
==== Front
J Adv Pharm Technol Res
J Adv Pharm Technol Res
JAPTR
J Adv Pharm Technol Res
Journal of Advanced Pharmaceutical Technology & Research
2231-4040
0976-2094
Wolters Kluwer - Medknow India

JAPTR-15-171
10.4103/japtr.japtr_525_23
Original Article
Potential antioxidant and antiradical agents from Allium ascalonicum: Superoxide dismutase and density functional theory in silico studies
Ajiati Dwipa
Sumiarsa Dadan
Amin Meiny Faudah 1
Kurnia Dikdik
Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang, Indonesia
1 Department of Dental Conservation, Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia
Address for correspondence: Prof. Dikdik Kurnia, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang, Indonesia. E-mail: dikdik.kurnia@unpad.ac.id
Jul-Sep 2024
22 7 2024
15 3 171176
03 12 2023
06 5 2024
02 5 2024
Copyright: © 2024 Journal of Advanced Pharmaceutical Technology & Research
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Antioxidants are compounds that can inhibit excessive free radical reactions in the body. Excessive free radicals can cause system imbalances in the body which can trigger oxidative stress and cause serious illness. The limitations of antioxidants in the body can be overcome by consuming safe natural additional antioxidants that can be obtained from natural products. Isolating compounds of Allium ascalonicum leaves as antioxidant and antiradical agents in inhibiting excessive free radicals by in vitro and in silico. The extracted compounds were purified by column chromatography. The compounds obtained were then characterized using ultraviolet, infrared, NMR, and mass spectrometry. Determination of antioxidant activity was carried out by in vitro using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the non-enzymatic superoxide dismutase (SOD) methods. The in silico study used the density functional theory (DFT) calculation method with global descriptive parameters (GDP), donor acceptor map (DAM), and frontier molecular orbitals (FMO) analysis. Three compounds have been isolated, of which compound 1 is a new compound. In the DPPH method, compound 1 has more strong antioxidant activity than others, as well as in the non-enzymatic SOD method. Whereas, in the DFT calculation shows that compound 1 has the best reactivity and stability between other compounds and was categorized as the best antiradical. Compound 1 has the highest antioxidant activity compared to the other compounds by in vitro both the DPPH and non-enzymatic SOD methods. In silico, compound 1 has the potential as the best antiradical.

Allium ascalonicum
antioxidant
density functional theory
oxidative stress
the nonenzymatic superoxide dismutase
==== Body
pmcINTRODUCTION

Shallot (Allium ascalonicum L.) is a plant which is widely cultivated and used as a spice and believed to be a traditional medicine.[1] According to previous studies, A. ascalonicum bulbs contain several important compounds that have potential as natural antioxidant compounds.[2]

Antioxidants are compounds that can inhibit the occurrence of free radical reactions in the body.[3] It is important to protect the body from free radicals that can harm and prevent the body from oxidative stress that causes several serious diseases such as premature aging and cancer.[4] In addition, it is also caused by excessive production of reactive oxygen species.[5] Natural antioxidants can be obtained from fruits and vegetables because they contain important compounds which have an antioxidant activity.[6]

The antioxidant activity of a compound is influenced by its structure. The more polar the compound, the greater its ability is as an antioxidant. The structure which contains many hydroxyl groups bound to aromatic rings or conjugated carbon double bonds that can easily donate hydrogen or electrons to stabilize radical atoms because it has good resonance ability.[7] In other studies, antioxidant activity assay is mostly conducted using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which is considered more effective, easy, and cheap.[8] DPPH is a stable radical compound which has reactive nitrogen species.[9] Another method used in this study is non-enzymatic superoxide dismutase (SOD) or known as mimic SOD because it mimics the workings of the antioxidant testing method using the SOD enzyme. In this method, the source of superoxide anion comes from riboflavin photoreduction and the role of SOD enzyme is replaced by secondary metabolite compounds.[10]

In recent years, many studies on antioxidants have used computational studies or better known as in silico studies to predict the antioxidant ability of a molecule because it is considered more effective and certainly more affordable. One method that is widely used to predict antioxidant reactivity and activity is density functional theory (DFT).[1112] This method involves electron transfer ability and electron affinity in determining antioxidant ability.[13] This study uses quantitative and qualitative approaches involving laboratory research. In addition to a new compound, the use of mimic SOD (mSOD) method in vitro and DFT in silico is a novelty of this study.

MATERIALS AND METHODS

Materials

The leaves of A. ascalonicum were obtained from Demak, Indonesia, which were then determined (No. 25/LBM/IT/12/2021) at the Taxonomy Laboratory, Padjadjaran University, Indonesia. Several organic solvents such as n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) as well as several other chemicals, namely, Silica Gel 60 (0.2–0.5 mm) and (0.063–0.200 mm), Silica ODS RP-18 (0.040–0.063 mm), KLT Gel 60 F254 plate and ODS RP-18 F254 S plate were used for extraction, fractionation and isolation processes. In the process of identifying compound spots with KLT, 10% H2SO4 in ethanol (v/v) spotting solution was used, which was then monitored under UV light at λ 254 and 365 nm. In vitro research, antioxidant activity test with DPPH and non-enzymatic SOD methods using several chemicals such as DPPH, methanol, riboflavin, phosphate buffer pH 7.4, TEMED, distilled water, and NBT.[1014] Materials for in silico study only require 3D-conformers of compounds 1–3 downloaded from PubChem in sdf format.

Extraction and isolation of Allium ascalonicum leaves

A. ascalonicum leaves were extracted using methanol. The methanol extract obtained was concentrated with a rotary evaporator at ± 50°C. The extract was partitioned using column chromatography (Silica G 60) with n-hexane, EtOAc, and MeOH to obtain fractions of each solvent. EtOAc and MeOH fractions were selected to be purified using Silica G 60 column chromatography (n-hexane-EtOAc) and ODS RP-18 (H2O-MeOH) to obtain pure compounds 1 and 2. Characterization of pure compounds was carried out using 1H-NMR (JEOL, 500 MHz), ultraviolet-visible, infrared, and mass spectrometry.

Antioxidant activity assays using the 2,2-diphenyl-1-picrylhydrazyl method

This method was carried out twice, in the preliminary test and the pure compounds. Solvent used is methanol and DPPH as a radical with variation of sample concentration. The absorbance measurement was conducted using a microplate reader at 510 nm after being left in a dark room for ± 30 min. Percentage inhibition was calculated using the absorbance value of measurement result using (Acontrol − Asample)/Acontrol × 100%. Whereas, the IC50 value was calculated using Y = ax + b, which is the result of the regression equation.[15]

Antioxidant activity assays using the mSOD method

Initially, we prepared sample solutions of compounds with varying concentrations. The concentration variation was done by microdilution. Then, the sample solution, MeOH, working solution A (aquabides, phosphate buffer pH 7.4, NBT, TEMED, Riboflavin) and B (aquabides, phosphate buffer pH 7.4, NBT, TEMED) were put into 96-well and then irradiated for 10–15 min in a closed box. Absorbance measurement was performed using a microplate reader at λ 550 nm to obtain percentage inhibition and IC50 value.

Study of antioxidant activity with density functional theory calculation by in silico

In this antioxidant activity study by in silico, we used DFT calculation with basis sets B3 LYP/6-31G (2d, 2p) using MarvinSketch, Gaussian 09 W, and GaussView 5.0. The calculation was carried out under gas phase conditions.[16] We used three analyses consisting of global descriptive parameters (GDP), donor acceptor map (DAM), and frontier molecular orbitals (FMO). The three analyses involved calculations and DAM diagrams as follows [Figure 1].

Figure 1 Donor acceptor map analysis diagram used to determine antioxidant activity in silico

RESULTS

Compounds 1 and 2 were obtained from the EtOAc fraction of the methanol extract of A. ascalonicum leaves. Meanwhile, compound 3 was obtained from the methanol fraction. Based on the characterization data, it shows that compound 1 belongs to the aromatic compound group and compounds 2–3 belongs to the steroid group.

2-((2-hydroxyphenanthrene-1-yl) oxy)-2-oxoacetic acid (1) is a white crystal-shaped compound and has the molecular formula of C16H10O5; IR, νmax 1094 (C-O), 1494 (C = C), 1683, 1701 (C = O) and 3453 cm − 1 (OH); MS, m/z 282 (M − H)+; It has the carbon sp2 at the shift δC 167.7 ppm which is a characteristic of the ester or carboxylic acid group. Then, there are several characteristics of alkene and aromatic carbon at δC 121.6–135.6 ppm.[17] According to the UV data of compound 1 at λmax 255 nm is the absorption band of the benzenoid indicating that compound 1 has a benzene ring. Based on the shift δC, compound 1 has eight methine (CH) and eight sp2 quaternary (Cq) carbons.[18] β-sitosterol (2), the white crystal-shaped compound, has the molecular formula of C29H50O; IR, νmax 1050 (C-O), 1381 (gem-dimethyl), 1465 (C = C), 2937 (C-H) and 3429 cm−1 (OH); MS, m/z 414 (M + H)+; These data provide information that compound 2 has six methyl, eleven methylene, nine methine and three quaternary (Cq) carbons.[19] Sitosterol-3-O-glucoside (3) is a white powder-shaped compound with the molecular formula of C35H60O6; IR, νmax 1021 (C-O), 1367 (gem-dimethyl), 1639 (C = C), 2932 (C-H) and 3400 (OH); MS, m/z 576 (M − H)+.[20]

Based on 1H and 13C-NMR analysis, compounds 2 and 3 have almost the same chemical shift. However, compound 3 has chemical shift that was the characteristics of glucoside at δC 61.5 (C-26), 73.9 (C-28), 77.2 (C-29, C-29a), 77.3 (C-30), and 101.2 ppm (C-31).[21] According to 13C-NMR, compound 1 has carboxyl group at δC 167.7 ppm (C-12, C-12a).[22] Structures of compounds 1-3 are shown in Figure 2.

Figure 2 Compounds 1–3 were obtained from Allium ascalonicum leaves

DISCUSSION

Antioxidant activity assays of Allium ascalonicum leave compounds by in vitro

Table 1 shows that compound 1 has the best antioxidant activity among the other compounds in DPPH and non-enzymatic SOD methods with IC50 of 76 and 40 µg/mL, respectively. The IC50 value of compound 1 indicates that the compound has strong antioxidant activity. Meanwhile, compounds 2–3 showed weak antioxidant activity in both methods. In this study, we used quercetin as a positive control in both methods because quercetin is known to have a strong antioxidant ability to inhibit free radicals.[14]

Table 1: Data of antioxidant activity of compounds[123]

Compounds	IC50 (μg/mL)	
	DPPH	SOD nonenzymatic	
1	76	40	
2	3273	3837	
3	2500	10,000	
Quercetin	3.8	5	
DPPH: 2-diphenyl-1-picrylhydrazyl, SOD: Superoxide dismutase, IC: Inhibitory concentration

Based on Table 1, compound 1 has good activity because it is influenced by its structure. There are several influential groups from compound 1 that can affect antioxidant activity, namely the aromatic benzene ring, carbonyl groups, and phenol hydroxyl groups attached to it.[23] Compound 1 could donate its electrons to stabilize radical atoms to become more stable.[24] Meanwhile, compounds 2–3 do not have an influential group to donate its electrons. Although compound 3 has some hydroxyl groups on the glucoside substituents in its structure, it cannot conjugate and delocalize electrons when donating or transferring electrons.[25] Those functional groups have a relationship with the polarity of compounds and effect on antioxidant activity.[26] Figure 2 also shows that compound 1 is more polar than compounds 2 and 3. The more polar the compound, the easier it is to break hydrogen bonds and donate electrons.[27]

Antioxidant activity assay of Allium ascalonicum leave compounds by in silico

In the DFT calculation in this study, we used three analysis methods to determine and validate the results of antioxidant activity in vitro, namely, GDP, DAM, and FMO.

Global descriptive parameters

GDP calculation was obtained from single point energy value of each charge (neutral, anion, and cation) using formulas that were mentioned above (method section). Those parameters are used to predict reactivities and stability of compounds.[28] They consist of ionization potential (I), electron affinity (A), hardness (η), softness (S), electronegativity (χ), chemical potential (µ), and electrophilicity index (ω).[29] Table 2 shows that compound 1 is more stable than compounds 2–3 as indicated by the higher hardness values than the others. The higher the hardness value, the more stable the compound. Stability can also be seen based on a more negative chemical potential value. Reactivity and stability greatly affect antioxidant activity as they are related to electron transfer in inhibiting radicals.[30]

Table 2: The result of global descriptive parameter calculation

Parameters (eV)	Compounds	
	1	2	3	Quercetin	
I	10.85	7.62	7.51	9.43	
A	1.12	–1.93	1.63	1.75	
η	4.86	4.77	4.57	3.83	
S	0.10	0.10	0.11	0.13	
χ	5.99	2.84	2.94	5.59	
μ	–5.99	–2.84	–2.94	–5.59	
ω	17.96	4.05	4.32	15.64	

Donor acceptor map

Determination of antioxidant activity based on DAM analysis is inseparable from the previous analysis, namely GDP. I and A values as in Table 2 were used to calculate electron donor (ω−) and electron acceptor (ω+) to obtain electron donor (Rd) and electron acceptor (Ra) index.[2931] We determined these values using the formulas mentioned above. In this analysis, florin and sodium were also involved to obtain the Ra and Rd values. Florin (F) represents an electron acceptor and sodium (Na) a good electron donor.[31] The Ra and Rd values that have been obtained as in Table 3, then plotted on the DAM diagram. Figure 3 shows that compound 1 is categorized as the best antiradical that acts as a good acceptor and donor based on the DAM diagram [Figure 1]. Meanwhile, compounds 2–3 were categorized as a good antiradical with poor acceptor, good donor, and good antioxidant. When a molecule acts as a good electron donor and acceptor, it has high reactivity, so that hydrogen bonds can be broken more easily and donate electrons or hydrogen.[32]

Figure 3 The results of compounds 1–3 were obtained using donor acceptor map diagram analysis

Table 3: The calculation results of compounds 1–3 using donor acceptor map analysis

Compounds	Ω– (eV)	ω+ (eV)	Ra	Rd	
1	7.29	1.30	0.62	2.08	
2	2.86	0.02	0.01	0.82	
3	2.98	0.04	0.02	0.85	
Quercetin	7.35	1.75	0.84	2.10	
Natrium	3.50	0.57	0.27	1.00	
Flourin	10.68	2.09	1.00	3.05	
Ra: Electron acceptor, Rd: Electron donor

Frontier molecular orbital

The third analysis is FMO which involves the highest occupied molecular orbital energy (HOMO)–the lowest unfilled molecular orbital energy (LUMO). In this analysis, the reactivity of the compound can be seen from the energy gap between the HOMO-LUMO energies of the molecule. The lower the energy gap, the more reactive the molecule is.[32] HOMO-LUMO visualization can also shed light on orbital distribution and electron density. In addition, this analysis can show the active sites or radical attack sites in the molecule, qualitatively.[33] As shown in Figure 4, of the three compounds, compound 1 has a lower energy gap than the other two compounds and even lower than the positive control. This shows that compound 1 has the highest reactivity with an energy gap of 0.11 eV.[13] Figure 2 shows that radicals can attack compound 1 at the aromatic ring and phenol hydroxyl group. Meanwhile, in compounds 2 and 3, radicals can attack the cyclohexanol site. This can be seen from the orbital distribution in the HOMO-LUMO analysis as shown in Figure 4.

Figure 4 Frontier molecular orbitals analysis of compounds 1–3 involved highest occupied molecular orbital-lowest unfilled molecular orbital energy. HOMO: Highest occupied molecular orbital, LUMO: Lowest unfilled molecular orbital

CONCLUSION

In this study, we have successfully isolated three compounds and one of them is a new compound. Based on the in vitro antioxidant activity test, compound 1 has strong antioxidant activity on DPPH and non-enzymatic SOD method. Based on in silico study, in GDP analysis, compound 1 has the highest reactivity and stability compared to other compounds. This is also reinforced by the results of FMO analysis which can be seen from the energy gap value. In DAM analysis, compound 1 is categorized as the best antiradical among the other. Thus, the antioxidant activity of compounds using in vitro methods does not correlate to certain analyses in in silico studies.

Financial support and sponsorship

The Academic Leadership Grant (ALG) Dikdik Kurnia, Indonesia (1439/UN6.3.1/PT.00/2024, March 18, 2024), Universitas Padjadjaran, Indonesia.

Conflicts of interest

There are no conflicts of interest.

Acknowledgement

The authors are grateful to the Academic Leadership Grant (ALG) Dikdik Kurnia, Indonesia (1439/UN6.3.1/PT.00/2024, March 18, 2024) and also grateful to Universitas Padjadjaran for supporting all research facilities.
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REFERENCES

1. Thuy NM Tuyen NT Cuong NP Huyen LT Phuong NP Nguyen LT , Identification and extraction method of quercetin from flesh and skin of shallot (Allium ascalonicum) cultivated in Soc Trang Province, Vietnam Food Res 2020 4 358 65
2. Deswati DA Dhina MA Mubaroq SR IOP Conference Series: Materials Science and Engineering Bristol, England Institute of Physics Publishing 2018
3. Sandhiutami NM Rahayu L Uji toksisitas akut, aktivitas antioksidan in vitro dan efek rebusan bunga kamboja merah (Plumeria rubra L.) terhadap kadar malondialdehid J Ilmu Kefarmasian Indones 2014 12 43 9
4. Batinić Haberle I Rebouças JS Spasojević I Superoxide dismutase mimics: Chemistry, pharmacology, and therapeutic potential Antioxid Redox Signal 2010 13 877 918 20095865
5. Gospodaryov D Lushchak V Oxidative Stress: Cause and Consequence of Diseases Oxid Stress Dis 2012 2 14 38 [doi: 10.5772/38093]
6. Rahal A Kumar A Singh V Yadav B Tiwari R Chakraborty S , Oxidative stress, prooxidants, and antioxidants: The interplay Biomed Res Int 2014 2014 761264 24587990
7. Ahmadi SM Farhoosh R Sharif A Rezaie M Structure-antioxidant activity relationships of luteolin and catechin J Food Sci 2020 85 298 305 31957877
8. Romulo A The Principle of Some in vitro Antioxidant Activity Methods: Review IOP Conf Ser Earth Environ Sci 2020 426 1 7 [doi: 10.1088/1755-1315/426/1/012177]
9. Tirzitis G Bartosz G Determination of antiradical and antioxidant activity: Basic principles and new insights Acta Biochim Pol 2010 57 139 42 20454707
10. Deawati Y Onggo D Mulyani I Hastiawan I Kurnia D Activity of Superoxide Dismutase Mimic of [Mn(salen)OAc] Complex Compound Non-enzymatically in Vitro Through Riboflavin Photoreduction Molekul 2017 12 61
11. Dupont S Fleurat Lessard P Cruz RG Lafarge C Grangeteau C Yahou F , Antioxidant properties of ergosterol and its role in yeast resistance to oxidation Antioxidants (Basel) 2021 10 1024 34202105
12. Chong DP Density functional theory study of allopurinol Can J Chem 2013 91 637 41
13. Farrokhnia M Density functional theory studies on the antioxidant mechanism and electronic properties of some bioactive marine meroterpenoids: Sargahydroquionic acid and sargachromanol ACS Omega 2020 5 20382 90 32832791
14. Ozgen S Kilinc OK Selamoğlu Z Antioxidant Activity of Quercetin: A Mechanistic Review Turk J Agric Food Sci Technol 2016 4 1134
15. Rivero Cruz JF Granados Pineda J Pedraza Chaverri J Pérez Rojas JM Kumar Passari A Diaz Ruiz G , Phytochemical constituents, antioxidant, cytotoxic, and antimicrobial activities of the ethanolic extract of Mexican brown propolis Antioxidants (Basel) 2020 9 70 31940981
16. Soliva R Luque FJ Orozco M Reliability of MEP and MEP-derived properties computed from DFT methods for molecules containing P, S and CL Theor Chem Acc 1997 98 42 9
17. Rakhmatullin IZ Efimov SV Klochkov AV Gnezdilov OI Varfolomeev MA Klochkov VV NMR chemical shifts of carbon atoms and characteristic shift ranges in the oil sample Pet Res 2022 7 269 74
18. Rawat DS Kumar D Nature of electronic transitions and factors affecting it Org Spectro 2017 12 1 21
19. Khan N Sagar Hossain MN Mofiz Uddin Khan Lecturer M Mofiz Uddin Khan N Scopoletin and β-sitosterol glucoside from roots of Ipomoea digitata J Pharmacogn Phytochem 2015 4 5 7
20. Klawun C Wilkins CL Chem J Optimization of functional group prediction from infrared spectra using neural networks Inf Comput Sci 1996 36 69 81
21. Khatun M Billah M Quader MA Sterols and Sterol Glucoside from Phyllanthus Species Dhaka Univ J Sci 2012 60 5 10
22. Zhang X Macmillan DW Alcohols as Latent Coupling Fragments for Metallaphotoredox Catalysis: sp3-sp2 Cross-Coupling of Oxalates with Aryl Halides J Am Chem Soc 2016 1 5
23. Muzolf-Panek M Gliszczyńska-Świgło A Szymusiak H Tyrakowska B The influence of stereochemistry on the antioxidant properties of catechin epimers Eur Food Res Technol 2012 235 1001 9
24. Heim KE Tagliaferro AR Bobilya DJ Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships J Nutr Biochem 2002 13 572 84 12550068
25. Godlewska Żyłkiewicz B Świsłocka R Kalinowska M Golonko A Świderski G Arciszewska Ż , Biologically active compounds of plants: Structure-related antioxidant, microbiological and cytotoxic activity of selected carboxylic acids Materials (Basel) 2020 13 4454 33049979
26. Saptarini NM Wardati Y Effect of extraction methods on antioxidant activity of papery skin extracts and fractions of Maja Cipanas onion (Allium cepa L. var. ascalonicum) ScientificWorldJournal 2020 2020 1 6
27. Rice Evans C Flavonoid antioxidants Curr Med Chem 2001 8 797 807 11375750
28. Safna Hussan KP Shahin Thayyil M Rajan VK Muraleedharan K DFT studies on global parameters, antioxidant mechanism and molecular docking of amlodipine besylate Comput Biol Chem 2019 80 46 53 30897526
29. Pottachola S Kaniyantavida A Karuvathodiyil M Density Functional Theory – Recent Advances, New Perspectives and Applications Croatia IntechOpen 2012 1 18
30. Xue Y Zheng Y An L Dou Y Liu Y Density functional theory study of the structure-antioxidant activity of polyphenolic deoxybenzoins Food Chem 2014 151 198 206 24423521
31. Hernandez DA Rodriguez Zavala JG Tenorio FJ DFT study of antioxidant molecules from traditional Japanese and Chinese teas: comparing allylic and phenolic antiradical activity Struct Chem 2020 31 359 69
32. Rajan VK Ragi C Muraleedharan K A computational exploration into the structure, antioxidant capacity, toxicity and drug-like activity of the anthocyanidin “petunidin” Heliyon 2019 5 e02115 32346622
33. Mahmoudi S Dehkordi MM Asgarshamsi MH Density functional theory studies of the antioxidants-a review J Mol Model 2021 27 271 34463834
