a

ISSN 0101-2061 (Print)

ISSN 1678-457X (Online) Food Science and Technology

DOI: https://doi.org/10.1590/fst.27720

Determination of some chemical compounds of bignay (Antidesma bunius) fruit juice

Gemala HARDINASINTA1, Mursalim MURSALIM2, Junaedi MUHIDONG2, Salengke SALENGKE2*

Abstract
Bignay fruits are produced from wild trees of Antidesma bunius Spreng which can be found in India, Ceylon, and South East
Asia countries. Each mature tree can produce hundreds of kilograms of fruits per year, so this fruit has the potential to be used
as a raw material in juice and beverage industries. In addition, recent trends in consumer demand indicates that consumers are
more inclined to demand products which can provide health benefits. In order to assess the potential of bignay fruit as a raw
material for juice production, it is important to determine the health-promoting bioactive compounds contained in fresh bignay
fruit juice. Therefore, the objective of this study was to determine the concentration of bioactive compounds in fresh bignay fruit
juice. Results obtained from this study showed that bignay fruit juice contained 1202.5 mg GAE/100 mL of total phenolic,
436.602 mg/100 mL of anthocyanin, 48.931 mg/100 mL of ascorbic acid, and 3.78 mg/100 mL of flavonoid. The scavenging
activities obtained from DPPH and ABTS methods were 0.110 mg/mL and 0.126 mg/mL respectively. The above results indicate
that bignay fruits contain health-promoting chemical compounds and can be used as a natural source of antioxidants.
Keywords: Antidesma bunius; color; phenolic compound; ascorbic acid; antioxidant activity.
Practical Application: Bignay fruit was found to contain active compounds with high antioxidant activity so it can provide
health benefits. The utilization of this fruit as a raw material for juice industries is viable, especially in its countries of origin.

1 Introduction
The utilization of exotic fruits as a raw material in the Studies on bioactive compounds for this fruit have been
food industry has become an emerging trend due to their conducted mainly for bignay from trees grown in northern
health-promoting functional compounds such as antioxidants. Thailand. The major flavonoid contents found in those studies
In addition, there is a trend in food industries where synthetic were (-)-epicatechin and (+)-catechin (flavan-3-ols), while
antioxidants were replaced with antioxidants from natural the major contents of anthocyanin were cyanidin, followed
sources (Caleja  et  al.,  2017; Kumar  et  al.,  2015). Among the by malvidin, pelargonidin, and delphinidin. Phenolic acids
organic compounds found in fruit that have shown health identified in bignay consist of two hydroxybenzoic acids i.e.
benefits for humans are vitamins and phenolic compounds gallic acid and vanillic acid and five hydroxycinnamic acids
such as flavonoids. These compounds have high antioxidant i.e. caffeic acid, p-coumaric acid, ferulic acid, sinapinic acid, and
activities and other biological functions. As a source of natural cinnamic acid (Butkhup & Samappito, 2008; Jorjong et al., 2015).
There has been no such study conducted for bignay fruits from
polyphenols and other health-promoting compounds, exotic
trees found in Indonesia.
fruits contain an abundant amount of phenolic acids and
flavonoids such as anthocyanin. These compounds have distinct Despite its potential beneficial impacts on health, the intake
properties such as strong antioxidant activity, anti-inflammatory, of bignay fruits in the producing countries such as Indonesia is
and antiproliferative activities which could inhibit cell cancer still very low due to its stone, tart taste, and purple stains when
growth and prevent cardiovascular diseases (Swami et al., 2012; eaten as fresh fruit. Therefore, to increase consumption of this
Timmers et al., 2015). fruit, processing into processed products such as juice and jams
need to be done. The increase in consumption of juice and other
Antidesma bunius is an indigenous species found mainly processed products from fruits with high bioactive compounds
in the South East Asia region such as Thailand, Philippine, and can help meet the recommended daily intake for fruits and
Indonesia but it can also be found in India and Ceylon. This fruit provide additional health benefits. For example, Asgary et al.
has different names such as “Maoberry” or “Mao-Luang” in (2014) reported that consumption of high-flavonoid sweetie juice
Thailand, “Bignay” in Philippine, and “Buni” in Indonesia. provided a higher reduction of Diastolic Blood Pressure (BDP)
In some countries, this fruit has been consumed in the form of compared to the consumption of low-flavonoid sweetie juice.
juice, jam, and wine (Chaikham, 2015; Chaikham et al., 2016; The objectives of this study were to determine total phenolic,
Jorjong et al., 2015; Samappito & Butkhup, 2008). total monomeric anthocyanin, total flavonoid, and vitamin C

Received 09 June, 2020

Accepted 30 July, 2020
1
Department of Agricultural Science, Graduate School of Hasanuddin University, Kampus Unhas Tamalanrea, Makassar, Indonesia
2
Department of Agricultural Engineering, Hasanuddin University, Kampus Unhas Tamalanrea, Makassar, Indonesia
*Corresponding author: salengke@g.unhas.ac.id

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 Hardinasinta et al.

contents of bignay juice and to determine the antioxidant activity (Jiang et al., 2013). Briefly, two dilutions of samples were prepared
of fresh bignay fruit juice. Besides, the color and pH of fresh by adding 50 μL of samples with 4950 μL of potassium chloride
bignay fruit juice were also measured. This is important since (0.025 M) buffer pH 1.0 and the other with sodium acetate (0.4 M)
the color of a fruit can be used as an indicator to predict the buffer pH 4.5. The absorbance of each sample was measured after
chemical compounds contained in the fruit. Several chemical 20 minutes at wavelengths of 520 and 700 nm. Total anthocyanin
compounds that have been proven to provide a beneficial impact content was determined based on the absorbance value obtained
on health also serve as a color pigment. Anthocyanin is used as from Equation 1.
a source for the blue-purple pigment, lycopene for red pigment,
and β-carotene for orange pigment (Jamal et al., 2017). A = ( A520 − A700 ) pH 1,0 − ( A520 − A700 ) pH 4 , 5 (1)

2 Materials and methods where A520 is the absorbance at the 520 nm wavelength, and A700
is the absorbance at the wavelength of 700 nm. Total anthocyanin
2.1 Sample preparation was represented as cyanidin-3-glucoside and calculated using
Equation 2:
Bignay fruits were bought from a local market in Makassar,
South Sulawesi, Indonesia. The fruits were washed and sorted
3
based on the maturity level. The ripe fruits (purple blackish  mg  A × MW × DF × 10
Anthocyanin  = (2)
color) were chosen and processed into juice using a commercial  L  ε ×l
juicer (Philips HR1832). The juice obtained was filtered through
where A is the absorbance value, MW is the molecular weight
cheesecloth to separate the pulp and the fresh juice obtained
of standard (445.2  g/mol), DF is the dilution factor, ε is the
was poured into a 500 mL PTFE bottle and stored in a freezer
molar absorption capacity (29,600 l/mol.cm), and l is the cuvette
maintained at -18 °C until used.
length (1 cm).

2.2 Determination of pH, Total Soluble Solids (TSS), and color 2.5 Ascorbic acid content
The pH measurement was carried out using a pH meter The ascorbic acid content was measured using Ultra-Fast
(LAQUAtwin pH-11, Horiba Scientific, Japan) calibrated with Liquid Chromatography (UFLC) based on the method represented
buffer solutions of pH 7.0 and 4.0. Total soluble solid (TSS, in literature (Mercali  et  al.,  2014) with some modifications.
expressed in °Brix) was measured using a digital refractometer The preparation of the stock sample was carried out by adding
(HI 96801, Hanna Instruments, Rhode Island, USA) calibrated 1.5 mL of sample into 7.5 mL of meta-phosphoric acid solution
with distilled water. The color of the juice was determined using a (4.5%). The mixture was filtered through a syringe filter of 0.45 μm,
colorimeter (CS-10 Colorimeter, Hangzhou CHN Spec Technology, and 40  μL was injected into the UFLC system (Prominence
China). Color parameters of the samples were determined based UFLC, Shimadzu, Japan).
on CIE L* a* b* parameters, where L* represents the lightness
with value ranged from 0-100 (black-white axis), a* represents The UFLC conditions were described as follows: The column
the green-red color with value ranged from -60 to +60 (green-red used was a reverse C18 (Shimadzu, Japan) and the separation
axis), and b* represents the blue-yellow color with ranged of -60 was carried out under isocratic condition using a mobile phase
to +60 (blue-yellow axis). of KH2PO4 (5%). The eluent flow rate was 0.7  mL/min and
the column temperature was set at 25 °C. The absorbance was
2.3 Total phenolic content measured at 254 nm and pure ascorbic acid was used as standard.

Total phenolic measurement was conducted according to the 2.6 Total flavonoid content
Folin Ciocalteu method described in Tezcan et al. (2009) with
some modification. The stock solution was made by diluting Total flavonoid content was measured using Thin Layer
0.1 mL of bignay fruit juice in 10 mL of methanol:water (6:4 v/v) Chromatography (TLC) based on the method used by several
solvent. The determination of phenolic compounds was performed researchers (Altemimi  et  al.,  2015) with some modification.
as follows. 0.5 mL of stock solution was mixed with 1.5 mL of A 20 × 20 cm TLC silica gel 60 F254 plates (Merck, Germany)
Folin Ciocalteu reagent (7.5%) and 1.2 mL of Na2CO3 (7.5%) and were divided into four equal pieces (5 × 20 cm) and then placed
then topped with distilled water to reach a total volume of 5 mL. into an oven at 110 °C for 20-30 min prior to use. The separation
The mixture was kept for 90 minutes at room temperature before was carried out using the mixture of ethyl acetate: formic acid:
the measurement of absorbance was conducted at 760 nm using water (10:2:3 v/v) as the solvent. 10 mL of solvents were saturated
UV-Vis Spectrophotometer (UV-1800, Shimadzu, Japan). The results inside the chromatography chamber for 30-40 min before used.
were expressed as gallic acid equivalent (mg GAE/100 mL juice) For the measurement, 2 μL of samples and standards were dotted
using calibration curved of gallic acid from 2-10 ppm. on the TLC plate with a 1 cm gap between each dotted sample.
The plate was placed inside the chamber until the separation
process was completed. The determination of flavonoid content
2.4 Total anthocyanin content
was performed by densitometer (Camag TLC scanner 3,
The total monomeric anthocyanin assay was determined Switzerland) at a wavelength of 254 nm and the concentration
using the pH differential method as described in the literature was expressed as mg rutin/100 mL of juice.

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 Chemical compounds of bignay fruit juice

2.7 Antioxidant activity The pH value obtained indicates that bignay fruit juice can be
categorized as a high acid food (pH < 3.6). The pH of foods and
2,2 Diphenyl-1-picrylhydrazyl (DPPH) assay beverages is an important factor that affects their shelf life and
processing condition. The lower pH level of the bignay fruit juice
The scavenging activity of DPPH radical was measured may permit short pasteurization time at relatively moderate
based on the method described in Kenny  et  al. (2013) with temperatures since pathogenic bacteria do not grow at low pH
modifications. DPPH solution was made by diluting 8 mg of (Achir  et  al.,  2016; Branco  et  al.,  2016; Park  &  Kang, 2013).
DPPH (Sigma-Aldrich) with 50 mL of methanol. A stock solution In addition, pH also influences the stability of compounds in the
of the sample was prepared by dilution of 5 mg juice in 5 mL food product. Anthocyanin has been reported to be susceptible
of 100% methanol. Five different concentrations (75-375 ppm) toward pH, where acidic condition provides higher stability of this
of each sample were performed by pipetting 15, 30, 45, 60, and compound (Fredes et al., 2018; Jiang et al., 2015). The pH value
75 μL of the stock sample across the 96-well plate. A methanol obtained in this study (3.45) was equivalent to the pH level of
blank was also used as a negative control. The DPPH solution Phuchong cultivars as reported in Butkhup & Samappito (2008).
(75 μL) was added to the plate and then topped with methanol The pH of bignay fruit and its products that have been reported in
to the final volume of 200 μL. The mixture was stored in the literature ranged from 3.40 to 3.50 (Butkhup & Samappito, 2008;
dark for 30 minutes at room temperature before its absorbance Chaikham, 2015; Chaikham et al., 2016; Sripakdee et al., 2015).
at 515  nm was measured using ELISA plate reader (ELx808
The total soluble solid (TSS) of fresh bignay juice obtained
BioTek, Vermont, USA). The results were expressed in IC50
in this study (14.67 °Bx) was comparable with those reported
scavenging activity.
in Butkhup  &  Samappito (2008) for Sangkrow No.2 cultivar
(14.50  °Bx) and Phuchong cultivar (14.80  °Bx). Higher TSS
2,20-azino-bis (3-ethylbenzthiazonline-6-sulfonic acid) (ABTS) assay value (16.50 °Bx) was reported by Sripakdee et al. (2015) and
The determination of ABTS scavenging was carried out Butkhup & Samappito (2008) for Sangkrow No.5 cultivar, while
according to the method described in (Jorjong et al., 2015) with lower values (12.77 and 12.35 °Bx) were respectively reported
some modification. The stock solution of the ABTS reagent was in Chaikham et al. (2016) and Butkhup & Samappito (2008)
prepared by mixing 7.4mM ABTS solution and 2.6mM potassium for Sangkrow No.4 cultivar. In terms of pH value and total
persulfate solution with the ratio of 1:1 (v/v). The ABTS solution soluble solid, bignay juice originated from Indonesia has similar
was made by diluting 0.018 g of ABTS with 5 mL of methanol, characteristics to the Phuchong cultivars grown in Thailand.
while the potassium persulfate solution was prepared by diluting
3.6 mg of potassium persulfate with 5 mL of methanol. The mixture 3.2 Chemical characteristics of bignay juice
of ABTS and potassium persulfate solutions was topped with Results of chemical analysis of bignay juice were presented
methanol to reach the total volume of 25 mL. The solution was in Table 2. Total phenolic (TP) content of bignay juice used in
then incubated in the dark for 12-16 hours. this study was much higher (1202.5 mg/100 mL) than the value
The stock solution of the sample that had been prepared for reported (274.65 mg GAE/100 mL) in Chaikham et al. (2016).
the measurement of scavenging activity using the DPPH method A  lower amount of TP was also reported in several studies
was also used for this ABTS scavenging test. The varying volume (Chaikham, 2015; Jorjong et al., 2015; Sripakdee et al., 2015).
of the stock solution was pipetted into a well plate to form five These authors reported TP contents of 390.67  mg/100  mL,
different concentrations. Methanol was used as the negative 345.68  mg GAE/100  g dry weight (DW), and 337.52  mg
blank. ABTS stock solution was added to the plate with a volume GAE/100  mL respectively. On the other hand, higher TP
of 125  μL and topped with methanol to the final volume of content (1978.38  mg/100  g DW) was reported in the crude
200 μL. The mixture was left in the dark for 30 minutes at room extract by Barcelo et al. (2016). It is important to note that the
temperature before absorbance measurement was conducted at TP content found in this study was within the range reported
650 nm using ELISA plate reader. by Butkhup & Samappito (2008) who reported the TP values
All the measurements were conducted in triplicates. The IC50
values and statistical analysis were determined using GraphPad Table 1. Physical characteristics of bignay juice.
Prism 8.0. All data except antioxidant activity will be express in Value
mean ± standard deviation value. Parameter
Mean ± SD
Color:
3 Results and discussions
L* 25.885 ± 0.035

3.1 Physical characteristics of bignay juice a* -0.470 ± 0.010

b* 1.460 ± 0.370
The physical characteristics measured were color parameters
of fresh juice, pH, and total soluble solid content, and the results Chroma (C*) 1.537 ± 0.354
are given in Table 1. From the color parameter data, bignay Hue angle ( h)
o
-1.243 ± 0.071
fruit juice provided a dark-colored liquid as shown by the low pH 3.453 ± 0.031
Lightness and Chroma value. Dark-colored fruits are generally
Total soluble solids ( Bx)
o
14.667 ± 0.094
known as a potential source for anthocyanin (Wu et al., 2012).

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 Hardinasinta et al.

of 8.37-13.56  mg GAE/g DW (837-1356  mg GAE/100  mL). for the human diet and can be used as a substitute for orange
The  addition of water during the extraction process might juice which can be expensive in certain regions.
become a contributing factor that caused the TP content of
In contrary to other compounds reported previously,
bignay juice was lower in several studies (Chaikham, 2015;
the flavonoid content of bignay juice found in this study
Jorjong et al., 2015). It is important to note that in our study,
(3.78 mg/100 mL) was significantly lower than those reported
the extraction of juice from bignay fruits was accomplished by others. A study conducted by Butkhup & Samappito (2008)
without the addition of water. reported that the amount of rutin in bignay fruit ranged from
The total phenolic content of bignay juice found in this 16.61-25.54 mg/100 g fruit weight (FW) and the total flavonoid
study is higher than those of other juices such as pomegranate content was in the range of 120.39-397.90 mg/100 g FW. A study
juice (230.86 mg GAE/100 mL) (Yildiz et al., 2009), grape juice by Jorjong et al. (2015) on bignay cultivars from Thailand reported
(2135-2647  mg/L) (Padilha  et  al.,  2017), and mulberry total flavonoid content ranged from 138.60-289.60 mg catechin
juice  (266.8  mg GAE/100  g DW) (Kamiloglu  et  al.,  2013a). (CE)/100 g DW. The significant difference in the total flavonoid
A high amount of TP found in bignay juice can be explained content found in this study and those reported by others might
by the major phenolic acid in this fruit, which is gallic acid and be attributed to the difference in the type of varieties and growing
vanillic acid (Butkhup & Samappito, 2008; Jorjong et al., 2015). conditions. Another contributing factor might be the analytical
In comparison, the major phenolic acid reported in mulberry method, mainly the use of different types of compounds as the
fruit was chlorogenic (Kamiloglu et al., 2013b). equivalent standard.
As mentioned before, the dark-colored fruit potentially contains The antioxidant activity of bignay juice (Figure 1) was measured
high anthocyanin content. This was in agreement with the result by DPPH and ABTS method. The principal of both methods
obtained in this study, where the anthocyanin content of bignay was based on the reaction between hydrogen donor antioxidants
juice was 426.602 mg/100 mL of juice. The anthocyanin content with free radicals that would result in a discoloration of those
found was significantly higher than those reported previously radical substances (Pisoschi & Negulescu, 2011). The antioxidant
by Chaikham (2015) (59.46 mg/100 mL) and Chaikham et al. activity of bignay juice was expressed by the half-maximum
(2016) (44.32 mg/100 mL) for bignay juice, Barcelo et al. (2016) inhibitory concentration (IC50) of a sample that measures the
(131.42 mg/100 g DW for extract), and Butkhup & Samappito concentration needed by a sample to inhibit 50% of the free
(2008) (141.94 mg/100 g DW for fruit). The varying level of radical reaction. The IC50 found in this study for bignay juice
anthocyanin can be due to the difference in maturity or ripeness was 0.11 mg/mL for the DPPH method and 0.126 mg/mL for the
during harvest, considering that at the earlier stage of maturity ABTS method. The value of IC50 has a negative correlation with
bignay fruits display bright red-color, while at the later stage the the antioxidant activity, which means that a smaller IC50 value
color shifts to dark-purple. corresponded to higher antioxidant activity. Previous research
evaluating the antioxidant activity of bignay fruit grown in
Vitamin C content of bignay juice is represented in ascorbic Manipur, India, exhibited lower antioxidant activity with an IC50
acid equivalent. The result obtained in this study (48.93 mg/100 mL) value of 1717 μg/mL (1.717 mg/mL) (Khomdram & Devi, 2010).
is higher than the ascorbic acid content of bignay juice reported However, relatively similar value have been reported with other types
by Chaikham (2015) and Chaikham et al. (2016). In these two of fruit such as Litchi chinensis fruit pulp extract (0.102 mg/mL)
studies, the vitamin C contents reported were 19.05 mg/100 mL (Prakash  et  al.,  2011), Radix Angelicae Sinensis extracted by
and 26.14 mg/100 mL respectively. The vitamin C content of ethyl acetate (0.13 mg/mL) (Li et al., 2009), Angelicae Sinensis
bignay juice found in this study was comparable to the ascorbic extract (0.093  mg/mL by water extraction and 0.15  mg/mL
acid content of orange juice (ranged from 249.81-612.75 mg/L) by 95% ethanol extraction). The antioxidant activity of bignay
(Sánchez-Moreno et al., 2003). Therefore, this result indicates juice found in this study is relatively similar to the antioxidant
that bignay juice could become a potential source of vitamin C activity of Radix Angelicae Sinensis extract which many consider

Table 2. Bioactive compounds in bignay juice.

Value
Compounds
Mean ± SD
Total Phenolic (mg/100 mL) a
1202.500 ± 0.001
Total Monomeric Anthocyanin (mg/100 mL)b 426.602 ± 3.723
Vitamin C (mg/100 mL) c
48.931 ± 0.001
Total Flavonoid (mg/100 mL) d
3.78 ± 2.114
Antioxidant activity:
DPPH method-IC50 (mg/mL) 0.110
ABTS method-IC50 (mg/mL) 0.126
a
gallic acid equivalent; cyanidin-3-glucoside equivalent; ascorbic acid equivalent;
b c Figure 1. Radical scavenging activity of bignay fruit with DPPH and
d
rutin equivalent. ABTS methods.

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 Chemical compounds of bignay fruit juice

as a superior traditional Chinese medicine with various health Chaikham, P., Prangthip, P., & Seesuriyachan, P. (2016). Ultra-sonication
benefits such as promoting the blood circulations, regulating effects on quality attributes of maoberry (Antidesma bunius L.) juice.
menstruations, and relieving pain (Li et al., 2009). Food Science and Technology Research, 22(5), 647-654. http://dx.doi.
org/10.3136/fstr.22.647.
Fredes, C., Osorio, M. J., Parada, J., & Robert, P. (2018). Stability and
4 Conclusions
bioaccessibility of anthocyanins from maqui (Aristotelia chilensis
Bignay juice was found to contain high amounts of total [ Mol. ] Stuntz) juice microparticles. Lebensmittel-Wissenschaft +
phenolic, anthocyanin, and vitamin C and has relatively high Technologie, 91, 549-556. http://dx.doi.org/10.1016/j.lwt.2018.01.090.
antioxidant activity. Therefore, bignay fruit can be used as a Jamal, P., Hashlamona, A., Jaswir, I., Akbar, I., & Nawawi, W. M. F.
natural source of antioxidant and as a potential source of raw (2017). Extraction of lycopene from tomato waste using solid state
material for juice processing industries. fermentation. International Food Research Journal, 25, 416-421.
Jiang, B., Mantri, N., Hu, Y., Lu, J., Jiang, W., & Lu, H. (2015). Evaluation of
bioactive compounds of black mulberry juice after thermal, microwave,
Acknowledgements
ultrasonic processing, and storage at different temperatures. Food
This research was funded by the Indonesian Ministry of Science & Technology International, 21(5), 392-399. http://dx.doi.
Research, Technology, and Higher Education under the PMDSU org/10.1177/1082013214539153. PMid:24917651.
Program managed by the Directorate of Human Resources Jiang, D. Q., Guo, Y., Xu, D. H., Huang, Y. S., Yuan, K., & Lv, Z. Q. (2013).
Qualification. Salaries and research support were provided by Antioxidant and anti-fatigue effects of anthocyanins of mulberry juice
Hasanuddin University. purification (MJP) and mulberry marc purification (MMP) from
different varieties mulberry fruit in China. Food and Chemical Toxicology,
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