Int J Pharm Pharm Sci, Vol 8, Issue 8, 177-181Original Article


IN VITRO AND IN SILICO ANTIOXIDANT ACTIVITY OF PURIFIED FRACTIONS FROM PURPLE SWEET POTATO ETHANOLIC EXTRACT

N. P. LINDA LAKSMIANI1*, N. L. P. VIDYA PARAMITA1, I M. A. G. WIRASUTA1

1Departement of Pharmacy, Faculty of Mathematics and Science, Udayana University
Email: lindalaksmiani@gmail.com

Received: 12 Apr 2016 Revised and Accepted: 20 June 2016

ABSTRACT

Objective: Purple sweet potato (Ipomoea batatas L.) contains antioxidant compounds like anthocyanins (cyanidin and peonidin). Therefore, the current study was conducted to obtain anthocyanins fractions from purple sweet potato with evaluation of its antioxidant activity following ferrous ion chelating (FIC) method and comparing its activity with Na2EDTA. Furthermore, the oxidative molecular mechanism of purified fractions was investigated by in silico molecular docking to superoxide dismutase (SOD) and glutathione peroxidase (GPX).

Methods: Evaluating antioxidant activity by using FIC method performed by observing the absorbance of a mixed solution of (NH4)2 Fe(SO4)2, ferrozine and purified fraction measured using a UV-Vis spectrophotometer. Linear regression was used to calculate the IC50 value. Molecular docking was performed using 4.2 Autodock program. Data obtained in the form of docking score. Lower binding energy value show the more stable bond between the active compound and its target protein, SOD and GPX.

Results: Anthocyanin purified fraction has strong antioxidant capabilities with IC50 value of 74.44±1.29 µg/ml, but was significantly lower with Na2EDTA (p<0.05). The other mechanism is its ability to induce the target protein such as SOD and GPX intracellular defense body to capture free radicals. But it still lower affinity than native ligand to the target protein GPX is-4.28 kcal/mol while the native ligand with GPX of-7.12 kcal/mol. While the bond between peonidin with SOD is greater affinity (-4.21 kcal/mol) than the native ligand (-0.86 kcal/mol).

Conclusion: Anthocyanin purified fraction of purple sweet potato has a very potent antioxidant activity through two mechanisms as well as the metal chelating by using in vitro assay and free radical scavenger by inducing SOD.

Keywords: Anthocyanin purified fraction, FIC, SOD, GPX, Antioxidant

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Antioxidant agents act as free radicals or reactive oxygen species (ROS) scavenger by terminating the radical chain reactions [1-2]. ROS can trigger lipid peroxidation as a beginning of the oxidation process. In the body, there is a system of intracellular ROS scavengers to inhibit further oxidation processes; and these include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and others [3-4]. In addition to the natural antioxidants, there are also some synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) which are commercially available and currently used in industrial processes. However, the use of these industrial antioxidants in food, cosmetics and pharmaceutical products has declined because of their alleged promotion of carcinogenesis and other side effects. Lately, various natural antioxidants derived from vegetables, fruits, leaves, plant cereals, bark, roots, spices and herbs become the people's choice [5].

Research on the antioxidant activities from plants began to multiply readily. Among the phytochemicals with potent antioxidative effect, phenolic compounds proved to be powerful [6]. Of special interest, flavonoids are phenolic compounds that have an antioxidant activity [7]. One of the plants with potential antioxidant activity is the purple sweet potato (Ipomoea batatas L.) due to its high content of anthocyanins particularly cyanidin and peonidin [8]. Anthocyanin levels were positively correlated with antioxidant activity referring to the possibility that peels and purple yam tubers of purple sweet potato may have higher antioxidant activity [9-10]. Thus, the current study aimed at screening the antioxidant activity of acidified ethanolic extract of purple potato peel and tuber following in vitro ferrous ion chelating (FIC) method. Moreover, the molecular mechanisms involved in such activity would be investigated using in silico molecular docking analysis to superoxide dismutase (SOD) and glutathione peroxidase (GPX) as protein target.

MATERIALS AND METHODS

Materials

Purple sweet potatoes (peels and yam tubers) were obtained from Sidemen Village on Karangasem Regency. The chemical agents such as ferrozine, toluene, ethyl acetate were purchased from the Sigma-Aldrich co. USA. (NH4)2 Fe(SO4)2, ethylene diamine tetra-acetic acid (EDTA) disodium salt, ethanol 70%, HCl 0,005%, AlCl3 5%, FeCl2 2%, NH3, n-buthanol, aqua dest, glacial acetic acid and formic acid were purchased from Kurniajaya Multisentosa, Surabaya, Indonesia. Silica gel 60, silica gel F254 TLC plate were purchased from Merck, Pvt. Ltd., Germany.

Extraction and purification of anthocyanin from peels and yam tubers of purple sweet potatoes

One kg small piece of purple sweet potatoes was ground and extracted with 1 liter 0,005% HCl in 70% ethanol solution (pH 3) for 24 h and the process was repeated twice. The extracts were filtered through Whatman No. 2 filter papers and concentrated in vacuum at 50°C using a rotary evaporator to obtain 110. 64 g of extract (yield 11,06% based on a sample).

Part of the extract (420.8 g) was separated by solid phase extraction (SPE) column on silica gel 60 by elution with ethyl acetate, toluene, water and formic acid (12:3:0.8:1.2) v/v and it was continued by elution using ethanol under acidic condition. The first eluent was used to separate the cyanidin and peonidin with the other classes of anthocyanin and eliminate the impurities compound from cyanidin and peonidin. Second elution using acidic ethanol was conducted 5 times with a volume of 5 ml to obtain the clear fraction.

The anthocyanin purified fraction identification and characterization were determined using thin layer chromatography with silica gel F254 as the stationary phase and n-buthanol: glacial acetic acid: water (4:1:2) v/v as mobile phase. The fraction was monitored at λ 210 nm by means of a scanning pass in λ range of 200-700 nm.

Metal chelating ability of ferrous ions

The chelating of ferrous ions by anthocyanin purified fraction of ethanol extract 0.005% HCl from purple sweet potato was estimated by the method of Hinneburg et al. [11]. The different concentration of fraction was added to a solution of 0.027 mg/ml (NH4)2 Fe(SO4)2 (0.75 ml). The reaction was initiated by the addition of 0.027 M (0.75 ml) and the mixture was shaken vigorously and left at 37 °C for 10 min and the absorbance was measured spectro-photometrically at 562 nm. The chelating activity of the extracts was evaluated using EDTA disodium salt as standard. This assay was held in triplicate. The lower absorbance at 562 nm indicated a stronger chelating effect. Those absorbance data were calculated chelating ability percentage by using formula:

Chelating ability (%) = [A0-A1] x 100%

Where A0 was the control absorbance (complex between ferrozine and Fe) and A1 was the mixture containing the fraction absorbance or the standard absorbance.

Molecular docking

Molecular docking was performed to get the drug-receptor binding energy. The PDB files obtained from Protein Data Bank (PDB) (www. rcsb. org) is a worldwide repository for processing and distribution of 3D biological macromolecular structure data [12]. The structure of SOD (1MFM) and GPX (2F8A) proteins were downloaded from PDB and prepared for docking by removing the heteroatoms and water molecules in them.

The software used were as follows: Autodock 4.2. for molecular docking, Chimera 1.10.1 for protein and ligand preparation. The three-dimensional structure optimization by using HyperChem 8 by semi-emphasis AM1 computation and calculate with a single point and geometry optimization. The downloaded PDB file of SOD and GPX were first read in Chimera 1.10.1, added waters removed, and polar hydrogens were added. The structure of peonidin was drawn by using Hyper Chem 8. Next running docking protein target and ligand in file Autodock 4.2. The configuration then the ligand’s binding energy to target protein score was compared to native ligands.

Statistical analysis

Each sample was tested first distribution by Shapiro-Wilk Test and homogeneity with Lavene Test. The data were normally distributed and homogeneous (p>0.05) can be analyzed statistically with Paired T-Test with a 95% confidence level. If Paired T-Test results provide significant value less than 0.05 (p<0.05), then proceed with the statistical analysis of test Independent T-Test to determine which of the test samples provide a significant difference (p<0.05). For data were not normally distributed and homogeneous, statistically analyzed with Kruskal-Wallis test followed by Mann-Whitney test [13].

RESULTS

Fractionation and identification of anthocyanin fraction from purple sweet potato

Anthocyanin fraction is the fraction of the ethanol elution with the acidic condition. The whole anthocyanin fraction was collected and the solvent was evaporated. Anthocyanin fraction obtained in brown colour with a combined weight fraction yield amounted to 51.5%. The result of identification assay was shown in spectra. The spectra resulting from measurements in the wavelength range of 200-700 nm is shown in fig. 1. The spectra showed that the maximum absorbance is at a wavelength of 280 nm, and there is also a peak at a wavelength of 325 nm and 540 nm. Depending on appropriate literature on the identification of anthocyanin extract, the spectra from fractions showed the spectrum anthocyanin acyl group [14-19].

Fig. 1: Fraction spectra of anthocyanin fraction from purple sweet potato on λ 200-700 nm

Maximum λ a band I is at 540 nm, and therefore the wavelength of 540 nm chosen as the maximum wavelength of the aglycone group. Further TLC plate is scanned at a wavelength of 540 nm and a wavelength range of 200-700. The scans at a wavelength of 540 nm were data chromatograms, and spectra could be seen in fig. 2. These results suggest that the peak of the chromatogram 1 is caused by anthocyanin compound.

Anthocyanin fraction of purple sweet potato chelates the ferrous ion by using ferrous ion chelating

FIC method is a method to measure the ability of antioxidant compounds to compete with ferrozine to form chelates with iron ions [20]. The formed Fe ferrozine complex can be characterized by the formation of intense purple color in the solution. At the time of addition of the metal chelate compounds capable of the intensity of the purple color will begin to decrease [21]. Metal chelating measurement results of Na2EDTA as a positive control and anthocyanin fraction with three repetitions show the chelating effects of anthocyanin fraction and Na2EDTA on ferrous ions increase with increasing concentrations (fig. 3).

Based on calculation (fig. 3), the average IC50 value of anthocyanin fraction from purple sweet potato amounted to 74.44±1.29 µg/ml and IC50 of Na2EDTA is 5.00±0.25 µg/ml. Then performed statistical analysis of the data to the IC50 anthocyanin fraction of purple sweet potato and Na2EDTA. However, after tests of normality and homogeneity, the data obtained is not homogeneous. So that continued analysis using nonparametric test of Mann-Whitney with a 95% confidence level. Results of the analysis states that the value of IC50 fraction of purple sweet potato anthocyanins different significantly with IC50 value of the positive control, namely Na2EDTA (P = 0.05).

Anthocyanin fraction with peonidin as active compound potentially induces the target protein SOD and GPX

Molecular docking was performed to explore the binding mechanism, and it’s docking score to predict the activity of an active compound that showed the binding affinity between the target protein and compound. In this study, the docking of peonidin as the active compound in anthocyanin fraction to SOD and GPX binding site was observed, using ascorbic acid as a standard.

The docking analysis described the interaction of peonidin with antioxidant enzyme SOD and GPX. Before the molecular docking analysis was done, the method of docking with Autodock 4.2 was confirmed to be good and valid to qualify the RMSD value 1-3 Å. SOD with ligands has RMSD value of 2.98 Å, while GPX with ligands shows RMSD value 2.63 Å (table 1). Lower RMSD value means better docking method that used. In terms of the value of RMSD the molecular interaction could be continued to the following step. The interaction between peonidin or native ligan with GPX and SOD showed in table 1 and 2 and fig. 4.

Fig. 2: Chromatograms and spectra of each peak in anthocyanin fraction of purple sweet potato at λ 540 nm


A


B

Fig. 3: Chelating activity of ferrous ions by Na2EDTA as a positive control (A) and anthocyanin fraction from purple sweet potato (B). Results are shown as mean±SD (n=3)

Table 1: Docking score of interaction between peonidin to the target protein

Score docking (kcal/mol)
SOD (1MFM)
RMSD (Root mean square deviation) 2.98 Å
Native ligan -0.86
Peonidin -4.21
Ascorbic acid -3.64

Table 2: Amino acid residue and bonding formation of interaction peonidin and the target protein

Target proteins Compound Amino acid residue Bonding formation
GPX (2F8A) Native ligan Arg 180 Hydrogen Bond
Arg 179 Hydrogen Bond
Peonidin Arg 180 Hydrogen Bond
Lig 1 Hydrogen Bond
Ascorbic acid Thr 143 Hydrogen Bond
SOD (1MFM) Native ligan Lig 1 Hydrogen Bond
Peonidin Lig 1 Hydrogen Bond
Ascorbic acid Lig 1 Hydrogen Bond

Fig. 4: Docking representations of ligan and peonidin inactive binding site to SOD and GPx target proteins using Autodock 4.2. The 3D interaction between native ligan and active site of SOD (1MFM)(A1); native ligan and active site of GPX (2F8A)(A2); peonidin and active site of SOD (1MFM) (B1); peonidin and active site of GPX (2F8A) (B2)

DISCUSSION

Ferrous ion chelating activity assay used as Na2EDTA a comparison or positive control. Na2EDTA is the salt form of EDTA where the compound EDTA is a chelating ligand which has a strong affinity for forming complexes with metal [22]. Fig. 3 shows the correlation between the concentrations of Na2EDTA with chelating ability percentage. This is because of EDTA which has a high affinity for forming complexes with metal.

In this study, the addition of the anthocyanin fraction to a solution of Fe-Ferrozine, purple color diminishing with increasing concentration of fraction added. So that it could be interpreted that the anthocyanin fraction of purple sweet potato able to compete with the chelate iron ions ferrozine. Anthocyanin fraction included in compounds that have potent antioxidant activity through the metal chelating mechanism. Antioxidant compounds said to be strong if IC50 value are in the range of 50-100 µg/ml.

Na2EDTA has a strong chelating ability, but Na2EDTA could not be used as a chelator agent on the body. Because It has a very strong ability to bind all metals, Including metals needed by the body. When all the metal in the body are bound by EDTA, of course, this will interfere with the body's metabolic processes. So that the compounds could be used as an alternative to natural materials such as anthocyanin fraction of purple sweet potato as a chelator agent.

Molecular docking analysis demonstrated that peonidin as the active compound in anthocyanin fraction of purple sweet potato potentially induced SOD and GPX. Both of these enzymes play a role in ROS scavenging. Peonidin has lower affinity than native ligand to the target protein GPX (4.28 kcal/mol) while the native ligand with GPX is-7.12 kcal/mol. Score docking between peonidin with SOD protein is higher affinity (-4.21 kcal/mol) than the native ligand is-0.86 kcal/mol. This shows the anthocyanin fraction of purple sweet potato has a very potent antioxidant potency through two mechanisms as well as the metal chelating by using in vitro assay and free radical scavenger by using in silico.

CONCLUSION

Peonidin and cyaniding, as the active compound in anthocyanin fraction of purple sweet potato, have potent antioxidant efficiency as ferrous ion chelating and ROS scavenger.

ACKNOWLEDGEMENT

This research was supported by Hibah Unggulan Program Studi PNBP Udayana University and Departement of Pharmacy, Mathematics and Science Faculty, Udayana University.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Kumaran A, Karunakaran RJ. Antioxidants activities of methanol extracts of five Phyllanthus urinaria. Food Chem Toxicol 2007;46:2485-92.
  2. Rajalakshmi M, Dan Anita R. In vitro and In silico evaluation of antioxidant activity of a sesquiterpene lactone, costunolide, isolated from Costus Speciosus rhizome on MCF-7 and MDA-MB-231 human breast cancer cell lines. World J Pharm Res 2014;12:1334-47.
  3. Lu JM, Lin PH, Yao Q, Chen C. Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J Cell Mol Med 2010;14 Suppl 4:840-60.
  4. Rahman Khalid. Studies on free radicals, antioxidants and co-factors. Clin Interv Aging 2007;2 Suppl 2:219-36.
  5. Rumbaoa RGO, DF Cornago, IM Geronimo. Phenolic content and antioxidant capacity of philippine sweet potato (Ipomoea batatas) varieties. Food Chem 2008;113 Suppl 4:1133-8.
  6. Fawole OA, NP Makunga UL. Opara antibacterial, antioxidant and tyrosinase inhibition activities of pomegranate fruit peel methanolic extract. BMC Complementary Altern Med 2012;200 Suppl 12:2-11.
  7. Heim KE, Tagliaferro AR, dan Bobilya DJ. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J Nutr Biochem 2002;13:572-84.
  8. Jiao Y, Jiang Y, Zhai W, Yang Z. Studies on the antioxidant capacity of anthocyanin extract from sweet potato (Ipomoea batatas L.). Afr J Biotechnol 2012;11 Suppl 27:7046-54.
  9. Castaneda-Ovando, Araceli Ma. De Lourdes Pacheco-Hernandez, Ma. Elena Paez-Hernandez, Jose A. Rodriguez, Carlos Andres Galan Vidal. Chemical studies of anthocyanins. Food Chem 2009;113:859–71.
  10. Cevallos-Casals BA, Cisneros-Zevallos LA. Bioactive and functional properties of purple sweet potato (Ipomoea batatas (L.) Lam). Acta Hort 2002;583:195–203.
  11. Hinneburg I, Dorman DHJ, Hiltuen R. Antioxidant activities of extracts from selected culinary herbs and spices. Food Chem 2006;97:122-9.
  12. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The protein data bank. Nucleic Acids Res 2000;28 Suppl1:235-42.
  13. Garson D. Testing Statistical Assumptions. Edition. David Garson Publishers; 2012.
  14. Harborne JB. Spectral methods of characterizing anthocyanins. Biochem J 1957;70:22-8.
  15. Harborne JB, Grayer RJ. The antocyanins. In: Harborne JB. ed. The flavonoids: advances in research since 1980. London: Chapman and Hall; 1988. p. 1-20.
  16. Markham KR. Techniques of flavonoid identification. London: Academic Press; 1982.
  17. Woodall GS, Stewart GR. Do anthocyanins play a role in UV protection of the red juvenile leaves of Syzygium? J Exp Bot 1998;49 Suppl 325:1447-50.
  18. Qin Y, Li Y, Ding Y, Zhang R, Shang X. Analysis and characterization of anthocyanins in mulberry fruit. J Food Sci 2010;2:117-26.
  19. Silva FL, Teresa SP, Gonzalo JR, Buelga CS. Identification of anthocyanin pigments in strawberry (cv camarosa) by LC using DAD and ESI-MS detection. Eur Food Res Technol 2002;214:248-53.
  20. Elmastas M, I Gulcin, O Isildak, OI Kufrevioglu, K Ibaoglu, HY Aboul-Enein. Radical scavenging activity and antioxidant capacity of bay leaf extracts. J Iranian Chem Soc 2006;3:258–66.
  21. Aboul-Enein AM, El Baz FK, El-Baroty GS, Youssef AM, Abd El-Baky HH. Antioxidant activity of alga extracts on lipid peroxidation. J Med Sci 2003;3:87-98.
  22. Campbell JL. The influence of oxyanions on the EDTA-promoted dissolution of goethite. Virginia: Faculty of Virginia Polytechnic Institute and State University; 2001.

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