a triterpenoid skeleton derivative and identified as ursolic acid as seen in [Figure 1].[14]

Antioxidant activity of extracts and compound 1

The data in [Table 1] presented that U. gambir extracts were active as antioxidant with IC50 values ≤50 ppm, and especially the ethyl acetate extract, were very active as seen in [Figure 2]a, for DPPH; in [Figure 2]b, for positive control; in [Figure 3] for SOD, respectively.[10]
Table 1: Antioxidant activity of Uncaria gambir extracts

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Figure 2: Graph of antioxidant activity against DPPH; Uncaria gambir extract (a); control positive: quercetin (b)

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Figure 3: Graph of antioxidant activity of Uncaria gambir and quercetin against superoxide dismutase

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Further evaluation of ursolic acid (1) indicates a low inhibition activity against DPPH with IC50 of 1721± μg/mL while for superoxide radical against SOD was more active with IC50 of 392 ± 53.57 μg/mL, respectively.

Antioxidant activity prediction of ursolic acid through molecular interaction with superoxide dismutase

Validation of docking parameters was done by docking on native ligands (naphthalene-catechol) and receptors (5YTO) to find whether the close-match docking pose can be predicted. For basic mode selection, the root-mean-square deviation (RMSD) value ≤2 Å is fairly good.[15] This docking parameter will be used in virtual screening of ursolic acid and quercetin. The structure with the lowest predicted free energy of binding (−7.1 kcal/mol), i.e., conformation no. 5 was selected. There were ten conformations, where the conformational differences of the ligands were also obtained. The results of this in silico experiment are shown in [Table 2] and [Figure 4], [Figure 5].
Table 2: Binding affinity and hydrogen bond in complex superoxide dismutase-compounds

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Figure 4: Ligand positions on superoxide dismutase: quercetin (a); ursolic acid (b); naphthalene-catechol (c)

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Figure 5: Interaction superoxide dismutase with ligands: quercetin (a), ursolic acid (b), and naphthalene-catechol (c)

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  Discussion Top


The discovery new antioxidant as an active agent for teeth whitening is an important research target in exploring new bioactive compound from medicinal plants, and recently in dentistry, the antioxidant agent is used to decrease bond strength after bleaching.[16] Many plants were reported as source of antioxidant agents with a function to prevent any free radical reaction in our body.[17]

The medicinal plant of Gambir (U. gambir) is a natural antioxidant sources that contain roxburghine B as a receptor adenosine diphosphate inhibitor, (+)-catechin and procyanidin B3 as antibacterial and antioxidant, (−)-epicatechine as antiviral and gambirine D as α-glycosidase inhibitor, respectively.[18]

Antioxidant-guided isolation of ethyl acetate extract resulted an antioxidant of ursolic acid, (1) which was isolated for the first time in this research from U. gambir Roxb.[19] The ursolic acid (1) was reported which shows pharmacological activity as anti-inflammatory, anticancer, and antioxidant, respectively, while the activity as an antioxidant against SOD is not reported yet.[20]

According to assay data, ursolic acid has weaker antioxidant activity than quercetin, it is predicted the absence of conjugated hydroxyl groups in the structure of ursolic acid cause DPPH and superoxide radicals not being optimally scavenged. Even that activity of ursolic acid (1) is very weak, it's activity against nonenzymatic SOD is new and interesting research data and its need to further study for use of ursolic acid (1) as an alternative antioxidant agent to assist SOD enzyme in radical scavenging.

Naphthalene catechol has a binding affinity of − 7.1 kcal/mol and shows hydrogen interactions on the residues of Lys23, Glu100, and Pro28, while ursolic acid has a lower binding affinity of − 5.4 kcal/mol and has a hydrogen interaction at the same residues of Glu100 while Lys23 with hydrophobic interactions. The absence of interactions with amino acids Pro28 allows a decrease in the affinity value of ursolic acid and quercetin (−5.1 kcal/mol). However, ursolic acid has a higher binding affinity value than quercetin, and it is known that quercetin has a stronger antioxidant value than ursolic acid. Although quercetin has quite a lot of hydrogen interactions with some of the same residues such as Glu21, Glu100, and Lys23, its affinity value is lower than ursolic acid.

The more negative values of binding affinity indicate that the bond is in the best bond strength condition because it is more stable, and the bond is stronger.[21] The SOD enzyme inhibitor was known to be naphthalene catechol, which has a lower free energy than other ligands; ursolic acid's low antioxidant action is expected due to the lack of hydrogen bonding.[22],[23]

Based on the location of the three complexes, all ligands are bound to SOD in the same position, it can be concluded that ursolic acid and quercetin have the same active site as naphthalene catechol competitively.[23]

According to in vitro and in silico studies, ursolic acid is deduced to have two “opposing faces” that mean ursolic acid which has two actions, inhibiting radicals and at the same time, attenuating the action of the SOD enzyme. In the other words, ursolic acid acts as “potentiation” (enhancement of the effects of one drug by another, but having dissimilar action) and “subtraction” agent (abolishing effect of another drug).


  Conclusion Top


The herbal of U. gambir containing antioxidant constituents of ursolic acid (1). The in vitro and in silico study of ursolic acid against DPPH and SOD presented interesting mode action mechanism those suggested ursolic acid as new natural antioxidant through a competitive inhibitor type. This antioxidant data can be used as preliminary bioactivity of interesting drugs candidate for applied to treat oral diseases caused by the toxic whitening agent and inflammation process. However, further research such as the synthesis of lead derivatives, in vivo method, and clinical studies is still needed.

Acknowledgment

The authors are grateful to Central Library of Universitas Padjadjaran for deposit of supplementary material and also grateful to Universitas Padjadjaran for all research facilities.

Financial support and sponsorship

This research was supported by Penelitian Dasar - KEMENRISTEK DIKTI/BRIN (PD: Dikdik Kurnia, Ph.D. with No. 1832/UN6.D/LT/2019) 2019 from the Indonesian Ministry of Research, Technology, and Higher Education, Jakarta-Indonesia.

Conflicts of interest

There are no conflicts of interest.


  Supplement Materials Top


Spectral data of compound 1 was UV: 201 nm. IR: 3434, 2926, 1749, 1465, 1375 and 1059 cm-1. The MS (negative ion mode): (m/z) 455.54.1H-NMR (CD3OD and CDCl3): δH 1.62 & 1.68 (2H, m, H-1), 1.64 (2H, m, H-2), 3.14 (2H, dd, 4.5 and 11.5Hz, H-3), 0.75 (1H, d, H-5), 1.43 & 1.40 (2H, m, 6HZ, H-6), 1.29 & 1.30 (2H, m, H-7), 1.56 (1H, m, H-9), 1.91 (2H, q, H-11), 5.22 (1H, t, H-12), 1.62 (2H, m, H-15), 1.59 & 1.63 (2H, m, H-16), 2.19 (1H, d, 11.5Hz, H-18), 1.37 (1H, m, H-19), 1.35 (1H, m, H-20), 1.48 (2H, m, H-21), 1.62 (2H, m, H-22), 0.96 (3H, s, H-23), 0.77 (3H, s, H-24), 0.97 (3H, s, H-25), 0.84 (3H, s, H-26), 1.11 (3H, s, H-27), 0.87 (3H, d, 6Hz, H-29), 0.95 (3H, d, 6Hz, H-30). 13C-NMR (CD3OD): δC39.9 (C-1), 27.8 (C-2), 79.6 (C-3), 39.8 (C-4), 56.7 (C-5), 19.4 (C-6), 34.3 (C-7), 43.2 (C-8), 48.9 (C-9), 40.7 (C-10), 24.2 (C-11), 126.8 (C-12), 139.6 (C-13), 48.5 (C-14), 29.2 (C-15), 25.3 (C-16), 49.9 (C-17), 54.3 (C-18), 40.3 (C-19), 40.4 (C-20), 31.8 (C-21), 38.1 (C-22), 28.8 (C-23), 16.4 (C-24), 16.1 (C-25), 17.8 (C-26), 24.3 (C-27), 180.3 (C-28), 17.7 (C-29), 21.6 (C-30).



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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