INTRODUCTION
Free radicals are chemical species with unpaired electrons, formed by homolytic cleavage of a covalent bond of a molecule by the loss of a single electron from a normal molecule or by the addition of a single electron to a normal molecule.[1] Bromelain, which is a protease enzyme, has antioxidant activity as lipid peroxidation inhibition and free radical scavenging.[2]
Bromelain is a mixture of different thiol endopeptidases, phosphatases, glucosidase, peroxidases, cellulases, glycoproteins, carbohydrates, and several protease inhibitors.[3] It is obtained from pineapple (Ananas comosus (L.) Merr), which is grown in several subtropical and tropical countries, including Indonesia.[4]
Bromelain is distributed in every part of pineapple fruit with different characters. The most studied bromelain are extracted from stem and fruit.[3] In this study, the pineapple was collected from Subang district, West Java, Indonesia, one of the pineapple production centers in Indonesia.[5] Most studies used Thailand pineapple.[3] The pineapple crown was chosen because of agricultural waste and not being used properly. In our previous study, the proteolytic activity of crude bromelain of pineapple crown was 7.72 ± 0.45 IU/mg, with pH 5.0 as optimum pH and 55°C as optimum temperature. Proteolytic activity of crown bromelain was higher than bromelain from stem (4.52 IU/mg) and fruit (4.71 IU/mg).[6] Therefore, pineapple crown was potential to be developed as a bromelain source. The purpose of this study was to determine the antioxidant activity of crude bromelain, which was extracted from pineapple crown. Antioxidant activity is important to determine because of it can be developed as a compound to prevent degenerative diseases.[1] An easy and rapid way to evaluate antiradical activities of bromelain as antioxidant activity was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.[7]
MATERIALS AND METHODS
Materials
The pineapple crowns were collected from 12-month-old fruits that were planted in Subang district, West Java, Indonesia. The plant was identified by Plant Taxonomy Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia with No. 519/HB/02/2018. Ethanol and DPPH were analytical grade and purchased from Sigma-Aldrich (St. Louis, USA).
Bromelain extraction
One part of pineapple crown was mashed with two parts of water, and then filtered. Ethanol was added to the filtrate (1:4) and stored for 8h at 4°C, and then centrifuged at 15,000rpm for 15min. The sediment was dried at 30°C and the yield was calculated.[8]
Qualitative test of protein
Crude bromelain was dissolved in water with ratio 1:2. The solution was tested with Millon,[9] ninhydrin,[10] and Biuret reagent,[11] then observed the reddish brown solution for millon test, purple-blue solution for ninhydrin test, and blue purple for Biuret test.
Determination of total protein content
Five concentrations of casein standard, 30 mg/mL of crude bromelain, and water as blank were prepared. Bradford reagent was added to each solution and incubated at room temperature for 5min. The absorbance was measured at 595nm.[12]
Antioxidant activity assay
The maximum wavelength of 40 μg/mL of DPPH solution was determined at 400–800nm. Five different concentrations of vitamin C as standard and crude bromelain were added in 40 μg/mL of DPPH solution. All mixtures were incubated in a dark chamber for 30min; the absorbances were then measured at 517nm using a visible spectrophotometer. The blank was 96% ethanol. Percentage scavenging activity was calculated using formula (1). Linear regression resulted from percent inhibition versus concentration. The concentration of a sample which required for 50% of inhibition was determined and expressed as IC50 value:[13]
% of inhibition = [(ADPPH - Asample / ADPPH] × 100% (1)
RESULTS
Bromelain extraction
An 11.92kg of pineapple crown produced 31.21 mg of crude bromelain, so the yield was 0.26%. Crude bromelain was light brown powder, tasteless, and slightly pineapple fragrance [Figure 1].
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Figure 1: Crude bromelain of pineapple crown
Qualitative test of protein
The main composition of bromelain is protein, so qualitative test was conducted with spot test for protein. Tyrosine residues of bromelain were detected with Millon reagent, which was observed from reddish brown solution.[9] Free amine groups of bromelain were detected with Ninhydrin, which was observed from purple solution.[10] Peptide bonds of bromelain were detected with Biuret reagent, which was observed from a blue solution.[11]
Determination of total protein content
The absorbance of crude bromelain was 0.692 ± 0.004, so total protein content was 13.23 ± 0.42 mg/mL when calculated with a linear regression equation.
Antioxidant activity assay
Crude bromelain contains 44.10% of protein. IC50 value of 3624 μg/mL of crude bromelain [Table 1] was equivalent to 1590.18 μg/mL of protein.
Table 1: Antioxidant activity of vitamin C and crude bromelain
DISCUSSION
Bromelain extraction
Subang district produced 1,633 tons of pineapple in 2017.[5] Its altitude is 0–1500 m above sea level with temperature 21–31°C, and relative humidity is 78–84,[5] which is suitable for pineapple growth.[14] This study used 12-months-old fruit, because one third of the fruits is yellow. This shows that pineapple is ready to be harvested.[15] Bromelain was extracted with water because globular protein is soluble in water. Ethanol as an organic solvent was added to precipitate bromelain because of a globular protein that was insoluble in an organic solvent. Ethanol interferes the hydrophobic interactions in the interior of globular proteins without damaging the covalent bonds in the peptide chain.[16] This precipitation will concentrate bromelain. In this study, the yield (0.26%) showed a low-protein content in the pineapple crown, which was similar to the literature, that is, 0.25%.[14]
Determination of total protein content
Bradford reagent contain the dye Coomassie Brilliant Blue G-250. This dye bind to protein’s amino groups rhrough electrostatic interaction and carboxyl group through van der Waals forces to form the blue solution.[17] This wavelength is the orange wavelength (580–620nm) as transmitted color.[18] A correlation coefficient of the casein calibration curve was 0.997 [Figure 2]. This value met the ICH criteria[19] and showed that instrument response is equal to the concentration. Crude bromelain contains 44.10% of protein and 55.90% of nonprotein compounds, these result was in accordance with the literature, i.e., the protein content in bromelain range from 36% to 45%.[20] This result has supported the statement that bromelain is a combination of dissimilar thiol endopeptidases and other components such as cellulases, glucosidases, peroxidases, phosphatases, glycoproteins, carbohydrates, and several protease inhibitors.[321] This value was higher than the total protein of fruit, that is, 30%–40%.[16] Total protein content was used for determining the ratio of antioxidant activity of crude bromelain.
Figure 2: Calibration curve of casein as standard protein
Antioxidant activity assay
The maximum wavelength of the DPPH solution was 517nm [Figure 3], according to the literature.[13] This wavelength is the green wavelength (500–520nm) as transmitted color.[18] DPPH method is a sensitive, rapid, and easy method to determine the antioxidant activity of plant extract.[22] Antioxidant activity was observed through alteration of the DPPH solution. The DPPH solution color changes from purple to yellow, and the intensity of the color is proportional to the number of moles of the stabilized molecule.[23] Plant-sourced antioxidant agents, including bromelain, protect the human body from free-radical-induced diseases with little or no side effects. Antioxidant agents decrease oxidative stress by ending the oxidative chain reaction, which inhibits oxidative damage.[24]
Figure 3: Maximum wavelength of DPPH solution
The IC50 value was different from the other study,[24] because of differences in pineapple source, used-plant part, and bromelain purity level. The difference in the growth place and the pineapple part affects the bromelain character.[25] The pineapple source in this study was obtained from Indonesia, whereas that of Lee et al.’[24] study was obtained from Del Monte (Philippines). The used-plant part in this study was the pineapple crown, whereas that of Lee et al.’s[24] study was mixed with stem and bark. Bromelain concentration is high in the pineapple stem.[26] The result’s Lee et al.[24] which using stem better than our result which using crown. The pineapple crown was chosen to determine the potential of a pineapple crown as a bromelain source, so that it can use the other parts of the pineapple waste. In this study, crude bromelain has not been purified and contains 55.90% of nonprotein components, which showed no contribution to antioxidant activity. In another study by Lee et al.,[24] bromelain was purified by chromatography using diethylaminoethyl-cellulose column, followed by a G-150 gel filtration column, thus increasing the bromelain purity by 17 times. Bromelain purity affects the strength of antioxidant activity. Crude bromelain was a medium antioxidant, whereas purified bromelain was a potent antioxidant.[24] The usage of crude bromelain was more profitable than purified bromelain, because crude bromelain no need expensive and long purification steps and higher yield compared to purified bromelain.
Financial support and sponsorship
This work was supported by Kemenristek Dikti Grants in 2019 [grant number 1123aq/UN6.O/LT/2061].
Conflicts of interest
There are no conflicts of interest.
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