Advances in Applied Chemistry and Biochemistry

ISSN: 2652-3175

Research Article

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Mohd Nazrul Hisham Daud1,2*, Rohaya Ahmad2,3, Noriham Abdullah2,4, Mohd Lip Jabit1,Agustono Wibowo5 and Wan Saidatul Syida Wan Kamarudin2

1Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor, Malaysia

2Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

3Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, 42300 Bandar Puncak Alam, Selangor, Malaysia

4Malaysia Institute of Transport, Universiti Teknologi MARA, 40450 Shah Alam, Selangor,Malaysia

5Faculty of Applied Sciences, Universiti Teknologi MARA, 26400 Jengka, Pahang, Malaysia

Received: 21 July 2019

Accepted: 21 August 2019

Version of Record Online: 10 September 2019

Citation

Daud MNH, Ahmad R, Abdullah N, Jabit ML, Wibowo A, et al. (2019) Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract. Adv Appl Chem Biochem 2019(1): 68-81.

Correspondence should be addressed to
Mohd Nazrul Hisham Daud, Malaysia

E-mail: nazrul@mardi.gov.my
DOI: 
10.33513/ACBC/1901-07

Copyright

Copyright © 2019 Mohd Nazrul Hisham Daudet al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.

Abstract

In this paper, we report the storage effect of Artocarpus heterophyllus J33 rind extract at room (25°C), chilled (4°C) and frozen (-18°C) conditions on its antioxidant activity, LC profile and quality of bioactive compound. The bioactive compound was isolated via bioassay-guided isolation identified as Protocatechuic Acid (PCA) using spectroscopic methods. After 6 months storage, it was found that the antioxidant activity value of the frozen and chilled extracts remain above 90%, while at room temperature the value was lower. The TIC and LC profiles of the extracts indicated a drop in the content of the bioactive compound. Quantification of the bioactive compound for the extracts (after 6 months storage) revealed that its content was in the order of room <chilled ≤ frozen indicating that, under chilled and frozen conditions, the antioxidant activity of the extracts were prolonged due to the stability of PCA. These findings indicate that A. heterophyllus J33 rind extract could be a potential source of antioxidants forfood and nutraceutical products utilizing the waste to wealth concept from agricultural source.

Keywords

Antioxidant Activity; Artocarpus heterophyllus J33; Protocatechuic Acid; Rind; Spectroscopic; Storage

Introduction

Antioxidant activity and bioactive constituent or known as biomarker have become one of the important indicators for the determination of quality in storage studies. Most of the target bioactive compounds in plant extracts belongs to phenolic groups, since it has been reported to possess many therapeutic values [1]. Curently, storage studies have been conducted by many researchers especially for monitoring production of natural plant extracts with therapeutic value, either for food-based applications or nutraceuticals.

Most of the time, storage studies involve temperature, which could significantly affect the quality of the extracts through certain periods. In many cases, three storage conditions which involved room, chilled and frozen were selected in order to evaluate the quality of the extract [2-4].Room temperature is suitable to store extracts within short periods [5], while for longer periods of storage, low temperature of chilled and frozen have been found to be more suitable to retain bioactive constituents [4]. A period of six months is usually considered a suitable length for storage [6,7].

Based on our previous findings, the rind maceration extract (RDM) of A. heterophyllus J33 (AhJ33) variety fruit possess highest antioxidant ability compared to other extracts [8] and 15 antioxidant constituents were identified viaTOF-LCMS [9]. Hence, in this paper we now report the effect of storage on RDM under room, chilled and frozen conditions at a storage period of six months. The bioassay-guided isolation of the bioactive compound was also conducted in order to facilate the quantification of it during storage. The crude extract from each condition was drawn for initial quality evaluation via its antioxidant activity, LC profile and quantification of major bioactive compound.The quality evaluation process was carried-out every month for each sample throughout the six month storage period.

Materials and Methods

General instrumentation

The extract was filtered through filter paper Whatman No. 1. Solvents were evaporated using Buchi Rotavapor R210 at 45°C. The UV-Vis was recorded on ELISA Spectrophotometer Spectramax Plus (Molecular Devices).The mass were obtained from mass analyzer 6224 TOF LC/MS Agilent Technologies consisting electrospray (ESI) source system. The HPLC was carried-out using Agilent HPLC 1200 series. The analytical grade Protocatechuic Acid (PCA) for quantification was purchase from Sigma Aldrich (CAS number : 99503)The 1H NMR and 13C NMR were analyzed on a Joel Resonance ECZ400S NMR spectrometer measured at 400 and 100 MHz, respectively. Deuterated acetone was used and chemical shifts (dHanddC) were given in ppm. The column chromatography fractions were analyzed using Analytical TLC utilized DC-Plastikfolien 60F254 (Merck 5735 and 5559). Silica gel 60 (Merck 7734) was used for open column chromatography.

Plant material

Non-seasoning type of Malaysian Artocarpus heterophyllus (AhJ33) fruit for antioxidant and LCMS analysis was obtained from commercial source at Taman Kekal Pengeluaran Makanan Lancang Pahang, of Peninsular Malaysia. The rind of the fruits was separated, cut into small pieces (2.0 mm) and oven-dried at 40°C for 48 hours.

Extraction process

The extraction approach was carried out using the maceration technique. As reported in our previous study [8], maceration produced the highest antioxidant activity, phenolic and polyphenolic contents for Artocarpus heterphyllus J33 variety from rind part evaluated using TPC and TFC assays, respectively. The sample (1.0 kg) was macerated with 2L 70% ethanol for 72 hours at room temperature (25°C) with occasional shaking. The extract was filtered and the marc was re-extracted with the same amount of solvent. The solvent was evaporated off to yield the crude Rind Maceration Extract (RDM).

Isolation of bioactive compound

The bioactive compound was isolated from the most active subfraction A2-1 of the active ethyl acetate fraction via a bioassay-guided isolation. As reported in our earlier study [9], subfraction A2-1 possessed a percent inhibition of 92% in the DPPH radical-scavenging assay. The scheme for the bioassay-guided is shown below with the percent of DPPH radical scavenging activity of the fractions and sub-fractions (Figure 1):

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 1: The scheme for the bioassay-guided is shown with the percent of DPPH radical scavenging activity of the fractions and sub-fractions.

Structural elucidation of isolated bioactive compound

The structural elucidation of the isolated bioactive compound was carried-out using Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (LCMS) technique. All 1H (400 MHz) and 13C NMR (100 MHz) experiments were recorded on a Bruker equipped with Pulsed Field Gradients (PFG) and Indirect Detection Probe. Deuterated acetone was used and chemical shifts (δand δC) were given in ppm. MS spectrum were recorded on 6224 TOF LC/MS Agilent Technologies equipped with electrospray (ESI) system in the negative mode.

Determination of scavenging activities

The antioxidant potential of the fractions of AhJ33 rind was assessed on the basis of their scavenging activity on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. The assay was selected based on its strong correlation coefficient value with phenolic and polyphenolic contents evaluated by TPC and TFC content, respectively, as reported previously [8]. All test samples were prepared by dissolving 1.0 mg of samples into 1.0 ml 70% ethanol. A solution of DPPH was prepared by dissolving 5.0 mg DPPH in 2.0 ml of methanol and the solution was kept in the dark at room temperature. Different concentrations of the test samples were prepared in 96-well microtitre plates. Five μl of methanolic DPPH solution was added to each well. The plate was shaken to ensure thorough mixing before being placed in the dark and wrapped with aluminum foil. After 30 minutes, the optical density of the solution was analyzed using an ELISA reader Spectramax Plus (Molecular Devices) at a wavelength of 517 nm. Percentage inhibition by sample treatment was determined by comparison with 70% ethanol treated control group. All test analyses were run in triplicates and the readings were averaged. Quercetin (Sigma, USA) was used as positive control. In the DPPH method, the antioxidant activity is described by percent inhibition;

Percent inhibition = [(Abs control-Abs sample)/Abs control)]100

Storage conditions

The active crude rind extract obtained via maceration (RDM) was divided into three portions and each was transfered into a glass amber bottle. The three extracts were then stored separately at room (25°C), chilled (4°C) and frozen (-18°C) conditions. Each sample was drawn for initial quality evaluation for its DPPH radical scavenging activity. Chemical profiling of phenolic compounds employed LCMS while quantification of the major bioactive compound employed HPLC. Analyses of samples were carried every month for 6 months.

Profiling of chemical constituents

Profiling of the crude extracts during storage period was carried out using Agilent HPLC 1200 series and 6224 TOF LC/MS Agilent Technologies as described by Daud et al., [8]. TheLiquid Chromatography (LC) parameters were as follows: Injection volume was set at 5ul; separation via GL Sciences - Inertsustain Column C-18 (250 mm x 4.6 mm, i.d., 5µm); binary pump 45.01 min with post time 4.99 min; flow rate 0.6ml/min; minimum pressure 1 bar; maximum pressure 400 bar; max flow gradient 100 ml/min; two solvents A and B was used. Solvent A: H2O: MeOH (8:2) with 0.1% formic acid; solvent B: ACN with 0.1% formic acid. The solvent system comprise 95:5 of solvent A:B with gradient elution: from 5% solvent B at 0 min, 55% solvent B at 30min to 100% solvent B at 40 min. The eluent was monitored with a diode array detector at 254, 280 and 310 nm which usually used to monitor phenolic constituents.

The acquisition method for mass spectrometer was set for dual ESI for ion source with minimum range MS 100 and maximum at 1000. The gas temperature was set at 350°C, drying gas flow at 8 L/min, nebulizer gas at 30 psi, capillary voltage at 4000 V, fragment voltage at 175V, skimmer voltage at 65V, and OCT 1 RF at 750 V. Scan segment was carried out for negative (-) mode. Data processing was performed using Agilent Mass Hunter Workstation software. The reference masses involved were TFA anion (112.985587) and TFA adduct (1033.988109).

Quantification of bioactive compound using HPLC

Quantification of the bioactive compound (PCA) in the samples during the 6 month storage was carried out using Agilent HPLC 1200. The parameters used were as follows: Injection volume 5ul; separation via Kinetex - Phenomenex Column C-18 (250 mm x 4.6 mm, i.d., 5µm); binary pump 30.0 min; flow rate 0.6ml/min; minimum pressure 1 bar; maximum pressure 400 bar; max flow gradient 100 ml/min. Two solvents A and B were used. Solvent A: H2O: MeOH (8:2) with 0.1% formic acid and solvent B: ACN with 0.1% formic acid. Solvent system comprise 95:5 of solvent with gradient elution: from 5% A:B solvent B at 0 min, 55% solvent B at 25 min. The eluent was monitored using diode array detector at wavelength of 280 nm [10]. The series of standard solution (125, 60.25, 30.12, 15.6, 7.8 and 3.9 ppm) was analyzed in triplicates via direct injection of 5 μL volumes into the HPLC. The calibration curve was constructed by plotting the peak area (y) against the corresponding concentration of the standard solutions [11].

Statististical analysis

All experiments were run in triplicates. Statistical analyses were conducted with the Statistical Analysis System (SAS) 9.1.3 software package. Analyses of variance were performed by ANOVA procedures. Significant differences (P<0.05) were determined by least square means comparison.

Results and Discussion

Isolation and structural elucidation of bioactive compound

Bioassay-guided isolation work-up of the ethyl acetate sub-fraction (A2-1) yielded 450 mg of transparent needles of compound 1.

The mass spectrum of the compound exhibited two major peaks with m/z of 109 and 153. The molecular ion peak at m/z of 153 identifies the molecular formula to be C7H6O4 by in-house database. The molecular formula corresponds to an Index of Hydrogen Deficiency (IHD) of 5 which indicates the possible presence of benzene ring with another double bond. The fragment ion peak at m/z 109 may be due to the loss of a carboxyl moiety. The presence of signals within δH6.5-8.0 ppm and δc 114 to 170 ppm in the 1H NMR and 13C NMR spectrum, respectively, indicating the presence of aromatic compound. Through comparison of 1H NMR and 13C NMR data with available literature [12], the compound is assigned as 3,4-dihydroxybenzoic acid or protocatechuic acid (1) (C7H6O4).

Protocatechuic acid (1): 1H NMR (acetone-d6, 400 MHz, TMS), δH(ppm), J (Hz): 7.53 (1H, d, J = 2.0, H-2), 6.9 (1H, d, J = 8.4, H-5), 7.49 (1H,dd, J = 8.0; 2.0, H-6), 13C NMR (acetone-d6, 100 MHz, TMS), δ(ppm): 122.0 (C-1), 116.6 (C-2), 144.8 (C-3), 150.1 (C-4), 115.0 (C-5), 122.9 (C-6), 167.8 (C-7), [M]- : 153 (C7H6O4), 109 (C6 H6 O2).

The structure of the compound is shown below (Figure 2):

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 2: The structure of the compound 1.

Antioxidant (DPPH Radical Scavenging) during storage

Table 1 shows the antioxidant activity of RDM stored at room, chilled and frozen conditions. The range of antioxidant activity for the three crude extracts from 0-6 months was 86-94% After six months of storage, the order of antioxidant activity for the extracts is RDM(25°C) RDM(-18°C) with values of 86.0, 90.2 and 90.8%, respectively.

Month /

Condition (°C)

DPPH % inhibition

Room (25°C)

Chilled (4°C)

Frozen (-18°C)

0

94.4±0.10Aa

94.4±0.10Aa

94.4±0.10Aa

1

93.0±0.12Ba

94.2±0.13Aa

94.4±0.11Aa

2

90.0±0.1Cb

93.1±0.12Bb

93.8±0.13Ab

3

88.7±0.11Cc

92.2±0.11Bc

92.8±0.12Ac

4

87.5±0.12Cd

91.2±0.14Bd

91.0.10Ad

5

86.8±0.12Ce

90.6±0.11Be

91.2±0.13Ae

6

86.0±0.10Cf

90.2±0.12Bf

90.8±0.12Af

Table 1: Antioxidant activity of rind crude extract (% DPPH inhibition) stored for a period of 0-6 months. Values are expressed as mean ± standard deviation (n = 3). Means with different capital letter within a raw were significantly different at the level p < 0.05. Means with different small letter within a column were significantly different at the level p < 0.05.

Between RDM stored at chilled and frozen conditions, there is only a slight difference in the antioxidant values. However the DPPH inhibition remains above 90% throughout the storage period. The same trend was reported by Genova et al., [13], in which the authors reported that the crude grape extracts stored at chilled and frozen conditions for a period of 2 weeks showed no significant differences in their antioxidant values. Their findings indicated that at low temperatures (chilled or frozen), the antioxidant activity for the crude extracts could be preserved. Similarly, Qiu et al., [14] reported that at low temperature degradation of compounds could be prolonged and the antioxidant activity could be preserved. Hence, this could justify the preservation of antioxidant activity for the crude extract stored at chilled and frozen conditions in this study. For RDM stored at room temperature (25°C), after two months of storage, the antioxidant activity began to drop slightly from 90% at the end of two month to 88.7% at the end of third month storage period. The antioxidant activity for the crude extract continued to decrease gradually after the fourth and fifth month of storage with values of 87.5 and 86.8%, respectively. Finally, after six months of storage, the antioxidant activity of the extract dropped by 8.4% down to 86%. However, the antioxidant activity of chilled and frozen extracts only dropped by 4.4 and 3.8%, respectively. This indicates that storage at room temperaturehas caused a significant loss of antioxidant activity compared to storage at chilled and frozen conditions due to the degradation of antioxidant compounds. This is supported by a study by Del-Toro-Sanchez et al., [15] on the storage stability of Anemopsis californica. commonly known as yerba mansa or hierba mansa.

Concentration of isolated bioactive compound during storage

Protocatechuic Acid (PCA) was used as a standard compound and its quantification in the three samples during storage was carried out using Agilent HPLC 1200 series. This compound showed a strong absorption and produced a sharp signal at wavelength 280 nm. The same finding was also observed in a many previous studies for the analysis of PCA [10,16,17]. Figure 3 showed the calibration curve for PCA with a correlation coefficient, rvalue of 0.999 indicates good linearity.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 3: Calibration curve for Protocatechuic Acid (PCA).

Table 2 shows the concentration of PCA calculated for the crude extract stored at room, chilled and frozen conditions at the end of each month throughout the 6-month storage period.

Month /

Condition (°C)

Protocatechuic acid concentration (ppm)

Room (25°C)

Chilled(4°C)

Frozen (-18°C)

0

58.9±0.12Aa

58.9±0.12Aa

58.9±0.12Aa

1

58.0±0.12Ba

58.8±0.12Aa

58.9±0.12Aa

2

57.3±0.06Cb

58.1±0.06Bb

58.2±0.06Ab

3

56.6±0.06Cc

57.8±0.06Bc

58.0±0.12Ac

4

56.3±0.06Bd

57.7±0.13Ad

57.7±0.11Ad

5

55.6±0.12Ce

57.4±0.06Be

57.5±0.06Ae

6

55.2±0.12Cf

57.3±0.06Be

57.4±0.06Ae

Table 2: Concentration of protocatechuic acid in crude extract during storage period of 0-6 months. Values are expressed as mean ± standard deviation (n = 3). Means with different capital letter within a raw were significantly different at the level p < 0.05. Means with different small letter within a column were significantly different at the level p < 0.05.

Overall, the concentration of PCA in the crude extract stored at the three conditions decreased gradually during the 6-month storage from 57.5 to 55.2 ppm. After six months of storage, RDM stored at room temperature shows the highest drop in PCA concentration, followed by chilled and frozen with 6.2, 2.7 and 2.5% compared to the control, respectively. The percent decrease of PCA concentration for RDM (room temperature) is almost three fold the value for chilled and frozen extract. As for RDM stored at chilled and frozen conditions, the difference between them was only 0.2% which was not considered significant. The decreasing trend in the concentration of PCA paralleled the decreasing trend in the antioxidant activity of the extract.

As reported by Chaudhary et al., [18], Mirsaeedghazi et al., [19] and Christopoulos et al., [20], in a study of the effect of temperature on phenolic content in Citrus paradise, Punica granatum and Juglans regia, respectively, for storage at low temperatures (chilled and frozen), phenolic compounds could be preserved. Hence, the small drop in the content of PCA in the crude extract stored at chilled and frozen conditions compared to room condition indicated its stability.

LCMS profile of phenolic constituents

The profiling of the extract during storage period was based on Total Ion Chromatogram (TIC) and LC profiles at wavelengths 254, 280 and 310 nm. Since the antioxidant activities of some extracts showed reduction below 90% after three months of storage, the results and discussion will focus on i) TIC profile and ii) LC profile at 0 month (control), 3 months, and 6 months of storage period.

TIC profiling of crude extracts

Figure 4(a) shows the TIC profile of each crude extract after three months of storage at frozen, chilled and room conditions. As seen for the control, five major peaks could be observed with peaks a-appearing as an unresolved peak, while peaks d and e were well separated. The slight deviation of the peaks d and e at room temperature could be due to matrix effect resulting from storage of the extract. As reported by many previous studies the matrix effect could cause significant shift in LC-MS peak retention time besides increasing baseline and chromatographic peak tailing [21-23]. Figure 4(b) shows the comparison of the intensity strength of peaks d and e stored at the three conditions identified as protocatechuic acid and chlorogenic acid, respectively as previously reported [8]. The intensity strength of peakd (protocatechuic acid) was in the order of frozen > chilled > room. Although peak e (chlorogenic acid) at the frozen condition showed almost the same intensity strength as the control, the antioxidant activity of the extract after three months of storage recorded a lower value. This indicates that chlorogenic acid did not contribute significantly to the antioxidant activity. Figure 4(c) shows the TIC profile for each crude extract after 6 months of storage at the three conditions. As seen in the TIC profile at 3-month storage, five peaks were observed with peak d and e completely resolved and isolated from peaks a, b and c. The intensity strength of protocatechuic acid with the order of frozen > chilled > room is shown in figure 4(d) Compared to the control, there was a decrease in the intensity strength for protocatechuic acid at all conditions. Similarly, chlorogenic acid showed the same trend as protocatechuic acid. The decrease in the quantity of both constituents could explain the decrease in the antioxidant activity of the extract at all three conditions.

 Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 4(a): TIC profile of rind maceration extract stored at frozen, chilled and room conditions at 3-month storage.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 4(b): Comparison of the intensity strength of protocatechuic acid and chlorogenic acid stored at frozen, chilled and room conditions at 3-month storage.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 4(c): TIC profile of rind maceration extract stored at frozen, chilled and room at 6 month storage.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 4(d): Comparison of the intensity strength of protocatechuic acid and chlorogenic acid stored at frozen, chilled and room conditions at 6-month storage.

LC profiling of RDM samples after storage

Three months of storage

Figure 5a (i), (ii) and (iii) shows the LC profiles of the three RDM samples at λ 254, 280 and 310 nm at the end of 3 months of storage. The profiles at the different wavelengths exhibited some similarity but with notable differences. At λ 254 nm (Figure 5a(i)), the LC profiles of all extracts exhibited peaks a -d indicating the presence of aromatic compounds and simple or unconjugated compounds [24] in general, the UV wavelength at λ 254 nm is useful for detection of aromatic compounds. At 0 month, protocatechuic acid showed the highest intensity compared to other peaks. However,chlorogenic acid was not detected although the compound contains the structural features needed for UV absorption.This may possibly be due to its presence in low amounts in the extract.The LC profiles at λ 280 nm (Figure 5a(ii)) at all three conditions resemble that of the control. Unlike the profile at λ 254 nm, chlorogenic acid was observed at 10.9 min indicating that the phenolic moiety in chlorogenic acid absorbs strongly at λ 280 nm. Figure 5a(iii) shows the LC profile for samples at λ 310 nm. At this wavelength, only four peaks (a, b, d and e) were observed, with peak undetected indicating that the compound are not simple ketones, organic acids, esters or double bonds conjugated with phenyl rings [24]. As observed from the chromatogram, peaks d and showed better resolution compared to peaks a and b, and both peaks were dominant in the chromatogram. The higher intensity of peak at this wavelength is justified by the presence of double bond conjugated with the aromatic ring of chlorogenic acid.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 5(a): LC profiles at i) λ 254 nm ii) λ 280 nm and iii) λ 310 nm of crude maceration extract stored at frozen, chilled and room conditions at 3-month storage.

Figure 5b shows the intensity strengths for peaks and e at the three wavelengths. As shown in figure 5b(i), peak d for the extract stored at frozen and chilled conditions showed only a slight difference in the intensity strength with frozen > chilled. Hence, this could justify the decreased antioxidant activity observed for the crude extract stored at room condition after 3 months of storage. The chromatogram at λ 280 nm (Figure 5b (ii)) resemble the LC at λ 254 nm (Figure 5a(i)) for peak d at all three storage conditions. Again, the major peak d at room condition showed the lowest intensity compared to other peaks. This indicates that the loss of phenolic compounds at high temperature is greater than at lower temperature. This finding agrees with a previous report by Camargo et al., [25] instudied the effect of temperature during storage on the phenolic content in Achras sapota. In this study, the authors found that storage at low temperature could prolong post-harvest life of A. sapota fruits and retain the phenolic content. In another study, Moldovan et al., [26] found that, after 2 month storage at room temperature, the total phenolic content in Cornus mas fruits decreased up to almost 30%.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 5(b): Comparison of i) peak d (protocatechuic acid) at λ 254 nm ii) peak (protocatechuic acid) and e(chlorogenic acid) at λ 280 nm and iii) peak d (protocatechuic acid) and e(chlorogenic acid) at λ 310nm stored at frozen, chilled and room conditions at 3-month storage.

Similarly for peak e, the extract stored at room condition showed the lowest instensity strength compared to chilled and frozen extracts. Figure 5b(iii) showed the intensity strength of peak d ande at λ 310 nm. Interestingly, at this wavelength chlorogenic acid (peak e) showed a higher intensity than protocatechuic acid (peak d). However, this may be due to poor detector response for protocatechuic acid (phenolic compound) which absorbs more strongly at 280 nm as reported by Xu et al., [27] and Zhang et al., [10].

For both peaks d and e, the intensities were still in the same order of frozen > chilled > room. The lower intensity of peak d which represents protocatechuic acid at room condition could justify the significant loss of antioxidant activity of crude extract at three-month storage as seen in the LC profiles at λ 254 and 280 nm.  Hence, it can be deduced that the intensity strength at λ 310 nm for peak d does not reflect the quantity of protocatechuic acid in the extract.

Six months of storage

The LC profile of the rind maceration extract after six months of storage showed the same pattern as that of the 3-month storage at λ 254, 280 and 310 nm (Figure 6a (i), (ii) and (iii)). However the intensity of peak (protocatechuic acid) and e (chlorogenic acid) are significantly different. Figure 6b showed the comparison of intensity strength of peaks d at the three wavelengths. At each wavelength, the order of the intensity strength of peak is the same: frozen > chilled > room. There is a further decrease in intensity strength for peak d at all three storage conditions compared to control.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 6(a): LC profile at i) λ 254, ii) λ 280 and iii) λ 310 nm of crude extract stored at frozen, chilled and room conditions at 6-month storage.

Effect of Storage on Antioxidant Activity and Bioactive Compound of Artocarpus heterophyllus J33 Rind Extract

Figure 6(b): Comparison of peak d (protocatechuic acid) at i) λ 254 nm, ii) λ 280 nm and iii) λ 310 nm stored at frozen, chilled and room conditions at 6-month storage.

In summary, the LC profiles of the samples at 6-month storage resemble the LC profiles at 3-month storage at all three conditions. The difference in the intensity strength of protocatechuic acid detected at all wavelengths was in the order of frozen > chilled > room. As clearly seen from the profile at 3-month storage, the lower intensity of protocatechuic acid at λ 254 and 280 nm at room condition indicated a significant loss of antioxidant activity of the samples after 6 months of storage.

Conclusion

The storage study of the active Rind Maceration Extraction (RDM) indicated that at 3-month storage period, the antioxidant activity of the extract at room condition dropped from 94.4% to 88.7%, while for frozen and chilled extracts, the antioxidant activity dropped from 94.4% to 90.8% and 90.2%, respectively. The antioxidant activity of the frozen and chilled extracts remained above 90% even after 6 months of storage while the extract stored at room condition lost its antioxidant activity by 8.4% compared to control. HPLC quantitative analysis of the stored extracts at 3 and 6-month storage each revealed that the content of protocatechuic acid was in the order of room < chilled ≤ frozen. The storage stability study found that at chilled (4°C) and frozen (-18°C) conditions, the antioxidant activity could be prolonged due to the presence and stability of the protocatechuic acid (1). The TIC profiles of the samples supported the drop in the content of protocatechuic acid at 3-month and 6-month storage, as observed in the HPLC analysis. The same trend was observed from the LC profiles at three different wavelengths (λ 254, 280 and 310 nm). Based on these findings, A.heterophyllus J33 rind extract could be a potential source of antioxidants either as a based or ingredient in food and nutraceutical products.

Acknowledgement

The authors would like to thank the Faculty of Pharmacy, UiTM for permission to use the TOF LC/MS instrument. MNHD would like to acknowledge MARDI for facilitating the research project.

Conflict of Interests

The authors declare no conflict of interest.

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