foods

Article
The Extraction Process, Separation, and Identification of
Curcuminoids from Turmeric Curcuma longa
Gal Slaček 1 , Petra Kotnik 1,2 , Azra Osmić 1 , Vesna Postružnik 1 , Željko Knez 1,2 , Matjaž Finšgar 1
and Maša Knez Marevci 1, *

1 Faculty of Chemistry and Chemical Engineering, University of Maribor, SI-2000 Maribor, Slovenia;
[email protected] (G.S.); [email protected] (P.K.); [email protected] (A.O.); [email protected] (V.P.);
[email protected] (Ž.K.); [email protected] (M.F.)
2 Faculty of Medicine, University of Maribor, Taborska 8, SI-2000 Maribor, Slovenia
* Correspondence: [email protected]

Abstract: Turmeric Curcuma longa is a well-known spice with various health benefits, attributed
primarily to curcumin. Soxhlet extraction, cold maceration, ultrasound-assisted extraction (UAE),
and supercritical fluid extraction were performed, and the content of total phenols, proanthocyani-
dins, and antioxidants was analysed by UV/VIS spectrophotometry. High-performance liquid
chromatography–tandem mass spectrometry (HPLC-MS/MS) was employed to identify and quan-
tify the curcumin content. Supercritical extracts had the highest total phenolic content (538.95 mg
GA/100 g material), while the Soxhlet extracts had the highest content of proanthocyanidins (4.77 mg
PAC/100 g of material). Extracts obtained by UAE and supercritical extraction have the highest
antioxidant potential. Antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH• ) was
64.27% and 1750.32 mg Trolox per g dry weight by 2,2-azinobis(3-ethylbenzothiazoline 6 sulphonic
acid) (ABTS+• ) for the extract obtained by supercritical extraction. The UAE resulted in the highest
amount of curcumin (1.91 mg curcumin/g material). A kinetic study showed that extraction yield in
supercritical extracts decreased with increasing temperature and that the content of isolated curcumin
was inversely proportional to solvent-to-feed ratio (S/F). The present study has confirmed that
Citation: Slaček, G.; Kotnik, P.; turmeric is an excellent source of antioxidants, such as curcumin, that play an important role in
Osmić, A.; Postružnik, V.; Knez, Ž.; reducing cellular stress by neutralising free radicals.
Finšgar, M.; Knez Marevci, M. The
Extraction Process, Separation, and Keywords: turmeric; curcumin; total phenols; proanthocyanidins; antioxidants
Identification of Curcuminoids from
Turmeric Curcuma longa. Foods 2023,
12, 4000. https://doi.org/10.3390/
foods12214000 1. Introduction
Academic Editors: Shahab A. Shamsi The steep rise in inflammatory and cancerous diseases has led to a massive increase
and Isabel Borrás-Linares in research on natural antioxidants or natural materials rich in antioxidants. One of the
promising natural materials rich in antioxidants is turmeric. Turmeric is a source of
Received: 31 August 2023
antioxidants, vitamins (B3, B6, C, and E), volatile components (turmerone, atlantone, and
Revised: 12 October 2023
Accepted: 30 October 2023
zingiberene), and mineral elements (Ca, Mg, P, Fe, Zn, and Na) which have a positive effect
Published: 1 November 2023
on health and various disease treatments [1–3].
Turmeric is derived from the rhizomes of the Curcuma longa plant, which belongs to the
ginger family (Zingiberaceae). Curcuminoids, which are fat-soluble polyphenols, give the
plant its orange colour and have many favourable properties for human health. The main
Copyright: © 2023 by the authors. curcuminoid in the plant is curcumin [4]. Curcumin was first isolated in 1815 by Vogel and
Licensee MDPI, Basel, Switzerland. Pelletier, but it took 98 years, when in 1913 Lampe and Miłob˛edzka validated its chemical
This article is an open access article structure (Figure 1) (Figure 1) [5,6]. Curcumin is a hydrophobic compound and is soluble in
distributed under the terms and organic solvents (methanol, ethanol, acetone, benzene, etc.). Curcumin is found in nature
conditions of the Creative Commons
in two forms: in the keto monoole and diketone form. Jayaprakasha et al. [7] confirmed
Attribution (CC BY) license (https://
that curcumin is the most potent antioxidant of all curcuminoids. Turmeric also contains
creativecommons.org/licenses/by/
dimethoxycurcumin and bis-dimethoxycurcumin as main compounds [8]. The proportion
4.0/).

Foods 2023, 12, 4000. https://doi.org/10.3390/foods12214000 https://www.mdpi.com/journal/foods

 tion, cold maceration, supercritical fluid extraction, and ultrasound-assisted extraction
(UAE)) for optimising curcuminoid content in water or fat-soluble extracts different sol-
vents (acetone, water, ethanol, triacetin, and diacetin) were used.
The water- and fat-soluble extracts of turmeric and its active components exhibit an-
tioxidant activity comparable to that of vitamins C and E. Recent studies have demon-
Foods 2023, 12, 4000 2 of 14
strated turmeric’s anti-inflammatory and anti-cancer activity [14,15]. However, the use of
curcumin as an active medicinal ingredient is limited by its poor bioavailability, as it is
insoluble in water and rapidly degrades in the intestine [16,17]. Proper treatment and pro-
ofcessing
all three
of curcuminoids depends
the plant are crucial on geographical
to isolate conditions
the biologically and
active or harvesting. Curcumin
medicinal components
(80%) presents the largest proportion of curcuminoids, followed by dimethoxycurcumin
of turmeric for human consumption. Antioxidants, such as curcumin, play an important
(17%)
role in and bis-dimethoxycurcumin
reducing (3%) [9,10]. free radicals [18].
cellular stress by neutralising

Figure1.1.Chemical
Figure Chemicalstructure
structureofofcurcumin.
curcumin.

The extraction
In this procedures
work, different for extracting
extraction techniques curcuminoids
(Soxhlet, UAE, from Curcuma
cold longaand
maceration) were
su-
already
percriticalreported [11–13]. Several
fluid extraction extraction to
were performed techniques
study the(Soxhlet,
influencemicrowave-assisted
of extraction technique ex-
traction,
and process cold parameters
maceration,(time,supercritical
pressure,fluid
andextraction,
temperature) andonultrasound-assisted extraction
the content of bioactive com-
(UAE))
ponents forinoptimising curcuminoid
turmeric extracts. content influid
Supercritical water or fat-soluble
extraction extracts
has been different
applied solvents
as a sustaina-
(acetone,
ble method water,
for ethanol, triacetin,
the extraction andtarget
of the diacetin) were used.
compounds. Herein, the simultaneous investi-
The water- and fat-soluble extracts of
gation of the influence of extraction technique and process turmeric and its active components
parameters exhibit
(time, pressure,
antioxidant activity comparable to that of vitamins C and E. Recent
and temperature) on the content of bioactive components in turmeric extracts was per- studies have demon-
strated
formedturmeric’s
followed by anti-inflammatory and anti-cancer
subsequent statistical analysis. The activity [14,15]. However,
supercritical the use
fluid extraction al-
oflowed
curcumin
facileas an activeof
separation medicinal
the solventingredient
from theisextracts,
limitedandby its poor
thus, bioavailability,
despite the lower as it
mass
isyield,
insoluble in water and rapidly degrades in the intestine [16,17].
the recovery of phenolic compounds was the highest. Additionally, the data on the Proper treatment and
processing
curcumin of the plantkinetics
extraction are crucial
haveto isolate the biologically
been provided. The massactive or medicinal
yield and curcumincomponents
content
ofinturmeric
the extract obtained by supercritical extraction at 25 MPa, 40, 50, and 60 °C asimportant
for human consumption. Antioxidants, such as curcumin, play an a function
role in reducing cellular
of solvent-to-feed stressbeen
ratio have by neutralising
studied. Thefree radicals
optimal [18]. parameters and extraction
process
In this work, different extraction techniques
time for the highest recovery of curcumin have been determined, (Soxhlet, UAE, coldwhichmaceration) and
will be valuable
supercritical fluid extraction were performed to study the influence of extraction technique
for future studies.
and process parameters
Using different extraction (time, pressure,
methodsand temperature)
(Soxhlet on UAE,
extraction, the content of bioactive
supercritical com-
extraction,
ponents in turmeric extracts. Supercritical fluid extraction has been applied
and cold maceration) the optimal extraction conditions of turmeric for the isolation of high as a sustainable
method
contents forofthe extraction
bioactive of the target
components compounds.proanthocyanidins,
(antioxidants, Herein, the simultaneous investigation
and total phenolics)
of the influence of extraction technique and process parameters (time, pressure, and temper-
have been determined. Increasing the temperature and pressure parameters had a favour-
ature) on the content of bioactive components in turmeric extracts was performed followed
able effect on the increase in the extraction yield and the content of bioactive components
by subsequent statistical analysis. The supercritical fluid extraction allowed facile separa-
of the individual samples.
tion of the solvent from the extracts, and thus, despite the lower mass yield, the recovery of
The aim of the manuscript is to compare different extraction techniques and the ana-
phenolic compounds was the highest. Additionally, the data on the curcumin extraction
lytical performance of extracts by high-performance liquid chromatography coupled with
kinetics have been provided. The mass yield and curcumin content in the extract obtained
tandem mass spectrometry (HPLC-MS/MS). In addition, the evaluation of experimental
by supercritical extraction at 25 MPa, 40, 50, and 60 ◦ C as a function of solvent-to-feed ratio
extraction results by statistical analyses was performed.
have been studied. The optimal process parameters and extraction time for the highest
recovery of curcumin have been determined, which will be valuable for future studies.
Using different extraction methods (Soxhlet extraction, UAE, supercritical extraction,
and cold maceration) the optimal extraction conditions of turmeric for the isolation of
high contents of bioactive components (antioxidants, proanthocyanidins, and total phe-
nolics) have been determined. Increasing the temperature and pressure parameters had
a favourable effect on the increase in the extraction yield and the content of bioactive
components of the individual samples.
The aim of the manuscript is to compare different extraction techniques and the ana-
lytical performance of extracts by high-performance liquid chromatography coupled with
tandem mass spectrometry (HPLC-MS/MS). In addition, the evaluation of experimental
extraction results by statistical analyses was performed.

2. Experimental
2.1. Materials
Curcuma longa was supplied by Alfred Galke GmbH (Samtgemeinde, Bad Grund, Ger-
many). The material was ground and dried. The moisture content of the turmeric powder
Foods 2023, 12, 4000 3 of 14

was determined using a halogen moisture analyser HX204 (Mettler Toledo, Greifensee,
Switzerland). Ethanol (CAS 64-17-5, Sigma-Aldrich Chemie GmbH, Steinheim, Germany)
with purity ≥ 99.9% was used as a solvent for conventional extractions and 2,2-azinobis(3-
ethylbenzothiazoline 6 sulphonic acid) (ABTS+• ) analysis. Methanol (CAS reg.nNo. 67-56-1,
Sigma-Aldrich Chemie GmbH) with purity ≥ 99.9% and 2,2-diphenyl-1-picrylhydrazyl
(DPPH• ) (Sigma-Aldrich Chemie GmbH) with purity ≥ 97.0% were used for DPPH• anal-
ysis. CO2 (CAS reg. no. 124-38-9) was purchased from MESSER (MG-Ruše, Slovenia),
with a purity of 99.99%. All analytical standards (curcumin, product no. 08511, with pu-
rity ≥ 98.0%; bisdemethoxycurcumin, product no. 90,594 with purity ≥ 95.0%; demethoxy-
curcumin, product no. 90,593 with purity ≥ 95.0%), ABTS+• (product no. 194430), di-
ammonium salt, potassium persulfate (K2 S2 O8 , product no. 216224), (±)-6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid (Trolox, 97.0%), Folin–Ciocalteu’s reagent (FC,
product no. 1.09001), anhydrous sodium carbonate (Na2 CO3 , product no. 223530), and
ferrous sulfate heptahydrate (FeSO4 ·7H2 O, product no. 215422) were purchased from
Sigma-Aldrich. Hydrochloric acid (HCl, 37.0%) and n-butanol with purity ≥ 99.5% were
supplied by Merck (Darmstadt, Germany) and used for proanthocyanidins content.

2.2. Moisture Content

A halogen moisture analyser HX204 was used to determine the moisture content of
turmeric powder, following the Elbl et al. [19] method with some modifications. To conduct
the analysis, 3 g of turmeric powder was weighed in a crucible. The thermogravimetric
principle was used to measure the sample. The samples were heated to 100 ◦ C until a
constant weight was obtained, and the weight difference was recorded. The measurement
was performed in triplicate, and the amount of moisture is expressed as mass % with
standard deviation.

2.3. Extraction Methods

Soxhlet extraction, UAE, cold maceration, and supercritical extraction were performed.
The solvents used were ethanol for conventional extractions and supercritical CO2 in
combination with ethanol as co-solvent for supercritical extractions. The extraction time
was determined based on previous reports [9,20–26] and previous extraction experiences.
Each extraction procedure was performed in triplicate.

2.3.1. Soxhlet Extraction

Soxhlet extraction was carried out in a water bath at the solvent’s boiling point under
ambient air pressure (laboratory conditions). The procedure has been described in detail
previously [27]. Briefly, 25.00 g material was extracted with 150 mL of solvent (ethanol)
using Soxhlet extraction apparatus. After 3 h of extraction, the solvent was evaporated
using vacuum rotavapor (BÜCHI Rotavapor R-114 and BÜCHI Vacuum Controller B-721,
BÜCHI Labortechnik AG, Flawil, Switzerland) at 40 ◦ C, and the mass of the extract was
determined gravimetrically. The extraction yield (η) was calculated by using Equation (1).
The extract was stored in a freezer until the spectrophotometric and HPLC-MS/MS analyses
were carried out.
mextract
η (wt.%) = × 100, (1)
mraw material

2.3.2. UAE
UAE was carried out at temperatures up to 40 ◦ C. The extraction process has been
described in detail previously [27]. Briefly, 25.00 g of material was extracted with 150 mL
of solvent (ethanol) using an ultrasound bath (VEVOR, Rancho Cucamonga, CA, USA).
The extraction was carried out at a frequency of 40 kHz. After 1.5 h of extraction, the
extraction solution was filtered through filter paper (MN-751, Macherey-Nagel, Düren,
Germany) and evaporated using rotavapor at 40 ◦ C. The yield of extraction was calculated
by using Equation (1). The extract was stored in a freezer until the spectrophotometric and
HPLC-MS/MS analyses were carried out.
 of solvent (ethanol) using an ultrasound bath (VEVOR, Rancho Cucamonga, California,
United States). The extraction was carried out at a frequency of 40 kHz. After 1.5 h of
extraction, the extraction solution was filtered through filter paper (MN-751, Macherey-
Nagel, Düren, Germany) and evaporated using rotavapor at 40 °C. The yield of extraction
was calculated by using Equation (1). The extract was stored in a freezer until the spectro-
Foods 2023, 12, 4000 4 of 14
photometric and HPLC-MS/MS analyses were carried out.

2.3.3. Cold Maceration

2.3.3. Cold Maceration
Cold maceration was carried out on a magnetic stirrer (TEHTNICA, Železniki, Slo-
venia)Cold maceration
at 450–500 rpmwasandcarried out on a magnetic
room temperature. stirrer
Briefly, (TEHTNICA,
25.00 g of materialŽelezniki, Slove-
was extracted
nia) at
with 450–500
150 mL of rpm and(ethanol).
solvent room temperature.
After 3 h of Briefly, 25.00
stirring, thegsolution
of material
waswas extracted
filtered with
(MN-751)
150 mL of solvent (ethanol). After 3 h of stirring, the solution was filtered
to separate the solid material from the extract solution. The extract solution was evapo- (MN-751) to
separate the solid material from the extract solution. The extract solution was
rated using rotavapor at 40 °C. The yield of extraction was calculated by using Eq. (1). Theevaporated
using rotavapor at 40 ◦
extract was stored in aC. The yield
freezer untilofthe
extraction was calculated
spectrophotometric andbyHPLC-MS/MS
using Equationanalyses
(1). The
extract was stored
were carried out. in a freezer until the spectrophotometric and HPLC-MS/MS analyses
were carried out.
2.3.4. Supercritical Extraction
2.3.4. Supercritical Extraction
The procedure employed in this work was described previously [28]. A high-pressure
The procedure employed in this work was described previously [28]. A high-pressure
autoclave (Figure 2) filled with 25.00 g of turmeric powder was immersed in a water bath
autoclave (Figure 2) filled with 25.00 g of turmeric powder was immersed in a water bath at
at a constant temperature of 60 °C and pressure of 25 MPa for a duration of 120 min. Dur-
a constant temperature of 60 ◦ C and pressure of 25 MPa for a duration of 120 min. During
ing the extraction process, ethanol was pumped via a high-performance liquid chroma-
the extraction process, ethanol was pumped via a high-performance liquid chromatography
tography (HPLC) pump at a flow rate of 2 mL/min to isolate polar and non-polar compo-
(HPLC) pump at a flow rate of 2 mL/min to isolate polar and non-polar components. The
nents. The pressurised
pressurised CO2 in thewas
CO2 in the autoclave autoclave
pumpedwas frompumped
the gas from the using
cylinder gas cylinder using a
a high-pressure
high-pressure
pump (model 260D,pumpISCO(model 260D,pump,
syringe ISCO syringe
Lincoln,pump, Lincoln,
NE, USA) NE,constant
and was USA) and was con-
throughout
stant throughout the experiment. Separation and expansion of supercritical
the experiment. Separation and expansion of supercritical CO2 from ethanol and extract CO 2 from eth-

anol
wereand extract
carried outwere
in a carried outatinatmospheric
glass trap a glass trap at atmospheric
pressure pressure
and room and room After
temperature. tem-
perature. After the extraction was completed, the co-solvent was further evaporated
the extraction was completed, the co-solvent was further evaporated from the extraction from
the extraction
solution usingsolution usingThe
a rotavapor. a rotavapor. The yield was
yield of extraction of extraction
calculatedwas
bycalculated by using
using Equation (1).
Equation
The extract(1).
wasThe extract
stored was stored
in a freezer until theinspectrophotometric
a freezer until the andspectrophotometric
HPLC-MS/MS analyses and
HPLC-MS/MS
were carried out. analyses were carried out.

Figure 2. High-pressure extraction apparatus; 1—CO2 gas supply, 2,4,6—inlet valves, 3—one-way
2. High-pressure
Figure5—HPLC
valve, extraction apparatus;
pump, 7—high-pressure pump,1—CO 2 gas supply,
8—heating 2,4,6—inlet valves,
coil, 9—autoclave, 3—one-way
10—thermostated
valve, 5—HPLC pump, 7—high-pressure pump, 8—heating coil, 9—autoclave, 10—thermostated
bath, 11—temperature indicator and regulator, 12—manometer, 13,14—outlet valves, 15—extract
bath, 11—temperature
release indicator
valve, 16—sampling trap, and regulator, 12—manometer, 13,14—outlet valves, 15—extract
17—rotameter.
release valve, 16—sampling trap, 17—rotameter.

2.4. Spectrophotometric Analysis

Antioxidant capacity, total phenolic content, and proanthocyanidins content were de-
termined using a UV/VIS spectrophotometer (Cary 50 Scan, Varian, Surrey, England). The
total phenolic content in the extracts was determined using the Folin–Ciocalteu method [29].
Proanthocyanidins content was determined using the UV spectrophotometric method [30]
based on acid hydrolysis and colour formation. The antioxidant activity was evaluated
quantitatively by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH• ) and 2,2-azinobis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS+• ) radicals [31,32]. The extract solution was
prepared using 10 mg of extract, which was diluted in 10 mL of methanol.
Foods 2023, 12, 4000 5 of 14

2.4.1. Determination of Total Phenols

The total phenols were determined using a Folin–Ciocalteu (FC) reagent, as described
previously [33]. Briefly, the extract solution (0.5 mL) was mixed with 2.5 mL of FC reagent,
which had been diluted 1:10 with distilled water. The resulting mixture was neutralised
with 2 mL of Na2 CO3 solution (7.5%, w/v), and the preparation time was not more than
2 min. The samples were thermostated in a water bath for 5 min at 50 ◦ C and then cooled
down. For the control sample, 0.5 mL of distilled water was used instead of the extract.
The absorbance was measured at a wavelength of 760 nm. The total phenolic content was
expressed as mg of gallic acid (GA) per 100 g of material.

2.4.2. Determination of Proanthocyanidins

The proanthocyanidins content was determined by a hydrolysis reaction in a reaction
mixture containing n-butanol and concentrated hydrochloric acid [33]. To prepare the
sample, 1 mL of the extract solution with a concentration of 5 mg/mL was used. Then,
10 mL of FeSO4 ·7H2 O (77 mg/500 mL) in a mixture of HCl and n-butanol (2:3) was added
to the prepared solution. The mixture was incubated for 15 min in a water bath at 95 ◦ C.
After incubation, the samples were cooled to ambient temperature, and the absorbance was
measured at a wavelength of 540 nm. The content of proanthocyanidins was expressed in
mg PAC per 100 g of material.

2.4.3. ABTS+• Method

The antioxidant activity of obtained extracts was determined using ABTS+• method [34].
A 7 mmol/L solution of ABTS+• (solid ABTS+• ) and 2.5 mmol/L solution of K2 O8 S2
(solid K2 O8 S2 ) were prepared in ultrapure water. Ultrapure water was obtained from
Elga PURELAB Classic system (ELGA LabWater, High Wycombe, United Kingdom). The
working solution was prepared by mixing ABTS+• and K2 O8 S2 solutions in a 1:1 volume
ratio and left in a dark room. The working solution was diluted with ethanol until the
absorbance 0.70 at wavelength 734 nm was obtained. The method protocol was to mix
3.950 mL of the ABTS+• solution with 50 µL of the sample (either a standard Trolox solution
or a crude extract solution) and incubate it for 30 min in the dark at room temperature.
The absorbance of the solution was then measured at 734 nm. A blank solution contained
only the ABTS+• solution and ethanol. The radical-scavenging activity was calculated
by Equation (2), where Ablank is the absorbance of the blank solution, and Asample is the
absorbance of the sample solution. Antioxidant activity was expressed as mg Trolox
equivalent per g of dry weight (mg Trolox/g DW).

Ablank − Asample
 
%Inhibition = × 100, (2)
Ablank

2.4.4. DPPH• Method

The antioxidant activity of obtained extracts was determined using the DPPH• method [31].
The extracts (10 mg) were dissolved in methanol solvent (10 mL). The extract was dissolved
by stirring the solution and by exposure to the ultrasound bath for a few minutes. A
0.06 mmol/L solution of DPPH• was prepared of solid DPPH• and methanol. For the
samples, 77 µL of the extract solution was mixed with 3 mL of the prepared DPPH•
solution in dark bottles, and the mixture was incubated for 15 min at room temperature
in a dark room. The absorbance of the solutions was measured at the wavelength of
517 nm. The radical-scavenging activity was calculated by Equation (3), where A0 is the
absorbance of the control solution (t = 0 min), and A15 is the absorbance of the sample
solution (t = 15 min). The results are presented as % of inhibition at concentration 1 mg
extract per mL of methanol.

A0 − A15
 
%Inhibition = × 100, (3)
A0
 tion (t = 15 min). The results are presented as % of inhibition at concentration 1 mg extract
per mL of methanol.
𝐴0 −𝐴15
% Inhibition = ( ) × 100, (3)
𝐴0
Foods 2023, 12, 4000 6 of 14

2.5. High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The concentration of active components in the turmeric extracts was determined by
2.5. High-Performance Liquid Chromatography–Tandem Mass Spectrometry
HPLC-MS/MS using Agilent 1200 HPLC and Agilent 6460 JetStream triple quadrupole
The concentration of active components in the turmeric extracts was determined by
mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The Agilent Poroshell
HPLC-MS/MS using Agilent 1200 HPLC and Agilent 6460 JetStream triple quadrupole
EC-C18 column (2.7(Agilent
mass spectrometer µm particle size, 100Santa
Technologies, × 2.1 Clara,
mm ID) CA,was employed
USA). for chromatographic
The Agilent Poroshell
analysis. The analysis was carried out at 35 °C using gradient elution.
EC-C18 column (2.7 µm particle size, 100 × 2.1 mm ID) was employed for chromatographic The quantification
was performed
analysis. basedwas
The analysis on the calibration
carried ◦ C using
out at 35curve methodology.
gradient elution. The quantification
was Mobile
performed phase A consisted of ultrapure water containing 0.1% formic acid, and mobile
based on the calibration curve methodology.
phaseMobile
B wasphase A consisted
acetonitrile of ultrapure
containing 0.1%water containing
formic 0.1% formic
acid (elution acid, and
gradients: mobile
0 min 50% B, 5
phase B was acetonitrile containing 0.1% formic acid (elution gradients: 0 min
min 85% B, 7 min 85.0% B, 8 min 50.0% B to 10 min). The mass spectrometer was operated 50% B, 5 min
85% B, 7 min 85.0% B, 8 min 50.0% B to 10 min). The mass spectrometer was operated
in negative ionisation mode with optimised parameters for nitrogen as carrier gas at the
in negative ionisation mode with optimised parameters for nitrogen as carrier gas at the
temperature of 300 °C and a flow rate of 5 L/min, sheath gas temperature of 250 °C, sheath
temperature of 300 ◦ C and a flow rate of 5 L/min, sheath gas temperature of 250 ◦ C, sheath
gas flow of 11 L/min, and
gas flow of 11 L/min, andcapillary
capillary and
and nozzle
nozzle voltages
voltages at 3500
at 3500 V and V 500
andV,500 V, respectively.
respectively.
The
Themultiple-reaction monitoring
multiple-reaction monitoring (MRM)
(MRM) mode mode
waswas applied
applied to determine
to determine curcumin
curcumin based based
on
onion
ion transitions from m/z
transitions from m/z367.1
367.1toto217.0
217.0and and173.0
173.0with
with collision
collision energy
energy 4 and
4 and 12 V, re-
12 V,
spectively.
respectively.Corresponding chromatogramand
Corresponding chromatogram and mass
mass spectrum
spectrum are shown
are shown in Figure
in Figure 3. 3.

Figure
Figure 3.
3. (a) Chromatogram
(a) Chromatogram ofof curcumin
curcumin showing
showing MRM MRM spectrum
spectrum in transition
in transition 217.0,→ 217.0,
m/z→367.1
m/z 367.1
and
and(b)
(b) mass spectraofofprecursor
mass spectra precursor[M [M −−
− H] H]and
− and product ions of curcumin. ** means all fragments of
product ions of curcumin. ** means all fragments of
parent
parent ion 367.1atatcollision
ion 367.1 collision energy
energy 4 and
4 and 12 V.
12 V.

2.6.Statistical
2.6. Statistical Analysis
Analysis
The differences in the bioactive activity of the extracts were examined with statistical
The differences in the bioactive activity of the extracts were examined with statistical
tests using the R programming language version 4.1.1. and RStudio version 2022.02.03.
tests using the R programming language version 4.1.1. and RStudio version 2022.02.03.
Data distributions were analysed with Shapiro–Wilk statistical tests. Differences in bioactive
activity among normally distributed variables were examined using analysis of variance
(ANOVA), whereas the Kruskal–Wallis nonparametric statistical test was used to analyse
differences among nonnormally distributed variables. Post hoc Tukey’s test was performed
for normally distributed variables, while post hoc Dunn’s test was performed for non-
normally distributed variables. Significant differences between groups were marked on
bar plots with different letters. For cases with no significant differences, the same letters
were used.
 ance (ANOVA), whereas the Kruskal–Wallis nonparametric statistical test was used to an-
alyse differences among nonnormally distributed variables. Post hoc Tukey’s test was per-
formed for normally distributed variables, while post hoc Dunn’s test was performed for
nonnormally distributed variables. Significant differences between groups were marked
Foods 2023, 12, 4000
on bar plots with different letters. For cases with no significant differences, the same letters
7 of 14
were used.

3. Results and Discussion

3. Results and Discussion
3.1. Moisture Content
3.1. Moisture Content
The moisture content in the turmeric powder was 9.15 ± 0.53% (w/w), which is com-
The moisture content in the turmeric powder was 9.15 ± 0.53% (w/w), which is com-
parable to the results obtained by Paulluci et al. [35] and is within the acceptable range
parable to the results obtained by Paulluci et al. [35] and is within the acceptable range
specified by the European Medicines Agency [36], which states that values below 13.1%
specified by the European Medicines Agency [36], which states that values below 13.1%
are acceptable. The residual moisture content in the turmeric powder is an indicator of the
are acceptable. The residual moisture content in the turmeric powder is an indicator of
processing andand
the processing preservation methods
preservation used
methods andand
used can can
impact the chemical
impact andand
the chemical microbiolog-
microbio-
ical stability of the final product. Each measurement of moisture has been performed
logical stability of the final product. Each measurement of moisture has been performed in
triplicate.
in triplicate.

3.2.
3.2. Extraction
Extraction
Different extraction
extractionmethods
methodswere wereperformed
performedtotoobtain
obtainextracts
extracts from
from Curcuma
Curcuma pow-
powder.
der. Figure
Figure 4 shows
4 shows the extraction
the extraction yieldsyields
(η) for(η) for different
different extraction
extraction methods.
methods. Among Among all
all these
these extraction methods, Soxhlet extraction showed the highest η (10.27
extraction methods, Soxhlet extraction showed the highest η (10.27 wt.%), followed by wt.%), followed
by supercritical
supercritical extraction
extraction (8.67
(8.67 wt.%),
wt.%), coldmaceration
cold maceration(7.62(7.62wt.%),
wt.%),and
and UAE
UAE (6.83 wt.%).
wt.%).
Temperature, as well as solvent density, influencesinfluences the the extraction
extraction yield. With increasing
temperature at at constant
constantdensity,
density,thetheextraction
extraction yield
yield increases,
increases, andand
withwith increasing
increasing sol-
solvent
vent density,
density, the extraction
the extraction yieldyield increases
increases at constant
at constant temperature.
temperature. Differences
Differences in extrac-
in extraction
tion yields
yields between
between methodsmethods
were were compared
compared using using
ANOVA, ANOVA,
whichwhich
showed showed significant
significant differ-
ences between
differences extract
between groups
extract (F(3)(F(3)
groups = 21.88, p < 0.001).
= 21.88, Sahne
p < 0.001). Sahneet al. [9][9]
et al. reported
reportedlower
lowerη
using
η usingUAE
UAE(3.92
(3.92wt.%)
wt.%)andandSoxhlet
Soxhlet(6.90
(6.90wt.%)
wt.%)extraction
extractioncompared
comparedto to ηη obtained
obtained in this
work (6.83 wt.% for UAE and 10.27 10.27 wt.%
wt.% for
for the
the Soxhlet
Soxhlet extraction).
extraction). Compared to our
experimental work,
experimental work, the authors used different parameters,parameters, such
such as time and solvent, for
the extraction methods.

Figure 4. Comparison of different extraction methods (Soxhlet, UAE, supercritical, and cold macer-
Figure 4. Comparison of different extraction methods (Soxhlet, UAE, supercritical, and cold macera-
ation) on extraction yields (η wt.%). The error bars represent the standard deviation. Different letters
tion) on extraction yields (η wt.%). The error bars represent the standard deviation. Different letters
indicate significant differences (p < 0.05) among different extraction methods.
indicate significant differences (p < 0.05) among different extraction methods.

For Soxhlet extraction, Marin et al. [37], compared to the work herein, used five times
less material (5 g), added the same amount of ethanol, and extracted for 6 h. Obtained
η were 1.8% lower compared to what was obtained in this work (10.27 wt.%). Perko
et al. [27] had very similar supercritical extraction parameters (20–30 MPa, 60 ◦ C) to our
experimental conditions. The authors did not use a co-solvent, which resulted in a lower
yield (5.2%) compared to our experimental results, where the use of a co-solvent allowed
for the extraction of both non-polar and polar components, resulting in a higher yield of
8.7 wt.%.
 the extraction of both non-polar and polar components, resulting in a higher yield of
wt.%.
Therefore, we can conclude that the yield % of extraction is influenced by differ
Foods 2023, 12, 4000 factors such as the amount of material used, the type of solvent [38], contact8 time of 14 [39], a
the extraction method [40]. Additionally, temperature [41] is a crucial variable that c
impact the efficiency of extraction, as both methods, Soxhlet and supercritical, have hig
efficiencies wherewe
Therefore, thecan
temperature
conclude thatisthe
higher than
yield % room temperature.
of extraction is influenced by different
factors such as the amount of material used, the type of solvent [38], contact time [39], and
the extraction method [40]. Additionally, temperature [41] is a crucial variable that can
3.3. Spectrophotometric Analysis
impact the efficiency of extraction, as both methods, Soxhlet and supercritical, have higher
3.3.1.efficiencies
The Content whereofthe
Total Phenolic
temperature Compounds
is higher than roomintemperature.
Extracts
Total
3.3. phenols in Analysis
Spectrophotometric turmeric extracts obtained by different extraction methods
shown inThe
3.3.1. Figure 5a.ofThe
Content Totalresults
Phenolicare shown as
Compounds a mass of gallic
in Extracts acid in mg per 100 g
material.Total
Thephenols
supercritical method
in turmeric extractsyielded
obtained the highestextraction
by different contentmethods
of totalarephenolic
shown com
nents,in i.e.,
Figure538.95 mgresults
5a. The GA/100 g of material.
are shown The
as a mass of lowest
gallic total
acid in phenolic
mg per 100 g ofcontent
material.was det
mined Theusing
supercritical
the coldmethod yielded extraction
maceration the highest content
(178.42ofmgtotal phenolic
GA/100 g components,
of material).i.e.,
When co
538.95 mg GA/100 g of material. The lowest total phenolic content was determined using
paring the phenolic content of the extracts between extraction methods, the Kruskal–W
the cold maceration extraction (178.42 mg GA/100 g of material). When comparing the
lis statistical test confirmed
phenolic content significant
of the extracts differences
between extraction between
methods, the extractsstatistical
the Kruskal–Wallis (χ(3) = 10.385,
0.016).
test confirmed significant differences between the extracts (χ(3) = 10.385, p = 0.016).

Figure
Figure 5. Characterisation
5. Characterisation ofof
thetheextracts
extracts obtained
obtained by bydifferent extraction
different methods;
extraction (a) the(a)
methods; total
the total p
phenols content (in GA equivalents), (b) the proanthocyanidins content (PAC), (c) antioxidant activity
nols content (in GA equivalents), (b) the proanthocyanidins content (PAC), (c) antioxidant activ
determined based on DPPH• method (in % inhibition), and (d) antioxidant activity determined
determined based+•on DPPH• method (in % inhibition), and (d) antioxidant activity determin
based on ABTS method (in Trolox equivalents). The error bars represent the standard deviations.
based on ABTS+• method (in Trolox equivalents). The error bars represent the standard deviatio
Different letters indicate significant differences (p < 0.05) among different extraction methods.
Different letters indicate significant differences (p < 0.05) among different extraction methods.
3.3.2. The Content of Proanthocyanidins in Extracts
3.3.2. TheFigure 5b presents
Content the fraction of proanthocyanidins
of Proanthocyanidins in Extracts in turmeric extracts. The results
are shown as a mass of proanthocyanidins in mg per 100 g of material (mg PAC/100 g ma-
FigureTurmeric
terial). 5b presents
extracts the fraction
were ofby
obtained proanthocyanidins in turmeric
different extraction methods. All theextracts.
extractionThe resu
are shown
methodsasused,
a mass
apartof fromproanthocyanidins
the UAE, resulted inin mg per
a similar 100 gofofproanthocyanidins.
number material (mg PAC/10
material). Turmeric
The highest extracts
proportion were obtained was
of proanthocyanidins by different
obtained byextraction methods.
Soxhlet extraction (4.77All
mgthe extr
PAC/100 g material), while the lowest proportion of proanthocyanidins
tion methods used, apart from the UAE, resulted in a similar number of proanthoc was obtained by
UAE (2.71 mg PAC/100 g material). ANOVA was used for comparing proanthocyanidin
nidins. The highest proportion of proanthocyanidins was obtained by Soxhlet extract
content between extraction methods and confirmed significant differences between extract
groups (F(3) = 75.92, p < 0.001).
Foods 2023, 12, 4000 9 of 14

3.3.3. Antioxidant Activity

Figure 5c shows that the sample prepared by the supercritical extraction method
using ethanol as co-solvent resulted in the highest antioxidant activity (64.27% inhibition
determined by the DPPH• method), whereas the sample prepared by UAE resulted in the
lowest antioxidant activity (33.41% inhibition determined by DPPH• method). Fernández-
Marín et al. [37] reported that the Soxhlet method resulted in lower inhibition (49.0%)
compared to our study (51.52% inhibition). The extraction methods, with a degree of
freedom of three, were analysed for their statistical differences using the Kruskal–Wallis
statistical test. The test confirmed significant differences between the antioxidant activity of
the extracts depending on the extraction method for the DPPH• method of the antioxidant
assay (χ(3) = 10.385, p = 0.016)
Figure 5d shows that there were significant differences between the samples, con-
firmed with the Kruskal–Wallis statistical test (χ(3) = 10.385, p = 0.016). The highest
proportion of antioxidant activity by the ABTS+• method was determined by the UAE
method (1750.32 mg Trolox/g DW), while the lowest proportion of antioxidant activity was
obtained by the supercritical method (1216.87 mg Trolox/g DW).
There was a visible discrepancy in the antioxidant activity of the samples determined
by the two different methods. There could be several reasons. One of them is the different
solubility in the two solvents. In the case of DPPH• , the samples were dissolved in
methanol, whereas in the case of ABTS+• , they were dissolved in ethanol. Certain bioactive
compounds may not be soluble in the reaction media and may not exert radical-scavenging
activities. More likely is the reaction mechanism. ABTS+• and DPPH• both act as stable
free radicals but have different reaction mechanisms. ABTS+• is oxidised by antioxidants,
resulting in a colour change from green to blue. DPPH• , on the other hand, is reduced by
antioxidants, resulting in a decolourisation of the purple solution [42,43].
The results are in contrast when comparing the two methods used, DPPH• and
ABTS+• , to determine antioxidant activity. In the DPPH• method, the sample obtained by
the supercritical method had the highest antioxidant activity, and the sample obtained by
the UAE method had the lowest antioxidant activity, whereas, in the ABTS+• method, the
sample obtained by UAE had the highest antioxidant activity, and the sample obtained by
supercritical extraction had the lowest antioxidant activity. The high content of antioxidant
activity in the ABTS+• method shows how potently antioxidant turmeric is, as, despite
the high dilution of the sample, a considerable proportion was still present in the solution.
Based on Figure 5c,d, it can be concluded that the DPPH• method is the most suitable for
compounds extracted by supercritical CO2 and ethanol, while the ABTS+• method is more
suitable for the analysis of extracts prepared with ethanol by UAE (polar components).

3.4. HPLC-MS/MS Analysis of Curcumin

Figure 6 presents the curcumin content in the extracts obtained by different extraction
methods. The results are given as milligrams of the component per g of material. Figure 6
shows that the extract obtained by UAE contains the highest content of curcumin (1.91 mg
curcumin/g material), while there are no significant differences between the other extrac-
tion methods (supercritical, cold maceration, Soxhlet), whereas the contents of curcuminoid
are somewhat lower. The reason for the high content is most likely the ultrasound waves
breaking down the cell walls and causing an intense secretion of curcumin and solubility
of compounds in ethanol. For curcumin content using different extraction methods, the
Kruskal–Wallis statistical test was performed, but significant differences between extraction
methods could not be confirmed (χ(3) = 7.051, p = 0.0702).
Foods 2023, 12, x FOR PEER REVIEW 10 of 14
Foods 2023, 12, x FOR PEER REVIEW 10 of 14

solubility of compounds in ethanol. For curcumin content using different extraction meth-
solubility of compounds in ethanol. For curcumin content using different extraction meth-
Foods 2023, 12, 4000 ods, the Kruskal–Wallis statistical test was performed, but significant differences between
10 of 14
ods, the Kruskal–Wallis statistical test was performed, but significant differences between
extraction methods could not be confirmed (χ(3) = 7.051, p = 0.0702).
extraction methods could not be confirmed (χ(3) = 7.051, p = 0.0702).

Figure 6. Content of curcumin in extracts using different extraction methods. The error bars repre-
Figure 6. Content of curcumin in extracts using different extraction methods. The error bars repre-
sent the6.standard
Figure Content deviation.
of curcuminDifferent letters
in extracts usingindicate significant
different differences
extraction (p < 0.05)
methods. The error among differ-
bars represent
sent the standard deviation. Different letters indicate significant differences (p < 0.05) among differ-
ent
the extraction methods. Different letters indicate significant differences (p < 0.05) among different
standard deviation.
ent extraction methods.
extraction methods.
3.5. Kinetics for the Extracts Obtained with Supercritical Fluids
3.5. Kinetics
3.5. Kinetics for
for the Extracts Obtained
the Extracts Obtained with
with Supercritical
Supercritical Fluids
Fluids
To determine the effect of temperature on extraction efficiency, a study was per-
To determine
To determinethe theeffect
effect
of of temperature
temperature on extraction
onand
extraction efficiency, a study was per-
formed at the temperature range between 40 60 °C atefficiency,
constant asolvent
study was
and performed
co-solvent
formed at the
at theattemperaturetemperature
rangeThe range
between between 40◦and
40 and 60 ratio 60 °C at
C at constant constant
solvent solvent and
and co-solventco-solvent
flow at
flow 10 °C intervals. solvent-to-feed (S/F) ratio was calculated for samples
flow
10 ◦ C at 10 °C intervals. The solvent-to-feed ratio (S/F) ratio was calculated for samples
taken after 15, 30, 60, and 120 min. The increase in temperature had a negative effectafter
intervals. The solvent-to-feed ratio (S/F) ratio was calculated for samples taken on
taken
15, 30,after 15, 120
60, yield,
and 30, 60, and 120increase
min. The increase in temperature had aeffect
negative effect on
extraction as min.
shown The
in Figure 7.in Attemperature
the pressurehad of 25a MPa,
negative on extraction
the extraction yield de-
extraction
yield, aswithyield, in
shown as shown
Figure in
7. Figure
At the 7. At the pressure
pressure of 25 MPa, the extractiondecreased
yield de-
creased increasing temperature, which canofbe25attributed
MPa, thetoextraction
a decreaseyield
in solvent den-
creased
with with increasing
increasing temperature, which can be attributed to a decrease in solvent den-
sity. The highesttemperature, which
extraction yield can
was be attributed
obtained after to a decrease
120 min at 40 in°C,
solvent
and density. The
it was 11.16
sity. Theextraction
highest highest extraction
yield was yield wasafter
obtained obtained
120 after
min at 120◦ C,
40 min
and atit40was
°C,11.16
and wt.%.
it was 11.16
wt.%.
wt.%.

Figure 7. Extraction yield (wt.%) as a function of solvent-to-feed ratio (S/F) for supercritical extrac-
7. Extraction
Figure 7. Extractionyield
yield(wt.%)
(wt.%)asasa afunction ofof
function solvent-to-feed ratio
solvent-to-feed (S/F)
ratio forfor
(S/F) supercritical extraction
supercritical extrac-
tion at pressure 25 MPa and temperatures 40, 50, and 60 °C.
◦ C.
tion
at at pressure
pressure 25 MPa
25 MPa and temperatures
and temperatures 40,and
40, 50, 50, 60
and 60 °C.

For the same samples from the study shown in Figure 7, the release of curcumin as a
function of S/F ratio was analysed. Figure 8 shows that the content of isolated curcumin
was inversely proportional with S/F ratio. Figure 8 shows that the extract obtained at 60 ◦ C
had higher curcumin contents than extracts obtained at lower temperatures. At an S/F
Foods 2023, 12, x FOR PEER REVIEW 11 of 14

For the same samples from the study shown in Figure 7, the release of curcumin as a
Foods 2023, 12, 4000 11 of 14
function of S/F ratio was analysed. Figure 8 shows that the content of isolated curcumin
was inversely proportional with S/F ratio. Figure 8 shows that the extract obtained at 60
°C had higher curcumin contents than extracts obtained at lower temperatures. At an S/F
ratio ◦
ratio of
of around
around33and
andaatemperature
temperatureofof6060C,
°C,the sample
the with
sample thethe
with highest curcumin
highest content
curcumin con-
(1.14 mg curcumin/g material) was obtained.
tent (1.14 mg curcumin/g material) was obtained.

Figure 8. Curcumin content in the extract obtained by supercritical extraction at 25 MPa and 40, 50,
Figure 8. Curcumin content in the extract obtained by supercritical extraction at 25 MPa and 40, 50,
60 °C as a function of solvent-to-feed ratio.
60 ◦ C as a function of solvent-to-feed ratio.
4. Discussion
4. Discussion
Tosummarise,
To summarise,the thehighest
highest extraction
extraction yield
yield waswas obtained
obtained by Soxhlet
by Soxhlet extraction
extraction (10.27
(10.27 wt.%),
followed by supercritical extraction and cold maceration, and the lowest yield waswas
wt.%), followed by supercritical extraction and cold maceration, and the lowest yield ob-
obtained
tained byby UAEUAE (6.83
(6.83 wt.%).DPPH
wt.%). DPPH • •and ABTS+•+• assays
andABTS assays resulted
resulted in in different
different values for
the antioxidant ability of the extracts. It is assumed that differences differences between
between DPPHDPPH•• and
ABTS +
+••
ABTS radical-scavenging
radical-scavenging activities can be ascribed to reaction mechanism. mechanism. When DPPH DPPH••
was used, the sample prepared by the supercritical extraction method using ethanol as a
co-solvent had the highest antioxidant activity, with with 64.27% inhibition. Since DPPH•• is a
64.27% inhibition.
method applicable to extracts with more more phenolic
phenolic compounds,
compounds, especially
especially water-soluble
water-soluble
ones, the inhibition is consequently higher in extracts with a higher content of phenolic
compounds, which is in the extract after supercritical extraction. The lowest total phenolic
content was measured by cold cold maceration
maceration (178.42
(178.42 mgmg GA/100
GA/100 g material) and showed
55.58% inhibition
inhibitionusing usingDPPHDPPH• •method.
method.Not Notonly
onlyphenolic
phenolic compounds,
compounds, butbut
also proantho-
also proan-
cyanidins, are components that affect the level of antioxidant activity
thocyanidins, are components that affect the level of antioxidant activity in the extract. in the extract. Thus,
extracts containing a higher content of proanthocyanidins show
Thus, extracts containing a higher content of proanthocyanidins show a higher % of inhi- a higher % of inhibition
when • method.
bition using
whenDPPH using DPPH This can
• method. alsocan
This be also
observed in the extract
be observed in the obtained by Soxhlet
extract obtained by
extraction, where the
Soxhlet extraction, content
where theof phenolic
content compounds
of phenolic is low (278.29
compounds is lowmg GA/100
(278.29 mggGA/100
materialg
and highand
material in proanthocyanidins
high in proanthocyanidins (4.77 mg (4.77
PAC/100 g material).
mg PAC/100 The extract
g material). Theobtained by
extract ob-
UAE
tainedcontained 191.94 mg191.94
by UAE contained GA/100 mg gGA/100
material and 2.71and
g material mg 2.71
PAC/100 g material,
mg PAC/100 which
g material,
were the lowest values among the extracts. The second antioxidant
which were the lowest values among the extracts. The second antioxidant activity method activity method used
was ABTS +• . Based on the ability of antioxidant compounds, the DPPH• and ABTS+•
used was ABTS . Based on the ability of antioxidant compounds, the DPPH and ABTS+•
+• •
have
have different
different reaction
reaction mechanisms.
mechanisms. The DPPH•• ability
The DPPH ability ofof free
free radical
radical scavenging
scavenging of of aa
compound is based on the compound’s ability to donate hydrogen
compound is based on the compound’s ability to donate hydrogen atoms. Meanwhile, the atoms. Meanwhile, the
ABTS + • ability of
ofantioxidant
antioxidant compounds
compounds is is based
based onon the
the ability
ability of
of antioxidant
antioxidant compounds
compounds
ABTS+• ability •
to
to stabilise+•free radical compounds by donating proton radicals [44]. As a the
stabilise free radical compounds by donating proton radicals [44]. As a result, DPPH
result, the
and
DPPH ABTS
• and ABTSprovide differentdifferent
+• provide information about free
information radical
about freeand antioxidant
radical activity [45].
and antioxidant ac-
In the case of the UAE
tivity [45]. In the case of +the extract,
UAE the differences
extract, the between
differences the antioxidant
between the activity
antioxidant DPPH•
by activity
(Figure 5c) and by ABTS • (Figure 5d) confirmed that assumption. As the extract obtained
by UAE contained the highest curcuminoid content (1.91 mg curcumin/g material), and the
other extraction methods (supercritical, cold maceration, Soxhlet) did not show significant
differences in curcuminoid content when comparing both, the differences in antioxidative
activity of those extracts represent minor components such as phenolic acids and proan-
Foods 2023, 12, 4000 12 of 14

thocyanidins. Studies have shown a correlation between antioxidant activity by DPPH•

and total phenolic content in many plants [46], and this has been shown to be the case for
turmeric. At the same time, the content of curcumin (Figure 6) had influence on higher
antioxidative activity by ABTS+• in extracts with low content of phenolic compounds
and proanthocyanidins.

5. Conclusions
The aim of this study was to obtain Curcuma longa extracts using different extraction
methods (Soxhlet extraction, UAE, supercritical extraction, and cold maceration) and to
determine the optimal extraction method for the isolation of bioactive components from
turmeric (antioxidants, proanthocyanidins, and total phenolics) with high antioxidative
value. The main component extracted was curcumin (analysed by HPLC-MS/MS). The
content of other components in turmeric extracts was determined by spectrophotometric
analysis. Ethanol proved to be an excellent solvent in all extraction methods used to isolate
valuable components from turmeric powder.
The study of kinetics was performed with pressurised CO2 extraction. For samples
taken after 15, 30, 60, and 120 min, extraction yield was calculated, and curcumin content
was measured as a function of solvent-to-feed ratio (S/F). Increasing the temperature and
pressure had a favourable effect on the increase in the extraction yield and the content of
bioactive components in the individual samples.
The present study has confirmed that turmeric is an excellent and potent antioxidant
containing a high content of total phenolics and curcuminoids. Because of all these proper-
ties, there is high potential to develop applications to facilitate the uptake of the extract
into the human body and to monitor its release.

Author Contributions: Conceptualisation, G.S. and M.K.M.; formal analysis, P.K., A.O. and M.F.;
investigation, G.S.; statistical software, V.P.; resources, G.S., M.K.M. and Ž.K.; writing—original
draft preparation, G.S.; writing—review and editing, M.K.M., Ž.K., P.K. and M.F.; visualisation, G.S.;
supervision, M.K.M. and Ž.K; funding acquisition, Ž.K. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was founded by the Slovenian Research and Innovation Agency programme,
grant numbers P2-0046 (Separation processes and production design) and P2-0118, and research
project grant numbers J2-1725, L2-3175, and L2-9199.
Data Availability Statement: The original contributions presented in the study are included in the
article; further inquiries can be directed to the corresponding author.
Acknowledgments: Special thanks are given to the Slovenian Research and Innovation Agency
(ARIS) for financial support of research program P2–0046: Separation processes and production
design, research projects J2-1725: Smart materials for bio-applications, L2-3175: Advanced Extraction
and Formulation of Functional Tannin Food Supplements with Beneficial Health Effects, and L2-9199:
Purification and formulation of chemicals using supercritical fluids, and the scholarship awarded
to G.S.
Conflicts of Interest: The authors declare no conflict of interest.

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