Preparative Biochemistry and Biotechnology

ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20

Extraction of curcuminoids from Curcuma longa:

comparative study between batch extraction and
novel three phase partitioning

Sujata S. Patil, Siddhant Bhasarkar & Virendra K. Rathod

To cite this article: Sujata S. Patil, Siddhant Bhasarkar & Virendra K. Rathod (2019):
Extraction of curcuminoids from Curcuma�longa: comparative study between batch extraction
and novel three phase partitioning, Preparative Biochemistry and Biotechnology, DOI:
10.1080/10826068.2019.1575859

To link to this article: https://doi.org/10.1080/10826068.2019.1575859

Published online: 01 Mar 2019.

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PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2019.1575859

Extraction of curcuminoids from Curcuma longa: comparative study between

batch extraction and novel three phase partitioning
Sujata S. Patil, Siddhant Bhasarkar, and Virendra K. Rathod
Chemical Engineering Department, Institute of Chemical Technology, Mumbai, India

ABSTRACT KEYWORDS
Curcuminoids, the active components of dried rhizome of Curcuma longa have been extracted Anti-inflammatory; batch
using batch extraction and three-phase partitioning (TPP) process. The effect of different process- extraction; curcuminoids;
ing parameters, namely different solvents, extraction time, ammonium sulfate concentration, slurry three phase partitioning;
turmeric rhizomes
to tert-butanol ratio, and solute to aqueous ratio on extraction efficiency of TPP, was studied to
attain maximum extraction yield. The highest yield of 58.38 mg/g was achieved at 40±2
C in
150 min, with saturated ammonium sulfate 30% (w/v), slurry to tert-butanol ratio 1:1 (v/v), and tur-
meric powder to water ratio 1:40 (w/v) in TPP. However, batch extraction using ethanol as a solv-
ent yielded 52.77 mg/g in 180 min extraction time at 40±2
C with 1:40 (w/v) turmeric powder to
water ratio and 400 rpm agitation speed. In view of reference method, i.e., Soxhlet extraction
(100%), TPP showed 65.63% yield in 150 min and batch exhibited 59.92% in 180 min. The turmeric
extracts obtained by different methods exhibited excellent antioxidant and anti-inflammatory
activities equivalent to their respective reference standards. Hence, TPP extraction process assures
a rapid and improved recovery of curcuminoids with excellent therapeutic properties.

GRAPHICAL ABSTRACT
Curcuminoids, the active components of dried rhizome of Curcuma longa has been extracted
using batch extraction and three-phase partitioning (TPP) process.

RESEARCH HIGHLIGHTS
 Batch and three phase partitioning (TPP) methods were successfully studied and compared for
curcuminoids extraction.
 Influence of different processing parameters on TPP and batch were optimized.
 Three phase partition gives more yield than the batch extraction process.
 The extracts obtained showed excellent antioxidant and anti-inflammatory properties.
 TPP was found to be rapid method of extraction compared to batch process.

CONTACT Virendra K. Rathod vk.rathod@ictmumbai.edu.in Chemical Engineering Department, Institute of Chemical Technology, Mumbai 400019, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.
ß 2019 Taylor & Francis Group, LLC
2 S. S. PATIL ET AL.
Introduction
The dried rhizome of Curcuma longa L. is also known as
turmeric and belongs to Zingiberaceae family. Curcuminoids
are the active components of dried rhizome of C. longa L.
which is widely cultured in tropical and subtropical regions
of the world, namely Asia and Central America.[1]
Curcuminoids consist of three most fundamental constitu-
ents, namely curcumin (75%), demethoxycurcumin
(10–25%), and bisdemethoxycurcumin (5%) which belongs
to the diferuloylmethane group of phenolic compounds.[2]
Amongst the various phenolic compounds, curcuminoids
have been extensively used as pigments in food processing
to augment the nutritional and sensory values of foods.[3]
Several studies have also been focused on the applications of
curcuminoids in the pharmaceutical industry to treat
numerous disorders such as cancer, hepatic ailments, inflam-
mation, oxidative stress, and diabetes.[4–6] They also exhibit
both in vitro and in vivo antimalarial activity.[7] Hence, in
recent years, tremendous research has been focused on the
extraction of curcuminoids and its applications.
Though chemical methods can be implemented to synthesize
curcumin, the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) specifications permits those curcuminoids as
food additives which are extracted from natural sources. In the
literature, application of various conventional techniques for the
extraction of curcuminoids from natural sources involves
organic solvents extraction,[8] steam distillation,[9–11] hot and
cold percolation,[12] use of hydrotrope,[13] and alkaline solu-
tion.[14] In addition, several advanced techniques have been
studied like supercritical fluid extraction,[15] microwave-assisted
extraction,[2,16] ultrasound-assisted extraction,[17,18] and enzyme
assisted extraction.[19] These advanced methods like ultra-
sound, supercritical, and microwave-assisted extraction are
still at an emerging stage in India for the extraction of nat-
ural biomolecules and also have scalability issues. Hence, it is
necessary to develop a simple extraction process which con-
sumes low energy and less space and requires simple setup
resulting in adequate extraction yield. The present study
demonstrated the extraction of curcuminoids by batch
extraction with ethanol as solvent. Also, batch extraction of
curcuminoids was compared with a novel technique like
three-phase partitioning and conventional Soxhlet method as
reference. Three-phase partitioning (TPP) process involves
ammonium sulfate addition to the slurry (aqueous) of raw
material or crude extract, subsequently tert-butanol addition.
Water and tert-butanol are miscible though, by the ammo-
nium sulfate addition at the adequate amount, the reaction
slurry gets distributed into immiscible liquid phases; lower
aqueous (water) and upper organic (tert-butanol).[20] TPP is
based on this simple theory which is recent bioseparation
technique for proteins. However, actual mechanism is not
familiar to the researchers, there are postulated key factors
for the targeted protein partitioning such as cosolvent pre-
cipitation, protein hydration shifts, kosmotropic effect,
isotonic precipitation, osmolytic, and salting out.[21] Usually,
pigments and lipids are extracted in the upper organic
phase (tert-butanol), whereas polar moieties get accumulated
in the lower aqueous phase. TPP is simultaneous extraction
and purification technique which is easily scalable,
inexpensive and carried out at room temperature and can be
directly used with crude suspension.[22] It has predominantly
been explored for the enzymes purification such as peroxid-
ase from Momordica charantia,[23] b-galactosidase,[24] invert-
ase,[25] and for natural products extraction, namely
mangiferin from Mangifera indica,[26] and acetyl 11-keto-
b-boswellic acid from oleo gum resin of Boswellia serrata.[27]
The detailed literature reveals that there have been no
reports available on conventional three phase partitioning
and comparison study between three-phase partitioning and
batch extraction process of curcuminoids from C. longa
except for earlier work by Kurmudle et al.[28] The reported
study focused on three-phase partitioning assisted by
enzyme to extract turmeric oleoresin under optimized fac-
tors, viz. enzyme concentration, ammonium sulfate concen-
tration, slurry to tert-butanol ratio, and pH. However, they
have not discussed the effect of each parameter in detail on
the TPP process and also not quantified curcuminoids using
high performance liquid chromatography (HPLC) as analyt-
ical technique.[28] The present work deals with the three-
phase partitioning of curcuminoids from C. longa and com-
pared with batch and Soxhlet extraction processes. Further,
optimization of various process parameters affecting TPP
and batch was successfully done to get optimal extraction
yield of curcuminoids.
Materials and methods
Materials
Dried turmeric rhizomes (C. longa) and pure curcumin
(95%) were obtained from Konark Herbals and Health Care,
Mumbai, India. Rhizomes of C. longa were ground in a
mixer and screened for 20 min to get the particle size
<425 mm. Ammonium sulfate, ethanol (99% purity) and sol-
vents of analytical grade were procured from Thomas
Bakers Pvt. Ltd. India. All other chemicals were of analytical
grade (AR) and procured from reliable sources which were
used without any further purification. For HPLC analysis,
tetrahydrofuran solvent of HPLC grade was purchased from
Rankem Chemicals, Mumbai, India.
Methods
Soxhlet extraction
In Soxhlet extraction, 5 g of C. longa powder was placed in a
thimble-holder to bring in contact with the fresh solvent
each time which is condensed from the solvent reservoir.
Two hundred and fifty milliliters of ethanol (99%) was used
as a solvent and added in distillation flask. The extraction
process was carried out for 12 h at 60
C. Samples were
withdrawn from the solvent reservoir, filtered subsequently
and analyzed using HPLC for curcuminoids content. The
88.96 mg/g yield of curcuminoids was obtained from C.
longa by Soxhlet extraction was considered to be maximum
yield and all experimental values obtained by TPP and batch

method were compared with this maximum yield of curcu-
minoids in the source.
Three phase partitioning of curcuminoids
TPP process was optimized using a baffled glass reactor of
50 mL capacity with 3.5 cm diameter and 7 cm height along
with four pitched bladed glass turbines for agitation. The
aqueous slurry of turmeric powder was formed by suspend-
ing 0.5 g of C. longa powder in 20 mL distilled water by gen-
tle stirring to obtain a ratio of solid to liquid to 1:40. Then,
30% (w/v) ammonium sulfate concentration was added into
turmeric powder slurry followed by addition of 20 mL of
tert-butanol (1:1 v/v). The extraction was carried out for 3 h
at 300 rpm at 30±2
C. The mixture was further centrifuged
at 8000  g for 15 min to form the partitioning of three
phases. The upper organic layer was collected and quantified
for its curcuminoids content using HPLC. TPP process
was optimized by varying different process parameters
such as solvents, ammonium sulfate concentration, namely
10, 20, 30, 40, 50% (w/v), solute to aqueous ratio from 1:20
to 1:60 (w/v) and slurry to tert-butanol ratio from 1:0.5
to 1:3 (v/v). Extraction time was evaluated from 5 min to
3 h to achieve the utmost extent of extraction yield of
curcuminoids.
Figure 1. HPLC Profiles of Curcuminoids: (a) standard curcuminoids, curcuminoids obtained after (b) Soxhlet extraction, (c) batch extraction, and (d) Three Phase
Partitioning.
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 3
Batch extraction
For batch extraction, 1.5 g quantity of C. longa powder was
taken in 50 mL capacity of glass reactor and 30 mL ethanol
as a solvent was added to it. The glass reactor was equipped
with four pitched bladed glass turbines for mixing. The mix-
ture was stirred using propeller of 3.5 cm diameter and 7 cm
height. The temperature of 30±2
C was maintained and agi-
tation speed of 400 rpm was provided to ensure homoge-
neous mixing of raw material and solvent. Samples were
collected at a regular interval of time, filtered, and further
analyzed using HPLC for curcuminoids content.
Quantification of curcuminoids
Curcuminoids was analyzed using Agilent 1260 infinity
high-performance auto sampler HPLC on C18 column
(5 mm  250 mm  4.6 mm). Initially, HPLC column was
cleaned up for 1 h by elution and subsequently, washing
was done for another 1 h using acetonitrile. The column was
then equilibrated by eluting mobile phase for 30 min and
simultaneously, the baseline run was observed. For HPLC
analysis, the mobile phase used was the mixture of tetra-
hydrofuran (THF) and acidified distilled water (40:60) which
was filtered through a membrane filter (0.22 mm) under vac-
uum before use. The analysis was performed with the flow
rate of 0.8 mL/min using isocratic elution at 27±2
C. The
4 S. S. PATIL ET AL.
samples of 5 mL were injected into the HPLC and eluted cur-
cuminoids were detected at 420 nm; subsequently quantified
from the standard curve.
The typical HPLC chromatogram of standard curcumi-
noids, curcuminoids after Soxhlet, batch, and TPP extraction
is shown in Figure 1a–d. The retention time of curcumi-
noids was 12.02 min for curcumin, 13.28 min for demethox-
ycurcumin, and 15.58 min bisdemethoxycurcumin.
Determination of therapeutic properties of the extracts
Total phenolic content and anti-oxidant activity
Total phenolic content in the extract from the batch process
and TPP process was estimated as per described method by
Rao and Rathod in 2015 using Folin–Ciocalteau reagent.[29]
Gallic acid was used as a reference standard. The phenolic
content was expressed as mg of gallic acid equivalent (GAE)
per gram of extract.
Anti-oxidant activity of the extracts obtained under opti-
mized conditions of batch and TPP process and Soxhlet
extraction was evaluated using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging assay proposed by Blois
(1958).[30] Gallic acid was used as the reference compound.
The percentage of free radical scavenging was determined
for the different concentrations of extracts (25–150 mg/mL)
using the following equation:
 
Asample
% scavenging ¼ 1   100;
ADPPH
where ADPPH = Absorbance of DPPH as a control; Asample =
Absorbance of test sample
The graph was plotted between the percentage of DPPH
scavenging along y-axis and concentration of samples along
the x-axis. From calibration curves, obtained from the differ-
ent concentration of extracts and gallic acid, the IC50
(inhibitory concentration 50%) can be calculated. The IC50
value denotes the concentration of antioxidant required to
scavenge 50% of the DPPH free radicals.[31]
Anti-inflammatory activity
In vitro anti-inflammatory activity of extracts obtained from
batch, TPP, and Soxhlet extraction was evaluated using three
different assays such as inhibition of albumin denaturation,
nitric oxide radical scavenging assay, and proteinase inhibi-
tory assay. Acetylsalicylic acid (ASA), i.e., aspirin, was used
as a reference anti-inflammatory standard.
The potential of the extracts to inhibit the albumin
denaturation was determined as per the protocol followed
by Nagavekar and Singhal.[32] The absorbance was recorded
at 660 nm on a spectrophotometer (JASCO, Japan). The per-
cent inhibition of albumin denaturation was calculated using
the following formula:
 
At
% inhibition ¼ 1  100;
Ac
where Ac = absorbance of control; At = absorbance of test
or reference drug.
The scavenging assay for nitric oxide (NO) radical was
evaluated using the method described by Boora et al.[33]
while Senanayake et al. reported method was used to deter-
mine proteinase inhibition in terms of antitrypsin activ-
ity.[34] The absorbance was recorded at 546 and 410 nm,
respectively on spectrophotometer with methanol as a
medium. The percent inhibition was obtained using the fol-
lowing formula:
 
ðAs  Asb Þ
% inhibition ¼ 1  100;
ðAc  Acb Þ
where Ac = absorbance of control; Acb = absorbance of con-
trol blank; As = absorbance of sample; Asb = absorbance of
sample blank.
Required percent inhibition was obtained at 100 mg/mL
concentration of extracts and aspirin.
Scanning electron microscopy
The morphology of the surface of C. longa powder before
extraction, after batch and TPP process, was determined
using scanning electron microscopy (JEOL – JSM –
6360 A, Japan).
Statistical analysis
All Experiments were performed in triplicates and the data
was analyzed using Microsoft Excel (2010) and SPSS version.
One way ANOVA was also incorporated to test the mean
differences amongst all the treatments. The statistical signifi-
cance of differences between the mean values was estab-
lished at p  0.05 and Duncan’s New Multiple Range Test
was applied for all statistical analyses. Results were expressed
as the mean ± SE.
Results and discussions
Three-phase partitioning of curcuminoids
Effect of solvents on three-phase partitioning of
curcuminoids
A suitable solvent for the extraction of a desired natural
ingredient from the matrix is a vital factor in any extraction
technique. In the present study, five different solvents, i.e.,
methanol, ethanol, tert-butanol, iso-propanol, and n-butanol
were used to understand their effect on the three-phase par-
titioning of curcuminoids at 30±2
C. The solvents were
selected on the basis of desired component’s solubility,
polarity, and overall cost and safety. Other constant experi-
mental conditions were 40% (w/v) ammonium sulfate load-
ing, 1:1 (v/v) slurry: tert-butanol ratio, 1:50 (w/v) solute to
aqueous ratio and time 120 min. Three phases formation
occurred in case of ethanol, tert-butanol, iso-propanol, n-
butanol while phase separation was not observed with
methanol as an extracting solvent. Table 1 depicts the
extraction yield of curcuminoids with ethanol, tert-butanol,
iso-propanol, and n-butanol. The maximum recovery of cur-
cuminoids was observed in tert-butanol solvent compared to
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 5

Table 1. Influence of different process parameters on TPP of curcuminoids from C. longa L.

Effect of Ammonium Effect of slurry to Effect of solute to
Effect of solvents sulfate conc. tert-butanol aqueous ratio Effect of temperature

Curcuminoids Ammonium Curcuminoids Slurry to Curcuminoids Solute to aqueous Curcuminoids Temperature Curcuminoids
Solvents yield (mg/g) sulfate conc. (%w/v) yield (mg/g) tert-butanol (v/v) yield (mg/g) ratio (w/v) yield (mg/g) (
C) yield (mg/g)
Ethanol 24.68 ± 0.95d 10 22.03 ± 2.12c 1:0.5 35.69 ± 1.17c 1:20 30.06 ± 1.68c 20 39.43 ± 2.37d
Iso-propanol 29.13 ± 0.56c 20 36.27 ± 1.82b 1:1 50.92 ± 3.00a 1:30 40.43 ± 2.03b 30 50.1 ± 1.87c
n-butanol 33.92 ± 0.56b 30 48.94 ± 3.11a 1:1.5 48.77 ± 1.05a 1:40 48.54 ± 1.72a 40 58.38 ± 1.83a
tert-butanol 44.19 ± 0.52a 40 50.09 ± 3.05a 1:2 40.22 ± 3.02b 1:50 50.2 ± 2.10a 50 55.22 ± 1.82ab
– – 50 49.93 ± 3.47a 1:2.5 35.85 ± 2.01c 1:60 49.4 ± 3.06a 60 51.82 ± 2.64bc
– – – – 1:3 32.08 ± 2.70c – – – –
Values in the same column with different superscript letters are significantly different (p  0.05) as measured using Duncan’s New Multiple Range Test.
All values are mean ± SE of three determinations.

other solvents. Most of the published reports explored tert-

butanol as a solvent in TPP as it showed kosmotropic and
crowding effect at room temperature or above while other
co-solvents such as methanol and ethanol behave only at
near or below zero temperature.[20] tert-butanol does not
allow co-solvent to permeate into the folded proteins
because of their branched structure and size and prevents
the loss of extracted compound from organic phase into
middle layer or in the aqueous phase. Moreover, tert-butanol
proved to be superior to other organic solvents in several
TPP of biomolecule separation.[26,28,35,36] Taken into consid-
eration kosmotropic basis of TPP, tert-butanol was chosen
as the most suitable solvent for extraction of curcuminoids.

Effect of time on three-phase partitioning of curcuminoids

For each process, time plays a vital role from an economic
point of view. Time has been optimized to achieve maximum
extraction of curcuminoids from turmeric. All experiments
were performed separately at different agitation time ranging
from 15 min to 180 at 30±2
C with other experimental condi-
tions, namely 40% (w/v) ammonium sulfate, (1:1 v/v) slurry
to tert-butanol ratio, and (1:50 w/v) solute to solvent ratio
with constant agitation speed. Figure 2a indicates gradual
increase in curcuminoids extraction with an increase in
extraction time from 15 min to 150 min. At the start, there is
high concentration gradient between surface of particle and
the bulk solvent which improves the mass transfer rate.
Further increase in time reduces the mass transfer rate due to
decrease in the concentration gradient. Thus, extraction yield
remained constant with an increase in time from 150 min to
180 min, which indicates that the system reached to equilib-
rium. Hence, all further experiments were carried out for
150 min to optimize other parameters. Figure 2. (a) Effect of Time on TPP extraction process of curcuminoids
(Solvent ¼ tert-butanol, Ammonium sulfate concentration ¼ 30% (w/v),
solute to aqueous ratio ¼ 1:50 (w/v), slurry to solvent ratio ¼ 1:1 (v/v),
Effect of ammonium sulfate loading Temperature ¼ 30 ± 2
C). (b) Effect of time on Batch extraction process of cur-
cuminoids (Solvent ¼ ethanol, solute to solvent ratio ¼ 1:40 (w/v), agitation
Ammonium sulfate concentration plays a crucial role in TPP. speed ¼ 400 rpm, Temperature ¼ 30 ± 2
C). All experiments were performed in
Ammonium sulfate densely hydrates itself because of its triplicates and error bar represent standard error in each set of readings.
effective size which helps to exclude or crowd proteins.[24]
Thus, effect of ammonium sulfate concentration was studied
at different concentrations such as 10, 20, 30, 40, 50% (w/v) at
30±2
C with slurry to tert-butanol ratio 1:1 (v/v), solute to forms the kosmotropic system which we proposed to extract
aqueous ratio 1:50 (w/v) for 150 min. Table 1 shows that with curcuminoids from C. longa in the upper organic phase (tert-
an increase in ammonium sulfate concentration, there is butanol) by precipitating proteins in the middle layer and
increase in curcuminoids yield till 30% (w/v) saturated ammo- leaving carbohydrates in the lower (aqueous) phase. Further
nium sulfate solution. In combination with tert-butanol, it increase in concentration beyond 30% (w/v), there was
6 S. S. PATIL ET AL.
insignificant difference in extraction yield of curcuminoids in
TPP. The results obtained are in accordance with the reported
data by Kurmudle et al. for extraction of turmeric oleoresin
using enzyme assisted three phase partitioning.[28]
Effect of slurry to tert-butanol ratio on three-phase parti-
tioning of curcuminoids
Slurry to tert-butanol ratio was varied from 1:0.5, 1:1, 1:1.5,
1:2, 1:2.5, and 1:3 (v/v) in TPP of curcuminoids. The experi-
mental parameters, namely 30% (w/v) ammonium sulfate
concentration, 1:50 (w/v) solute to aqueous ratio, were used
for 150 min at 30±2
C. In TPP process, highest content of
curcuminoids was obtained with 1:1 (v/v) slurry to tert-buta-
nol ratio; after that, decrease in extraction yield was
observed (Table 1). At lower ratio, i.e., 1:0.5, the less quan-
tity of tert-butanol may not be proficient to effectively syn-
ergize with saturated ammonium sulfate solution.[20]
Whereas with an increase in the amount of tert-butanol
with respect to slurry, i.e. 1:2 and 1:2.5, we observed
decrease in curcuminoids yield. This may be attributed to
the transfer of tert-butanol into aqueous phase which
resulted in the distribution of curcuminoids into both the
phases even though it is water insoluble. This may resulted
into less recovery of curcuminoids content from organic
phase. Thus, 1:1 (v/v) ratio of slurry to tert-butanol was
used to achieve maximum curcuminoids recovery.
Effect of solute to solvent ratio on three-phase partition-
ing of curcuminoids
Effect of solute to aqueous ratio (amount of turmeric pow-
der to water) on TPP was successfully demonstrated at
30±2
C by varying this ratio as 1:20, 1:30, 1:40, 1:50, and
1:60 (w/v) with above mentioned experimental conditions
like 30% (w/v) ammonium sulfate concentration, 1:1 (v/v)
slurry to tert-butanol ratio were used, and 150 min stirring
time was provided in TPP process. The maximum curcumi-
noids extraction was attained with 1:30 (w/v) solute to aque-
ous ratio. Table 1 depicts the obtained results by TPP
process. The reduction in yield at lower solute to aqueous
ratio is might be due to the less amount of water was avail-
able for hydration of sulfate ions.[20] A kosmotropic system
forms only with optimum amount of sulfate and tert-
butanol which leads to the extraction of valuable products
into organic layer, i.e., tert-butanol. On the other hand,
varying solute to aqueous ratio beyond 1:40 (w/v) did not
enhance the extraction yield due to equilibrium. Thus, 1:40
(w/v) ratio of solute to aqueous was selected as optimum for
further experiments.
Effect of temperature on three-phase partitioning of
curcuminoids
To study the effect of temperature on TPP process, experi-
ments were carried out over the range of 20–60
C for the
time period of 150 min with tert-butanol as extracting solv-
ent, 30% (w/v) ammonium sulfate concentration, 1:40 (w/v)
solute to solvent ratio, 1:1 (v/v) slurry to tert-butanol
ratio, and 300 rpm agitation speed. Table 1 illustrates the
extraction yield of curcuminoids at different temperatures.
The highest yield of 58.38 mg/g was obtained at temperature
40
C. This is due to the temperature rise which helps the
greater solvent penetration into cellular matrix and increases
rate of extraction. In addition, solvent density and viscosity
decrease with an increase in temperature which causes mass
transfer intensification by the better way of solvent diffusion
through the opened cell matrix of plant material. As a result
of these effects, availability of curcuminoids increases for the
extraction. On the other hand, with an increase in tempera-
ture beyond 40
C, reduction in the extraction yield of cur-
cuminoids was observed. The volatility of tert-butanol
increases gradually with an increase in temperature resulting
in less amount of solvent available for extraction. Moreover,
1:1 (v/v) ratio of slurry to tert-butanol gets altered and thus
less amount of tert-butanol hampers the combined effect of
kosmotropic system with ammonium sulfate which causes
reduction in the extraction efficiency of TPP.[28,29] However,
it is difficult to hypothesize the cause behind 20–40
C is a
better range to perform TPP because of the convolution
involved in the process parameters.[37] Hence, 40
C
was used as optimum temperature for extraction of
curcuminoids.
Batch extraction
Selection of solvent for batch extraction of curcuminoids
Interaction between extracting solvent and natural ingredi-
ent is very difficult to predict due to the different chemical
structure of natural product. Composition of extract and
amount of yield may vary with different types of solvents.[38]
Like TPP, solvent study was also carried out for batch
extraction of curcuminoids for 180 min of stirring time, 1:40
(w/v) of solute to solvent ratio and 400 rpm agitation speed
at 30±2
C. Figure 3 shows that maximum extraction of cur-
cuminoids was attained in ethanol solvent followed by
Figure 3. Effect of solvents on batch extraction of curcuminoids (solute to solv-
ent ratio ¼ 1:40 (w/v), agitation speed ¼ 400 rpm, Temperature ¼ 30 ± 2
C).
All experiments were performed in triplicates and error bar represent standard
error in each set of readings.
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 7

methanol, tert-butanol, iso-propanol, and n-butanol. This

might be due to the high polarity and vapor pressure, low
viscosity and surface tension of ethanol. Moreover, its high
molecular affinity and solubility towards curcuminoids
resulted in ethanol being a suitable solvent compared
to others.[18]

Effect of agitation time on batch extraction of

curcuminoids
Effect of agitation time on batch extraction of curcuminoids
was performed from 5 to 240 min. All other parameters
such as ethanol as solvent, (1:40 w/v) solute to solvent ratio,
400 rpm agitation speed, and temperature 30±2
C were kept
constant and results obtained are depicted in Figure 2(b).
Similar to TPP, the extraction yield of curcuminoids
increased gradually with an increase in time from 5 min to
180 min and remains steady up to 240 min. Initially, the
increase in curcuminoids extraction is attributed to the large
concentration gradient between curcuminoids present on the Figure 4. Effect of solute to solvent ratio on batch extraction of curcuminoids
surface of cell wall and extracting solvent. Concentration (Solvent ¼ ethanol, agitation speed ¼ 400 rpm, Temperature ¼ 30 ± 2
C). All
experiments were performed in triplicates and error bar represent standard
gradient decreases as time increases which leads to the error in each set of readings.
reduction in extraction rate. Moreover, after 180 min, curcu-
minoids which are available on the surface of turmeric cell
wall are not easy to get diffused into solvent and hence the
extent of extraction yield does not change up to 240 min.
Thus, subsequent studies were carried out till 180 min.

Effect of solute to solvent ratio on batch extraction of

curcuminoids
The solute to solvent ratio, i.e., amount of turmeric powder
to solvent (ethanol), was varied from 1:10 to 1:50 to study
its effect on the extent of curcuminoids extraction using
batch method. This is necessary to optimize the solute to
solvent ratio to avoid excessive usage of solvent. Other
parameters such as ethanol as solvent, 400 rpm agitation
speed, time 180 min, and temperature 30±2
C were kept
constant. From Figure 4, it can be observed that the extent
of curcuminoids extraction increases with an increase in
amount of solvent from 1:10 to 1:40. At higher quantity of
solvent, the concentration gradient between cell matrix and
external solvent is more and thus extraction rate can be
higher.[39] However, further increase in the quantity of solv- Figure 5. Effect of agitation speed on batch extraction of curcuminoids
ent would lead to limited increase in extraction yield. (Solvent ¼ ethanol, solute to solvent ratio ¼ 1:40 (w/v), agitation speed ¼
400 rpm, Temperature ¼ 30 ± 2
C). All experiments were performed in tripli-
Moreover, Lou et al. demonstrated that limited mass transfer cates and error bar represent standard error in each set of readings.
restricts to the only solid interior; hence, driving force
would not change with large quantity of solvent.[40]
Additionally, use of high amount of solvent is not econom-
ical due to high energy requirement during separation. All experiments were performed from 200 to 500 rpm at 1:40
subsequent experiments were performed with 1:40 (w/v) sol- (w/v) solute to solvent ratio and 30±2
C temperature with
ute to solvent ratio. ethanol as solvent for 180 min. It can be observed from
Figure 5 that as speed of agitation increases; the extraction
yield also increases due to decrease in mass transfer resist-
Effect of agitation speed on batch extraction of ance. Further increase in agitation speed has no significant
curcuminoids effect on extraction yield which corroborates that there is
In the batch extraction, turbulence is generated through agi- negligible external mass transfer resistance at 400 rpm. Thus,
tation which leads to high mass transfer rate between solute sufficient agitation speed for extraction of curcuminoids was
and solvent. Hence, to optimize the speed of agitation, found to be 400 rpm.
8 S. S. PATIL ET AL.
Figure 6. Effect of temperature on batch extraction of curcuminoids
(Solvent ¼ ethanol, solute to solvent ratio ¼ 1:40 (w/v), agitation
speed ¼ 400 rpm). All experiments were performed in triplicates and error bar
represent standard error in each set of readings.
Effect of temperature on batch extraction of curcuminoids
Many physical properties of solvent, namely solubility, diffu-
sivity, viscosity, and surface tension change with an increase
in temperature.[26] To study the effect of temperature on
batch extraction, experiments were carried out over the
range of 30–60
C for the time period of 180 min with etha-
nol as extracting solvent, 1:40 (w/v) solute to solvent ratio
and 400 rpm agitation speed. From Figure 6, it can be seen
that the extraction of curcuminoids increases with an
increase in temperature from 30 to 60
C. The diffusivity of
solvent into the plant material improved at higher tempera-
ture resulting in the availability of curcuminoids for extrac-
tion. Moreover, due to high temperature, solvent viscosity
decreases, and solubility of curcuminoids increases, hence
extraction efficiency increases.[26] On the other hand, high
temperature extraction process may lead to degradation of
natural ingredients, energy cost, and volatilization of solvent.
In point of this, though maximum extraction was attained at
50
C, further experiments were performed at 40
C to avoid
the possible degradation of curcuminoids. Also, there is
marginal difference in extraction yield from 40
C to 50
C.
Determination of therapeutic properties of the extracts
Total phenolic content and anti-oxidant activity
Curcuma longa contains other phenolic compounds as well
along with curcucuminoids such as ascorbic acid, 2-Methoxy-
4-vinylphenol, and m-eugenol.[41] From the results, it was
found that total phenolic content of turmeric was highest in
Soxhlet extraction method 143.67 mg GAE/g extract followed
by batch extraction process 82.24 mg GAE/g and TPP process
76.34 mg GAE/g, respectively. The higher yield in Soxhlet
extraction was might be because of higher content of poly-
phenols in the extract compared to TPP and batch process.
Similar results were reported by Andrade et al. in which they
got higher polyphenolic content and antioxidant activity in
Figure 7. Plot of the concentration of extract vs. % DPPH scavenging of
Soxhlet, TPP, and batch process. All experiments were performed in triplicates
and error bar represent standard error in each set of readings.
Table 2. Total phenolic content (TPC), anti-oxidant properties and IC50 values
of extracts, gallic acid, and standard curcumin.
TPC % DPPH scavenging IC50
Soxhlet extract 143.67 ± 0.04a 86.03 ± 0.98a 8.46 ± 0.57
TPP extract 76.34 ± 0.01d 69.7 ± 1.07e 59.04 ± 0.04
Batch extract 82.24 ± 0.96c 73.56 ± 2.64d 47.83 ± 1.21
Gallic acid – 81.04 ± 0.85b 16.24 ± 0.34
Standard curcumin 93.8 ± 0.08b 76.6 ± 1.65c 38.74 ± 0.21
Values in the same column with different superscript letters are significantly
different (p  0.05) as measured using Duncan’s New Multiple Range Test.
All values are mean ± SE of three determinations.
conventional Soxhlet extraction method compared to super-
critical CO2 and supercritical CO2 þ ethanol.[42]
Antioxidant property of C. longa extracts was examined
using DPPH free radical scavenging capability. Serial dilu-
tions ranging from 25 to 150 mg/mL of gallic acid, standard
curcumin, turmeric extracts obtained by Soxhlet, batch and
TPP process exhibited different percentage of inhibition.
The preeminent antioxidant activity was found to be at
150 mg/mL concentration and amongst them, the Soxhlet
extract was the uppermost (86.03%), subsequently batch
(73.56%) and TPP extract (69.7%). The graph of % scaveng-
ing versus concentration of extracts of Soxhlet, TPP, and
batch was depicted in Figure 7. Soxhlet extract showed
higher, whereas TPP and batch extract showed lower anti-
oxidant activity compared to gallic acid and standard curcu-
min. Table 2 illustrated the IC50 values of gallic acid,
standard curcumin, Soxhlet, TPP, and batch extracts and
their respective % scavenging of DPPH free radicals. The
concentration of extract at which half of DPPH radicals will
be scavenged is known as IC50. A lower IC50 value indi-
cates the potential antioxidant activity.[29] The IC50 of
Soxhlet, TPP, and batch extract was observed to be 8.46,
59.04, and 47.83 mg/mL. Soxhlet and batch extract was
obtained using ethanol as a solvent. Qader et al. reported
that ethanolic extracts of C. longa exhibited higher percent

Figure 8. Anti-inflammatory activity of C. longa extracts obtained by different
techniques using (a) albumin denaturation assay, (b) Proteinase inhibition assay,
and (c) NOX radical scavenging assay. All experiments were performed in tripli-
cates and error bar represent standard error in each set of readings.
DPPH inhibition and also showed the positive correlation
between percent DPPH inhibition and total phenolic con-
tent.[43] Thus, Soxhlet extract showed higher percent DPPH
scavenging and then batch and TPP extracts.
Anti-inflammatory activity
The anti-inflammatory activity of the extract was studied by
preventing heat denaturation of protein like albumin.
Damage associated molecular patterns (DAMPs) are pro-
duced after protein denaturation which is due to damage to
the tissue. Scavenging receptors (SRs) identifies these pro-
duced DAMPs. Both DAMPs and SRs together activate
inflammatory mediators like cytokines spreading the inflam-
mation further. For this, albumin which is a frequent skin
protein was considered for denaturation of protein study.
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 9
Therefore, the extent of inhibition of albumin denaturation
is directly related to anti-inflammatory activity of
the extract.
Another important assay for proteinase inhibition can
also be carried out to study the ability of extract to hamper
activity of trypsin. Trypsin plays a crucial role at different
phases of inflammation. When body gets susceptible with a
pathogenic attack, immediately the neutrophils get activated.
During this period of inflammation, these neutrophils
secrete several serine proteases like proteinases-3 (PR3).
However, these proteases protract inflammatory response by
the degradation of opsonins and phagocytic receptors.
Moreover, trypsin along with protease activated receptor-2
(PAR2) activates on their surface. Accumulation of PARs
results into higher inflammation due to the synthesis of
inflammatory mediators from interstitial cells and platelets.
Also, trypsin causes degranulation of eosinophils which lead
to tissue damage and dysfunction.
Formation of NO radicals by NO syntheses is construct-
ive mechanism of activated neutrophils phagocytes toward
tissue damage. There is larger production of NO, which
causes nitration of protein molecules which reacts with
superoxide ions to form highly reactive peroxynitrite radicals
(ONOO–) which cause DNA damage. In addition, COX-2
enzymes are required to convert arachidonic acid to prosta-
glandin H2 in presence of NO, which induces pain during
inflammation. Hence, to reduce inflammation and pain,
NO radical scavenging is considered to be promis-
ing mechanism.
From the results, it has been observed that the anti-
inflammatory activity of turmeric extract was found to be
highest in Soxhlet extraction followed by TPP and batch
extraction process (Figure 8). Curcuminoids yield obtained
by Soxhlet method is highest as compared to TPP and batch
which ultimately shows different anti-inflammatory activity
obtained from different extraction methods. The results of
anti-inflammatory activities were comparable with standard
drug aspirin. Along with curcuminoids, others contents such
as oil, oleoresin, and flavonoids may also add to the anti-
inflammatory property of turmeric extract. This study will
definitely contribute to the replacement of aspirin with tur-
meric extract in many pharmaceutical formulations.
Scanning electron microscopy for surface
characterization of C. longa powder
The surface morphology of C. longa powder before and after
extraction by batch and TPP processes was observed using
scanning electron microscopy (SEM). From Figure 9a–c, it
clearly indicates that the surface of C. longa powder is intact
and non-porous before extraction and that was changed into
perforated one by batch and TPP extraction processes.
Moreover, in case of TPP, the surface of C. longa is more
perforated than batch extraction process as seen in Figure 9c
which led to enhancement in extraction yield and reduction
in extraction time. TPP results into bursting of cell walls by
dehydrating them with the use of ammonium sulfate salt
and solvent (tert-butanol).
10 S. S. PATIL ET AL.

Figure 9. Scanning Electron Microscopy images of turmeric powder (a) before extraction, (b) after extraction by batch process, and (c) after extraction TPP process.

Table 3. Comparison of extraction yield of curcuminoids (mg/g) and process Conclusion

time during Soxhlet, batch, and TPP.
Extraction yield of curcuminoids The present work demonstrated the advanced three-phase
System (mg/g) Process time (h) partitioning and batch extraction as an effective alternative
Soxhlet 88.96 ± 0.6a 12 to the conventional technique for the extraction of curcumi-
Batch 52.77 ± 0.2c 3 noids from C. longa. Optimization of TPP and stirred batch
TPP 58.38 ± 0.3b 2.5
extraction of curcuminoids was successfully performed and
Values in the same column with different superscript letters are significantly
different (p  0.05) as measured using Duncan’s New Multiple Range Test. compared for the first time. The maximum extent of extrac-
All values are mean ± SE of three determinations. tion was achieved in TPP process and highest yield of cur-
cuminoids was found to 58.40 mg/g. Influence of various
parameters such as different solvent, ammonium sulfate
Comparison of TPP and batch with Soxhlet extraction concentration, slurry to tert-butanol ratio, solute to aqueous
ratio, temperature, and time was studied on three phase par-
The obtained results for extraction of curcuminoids using titioning of curcuminoids. However, in view of reference
Soxhlet, optimized TPP, and batch process were compared method, i.e., Soxhlet extraction (100%), TPP and batch
(Table 3). In accordance with Table 3, Soxhlet extraction exhibited 65.62% and 59.92% curcuminoids extraction,
yielded maximum, i.e., 88.96 mg/g while TPP and batch respectively. On evaluating therapeutic properties of tur-
resulted in 58.40 and 52.78 mg/g extraction of curcuminoids. meric extracts, they exhibited excellent antioxidant and anti-
Soxhlet process was carried out for 12 h at 60
C, whereas inflammatory capacities equivalent to gallic acid and aspirin
TPP and batch processes were carried out for 150 and respectively. TPP is a better technique acquiring short
180 min at 40
C, respectively. In view of reference method, period of time in comparison with conventional solvent
i.e., Soxhlet extraction (100%), TPP showed 65.62% yield extraction method, i.e., Soxhlet extraction. With respect to
and batch exhibited 59.92%. Conventional extraction meth- batch study, TPP showed enhancement in yield alongwith
ods like Soxhlet and batch possess numerous disadvantages reduction in extraction time.
compared to TPP. There was requirement of large amount
of solvent, more time, and high temperature in case of
Soxhlet extraction, whereas in case of batch process, large List of Abbreviations
amount of energy was consumed. TPP has the benefit
C. longa L Curcuma longa L.
because of high selectivity and high extraction yield for the TPP Three phase partitioning
target biomolecules.[29] At small scale, TPP method for cur- FAO Food and Agriculture Organization of the United States
cuminoids extraction is relatively simple, less time consum- WHO World Health Organization
ing, and cost effective. Though, additional experiments may JECFA Joint FAO/WHO Expert Committee on Food Additives
HPLC High Pressure Liquid Chromatography
require for economic assessment. In spite of the fact that GAE Gallic acid per gram of extract
TPP is an effective process for extraction as well as purifica- DPPH 2,2-diphenyl-1-picrylhydrazyl
tion of natural compounds, it endures the problem of mass IC50 Inhibitory concentration 50%
transfer which led to the difficulty in utmost recovery of DAMPs Damage associated molecular patterns
SRs Scavenging receptors
natural compounds. To conquer these drawbacks, micro- PR3 Proteinases-3
wave and ultrasound can be used as process intensifying PAR2 Protease activated receptor-2
tool combined with TPP. NO Nitric oxide

Acknowledgment
The authors would like to gratefully acknowledge Konark Herbals and
Health Care and the Prime Minister’s Fellowship Scheme for Doctoral
Research, a public-private partnership between Science & Engineering
Research Board (SERB), Department of Science & Technology,
Government of India and Confederation of Indian Industry (CII).
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