Heliyon 9 (2023) e14426

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Effect of hot water, ultrasound, microwave, and pectinase-assisted

extraction of anthocyanins from black goji berry for
food application
Gayan Chandrajith Vidana Gamage, Wee Sim Choo *
School of Science, Monash University Malaysia, 47400, Bandar Sunway, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Lycium ruthenicum, commonly known as black goji berry, is a rich anthocyanin source containing
Black wolfberry a high amount of monoacylated anthocyanins. This study investigates the effect of different
Enzyme-assisted extraction extraction methods to extract anthocyanins from black goji berry for food application. Different
Lycium ruthenicum
hot water extraction conditions were applied to investigate the effect of specific substrate: solvent
Monoacylated anthocyanin
Petunidin
ratio (1:15 and 1:20 (w/v)), extraction time (30 and 60 min) and extraction temperature (40, 50
and 60 ◦ C) on the extraction yield, total anthocyanin content (TAC) and the total phenolic content
(TPC) of the anthocyanin extracts. Best hot water extraction conditions for obtaining an antho­
cyanin extract with high TAC (13.8 ± 1.14 mg CGE/g), TPC (69.7 ± 2.50 mg of GAE/g), and
extraction yield (48.3 ± 3.25%) consuming less solvent, time and heat were substrate: solvent
ratio of 1: 15 (w/v), extraction temperature of 50 ◦ C, and extraction time of 30 min. The effect of
pectinase, ultrasound, and microwave on hot water extraction of anthocyanins from black goji
berry was investigated using the best conditions for hot water extraction. Pectinase-assisted
extraction [1.5% (w/v) pectinase, substrate: solvent ratio of 1:15 (w/v) at 50 ◦ C for 30 min]
was the best extraction method to extract black goji berry anthocyanins demonstrating higher
extraction yield, TAC, TPC, and the highest percentage of petunidin-3-O-(trans-p-coumaroyl)-
rutinoside-5-O-glucoside.

1. Introduction

Lycium ruthenicum is a perennial shrub belonging to the family Solanaceae and is commonly known as black goji or wolfberry. They
are naturally found abundantly in the Qinghai-Tibet plateau (China), with hard conditions such as less soil moisture, high soil salinity
and sunlight [1]. Black goji berry is a herb used in traditional Chinese medicine with fruits containing high amounts of anthocyanins
and other bioactive compounds [2–5]. The anthocyanin content may range between 700 and 900 mg cyanidin-3-glucoside equivalents
per 100 g of dry black goji berries [6]. Nearly 37 different anthocyanins have been identified in black goji berries [7]. Anthocyanins
derived from petunidin are the most abundant anthocyanins present in black goji berry [4,8–11], that are rarely found anthocyanins in
berries [12]. Petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside is the major monoacylated anthocyanin present in black goji
berry, accounting for more than 80% of total anthocyanins [13]. Other monoacylated anthocyanins derived from petunidin, del­
phinidin, and malvidin with caffeic, ferulic, and coumaric acyl moieties are also found in black goji berry [14]. Studies have revealed

* Corresponding author.
E-mail address: choo.wee.sim@monash.edu (W.S. Choo).

https://doi.org/10.1016/j.heliyon.2023.e14426
Received 1 June 2022; Received in revised form 14 February 2023; Accepted 6 March 2023
Available online 10 March 2023
2405-8440/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

that the anthocyanins from black goji berries deliver various health benefits such as antilipidemic, antioxidant, and anti-inflammatory
activities. Therefore, black goji berry anthocyanins would be a potential ingredient in functional foods [15].
Extraction is a critical step in recovering active ingredients from plant materials [16]. An effective extraction method is needed to
obtain the maximum yield with a high concentration of target compounds. Anthocyanins could be negatively affected by light, heat,
acids, and alkalis. Therefore, to get the maximum amount of anthocyanins and other bioactive molecules without degradation,
selecting an extraction method causing the least damage to anthocyanins is critical.
Several innovative extraction techniques employing enzymes, ultrasonic, microwave waves, and supercritical fluid to extract
bioactive compounds like anthocyanins are being popularised [17]. Among all extraction techniques, solvent extraction is considered
the simplest and most widely used extraction technique in the food industry. However, extraction with organic solvents is associated
with the disadvantage of toxic solvent residue left in the final extract, limiting the applications of the extract [18]. Water could be
considered the best medium to extract water-soluble anthocyanins for food applications [19]. Therefore, the present work was
intended to investigate the effects of different extraction conditions on hot water extraction of anthocyanins from black goji berry and
compare ultrasound, microwave, and pectinase-assisted extraction on anthocyanin extraction from the black goji berry.

2. Materials and methods

2.1. Materials

Dried black goji berries from Qinghai, China, were purchased using a local distributor. Folin Ciocalteu reagent, gallic acid, sodium
acetate, sodium carbonate anhydrous, sodium hydroxide, hydrochloric acid, potassium chloride, pectinase from Aspergillus aculeatus,
absolute ethanol, 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, potassium persulfate, phosphate-buffered sa­
line (PBS) tablets, potassium ferricyanide, trichloroacetic acid, ferric chloride, gradient grade acetonitrile, and formic acid were
purchased from Sigma-Aldrich (St. Louis, USA).

2.2. Extraction of anthocyanins

2.2.1. Hot water extraction

Dried black goji berries were blended with water at a specific substrate-solvent ratio [1:15 or 1:20 (w/v)] using a commercial
blender (Waring MX1100XT11CE, USA) at low speed for 40 s. Then the mixture was placed in a thermostatic water bath set at a specific
temperature (40, 50 or 60 ◦ C) for a specific time (30 or 60 min) with continuous shaking. Then the mixture was vacuum filtered and
vacuum dried at 40 ◦ C for 36–48 h. Hereafter, the vacuum-dried extracts are referred to as anthocyanin extracts. Subsequent ultra­
sound, microwave, pectinase-assisted extractions, and 70% ethanolic extraction were carried out using the optimum substrate: solvent
ratio and extraction temperature from hot water extraction.

2.2.2. Ultrasound-assisted extraction

Ultrasound-assisted extraction equipment employed was DAIHAN Scientific Ultrasonic Cleaner Set WUC-A03H (Gangwon, Korea)
at a frequency of 60 Hz with a power of 230 W. Substrate: solvent ratio used for extraction was 1:15 (w/v). The extraction temperature
was 50 ◦ C and the extraction times were 30 min and 60 min. Blending, filtering and drying steps were the same as in section 2.2.1.

2.2.3. Microwave-assisted extraction

Microwave-assisted extraction was performed in Anton Paar Monowave 400, 800 W (Austria). Substrate: solvent ratio used for
extraction was 1:15 (w/v). The extraction temperature was 50 ◦ C and the extraction times were 30 min and 60 min. Blending, filtering
and drying steps were the same as in section 2.2.1.

2.2.4. Pectinase-assisted extraction

Pectinase from Aspergillus aculeatus (enzyme activity of 3800 PGU/mL) at a 1.5% and 3% (w/v) concentration was added to the
mixture prepared with black goji berry blended with water. Substrate: solvent ratio and extraction time were 1:15 (w/v) and 30 min,
respectively. Both 40 and 50 ◦ C were used as extraction temperatures to investigate the temperature dependence on pectinase activity.
Blending, filtering and drying steps were the same as in section 2.2.1.

2.2.5. Ethanolic extraction

Ethanolic extraction was carried out using 70% ethanol based on the method of He et al. [20] as a reference sample to compare with
hot water extraction. The 70% ethanolic extraction was the optimum concentration obtained by He et al. [20]. Substrate: solvent was
1:15 (w/v). The extraction temperature and time used were 50 ◦ C for 30 min. Blending, filtering and drying steps were the same as in
section 2.2.1.

2.3. Yield

The extract yield was calculated according to the formula:

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G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

Weight of vacuum − dried extract (g)

Yield (%) = × 100 (1)
Weight of dried black goji berry (g)

2.4. Measurement of total phenolic content (TPC)

TPC in each extract was determined with some modification of the Folin-Ciocalteu method previously described [21]. For the
analysis, the vacuum-dried extract was dissolved at a concentration of 1 mg/mL in distilled water. Then, 0.5 mL of the extract was
added to 2.5 mL Folin-Ciocalteu reagent (prepared by 10-fold dilution with distilled water) and vortexed for 3 min. Then, 2 mL of 7.5%
(w/v) sodium carbonate was added, and the mixture was allowed to stand at room temperature for 30 min. After 30 min, the
absorbance was measured at 760 nm using a UV-VIS spectrophotometer. TPC was expressed as mg gallic acid equivalents/g dry weight
of extract (mg GAE/g extract).

2.5. Measurement of total anthocyanin content (TAC)

The TAC was analysed by a pH differential method [22]. The vacuum-dried extract was dissolved in pH 1 potassium chloride buffer
(0.025 M) and pH 4.5 sodium acetate buffer (0.4 M) at a concentration of 1 mg/mL. The absorption of the samples in pH 1 buffer and
pH 4.5 buffer was measured at 520 nm and 700 nm using a UV-VIS spectrophotometer. The anthocyanin concentration was expressed
as mg cyanidin-3-glucoside equivalents/g dry weight of extract (mg CGE/g extract) and calculated as follows:
Anthocyanin pigment (mg / L) = (A × MW × DF × 1000)/(ε × 1) (2)

where A = [(A520 - A700) at pH 1.0]–[(A520 - A700) at pH 4.5],

MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol),
ε is the molar absorptivity (26,900 L/mol cm); l is the pathlength of the cuvette in cm.
DF is the dilution factor.

2.6. Antioxidant activity

2.6.1. ABTS radical scavenging capacity

The ABTS activity was determined by the method of Yan et al. [23]. Briefly, ABTS radical stock solution was prepared by adding
ABTS (7 mM) with potassium persulfate and storing the mixture in the dark for 16 h. The ABTS radical solution was diluted with PBS
(pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Then, 100 μL of sample was reacted with 3.9 mL of ABTS radical working solution
in the dark and absorbance was measured at 734 nm after 10 min. Antioxidant activity was measured as free radical scavenging
activity using the following equation:
Free radical scavenging activity (%) = [(Acontrol − Asample) / Acontrol] × 100 (3)

where Acontrol is the absorbance of the control and Asample is the absorbance of the sample.

2.6.2. Ferric reducing antioxidant power

Ferric reducing antioxidant power (FRAP) was analysed according to the method of Chong and Lim [24]. Briefly, 400 μL samples
were added to 1 mL phosphate buffer (0.2 M, pH 6.6) and 1 mL potassium ferricyanide (1% w/v). The mixture was left to stand for 20
min at 50 ◦ C before 1 mL of trichloroacetic acid (10% w/v) was added. The mixture was separated into aliquots of 1 mL and diluted
with 1 mL of water. Then 200 μL of ferric chloride (0.1% w/v) was added to the mixture. The mixture was left in the dark for 30 min,
and the absorbance was measured at 700 nm. FRAP was expressed as mg gallic acid equivalent/mL.

2.7. Colour measurement

Black goji berry anthocyanin extracts from hot water extraction, ultrasound, microwave, 1.5% (w/v) pectinase-assisted extraction,
and 70% ethanolic extraction at optimal conditions (1:15 (w/v) substrate: solvent ratio at 50 ◦ C for 30 min) were made to a con­
centration of 10 mg/mL dissolving in water, and the colour was measured using a colourimeter (HunterLab Associates Laboratory,
USA). The total colour change (ΔE*) for anthocyanin extracts obtained from ultrasound, microwave, pectinase (1.5% (w/v) at 50 ◦ C)-
assisted extractions, and 70% ethanolic extraction compared to the anthocyanin extract obtained from hot water extraction (1:15 (w/
v) and 30 min at 50 ◦ C) was calculated using L*, a* and b* coordinates by means of the following equation:

ΔE ∗ = (ΔL ∗ 2 + Δa ∗ 2 + Δb ∗ 2)1/2 (4)

where ΔL* = L* - L0*, Δa* = a* - a0*, Δb* = b* - b0*; L0*, a0*, and b0*are values of anthocyanin extract obtained from hot water
extraction (1:15 (w/v) and 30 min at 50 ◦ C).

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2.8. Liquid chromatography - mass spectrometry (LC-MS) analysis

The LC-MS analysis was carried out for black goji berry anthocyanin extracts obtained from hot water extraction, ultrasound,
microwave, 1.5% (w/v) pectinase-assisted extraction and 70% ethanolic extraction with optimum extraction conditions (substrate:
solvent ratio of 1: 15 (w/v), extraction temperature of 50 ◦ C, and extraction time of 30 min). Individual compounds were identified
based on retention time and mass-to-charge ratio using LC-MS according to Luo et al. [25] with slight modifications. Chromatographic
separations were performed on an Agilent 1290 Infinity LC system coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer
with a dual ESI source (Thermo Finnigan, San Jose, CA, USA) equipped with an autosampler, a Surveyor 2000 quaternary pump, and a
Surveyor UV 2000 PDA detector using an Agilent ZORBAX Eclipse XDB-C18 Analytical, 150 mm × 4.6 mm, 5 μm column. Electrospray
ionization was set to positive mode, the scan range of 600 and 1100 m/z and the data were analysed using Agilent MassHunter
Qualitative Analysis (B.07.00). The relative proportion of identified compounds was presented as the percentage of peak area. The
applied elution conditions were as follows: 0.1% formic acid in water (A) and acetonitrile (B); gradient elution conditions were 0–10
min, 5%–20% B; 10–22 min, 20%–40% B; 22–28 min, 40%–60% B; 28–38 min, 60%–65% B. Nitrogen was used as desolvation gas, at
300 ◦ C and a flow rate of 60 L/h, and He gas was used as the damping gas, with a declustering potential of 40 eV, collision energy of 5
eV, and a collision cell entrance potential of 10 eV.

2.9. Statistical analysis

All experiments were carried out in independent triplicates. The results were expressed as mean ± standard deviation. The data
obtained were analysed with SPSS Statistics 26.0 software. One-way ANOVA (analysis of variance) followed by post-hoc Tukey’s test
was used, and significance was set at p < 0.05 using SPSS 26 software (New York, USA).

3. Results and discussion

3.1. Effect of extraction conditions on extract yield of black goji berry anthocyanin extract

The effect of different treatments on the extraction yield of black goji berry anthocyanin extract from hot water extraction is shown
in Table 1. The highest extraction yield obtained in this study was 56.2 ± 2.2%, which was higher than that of previous studies. Wang
et al. [10] obtained an extraction yield of 26.3% using sub-critical water extraction at 170 ◦ C with a flow rate of 3 mL/min and 55 min
of extraction time. The difference may be attributed to the blending step that was used in the extraction procedure in this study and
differences in raw materials. Blending helped to macerate black goji berries reducing the particle size and allowing effective mass
transfer. In another study, the highest extraction yield of black goji berry anthocyanins obtained by the soaking extraction method
using 90% ethanol for 4 h at room temperature was 16.34% [26]. The reason for obtaining a lower extraction yield could be the low
extraction temperature and the difference in the solvent. When extraction time was 30 min and the temperature was 60 ◦ C, respec­
tively, extraction yield significantly increased (p < 0.05) when the substrate: solvent ratio changed from 1:15 to 1:20 (w/v) (Table 1).
The higher solvent: substrate ratio caused a higher-density gradient resulting in a faster release of soluble matter from tissue [27].
Contrastingly, when extraction time was 60 min and the temperature was 50 ◦ C, respectively, extraction yield significantly decreased
(p < 0.05) when the substrate: solvent ratio increased to 1:20 (w/v) from 1:15 (Table 1). This finding does not comply with mass
transfer principles. Generally, the extraction yield increases with increasing solvent volume [28]. The variation of the internal
structure of fruits within a single variety, or even single fruit, might cause the plant tissue to be susceptible to different thermal
processes, thus lowering the extraction [29].
Extraction time did not significantly affect the extraction yield when the substrate: solvent ratio and extraction temperatures were
constant (Table 1). This finding does not agree with Liu et al. [5]. The extraction yield of black goji berry anthocyanin extract increased

Table 1
Extraction yield, TAC and TPC of vacuum-dried Lycium ruthenicum extracts obtained from hot water extraction.
Treatment Extraction conditions Extraction yield (%) TAC (mg CGE/g) TPC (mg GAE/g)

1 1:15, 30 min, 40 ◦ C 44.4 ± 0.56bce 11.9 ± 2.51acd 38.4 ± 7.57d

2 1:15, 60 min, 40 ◦ C 52.3 ± 4.68abde 12.1 ± 0.41acd 41.0 ± 3.77d
3 1:15, 30 min, 50 ◦ C 48.3 ± 3.25abc 13.8 ± 1.14ac 69.7 ± 2.50ab
4 1:15, 60 min, 50 ◦ C 52.9 ± 3.25ad 10.3 ± 2.26acde 32.6 ± 2.52d
5 1:15, 30 min, 60 ◦ C 45.0 ± 2.55bcd 15.6 ± 1.77a 39.4 ± 5.34d
6 1:15, 60 min, 60 ◦ C 45.9 ± 3.71bcd 13.7 ± 1.95ac 62.9 ± 5.05b
7 1:20, 30 min, 40 ◦ C 44.2 ± 4.15bc 13.6 ± 1.05ac 47.2 ± 9.75d
8 1:20, 60 min, 40 ◦ C 46.5 ± 0.63bc 6.77 ± 1.24bd 35.2 ± 1.91d
9 1:20, 30 min, 50 ◦ C 47.7 ± 2.67bcd 8.45 ± 2.55bcd 82.7 ± 3.54a
10 1:20, 60 min, 50 ◦ C 40.1 ± 0.98c 5.33 ± 0.45be 43.1 ± 3.37d
11 1:20, 30 min, 60 ◦ C 56.2 ± 2.28a 4.23 ± 0.75b 75.7 ± 5.30a
12 1:20, 60 min, 60 ◦ C 49.9 ± 1.37abd 14.6 ± 3.94a 37.3 ± 4.24d

GAE = GAE; CGE = cyanidin-3-glucoside equivalent.

TAC = total anthocyanin content; TPC = total phenolic content.
abc
Values with different superscript letters within a column are significantly different (p < 0.05).

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G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

when the extraction period increased from 10 to 30 min but again reduced after 30 min [5]. This may be due to the structural
destruction and deformation of phenolic compounds with prolonged extraction time [30]. When substrate: the solvent ratio was 1:20
(w/v) and extraction time was 30 min, extraction yield at 60 ◦ C was significantly higher (p < 0.05) than at 40 ◦ C and 50 ◦ C, but the
extraction yield did not significantly differ between 40 and 50 ◦ C (Table 1). Similarly, when substrate: the solvent ratio was 1:20 (w/v)
and extraction time was 60 min, the extraction yield at 60 ◦ C was significantly higher (p < 0.05) than the extraction yield at 50 ◦ C, but
there was no significant difference between 40 and 50 ◦ C (Table 1). This is in accordance with the study, which reported that the
extraction yield of phenolics from litchi fruit pericarp increased significantly when extraction temperature increased from 40 to 60 ◦ C
[31]. Vergara-Salinas et al. [32] stated that higher anthocyanin extraction yields were obtained at higher temperatures when pres­
surised hot water extraction was applied to extract anthocyanins from grape pomace. The increase in temperature makes plant tissues
much permeable and facilitates the diffusion of more soluble material during extraction [33].

3.2. Effects of extraction conditions on TAC of black goji berry anthocyanin extract

The effect of different treatments on TAC of the black goji berry anthocyanin extract is shown in Table 1. The highest TAC of black
goji berry extract in this study was 15.6 ± 1.7 mg CGE/g. Previous work reported a TAC of 19.51 ± 0.21 mg CGE/g in a black goji berry
anthocyanin extract [34], which is higher than the highest TAC obtained in this study. Shen et al. [34] used pectinase-assisted
extraction at 38 ◦ C for 55 min. The effect of the enzyme and the longer extraction time in the study of Shen et al. [34] might be
the reasons for the difference in results in the two studies. He et al. [20] obtained a black goji berry extract with TAC of 6.22 ± 0.35 mg
CGE/g by applying accelerated solvent extraction with 70% ethanol at 89 ◦ C for 13 min. The higher temperature used by He et al. [20]
could be the reason for obtaining a lower TAC because anthocyanins tend to degrade at temperatures above 50 ◦ C [35]. When
extraction time was 30 min and the temperature was 60 ◦ C, respectively, TAC significantly decreased (p < 0.05) when the substrate:
solvent ratio was changed from 1:15 to 1:20 (w/v) (Table 1). Khazaei et al. [27] stated that the TAC of the anthocyanin extract of
saffron decreased when the substrate: solvent ratio increased. Aryanti et al. [36] also observed that TAC was reduced when the
substrate: solvent ratio increased in the ultrasound-assisted extraction of anthocyanins from Roselle (Hibiscus sabdariffa L.) calyces. The
reduction of TAC of the anthocyanin extract with increasing substrate: solvent ratio could be explained by the higher occurrence of
hydrolysis of anthocyanins when more water molecules are present in the matrix [37]. In the presence of water and heat, anthocyanin
degradation begins by breaking down the covalent bond between the aglycon and sugar moiety. This results in the opening of the
pyrilium ring and chalcone formation which ultimately affects the colour of anthocyanins [38]. When substrate: the solvent ratio was
1:20 (w/v) and extraction temperature was 40 ◦ C, the TAC of the anthocyanin extract significantly decreased (p < 0.05) when the
extraction time was raised from 30 min to 60 min (Table 1). Lee et al. [39] observed that the stability of anthocyanin from blue pea
anthocyanins showed low stability at 37 ◦ C. This low stability could be explained by the enzymatic hydrolysis of anthocyanins as this
temperature is the optimum temperature for many hydrolytic enzymes [40]. However, when the substrate: solvent ratio was 1:20
(w/v) and the extraction temperature was 60 ◦ C, the TAC of the anthocyanin extract significantly increased (p < 0.05) when the
extraction time increased to 60 min from 30 min (Table 1). This is in accordance with Anuar et al. [41], whereby when the extraction
temperature was 60 ◦ C, the TAC of ethanolic and methanolic anthocyanin extracts were obtained from Melastoma malabathricum fruit
increased with extraction time. When substrate: solvent ratio was 1:20 (w/v) and extraction time was 30 min, the TAC at 40 ◦ C was
significantly higher (p < 0.05) compared to 60 ◦ C (Table 1), but there was no significant difference between TAC at 50 and 60 ◦ C.
Contrastingly, when the substrate: solvent ratio was 1:20 (w/v) and extraction time was 60 min, the TAC of the anthocyanin extract at
60 ◦ C was significantly higher (p < 0.05) than at 40 and 50 ◦ C, but there was no significant difference between 40 and 50 ◦ C (Table 1).
The increase in TAC with temperature could be explained by the increase in kinetic energy of the molecules, which eventually en­
hances the diffusion and solubility of the target compound into the surrounding solvent [42].

3.3. Effects of extraction conditions on TPC of black goji berry anthocyanin extract

The effect of different treatments on the TPC of black goji berry anthocyanin extract is shown in Table 1. The highest TPC in this
study was 82.7 ± 3.5 mg GAE/g. He et al. [20] used accelerated solvent extraction and obtained a maximum TPC of 17.92 mg GAE/g,
which is lower than the highest TPC in this study. He et al. [20] used an extraction temperature of 89.38 ◦ C, an extraction time of 13
min, and an ethanol concentration of 70%. Obtaining a lower TPC could be attributed to the higher temperature and lower time used
by He et al. [20] because the high temperature could destroy phenolic compounds [43]. The TPC of the black goji berry extract ob­
tained by shaking dried black goji berry powder in 70% acetone for 3 h was 9.01 ± 0.77 GAE/g [44]. Anthocyanins are the main
phenolic compounds in black goji berry [45]. Anthocyanins are water-soluble compounds. The lower polarity of acetone and lower
extraction temperature may reduce the extraction of phenolic compounds. When extraction time was 30 min and the temperature was
60 ◦ C, the TPC of the anthocyanin extract significantly increased (p < 0.05) when the substrate: solvent ratio increased from 1:15 to
1:20 (w/v) (Table 1). The TPC of the ethanolic anthocyanin extract of black goji berry obtained from ultrasound-assisted extraction
increased with increasing substrate: solvent ratio from 1:20 to 1:40 (w/v) [46]. The increase in solvent volume relative to the amount
of substrate causes an increase in concentration gradient resulting diffusion of more phenolic compounds [47].
When substrate: solvent ratio was 1:15 (w/v) and extraction temperature was 50 ◦ C, the TPC of the anthocyanin extract signifi­
cantly decreased (p < 0.05) when extraction time increased from 30 min to 60 min (Table 1). Similarly, when the substrate: solvent
ratio was 1:20 (w/v) and extraction temperature was 60 ◦ C, the TPC of the anthocyanin extract significantly decreased (p < 0.05) when
extraction time increased from 30 min to 60 min (Table 1). Eadmusik et al. [48] observed a reduction of the phenolic content of yanang
leaves (Tiliacora triandra) extract with increasing extraction time at 60 ◦ C. When substrate: solvent ratio was 1:15 (w/v) and extraction

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time was 60 min, TPC of the anthocyanin extract anthocyanin at 60 ◦ C was significantly higher (p < 0.05) compared to 50 ◦ C. However,
the TPC of the anthocyanin extract did not significantly differ at 40 and 50 ◦ C (Table 1). Similarly, when the substrate: solvent ratio was
1:20 (w/v) and extraction time was 30 min, the TPC of the anthocyanin extract at 60 ◦ C was significantly higher (p < 0.05) compared
to 40 ◦ C. However, there was no significant difference in TPC of the anthocyanin extract obtained at 40 and 50 ◦ C (Table 1). The TPC of
the anthocyanin extract obtained from grape pomace increased when the temperature increased from 25 to 50 ◦ C [49]. Increased
temperature results in greater diffusion of the phenolic compounds to the solvent from the substrate matrix [50].

3.4. Selection of the best treatment

Considering all treatments, treatment number 3 with substrate: solvent ratio of 1:15 (w/v), 50 ◦ C for 30 min (Table 1) was selected
as the best treatment to obtain a high TAC (13.8 ± 1.14 mg CGE/g), TPC (69.7 ± 2.50 mg of GAE/g), and extraction yield (48.3 ±
3.25%). This extraction condition was used for subsequent pectinase, ultrasound and microwave-assisted extractions.

3.5. Effect of pectinase, ultrasound and microwave-assisted extraction on extraction yield

Extraction with pectinase 3% (w/v) with substrate: solvent ratio of 1:15 (w/v) at 30 min for 50 ◦ C resulted in the highest extraction
yield (Table 2). Black goji berry contains many water-soluble compounds, including carbohydrates [30]. Pectinase degrades cell wall
material and facilitates the diffusion of intracellular compounds during extraction [51]. These could be considered as the reasons for
the high extraction yield in pectinase-assisted extraction. The extraction yield of ultrasound-assisted extraction in this study (45.3 ±
0.73%) was higher than the extraction yield obtained by Wang et al. [10] using ultrasound-assisted extraction. Wang et al. [10]
obtained an extraction yield of 15.76% with substrate: solvent ratio, extraction time and the temperature 1: 15 (w/v), 90 min and
70 ◦ C, respectively. The substrate: solvent ratio used in this study is the same as Wang et al. [10]. The extraction time and temperature
used in Wang et al. [10] are higher compared to this study. The extraction yield of microwave-assisted extraction in this study was 62.1
± 5.60% which is higher than the extraction yield (16.8%) obtained by Liu et al. [26] using microwave-assisted extraction with
substrate: solvent ratio, extraction time and solvent of 1:20 (w/v), 25 min, and 90% ethanol, respectively. The reason for the difference
may be the differences in solvent and temperature used in the two studies. Liu et al. [26] used ethanol and room temperature for
extraction, whereas the solvent in this study was distilled water, and the extraction temperature was 50 ◦ C.

3.6. Effect of pectinase, ultrasound and microwave-assisted extraction on TAC

TAC of the anthocyanin extract obtained using pectinase extraction with 3% (w/v) pectinase was significantly lower (p < 0.05)
than TAC obtained using ultrasound, microwave-assisted extractions, and control methods (Table 2). The TAC of the anthocyanin
extract obtained using 3% (w/v) pectinase extraction in this study (Table 2) was lower than the TAC of the anthocyanin extract of black
goji berry (19.51 ± 0.2 mg CGE/g) reported by Shen et al. [34] obtained using pectinase-assisted extraction (extraction time: 55 min,
temperature: 38 ◦ C and enzyme dosage: 52.04 mg/100 g). The efficiency of enzyme-assisted extraction depends on operating con­
ditions such as temperature and pH, extraction duration, type and enzyme concentration, and particle size of the substrate [52]. This

Table 2
Extraction yield, TAC and TPC of vacuum-dried Lycium ruthenicum extracts obtained from various extraction methods at different extraction
conditions.
Treatment Extraction conditions Extraction yield TAC (mg TPC (mg ABTS assay IC50 FRAP (mg gallic acid equivalent/
(%) CGE/g) GAE/g) (mg/mL) g of extract)

1 1:15, 30 min, 40 ◦ C, pectinase 60.7 ± 1.83b 12.0 ± 57.6 ± 0.75 ± 0.02ab 69.6 ± 1.96b
1.5% (w/v) 1.24ab 4.12bc
2 1:15, 30 min, 50 ◦ C, pectinase 60.0 ± 4.17b 12.5 ± 69.9 ± 0.67 ± 0.01a 75.5 ± 0.45a
1.5% (w/v) 2.84ab 4.63ab
3 1:15, 30 min, 50 ◦ C, pectinase 73.6 ± 1.65a 9.09 ± 1.56b 49.6 ± 0.20c 0.72 ± 0.02ab 62.9 ± 1.45d
3% (w/v)
4 1:15, 30 min, 50 ◦ C, ultrasound- 45.3 ± 0.73c 15.7 ± 0.58a 70.6 ± 0.93 ± 0.04bf 74.4 ± 2.29ab
assisted 5.79ab
5 1:15, 60 min, 50 ◦ C, ultrasound- 41.9 ± 5.47c 10.7 ± 2.82b 29.5 ± 2.40d 1.40 ± 0.19c 38.8 ± 1.42c
assisted
6 1:15, 30 min, 50 ◦ C, 62.1 ± 5.60b 15.6 ± 0.15a 77.6 ± 4.63a 0.84 ± 0.04af 71.0 ± 1.82ba
microwave-assisted
7 1:15, 60 min, 50 ◦ C, 51.5 ± 1.79b 10.6 ± 0.69b 46.2 ± 2.15c 0.94 ± 0.03bf 44.2 ± 1.60e
microwave-assisted
8 1:15, 30 min, 50 ◦ C, 46.6 ± 8.06c 16.5 ± 1.64a 49.6 ± 4.51c 0.87 ± 0.05bf 66.3 ± 1.59b
70% ethanolic
Control 1:15, 30 min, 50 ◦ C 49.0 ± 2.37b 15.3 ± 0.66a 67.9 ± 0.70 ± 0.04a 72.3 ± 2.08bd
7.11ab

GAE = gallic acid equivalent; CGE = cyanidin-3-glucoside equivalent.

TAC = total anthocyanin content; TPC = total phenolic content.
abc
Values with different superscript letters within a column are significantly different (p < 0.05).

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G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

difference could be attributed to the longer extraction time and lower enzyme dosage used by Shen et al. [34]. The TAC of anthocyanin
extract did not significantly differ (p > 0.05) among ultrasound, microwave and 1.5% pectinase-assisted extraction (at 50 ◦ C) and the
control extraction (Table 2). Furthermore, the TAC of the anthocyanin extract from 70% ethanolic extraction did not significantly
differ (p > 0.05) from that of hot water extraction. In a previous study, the ultrasound-assisted ethanolic extraction of anthocyanins
from red araçá peel (Psidium cattleianum Sabine) increased the TAC of the extract by 12% compared to conventional extraction [53].
Extraction was done by 90% ethanol with 50 kHz frequency at 40 ◦ C for 120 min. Similarly, microwave-assisted extraction increased
the anthocyanin content of blackcurrant marc by 20% when employed at 700 W for 10 min [54]. The difference in extraction time,
solvent, and plant material’s internal structure might cause differences in outcomes.

3.7. Effect of pectinase, ultrasound and microwave-assisted extraction on TPC

The highest TPC (77.6 ± 4.63 GAE/g) was obtained in microwave-assisted extraction. The highest TPC of the black goji berry
anthocyanin extract obtained by He et al. [20] using accelerated solvent extraction with 70% ethanol at 89.38 ◦ C for 13 min was 17.92
mg GAE/g, which was lower than the highest TPC of the anthocyanin extract in this study (Table 2). High extraction temperature and
short time used in the study of He et al. [20] could be the reason for obtaining a lower TPC compared to this study. High temperatures
can destroy heat-sensitive phenolic compounds [55]. Demonstrating similarity to the results of TAC (Table 2), black goji berry
anthocyanin extracts obtained from 1.5% pectinase (substrate: solvent ratio of 1:15 (w/v) at 30 min for 50 ◦ C), ultrasound,
microwave-assisted extractions, and hot water extraction (control) did not significantly differ (p > 0.05) in TPC (Table 2). Generally,
ultrasound and microwave-assisted extractions increase the extraction of phenolic compounds from plant material [56].
Ultrasound-assisted extraction causes physical and mechanical damage to plant materials and enhances extraction recover through
cavitation effect [57]. Microwave irradiation disrupts and penetrates cell structures by volume heating. The diffusion capacities of
cellular components improve by raising solvent temperature under microwave heating [58]. However, the particle size of the sample,
the moisture content of the sample, plasticity, and internal structure may have hindered the effect of ultrasound [59] and use of
solvents like water with high dielectric constant may cause fast heating rates and degrade heat liable phenolics [60]. The TPC of 70%

Fig. 1. Compounds assigned from LC-MS analysis of the vacuum-dried anthocyanin extracts of Lycium ruthenicum obtained from different
extraction methods.

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G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

ethanolic extract did not significantly differ (p > 0.05) from that of the hot water extract indicating that both water and 70% ethanol
are equally effective in extracting phenolic compounds from black goji berry (Table 2). The TPC of the anthocyanin extract obtained
from using pectinase 3% (v/w) (1:15, 50 ◦ C and 30 min) was significantly lower (p < 0.05) than the TPC of the anthocyanin extract
obtained in all other treatments except in the treatment using pectinase 1.5% (w/v) with the substrate to solvent ratio of 1:15 at 40 ◦ C
for 30 min (Table 2). Extracting more non-phenolic compounds may reduce the phenolics in unit mass in the extract obtained by 3%
pectinase-assisted extraction.

3.8. Effect of pectinase, ultrasound and microwave-assisted extraction on antioxidant activity

The antioxidant activities of anthocyanin extracts from black goji berry obtained from different extraction methods are shown in
Table 2. The anthocyanin extract obtained from 1.5% pectinase-assisted extraction at 50 ◦ C had the lowest IC50 value (0.67 ± 0.01 mg/
mL) in ABTS assay, but it was not significantly different (p > 0.05) from the IC50 value of the anthocyanin extracts obtained from
microwave-assisted extraction (1:15, 30 min at 50 ◦ C) and the control hot water extraction (Table 2). Similarly, the anthocyanin
extract obtained from 1.5% pectinase-assisted extraction at 50 ◦ C had the highest FRAP value, but it did not significantly differ (p >
0.05) from that of the extracts obtained from microwave and ultrasound-assisted extraction [1:15, 30 min at 50 ◦ C] (Table 2). The
higher antioxidant activity of the extract from 1.5% pectinase-assisted extraction is also evident from the LC-MS results whereby the
extract contained the highest percentage of petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside (Fig. 1). Shen et al. [34]
applied pectinase-assisted extraction [37 min at 38 ◦ C with a pectinase dose of 52.04 mg/100 g dried fruit] and the IC50 value obtained
in the ABTS assay for black goji berry anthocyanins was 0.7623 ± 0.017 mg/mL. This value is comparable with the IC50 value (0.67 ±
0.01 mg/mL) obtained for the extract obtained from 1.5% (w/v) pectinase-assisted extraction (1:15, 30 min at 50 ◦ C) in this study
(Table 2). Tang et al. [6] isolated petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside from black goji berry and investigated
the ABTS and DPPH radical scavenging activities. The IC50 of the compound for ABTS and DPPH assays were 82.4 ± 0.15 and 106.53
± 0.23 μg/mL respectively. The IC50 value of the ABTS assay obtained by Tang et al. [6] is lower compared to this study. Lower IC50
values indicate higher antioxidant activity [61]. Unlike the isolated compound, crude extracts may contain several non-reducing
compounds as well. The presence of higher content of electron-withdrawing groups in the pure compounds weakened the dissocia­
tion energy of O–H bonds [6]. The IC50 value of the DPPH assay of black goji berry anthocyanin extracts obtained from
ultrasound-assisted extraction [ultrasound power-348 W, solvent: substrate-25 mL/g, temperature-42 ◦ C, time-29 min] ranged be­
tween 0.43 and 0.77 mg/mL [26]. This value is lower than the IC50 of ABTS assay of the anthocyanin extract (0.93 ± 0.04 mg/mL)
obtained from ultrasound-assisted extraction (1:15, 30 min, 50 ◦ C) in this study (Table 2). Applying a lower ultrasound power (230 W)
and higher extraction temperature could be the reason for the lower antioxidant activity obtained in this study. Furthermore, the
difference in ABTS and DPPH assays might affect the results. The extract obtained from ultrasound-assisted extraction carried out for
60 min had the highest IC50 value in the ABTS assay (Table 2). Similarly, the extract obtained from 60 min ultrasound-assisted
extraction (1:15, 50 ◦ C for 60 min) demonstrated the lowest FRAP value (Table 2). The FRAP value of the anthocyanin extract ob­
tained from ultrasound-assisted extraction was significantly reduced (p < 0.05) when the extraction time increased from 30 to 60 min
(Table 2). Annegowda et al. [62] observed that a prolonged sonication time of more than 40 min significantly reduced the antioxidant
activity of Terminalia catappa L. leaves. Prolonged sonication could degrade the antioxidants. Furthermore, prolonged sonication
decreases the diffusion area, diffusion rate, and increased diffusion distance leading to lower extraction of bioactive compounds [62].

3.9. Effect of pectinase, ultrasound and microwave-assisted extraction on colour

The total colour change (ΔE*) of the black goji berry anthocyanin extracts obtained from pectinase [1.5% (w/v) at 50 ◦ C for 30
min], ultrasound, microwave-assisted extractions and 70% ethanolic extraction were less than 1.5 compared to the hot water extract
indicating that the colour change among the extracts was not distinct (Table 3). The total colour changes can be categorized as ΔE*
more than 3, less than 3 but more than 1.5, and less than 1.5 that indicating a very distinct, distinct, and small difference, respectively
[63]. The TAC of black goji berry anthocyanin extracts did not significantly differ as discussed previously. This might be the reason for
not having a significant difference in colour among different extracts (Table 3). However, the b* of the anthocyanin extract obtained
from pectinase-assisted extraction was significantly lower (p < 0.05) compared to other extracts (Table 3) indicating lower blue colour.
This could be explained by the lack of delphinidin-3-O-rutinoside(coumaroyl)-5-O-glucoside in the extract obtained from
pectinase-assisted extraction as evident from LC-MS results (Fig. 1). Blue hues are mainly caused by delphinidin [64]. L* (lightness) of

Table 3
The L*, a*, b* values of anthocyanin extracts of Lycium ruthenicum obtained from various extraction methods at optimum conditions and the total
colour change (ΔE*).
Extraction method L* a* b* ΔE*

Hot water extraction 1.77 ± 0.01a 1.15 ± 0.01b − 1.16 ± 0.08a –

Ultrasound-assisted extraction 1.46 ± 0.01bc 1.47 ± 0.02a − 1.23 ± 0.00a 0.52 ± 0.05a
Microwave-assisted extraction 1.47 ± 0.01b 1.57 ± 0.03a − 1.28 ± 0.08a 0.54 ± 0.04a
Pectinase-assisted extraction 1.43 ± 0.00c 1.20 ± 0.08b − 0.82 ± 0.19b 0.49 ± 0.06a
70% ethanolic extraction 1.27 ± 0.00d 1.34 ± 0.04c − 0.85 ± 0.04c 0.61 ± 0.02c
abc
Values with different superscript letters within a column are significantly different (p < 0.05).

8
G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

the anthocyanin extract obtained from 1.5% pectinase-assisted extraction [1.5% (w/v) at 50 ◦ C for 30 min) was significantly lower (p
< 0.05) than hot water extract (Table 3). This does not agree with Kalcheva–Karadzhova et al. [65]. The L* of rose (Rosa damascena
Mill) petal extract obtained from hot water extraction did not significantly differ from the extract obtained from enzyme-assisted
extraction [65]. The difference could be attributed to the different compositions of the two raw materials. Black goji berries
contain many polysaccharides, phenolics, and enzymes [11]. Enzymes and other phenolic compounds can react with anthocyanins and
form brown pigments during extractions and reduce the L* of the anthocyanin extract [66].

3.10. Characterisation and identification of anthocyanins in extracts obtained from different extraction methods

LC-MS analyses were carried out to compare the effect of different extraction methods on the anthocyanin composition of black goji
berry anthocyanin extracts. The identified anthocyanins and the peak area percentage of each anthocyanin at different extraction
techniques are shown in Fig. 1. Eight anthocyanins were identified in each extract obtained from hot water, ultrasound, and
microwave-assisted extractions and 70% ethanolic extraction (Fig. 1). Liu et al. [5] and Wang et al. [67] identified seven anthocyanins
from anthocyanins in a black goji berry anthocyanin extract from sub-critical water extraction and ultrasound-assisted extraction,
respectively. However, the identified anthocyanins in previous studies are not the same as in this study. For example,
petunidin-3-O-glucoside-5-O-glucoside and delphinidin-3-O-rhamnoside-5-O-glucoside were not identified in the studies of Liu et al.
[5] and Wang et al. [67] but in this study. Only five anthocyanins were identified in the extract obtained from pectinase-assisted
extraction. Shen et al. [34] identified 13 anthocyanins in the black goji berry anthocyanin extract obtained using pectinase-assisted
extraction. Shen et al. [34] used a purified anthocyanin extract for LC-MS analysis, whereas a crude extract was used in this study.
Purification enhances the efficient identification of the intended compound [68].
Petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside was the most prominent anthocyanin (Fig. 1). Black goji berries
possess a high amount of monoacylated anthocyanins and more than 80% of monoacylated anthocyanins are petunidin-3-O-rutinoside
(trans-p-coumaroyl)-5-O-glucoside [13]. The anthocyanin extract obtained using pectinase-assisted extraction [1.5% (w/v) pectinase,
1:15 substrate: solvent at 50 ◦ C for 30 min] had the highest petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside content
(Fig. 1). Shen et al. [34] also reported that pectinase-assisted extraction effectively extracted anthocyanins from black goji berries.
Pectinases digest plant cell walls by degrading pectin matrices. This enhances cell permeability which eventually increases the sol­
ubility of anthocyanins and other bio-active compounds [69]. Similarly, Syrpas et al. [70] reported that the cyanidin-3-glucoside
content in the bilberry pomace anthocyanin extract was significantly higher when extracted using enzyme-assisted extraction with
viscozyme L, than in water extraction carried out at similar extraction conditions. However, González et al. [71] found that the
anthocyanin composition of the blackcurrant extracts obtained from pectinase enzyme and ultrasound-assisted did not significantly
differ. The extraction time, temperature and the state of raw material might influence the effect of enzymes on the substrate [72].
Despite the higher content of petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside content in pectinase-assisted extraction
(Fig. 1), the TAC of the extracts obtained from different extraction methods did not differ (Table 2). Anthocyanins such as
petunidin-3-O-rutinoside(coumaroyl-(4-O-glucoside))-5-O-glucoside and delphinidin-3-O-rutinoside(coumaroyl)-5-O-glucoside were
found in smaller amounts in the extracts obtained from pectinase-assisted extraction compared to other extracts (Fig. 1). This might be
the reason for the final TAC to be not significantly different among the extracts. In this study, the peak area percentage of petuni­
din-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside was 6.81% more in ultrasound-assisted extraction than in microwave-assisted
extraction (Fig. 1). However, a significant difference was not observed between the petunidin-3-O-rutinoside(trans-­
p-coumaroyl)-5-O-glucoside contents of black goji berry anthocyanin extract obtained by ultrasound and microwave-assisted ex­
tractions [26]. Liu et al. [26] employed ultrasound-assisted extraction at 40 ◦ C. The lower extraction temperature used in Liu et al. [26]
might reduce the extraction of anthocyanins.

4. Conclusions

This study demonstrated the effects of substrate: solvent ratio, extraction time and extraction temperature on the extraction yield,
TAC and TPC of black goji berry anthocyanin extracts. Based on the best set of extraction conditions for hot water extraction, the effects
of ultrasound, microwave, and pectinase-assisted extractions on the properties of the black goji berry extract were also investigated.
Hot water extraction, microwave-assisted extraction, ultrasound-assisted extraction, and 1.5% (w/v) pectinase-assisted extraction
[50 ◦ C, 1:15 (w/v) and 30 min] were equally effective techniques to obtain an extract with high TAC and TPC. The highest percentage
(80.9%) of petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside was obtained in pectinase-assisted extraction [1.5% (w/v)
pectinase, substrate: solvent ratio of 1:15 (w/v) at 50 ◦ C for 30 min], thus could be considered as the best extraction method. However,
3% pectinase extraction [50 ◦ C, 1:15 (w/v) and 30 min] was the best method to obtain a black goji berry extract with a high extraction
yield. The findings of this study provide an effective and safe method to extract black goji berry anthocyanin that could be used as a
potential natural food ingredient in the food industry.

Declarations

Author contribution statement

Wee-Sim Choo: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Gayan Chandrajith Vidana Gamage: Conceived and designed the experiments; Performed the experiments; Analysed and

9
G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

interpreted the data; Wrote the paper.

Funding statement

This work was supported by Monash University, Malaysia.

Data availability statement

Data will be made available on request.

Declaration of competing interest

The authors declare no conflict of interest.

References

[1] J. Zheng, C. Ding, L. Wang, G. Li, J. Shi, H. Li, H. Wang, Y. Suo, Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr. from
Qinghai-Tibet Plateau, Food Chem. 126 (3) (2011) 859–865, https://doi.org/10.1016/j.foodchem.2010.11.052.
[2] X.B. Zhu, J.M. Li, Y. Zong, X.M. Sun, S.M. Li, D. Cao, B. Zhang, W.J. Chen, B.L. Liu, Transcriptome analysis identifies the key genes responsible for high
anthocyanin content in the fruits of Lycium ruthenicum Murray, Curr. Sci. 116 (2) (2019) 256–263, https://doi.org/10.18520/cs/v116/i2/256-263.
[3] Q. Wang, S.H. Zeng, X.Y. Wu, H.H. Lei, Y. Wang, H.R. Tang, Interspecies developmental differences in metabonomic phenotypes of Lycium ruthenicum and L-
barbarum fruits, J. Proteome Res. 17 (9) (2018) 3223–3236, https://doi.org/10.1021/acs.jproteome.8b00349.
[4] S.S. Chen, H.N. Zhou, G. Zhang, J. Meng, K. Deng, W. Zhou, H.L. Wang, Z.H. Wang, N. Hu, Y.R. Suo, Anthocyanins from Lycium ruthenicum Murr. ameliorated d-
galactose-induced memory impairment, oxidative stress, and neuroinflammation in adult rats, J. Agric. Food Chem. 67 (11) (2019) 3140–3149, https://doi.org/
10.1021/acs.jafc.8b06402.
[5] P. Liu, W. Li, Z. Hu, X. Qin, G. Liu, Isolation, purification, identification, and stability of anthocyanins from Lycium ruthenicum Murr, LWT 126 (2020), 109334,
https://doi.org/10.1016/j.lwt.2020.109334.
[6] J. Tang, Y. Yan, L. Ran, J. Mi, Y. Sun, L. Lu, Y. Gao, X. Zeng, Y. Cao, Isolation, antioxidant property and protective effect on PC12 cell of the main anthocyanin in
fruit of Lycium ruthenicum Murray, J. Funct.Foods 30 (2017) 97–107, https://doi.org/10.1016/j.jff.2017.01.015.
[7] H. Wang, J. Li, W. Tao, X. Zhang, X. Gao, J. Yong, J. Zhao, L. Zhang, Y. Li, J.A. Duan, Lycium ruthenicum studies: molecular biology, phytochemistry and
pharmacology, Food Chem. 240 (2018) 759–766, https://doi.org/10.1016/j.foodchem.2017.08.026.
[8] G. Zhang, S.S. Chen, W. Zhou, J. Meng, K. Deng, H.N. Zhou, N. Hu, Y.R. Suo, Anthocyanin composition of fruit extracts from Lycium ruthenicum and their
protective effect for gouty arthritis, Ind. Crop. Prod. 129 (2019) 414–423, https://doi.org/10.1016/j.indcrop.2018.12.026.
[9] P.P. Tang, M. Giusti, Black goji as a potential source of natural color in a wide pH range, Food Chem. 269 (2018) 419–426, https://doi.org/10.1016/j.
foodchem.2018.07.034.
[10] Z. Wang, Y. Yan, T. Nisar, L. Zou, X. Yang, P. Niu, L. Sun, Y. Guo, Comparison and multivariate statistical analysis of anthocyanin composition in Lycium
ruthenicum Murray from different regions to trace geographical origins: the case of China, Food Chem. 246 (2018) 233–241, https://doi.org/10.1016/j.
foodchem.2017.11.030.
[11] X. Yang, S. Lin, Y. Jia, F. Rehman, S. Zeng, Y. Wang, Anthocyanin and spermidine derivative hexoses coordinately increase in the ripening fruit of Lycium
ruthenicum, Food Chem. 311 (2020), 125874, https://doi.org/10.1016/j.foodchem.2019.125874.
[12] X. Wu, G.R. Beecher, J.M. Holden, D.B. Haytowitz, S.E. Gebhardt, R.L. Prior, Concentrations of anthocyanins in common foods in the United States and
estimation of normal consumption, J. Agric. Food Chem. 54 (11) (2006) 4069–4075, https://doi.org/10.1021/jf060300l.
[13] C. Chen, S. Yun, Y. Tao, L. Mei, Q. Shu, L. Wang, Main anthocyanins compositions and corresponding H-ORAC assay for wild Lycium ruthenicum Murr. fruits from
the Qaidam Basin, J. Pharmaceut. Technol. Drug Res. 2 (1) (2013) 1, https://doi.org/10.7243/2050-120X-2-1.
[14] H.L. Jin, Y.F. Liu, F. Yang, J.X. Wang, D.M. Fu, X.L. Zhang, X.J. Peng, X.M. Liang, Characterization of anthocyanins in wild Lycium ruthenicum Murray by HPLC-
DAD/QTOF-MS/MS, Anal. Methods 7 (12) (2015) 4947–4956, https://doi.org/10.1039/c5ay00612k.
[15] G.C. Vidana Gamage, Y.Y. Lim, W.S. Choo, Black goji berry anthocyanins: extraction, stability, health benefits, and applications, ACS Food Sci. Technol. 1 (8)
(2021) 1360–1370, https://doi.org/10.1021/acsfoodscitech.1c00203.
[16] E.J. Jeyaraj, Y.Y. Lim, W.S. Choo, Extraction methods of butterfly pea (Clitoria ternatea) flower and biological activities of its phytochemicals, J. Food Sci.
Technol. 58 (6) (2020) 2054–2067, https://doi.org/10.1007/s13197-020-04745-3.
[17] R. Sasikumar, D. Das, A.K. Jaiswal, Effects of extraction methods and solvents on the bioactive compounds, antioxidant activity, and storage stability of
anthocyanin rich blood fruit (Haematocarpus validus) extracts, J. Food Process. Preserv. 45 (5) (2021), e15401, https://doi.org/10.1111/jfpp.15401.
[18] M.D.A. Saldaña, E.R. Martinez, J.K. Sekhon, H. Vo, The effect of different pressurized fluids on the extraction of anthocyanins and total phenolics from cranberry
pomace, J. Supercrit. Fluids 175 (2021), 105279, https://doi.org/10.1016/j.supflu.2021.105279.
[19] G.C. Vidana Gamage, Y.Y. Lim, W.S. Choo, Anthocyanins from Clitoria ternatea flower: biosynthesis, extraction, stability, antioxidant activity, and applications,
Front. Plant Sci. 12 (2021), https://doi.org/10.3389/fpls.2021.792303.
[20] Q. He, B. Du, B. Xu, Extraction optimization of phenolics and antioxidants from black goji berry by accelerated solvent extractor using response surface
methodology, Appl. Sci. 8 (10) (2018) 1905, https://doi.org/10.3390/app8101905.
[21] E.J. Jeyaraj, Y.Y. Lim, W.S. Choo, Effect of organic solvents and water extraction on the phytochemical profile and antioxidant activity of Clitoria ternatea
flowers, ACS Food Sci. Technol. 1 (9) (2021) 1567–1577, https://doi.org/10.1021/acsfoodscitech.1c00168S.
[22] E.J Jeyaraj, Y.Y. Lim, WS. Choo, Antioxidant, cytotoxic, and antibacterial activities of Clitoria ternatea flower extracts and anthocyanin-rich fraction, Sci. Rep. 12
(1) (2022) 14890, https://doi.org/10.1038/s41598-022-19146-z.
[23] F. Yan, X. Yu, Y. Jing, Optimized preparation, characterization, and antioxidant activity of chitooligosaccharide-glycine Maillard reaction products, J. Food Sci.
Technol. 55 (2) (2018) 712–720, https://doi.org/10.1007/s13197-017-2982-0.
[24] K.L. Chong, Y.Y. Lim, Effects of drying on the antioxidant properties of herbal tea from selected Vitex species, J. Food Qual. 35 (1) (2012) 51–59, https://doi.
org/10.1111/j.1745-4557.2011.00422.x.
[25] J. Luo, L.H. Bian, Z.W. Yao, X.M. Wang, Q.Y. Li, J.Y. Guo, J.L. Shi, Anthocyanins in Lycium ruthenicum Murray reduce nicotine withdrawal-induced anxiety and
craving in mice, Neurosci. Lett. 763 (2021), 136152, https://doi.org/10.1016/j.neulet.2021.136152.
[26] Z. Liu, X. Tang, C. Liu, B. Dong, Y. Shao, B. Liu, H. Yue, Ultrasonic extraction of anthocyanins from Lycium ruthenicum Murr. and its antioxidant activity, Food
Sci. Nutr. 8 (6) (2020) 2642–2651, https://doi.org/10.1002/fsn3.1542.
[27] K.M. Khazaei, S.M. Jafari, M. Ghorbani, A.H. Kakhki, M. Sarfarazi, Optimization of anthocyanin extraction from saffron petals with response surface
methodology, Food Anal. Methods 9 (7) (2016) 1993–2001, https://doi.org/10.1007/s12161-015-0375-4.
[28] S. Şahin, R. Şamlı, Optimization of olive leaf extract obtained by ultrasound-assisted extraction with response surface methodology, Ultrason. Sonochem. 20 (1)
(2013) 595–602, https://doi.org/10.1016/j.ultsonch.2012.07.029.

10
G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

[29] M. Janowicz, A. Lenart, The impact of high pressure and drying processing on internal structure and quality of fruit, Eur. Food Res. Technol. 244 (8) (2018)
1329–1340, https://doi.org/10.1007/s00217-018-3047-y.
[30] Z. Liu, J. Dang, Q. Wang, M. Yu, L. Jiang, L. Mei, Y. Shao, Y. Tao, Optimization of polysaccharides from Lycium ruthenicum fruit using RSM and its anti-oxidant
activity, Int. J. Biol. Macromol. 61 (2013) 127–134, https://doi.org/10.1016/j.ijbiomac.2013.06.042.
[31] N. Ruenroengklin, J. Zhong, X. Duan, B. Yang, J. Li, Y. Jiang, Effects of various temperatures and pH values on the extraction yield of phenolics from litchi fruit
pericarp tissue and the antioxidant activity of the extracted anthocyanins, Int. J. Mol. Sci. 9 (7) (2008) 1333–1341, https://doi.org/10.3390/ijms9071333.
[32] J.R. Vergara-Salinas, P. Bulnes, M.C. Zúñiga, J. Pérez-Jiménez, J.L. Torres, M.L. Mateos-Martín, E. Agosin, J.R. Pérez-Correa, Effect of pressurized hot water
extraction on antioxidants from grape pomace before and after enological fermentation, J. Agric. Food Chem. 61 (28) (2013) 6929–6936, https://doi.org/
10.1021/jf4010143.
[33] S. Berenji Ardestani, M.A. Sahari, M. Barzegar, Effect of extraction and processing conditions on anthocyanins of barberry, J. Food Process. Preserv. 40 (6)
(2016) 1407–1420, https://doi.org/10.1111/jfpp.12726.
[34] M. Shen, K. Liu, Y. Liang, G. Liu, J. Sang, C. Li, Extraction optimization and purification of anthocyanins from Lycium ruthenicum Murr. and evaluation of
tyrosinase inhibitory activity of the anthocyanins, J. Food Sci. 85 (3) (2020) 696–706, https://doi.org/10.1111/1750-3841.15037.
[35] G.C. Vidana Gamage, Y.Y. Lim, W.S. Choo, Sources and relative stabilities of acylated and nonacylated anthocyanins in beverage systems, J. Food Sci. Technol.
59 (2021) 831–845, https://doi.org/10.1007/s13197-021-05054-z.
[36] N. Aryanti, D.H. Wardhani, A. Wasi, G.A. Ramadhan, A. Purbasari, Extraction characteristics of anthocyanin from roselle (Hibiscus sabdariffa L.) calyces by
ultrasound-assisted extraction, Adv. Sci. Lett. 23 (6) (2017) 5626–5628, https://doi.org/10.1166/asl.2017.8785.
[37] H. Matsufuji, H. Kido, H. Misawa, J. Yaguchi, T. Otsuki, M. Chino, M. Takeda, K. Yamagata, Stability to light, heat, and hydrogen peroxide at different pH values
and DPPH radical scavenging activity of acylated anthocyanins from red radish extract, J. Agric. Food Chem. 55 (9) (2007) 3692–3701, https://doi.org/
10.1021/jf063598o.
[38] N. Jiménez, P. Bohuon, M. Dornier, C. Bonazzi, A.M. Pérez, F. Vaillant, Effect of water activity on anthocyanin degradation and browning kinetics at high
temperatures (100–140 ◦ C), Food Res. Int. 47 (1) (2012) 106–115, https://doi.org/10.1016/j.foodres.2012.02.004.
[39] P.M. Lee, R. Abdullah, L.K. Hung, Thermal degradation of blue anthocyanin extract of Clitoria ternatea flower, in: L. Xuan (Ed.), Biotechnology and Food Service.
International Proceedings of Chemical Biological and Environmental Engineering. 7. Singapore: Int Assoc. Comput. Sci. Inform. Technol, . Press-Iacsit Press,
2011, pp. 49–53.
[40] R.M. Daniel, M.J. Danson, Temperature and the catalytic activity of enzymes: a fresh understanding, FEBS (Fed. Eur. Biochem. Soc.) Lett. 587 (17) (2013)
2738–2743, https://doi.org/10.1016/j.febslet.2013.06.027.
[41] N. Anuar, A.F. Mohd Adnan, N. Saat, N. Aziz, R. Mat Taha, Optimization of extraction parameters by using response surface methodology, purification, and
identification of anthocyanin pigments in Melastoma malabathricum fruit, Sci. World J. 2013 (2013), 810547, https://doi.org/10.1155/2013/810547.
[42] J.E. Cacace, G. Mazza, Optimization of extraction of anthocyanins from black currants with aqueous ethanol, J. Food Sci. 68 (1) (2003) 240–248, https://doi.
org/10.1111/j.1365-2621.2003.tb14146.x.
[43] K. Sharma, E.Y. Ko, A.D. Assefa, S. Ha, S.H. Nile, E.T. Lee, S.W. Park, Temperature-dependent studies on the total phenolics, flavonoids, antioxidant activities,
and sugar content in six onion varieties, J. Food Drug Anal. 23 (2) (2015) 243–252, https://doi.org/10.1016/j.jfda.2014.10.005.
[44] T. Islam, X. Yu, T.S. Badwal, B. Xu, Comparative studies on phenolic profiles, antioxidant capacities and carotenoid contents of red goji berry (Lycium barbarum)
and black goji berry (Lycium ruthenicum), Chem. Cent. J. 11 (1) (2017) 59, https://doi.org/10.1186/s13065-017-0287-z.
[45] Y. Jiang, Z. Fang, W. Leonard, P. Zhang, Phenolic compounds in Lycium berry: composition, health benefits and industrial applications, J. Funct.Foods 77
(2021), 104340, https://doi.org/10.1016/j.jff.2020.104340.
[46] S. Chen, Z. Zeng, N. Hu, B. Bai, H. Wang, Y. Suo, Simultaneous optimization of the ultrasound-assisted extraction for phenolic compounds content and
antioxidant activity of Lycium ruthenicum Murr. fruit using response surface methodology, Food Chem. 242 (2018) 1–8, https://doi.org/10.1016/j.
foodchem.2017.08.105.
[47] M. Simsek, G. Sumnu, S. Sahin, Microwave assisted extraction of phenolic compounds from sour cherry pomace, Separ. Sci. Technol. 47 (8) (2012) 1248–1254,
https://doi.org/10.1080/01496395.2011.644616.
[48] S. Eadmusik, C. Phungamngoen, N. Choosuk, Kinetic degradation of total phenolic content, DPPH radical scavenging and xanthine oxidase inhibitory activities
in yanang (Tiliacora triandra) leaf extract during preparation process, Malaysian J. Anal. Sci. 23 (3) (2019) 516–523, https://doi.org/10.17576/mjas-2019-
2303-16.
[49] M.R. González-Centeno, F. Comas-Serra, A. Femenia, C. Rosselló, S. Simal, Effect of power ultrasound application on aqueous extraction of phenolic compounds
and antioxidant capacity from grape pomace (Vitis vinifera L.): experimental kinetics and modeling, Ultrason. Sonochem. 22 (2015) 506–514, https://doi.org/
10.1016/j.ultsonch.2014.05.027.
[50] T. Stéphanie Martins de Freitas, G. Menezes de Rodrigues, F. Matta Fakhouri, C. da Silva, C. Andréa Lima Cardoso, J. Ignacio Velasco, C. Tostes Filgueiras,
V. Augusto dos Santos Garcia, Application of the Box–Behnken experimental design for the extraction of phenolic compounds from araçá-roxo (Psidium
myrtoides), J. Food Process. Preserv. 45 (3) (2021), e15260, https://doi.org/10.1111/jfpp.15260.
[51] A. Gengatharan, G. Dykes, W.S. Choo, Betacyanins from Hylocereus polyrhizus: pectinase-assisted extraction and application as a natural food colourant in ice
cream, J. Food Sci. Technol. 58 (2020) 1401–1410, https://doi.org/10.1007/s13197-020-04651-8.
[52] J. Malik, S.C. Mandal, Chapter 2 - extraction of herbal biomolecules, in: S.C. Mandal, A.K. Nayak, A.K. Dhara (Eds.), Herbal Biomolecules in Healthcare
Applications, Academic Press, 2022, pp. 21–46.
[53] M.M. Meregalli, B.M.S. Puton, F.D.M. Camera, A.U. Amaral, J. Zeni, R.L. Cansian, M.L. Mignoni, G.T. Backes, Conventional and ultrasound-assisted methods for
extraction of bioactive compounds from red araçá peel (Psidium cattleianum Sabine), Arab. J. Chem. 13 (6) (2020) 5800–5809, https://doi.org/10.1016/j.
arabjc.2020.04.017.
[54] N. Pap, S. Beszédes, E. Pongrácz, L. Myllykoski, M. Gábor, E. Gyimes, C. Hodúr, R.L. Keiski, Microwave-assisted extraction of anthocyanins from black currant
marc, Food Bioprocess Technol. 6 (10) (2013) 2666–2674, https://doi.org/10.1007/s11947-012-0964-9.
[55] G. Xu, X. Ye, J. Chen, D. Liu, Effect of heat treatment on the phenolic compounds and antioxidant capacity of citrus peel extract, J. Agric. Food Chem. 55 (2)
(2007) 330–335, https://doi.org/10.1021/jf062517l.
[56] M. Hanula, J. Wyrwisz, M. Moczkowska, O.K. Horbańczuk, E. Pogorzelska-Nowicka, A. Wierzbicka, Optimization of microwave and ultrasound extraction
methods of açai berries in terms of highest content of phenolic compounds and antioxidant activity, Appl. Sci. 10 (23) (2020) 8325, https://doi.org/10.3390/
app10238325.
[57] S.B. Zimare, G.D. Mankar, R.B. Barmukh, Optimization of ultrasound-assisted extraction of total phenolics and flavonoids from the leaves of Lobelia nicotianifolia
and their radical scavenging potential, Curr. Res. Green Sustain. Chem. 4 (2021), 100109, https://doi.org/10.1016/j.crgsc.2021.100109.
[58] K. Kaderides, L. Papaoikonomou, M. Serafim, A.M. Goula, Microwave-assisted extraction of phenolics from pomegranate peels: optimization, kinetics, and
comparison with ultrasounds extraction, Chem. Eng. Process: Process Intensif. 137 (2019) 1–11, https://doi.org/10.1016/j.cep.2019.01.006.
[59] J.O. Chaves, M.C. de Souza, L.C. da Silva, D. Lachos-Perez, P.C. Torres-Mayanga, A.P.d.F. Machado, T. Forster-Carneiro, M. Vázquez-Espinosa, A.V. González-de-
Peredo, G.F. Barbero, M.A. Rostagno, Extraction of flavonoids from natural sources using modern techniques, Front. Chem. 8 (2020), https://doi.org/10.3389/
fchem.2020.507887.
[60] F. Zhang, Y. Yang, P. Su, Z. Guo, Microwave-assisted extraction of rutin and quercetin from the stalks of Euonymus alatus (Thunb.) Sieb, Phytochem, Anal 20 (1)
(2009) 33–37, https://doi.org/10.1002/pca.1088.
[61] Y.Y. Lim, T.T. Lim, J.J. Tee, Antioxidant properties of several tropical fruits: a comparative study, Food Chem. 103 (3) (2007) 1003–1008, https://doi.org/
10.1016/j.foodchem.2006.08.038.
[62] H.V. Annegowda, L.N. Anwar, M.N. Mordi, S. Ramanathan, S.M. Mansor, Influence of sonication on the phenolic content and antioxidant activity of Terminalia
catappa L. leaves, Pharmacogn. Res. 2 (6) (2010) 368–373, https://doi.org/10.4103/0974-8490.75457.

11
G.C. Vidana Gamage and W.S. Choo Heliyon 9 (2023) e14426

[63] A. Gengatharan, G.A. Dykes, W.S. Choo, Stability of betacyanin from red pitahaya (Hylocereus polyrhizus) and its potential application as a natural colourant in
milk, Int. J. Food Sci. Technol. 51 (2) (2016) 427–434, https://doi.org/10.1111/ijfs.12999.
[64] H.E. Khoo, A. Azlan, S.T. Tang, S.M. Lim, Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health
benefits, Food Nutr. Res. 61 (1) (2017), 1361779, https://doi.org/10.1080/16546628.2017.1361779.
[65] K. Kalcheva–Karadzhova, K. Mihalev, D. Ludneva, V. Shikov, R. Dinkova, I. Bakalov, Effect of pectolytic enzyme preparation on antioxidant capacity and color
characteristic of rose petals extract (Rosa Damascena Mill.), Bulg. Chem. Commun. 48 (2016) 464–467.
[66] X.L. He, X.L. Li, Y.P. Lv, Q. He, Composition and color stability of anthocyanin-based extract from purple sweet potato, Food Sci. Technol. 35 (2015) 468–473,
https://doi.org/10.1590/1678-457X.6687.
[67] Y.W. Wang, G.X. Luan, W. Zhou, J. Meng, H.L. Wang, N. Hu, Y.R. Suo, Subcritical water extraction, UPLC-Triple-TOF/MS analysis and antioxidant activity of
anthocyanins from Lycium ruthenicum Murr, Food Chem. 249 (2018) 119–126, https://doi.org/10.1016/j.foodchem.2017.12.078.
[68] L. Tuli, H.W. Ressom, LC-MS based detection of differential protein expression, J. Proteonomics Bioinf. 2 (2009) 416–438, https://doi.org/10.4172/
jpb.1000102.
[69] M. Kim, D.G. Nam, J.S. Choe, K.A. Hwang, A.J. Choi, Optimization of pectinase-assisted extraction condition of mulberry (Morus alba L.) fruit using response
surface methodology and its effect on anthocyanin synthesis pathway-related metabolites, J. Food Sci. 86 (9) (2021) 3926–3938, https://doi.org/10.1111/
1750-3841.15853.
[70] M. Syrpas, E. Valanciene, E. Augustiniene, N. Malys, Valorization of bilberry (Vaccinium myrtillus L.) Pomace by enzyme-assisted extraction: process
optimization and comparison with conventional solid-liquid extraction, Antioxidants 10 (5) (2021) 773, https://doi.org/10.3390/antiox10050773.
[71] M. José Aliaño González, C. Carrera, G.F. Barbero, M. Palma, A comparison study between ultrasound–assisted and enzyme–assisted extraction of anthocyanins
from blackcurrant (Ribes nigrum L.), Food Chem. X. 13 (2022), 100192, https://doi.org/10.1016/j.fochx.2021.100192.
[72] P.K. Robinson, Enzymes: principles and biotechnological applications, Essays Biochem. 59 (2015) 1–41, https://doi.org/10.1042/bse0590001.

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