Note: Descriptions are shown in the official language in which they were submitted.
1
METHOD FOR ISOLATING CURCUMINOIDS FROM TURMERIC
RHIZOME
FIELD
The disclosure relates to a method for isolating
curcuminoids, and more particularly to a method for
isolating curcuminoids from turmeric rhizome.
BACKGROUND
Turmeric (Curcuma longa L.) is a perennial plant
of genus Curcuma in the ginger family (Zingiberaceae),
and is usually 1 to 1.5 meters tall. Turmeric roots
are stout with dilated tuberous ends. Turmeric leaves
are oblong or elliptic with short tapering tips.
Turmeric bracts are ovate or oblong with blunt tips,
and are light green. Turmeric corolla is yellowish.
Turmeric is mainly distributed in southeastern India,
Jamaica, China, Bangladesh, Caribbean, South America,
etc.
The main useful part of turmeric is rhizome
thereof. In folk medicine, turmeric has effects of
stimulating blood circulation and relieving
menstrual pain. In addition, it has been reported
that curcuminoids (i.e., one of the main active
ingredients of turmeric, including curcumin,
bisdemethoxycurcumin, and demethoxycurcumin) exerts
a variety of beneficial effects on human bodies,
including antioxidant, anti-inflammatory, antiviral,
Date Recue/Date Received 2021-10-20
2
antibacterial, antifungal, and anticancer effects.
Therefore, mass production of curcuminoids has become
a major research focus for those skilled in the art.
The conventional methods for producing curcumin
include extraction with various organic solvents
(e.g., acetone or isopropanol) and hydrolysis with
acidic and/or alkaline solvents (see, e.g., CN
102617316 B and CN 1566215 A). However, use of these
solvents can be harmful to environment or human
health. In addition, extraction efficiency of some
solvents is poor. Therefore, multiple extractions or
further use of ultrasonic extraction and
supercritical fluid extraction are required to
improve curcuminoid yield (see, e.g., CN 105669410 B
and CN 1150924 C), which is not only cumbersome and
time consuming, but also increases the cost required
for practical application in the industry.
Therefore, it is desirable to develop a method
for producing curcuminoids with a high yield, a
simple operation procedure, and a low cost.
SUMMARY
Therefore, an object of the disclosure is to
provide a method for efficiently isolating
curcuminoids from turmeric rhizome using a simple
process.
According to the disclosure, there is provided a
method for isolating curcuminoids from turmeric
Date Recue/Date Received 2021-10-20
3
rhizome. The method includes the steps of:
a) subjecting the turmeric rhizome to extraction
with a first ethanol solution having an ethanol
concentration ranging from 90% to 100% at a stirring
speed ranging from 100 rpm to 300 rpm so as to obtain
an ethanol-extracted product; and
b) subjecting the ethanol-extracted product to
crystallization with a second ethanol solution
having an ethanol concentration ranging from 90% to
100% at a temperature ranging from 2 C to 8 C and a
stirring speed ranging from 40 rpm to 300 rpm so as
to obtain the curcuminoids.
DETAILED DESCRIPTION
A method for isolating curcuminoids from turmeric
rhizome according to the disclosure includes the
steps of:
a) subjecting the turmeric rhizome to extraction
with a first ethanol solution having an ethanol
concentration ranging from 90% to 100% at a stirring
speed ranging from 100 rpm to 300 rpm so as to obtain
an ethanol-extracted product; and
b) subjecting the ethanol-extracted product to
crystallization with a second ethanol solution
having an ethanol concentration ranging from 90% to
100% at a temperature ranging from 2 C to 8 C and a
stirring speed ranging from 40 rpm to 300 rpm so as
to obtain the curcuminoids.
Date Recue/Date Received 2021-10-20
4
In some embodiments of the disclosure, in step
a), the extraction is performed with the first
ethanol solution having the ethanol concentration of
95% at the stirring speed of 280 rpm.
In some embodiments of the disclosure, in step
b), the crystallization is performed with the second
ethanol solution having the ethanol concentration of
95% at the temperature of 4 C and the stirring speed
of 60 rpm.
Various turmeric cultivars can be used in the
method of the disclosure. In some embodiments of the
disclosure, the turmeric cultivars include red
turmeric, yellow turmeric, Curcuma zedoaria (also
known as white turmeric), Curcuma xanthorrhiza (also
known as Java turmeric), and combinations thereof.
In an exemplary embodiment of the disclosure, the
turmeric cultivar is red turmeric.
In some embodiments of the disclosure, in step
a), the first ethanol solution is present in an
amount ranging from 5 mL per gram to 10 mL per gram
of the turmeric rhizome. In an exemplary embodiment
of the disclosure, in step a), the first ethanol
solution is present in an amount of 5 mL per gram of
the turmeric rhizome.
In some embodiments of the disclosure, in step
a), the extraction is performed at a temperature
ranging from 10 C to 100 C for a time period ranging
Date Recue/Date Received 2021-10-20
5
from 15 minutes to 150 minutes. In an exemplary
embodiment of the disclosure, in step a), the
extraction is performed at a temperature of 25 C for
a time period of 30 minutes.
In some embodiments of the disclosure, in step
b), the second ethanol solution is present in an
amount ranging from 1 g per gram to 20 g per gram of
the ethanol-extracted product.
In some embodiments of the disclosure, in step
b), the crystallization is performed at the
temperature ranging from 3 C to 6 C for the time
period ranging from 12 hours to 72 hours. In an
exemplary embodiment of the disclosure, in step b),
the crystallization is performed at the temperature
of 4 C for the time period of 24 hours.
In some embodiments of the disclosure, the
curcuminoids include curcumin, bisdemethoxycurcumin,
demethoxycurcumin, or combinations thereof. In an
exemplary embodiment of the disclosure, the
curcuminoids include a combination of curcumin,
bisdemethoxycurcumin, and demethoxycurcumin.
In some embodiments, the first ethanol solution
used in step a) is a recycled ethanol solution
obtained from the extraction, and the method of the
disclosure further includes the steps of leaving the
ethanol-extracted product to stand at a temperature
ranging from -20 C to 8 C for a time period of 24
Date Recue/Date Received 2021-10-20
6
hours prior to step b), and siveing the curcuminoids
through a sieve of from 20 mesh to 100 mesh after
step b).
In some embodiments, the method of the disclosure
further includes, prior to step a), subjecting the
turmeric rhizome to lyophilization for a time period
ranging from 12 hours to 48 hours. Specifically, the
lyophilization is performed by freezing the turmeric
rhizome after washing with deionized water to a
temperature ranging from -20 C to -10 C, followed by
drying the turmeric rhizome at a first raised
temperature ranging from 30 C to 40 C for a first
time period ranging from 3 hours to 6 hours and then
at a second raised temperature ranging from 50 C to
60 C for a second time period ranging from 18 hours
to 21 hours.
Examples of the disclosure will be described
hereinafter. It is to be understood that these
examples are exemplary and explanatory and should
not be construed as a limitation to the disclosure.
Examples:
General Experimental Materials:
1. Preparation of turmeric rhizome powder
Turmeric rhizome powder used in following
examples was prepared by washing fresh red turmeric
rhizome purchased from Zuojhen District, Tainan City,
Taiwan with deionized water, followed by
Date Recue/Date Received 2021-10-20
7
lyophilization for 24 hours, grinding using a grinder
(Manufacturer: Hsiang Tai Machinery Industry Co.,
Ltd.; Model: SM-3C), and sieving using a sieve with
an aperture of 0.18 mm.
General Experimental Procedures:
1.Quantitative Analysis of Curcuminoids
In the following examples, contents of
curcuminoids (including
curcumin,
bisdemethoxycurcumin, and demethoxycurcumin) in each
test sample were determined by reference to a method
described in "Method of Test for Curcuminoids in
Foods in Capsule and Tablet Form" published by the
Ministry of Health and Welfare, Taiwan on March 19,
2015, and by performing an ultra performance liquid
chromatography (UPLC) analysis using an ACQUITY
UPLC photodiode array (PDA) e. detector from Waters
Corporation. The operating conditions for performing
the UPLC analysis are shown in Table 1 below.
Date Recue/Date Received 2021-10-20
8
Table 1
Operating conditions for UPLC analysis
Operating conditions Details
Type of chromatograhy
Acclaim C30 3 pm column
column
Size of chromatograhy
2.1 mm x 15 cm
column
Temperature of
35 C
chromatography column
Detection wavelength 420 nm
2% acetic acid solution/
Mobile phase
acetonitrile, 60/40 (v/v)
Flow rate 0.4 mL/min
Injection volumn of
pL
test sample
In addition, for comparison, proper amounts of
curcumin, bisdemethoxycurcumin, and
5 demethoxycurcumin purchased from Sigma-Aldrich were
respectively dissolved in 100% methanol to prepare
curcumin, bisdemethoxycurcumin and demethoxycurcumin
solutions with concentrations ranging from 0.1 pg/mL
to 20 gg/mL as control standards, and then subjected
to the UPLC analysis.
The total curcuminoid content was calculated by
adding up the contents of
curcumin,
bisdemethoxycurcumin, and demethoxycurcumin.
Example 1: Preparation of ethanol-extracted products
of turmeric (Curcuma longa L.) rhizomes
A. Effects of different extraction methods on total
curcuminoid content:
Turmeric rhizome powder prepared in section 1 of
Date Recue/Date Received 2021-10-20
9
the General Experimental Materials was divided into
one experimental group (i.e., Experimental group 1)
and two comparative experimental groups (i.e.,
Comparative experimental groups 1 and 2), and then
subjected to extraction processes shown in Table 2
below.
The extraction process for the turmeric rhizome
powder of Experimental group 1 was performed
according to the following steps: First, 30 g of the
turmeric rhizome powder was added with 150 mL of 95%
ethanol and mixed thoroughly. Then, extraction was
performed at a temperature of 25 C and a stirring
speed of 280 rpm for a time period of 30 minutes.
Extraction process for the turmeric rhizome
powder of Comparative experimental group 1 was
performed according to the following steps. First,
30 g of the turmeric rhizome powder was added with
150 mL of 95% ethanol and mixed thoroughly. Then,
ultrasonic extraction was performed at a temperature
of 25 C for a time period of 30 minutes using an
ultrasonicator (purchased from Taiwan Supercritical
Technologies Co., Ltd., Model: ES-600N) at a
frequency of 25 KHz to 30 KHz.
Extraction process for the turmeric rhizome
powder of Comparative experiment group 2 was
performed according to the following steps. First,
g of the turmeric rhizome powder was placed in an
Date Recue/Date Received 2021-10-20
10
extraction vessel of a supercritical fluid
extraction system (purchased from
Taiwan
Supercritical Technologies Co., Ltd., Model: OV-SCF-
B). Then, supercritical CO2 was introduced into the
extraction vessel and 95% ethanol was added as a co-
solvent. After that, supercritical fluid extraction
was performed at a temperature of 40 C and a pressure
of 4350 psi for a time period of 30 minutes.
A 250 mesh filter screen was then used to filter
each of ethanol-extracted semi-products of
Experimental group 1 and Comparative experimental
groups 1 and 2 so as to collect each of filtrates of
Experimental group 1 and Comparative experimental
groups 1 and 2. Ethanol contained in each of the
filtrates was removed by a concentration process
under reduced pressure to obtain an ethanol-
extracted product of the turmeric rhizome of each of
Experimental group 1 and Comparative experimental
groups 1 and 2.
Table 2
Extraction processes
Groups Extraction processes
Experimental group Stirring extraction (25 C,
1 280 rpm)
Comparative
Ultrasonic extraction (25 C,
experimental group
25-30 KHz)
1
Comparative
Supercritical fluid
experimental group
extraction (40 C, 4350 psi)
2
Date Recue/Date Received 2021-10-20
11
Thereafter, 10 mg to 20 mg of the ethanol-
extracted product of the turmeric rhizome of each of
Experimental group 1 and Comparative experimental
groups 1 and 2 was mixed in 10 mL of methanol to
prepare a test sample. The curcuminoid content in
the test sample of each of Experimental group 1 and
Comparative experimental groups 1 and 2 was analyzed
according to the procedures described in section 1
of the General Experimental Procedures. The results
are shown in Table 3 below.
Table 3
Total curcuminoid contents
Total curcuminoid
Groups
contents (wt)
Experimental group 1 48.65
Comparative experimental
35.55
group 1
Comparative experimental
37.99
group 2
As shown in Table 3, the total curcuminoid
content in Experimental group 1 is significantly
higher than those in Comparative experimental groups
1 and 2. These results show that the total
curcuminoid content in the ethanol-extracted product
of the turmeric rhizome obtained by the stirring
extraction at a stirring speed of 280 rpm is
significantly higher than those obtained by the
ultrasonic extraction and the supercritical fluid
extraction.
Date Recue/Date Received 2021-10-20
12
B. Effects of various concentrations of extracting
solvents on total curcuminoid contents:
The turmeric rhizome powder was divided into one
experimental group (i.e., Experimental Group 1) and
two comparative experimental groups (i.e.,
Comparative experimental groups 1 and 2). The
turmeric rhizome powder of Experimental group 1 was
subjected to the stirring extraction in accordance
with the procedures described for Experimental group
1 in the abovementioned section A. The turmeric
rhizome powder of each of Comparative experimental
groups 1 and 2 was also subjected to the similar
procedures as described for Experimental group 1 in
the abovementioned section A, except that 50% ethanol
and 75% ethanol were used in Comparative experimental
groups 1 and 2, respectively, to replace 95% ethanol.
Thereafter, the ethanol-extracted product of the
turmeric rhizome of each of Experimental group 1 and
Comparative experimental groups was prepared into a
test sample as described in the abovementioned
section A. The total curcuminoid content in the test
sample of each of Experiment group 1 and Comparative
experimental groups 1 and 2 was analyzed according
to the procedures as described in section 1 of the
General Experimental Procedures. The results are
shown in Table 4 below.
Date Recue/Date Received 2021-10-20
13
Table 4
Total curcuminoid contents
Total curcuminoid
Groups
contents (wt)
Experimental group 1 42.60
Comparative experimental
7.29
group 1
Comparative experimental
24.20
group 2
As shown in Table 4, the total curcuminoid
content in Experimental group 1 is significantly
higher than those in Comparative experimental groups
1 and 2. These results show that the total
curcuminoid content in the ethanol-extracted product
of the turmeric rhizome obtained by the stirring
extraction using 95% ethanol is significantly higher
than those obtained by the stirring extractions using
50% ethanol and 75% ethanol.
Example 2: Purification of curcuminoids in ethanol-
extracted product of turmeric rhizome by
crystallization
To evaluate the effect of various crystallization
conditions on purification of curcuminoids
(including curcumin, bisdemethoxycurcumin, and
demethoxycurcumin) from the ethanol-extracted
product of the turmeric rhizome, the following
experiments were performed.
Experimental Procedures:
The ethanol-extracted product of the turmeric
Date Recue/Date Received 2021-10-20
14
rhizome of Experimental group 1 as described in the
abovementioned section A of Example 1 was divided
into one experimental group (i.e., Experimental
group 1) and two comparative experimental groups
(i.e., Comparative experimental groups 1 and 2). The
ethanol-extracted product of the turmeric rhizome of
each of Experimental group 1 and Comparative
experimental groups 1 and 2 was subjected to a
crystallization process at 4 C with the conditions
shown in Table 5 below.
Table 5
Crystallization conditions
Crystallization conditions
Groups Stirring speeds
Solvents
(rpm)
Experimental
Ethanol 60
group 1
Comparative
experimental Ethanol -
group 1
Comparative
experimental Isopropanol 60
group 2
Specifically, the crystallization process for
Experimental group 1 was performed according to the
following steps. First, 3 g of the ethanol-extracted
product of the turmeric rhizome was added with 5 g
of 95% ethanol and mixed thoroughly, followed by
continuously stirring at a temperature of 4 C and a
stirring speed of 60 rpm for a time period of 24
hours, and then left to stand at a temperature of
Date Recue/Date Received 2021-10-20
15
4 C for a time period of 24 hours to form a
precipitate. The precipitate was collected by
filtration and dried in an oven at a temperature set
to 60 C.
The crystallization process for Comparative
experimental group 1 was performed using the steps
substantially the same as those described above for
Experimental group 1, except that stirring was not
performed.
The crystallization process for Comparative
experimental group 2 was performed using the steps
substantially the same as those described above for
Experimental group 1, except that isopropanol was
used to replace ethanol.
Thereafter, 10 mg to 20 mg of the precipitate of
each of Experimental group 1 and Comparative
experimental groups 1 and 2 was taken mixed in 10 ml
of methanol to prepare a test sample. The total
curcuminoid content in the test sample of each of
Experimental group 1 and Comparative experimental
groups 1 and 2 was analyzed according to the
procedures described in section 1 of the General
Experimental Procedures. The results are shown in
Table 6 below.
Results:
Date Recue/Date Received 2021-10-20
16
Table 6
Total curcuminoid contents
Total curcuminoid
Groups
contents (wt)
Experimental group 1 95.92
Comparative experimental
88.41
group 1
Comparative experimental
74.28
group 2
As shown in Table 6, the total curcuminoid
content in Experimental group 1 is significantly
higher than those measured in Comparative
experimental groups 1 and 2. These results show that
curcuminoids with a higher purity can be obtained by
crystallizing the ethanol-extracted product of the
turmeric rhizome using ethanol at a low temperature
with stirring during the crystallization process.
Example 3: Use of a recycled ethanol solution for
preparation of ethanol-extracted products
of turmeric rhizomes
The turmeric rhizome powder was divided into one
experimental group (i.e., Experimental Group 1) and
two comparative experimental groups (i.e.,
Comparative experimental groups 1 and 2). The
turmeric rhizome powder of Experimental group 1 was
subjected to the stirring extraction in accordance
with the procedures described for Experimental group
1 in the abovementioned section A of Example 1 except
that a recycled 90% ethanol obtained from a previous
Date Recue/Date Received 2021-10-20
17
extraction process was used to replace 95% ethanol.
A concentrate obtained by the concentration process
was left to stand at a temperature of from 2 C to
8 C for a time period of 24 hours to form a
precipitate (an oleoresin) .
The precipitate was subjected to the
crystallization process in accordance with the
procedures described for Experimental group 1 in
Example 2. The curcuminoids obtained by the
crystallization process was sieved through a sieve
of from 20 mesh to 100 mesh.
The turmeric rhizome powder of Comparative
experimental group 1 was used to isolate the
curcuminoids in accordance with the processes
substantially the same as those described above for
Experimental group 1, except that the curcuminoids
obtained by the crystallization process was not
sieved.
The turmeric rhizome powder of Comparative
experimental group 2 was used to isolate the
curcuminoids in accordance with the processes
substantially the same as those described above for
Experimental group 1, except that the concentrate
obtained by the concentration process was not left
to stand and the curcuminoids obtained by the
crystallization process was not sieved.
Date Recue/Date Received 2021-10-20
18
Preparation of the test sample of each of
Experimental group 1 and Comparative experimental
groups 1 and 2 was performed according to the
procedures described in the section entitled
"Experimental Procedures" of Example 2. The total
curcuminoid content in the test sample of each of
Experimental group 1 and Comparative experimental
groups 1 and 2 was analyzed according to the
procedures described in section 1 of the General
Experimental Procedures. The results are shown in
Table 7 below.
Results:
Table 7
Total curcuminoid contents
Total curcuminoid
Groups
contents (wt)
Experimental group 1 100.00
Comparative experimental
93.31
group 1
Comparative experimental
88.14
group 2
As shown in Table 7, the total curcuminoid
content in Experimental group 1 is significantly
higher than those measured in Comparative
experimental groups 1 and 2. These results show that
when a recycled ethanol solution is used for the
extraction process, curcuminoids with a higher
purity can be obtained by leaving the concentrate
obtained by the concentration process to stand for a
Date Recue/Date Received 2021-10-20
19
time period prior to the crystallization process and
sieving the curcuminoids obtained by the
crystallization process.
Example 4: Compositional analysis of the
curcuminoids
Experimental Procedures:
The turmeric rhizome powder was subjected to
stirring extraction according to the procedures
described for Experimental group 1 in section A of
Example 1. Crystallization of the ethanol-extracted
product of the turmeric rhizome and preparation of
the test sample were performed according to the
procedures described for Experimental group 1 in the
section entitled "Experimental Procedures" of
Example 2. Thereafter, contents of curcumin,
bisdemethoxycurcumin, and demethoxycurcumin in the
test samples were analyzed according to the
procedures described in section 1 of the General
Experimental Procedures, and the total curcuminoid
content was further calculated. The experiments were
repeated three times, and the obtained data were
expressed as mean standard deviation (S.D.).
The results are shown in Table 8 below.
Results:
Date Recue/Date Received 2021-10-20
20
Table 8
Curcuminoid contents
Curcumin content (wt%) 84.45 1.03
Bisdemethoxycurcumin content
1.45 0.07
(wt%)
Demethoxycurcumin content
12.04 1.55
(wt)
Total curcuminoid content
97.94 1.1
(wt)
As shown in Table 8, it is possible to obtain a
product containing three different types of
curcuminoids, in which the content of curcumin is
the highest.
In the description above, for the purposes of
explanation, numerous specific details have been set
forth in order to provide a thorough understanding
of the embodiments. It will be apparent, however, to
one skilled in the art, that one or more other
embodiments may be practiced without some of these
specific details. It should also be appreciated that
reference throughout this specification to "one
embodiment," "an embodiment," an embodiment with an
indication of an ordinal number and so forth means
that a particular feature, structure, or
characteristic may be included in the practice of
the disclosure. It should be further appreciated that
in the description, various features are sometimes
grouped together in a single embodiment, figure, or
description thereof for the purpose of streamlining
Date Recue/Date Received 2021-10-20
21
the disclosure and aiding in the understanding of
various inventive aspects, and that one or more
features or specific details from one embodiment may
be practiced together with one or more features or
specific details from another embodiment, where
appropriate, in the practice of the disclosure.
Date Recue/Date Received 2021-10-20
