Mahidol University Journal of Pharmaceutical Sciences 2005; 32(1-2): 23-30.

Original Article

Quantitative Analysis of Curcumin, Demethoxycurcumin

and Bisdemethoxycurcumin in the Crude Curcuminoid
Extract from Curcuma longa in Thailand by TLC-
Densitometry

Werayut Pothitirat and Wandee Gritsanapan*

Department of Pharmacognosy, Faculty of Pharmacy,

Mahidol University, Bangkok, Thailand.

Abstract Curcuminoids, the principal natural yellow pigments comprising curcumin, demethoxycurcumin
and bisdemethoxycurcumin, in Curcuma longa rhizome have been popularly used in drugs and cosmetics as
potent antioxidants and coloring agents. This study, C. longa rhizomes were collected from 10 locations in
the northern, northeastern, central and southern parts of Thailand. The powdered samples were continuously
extracted using a soxhlet apparatus with hexane and 95% ethanol, respectively. Hexane was used to defat
the sample. Curcuminoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were isolated from
the ethanol extract using column chromatography. Yields of crude curcuminoids in all samples were found
in the range of 2.7-4.7% dry weight. Individual curcuminoid content was analyzed using a TLC-
densitometry. The content of curcumin in all extracts was found in the range of 46.45 ± 3.21% to 67.31 ±
0.97% w/w while the contents of demethoxycurcumin and bisdemethoxycurcumin were found in the ranges
of 11.47 ± 0.61% to 23.81 ± 0.28% w/w and 5.97 ± 0.41% to 13.88 ± 0.86% w/w, respectively. The highest
average contents of curcumin (60.16 ± 3.23% w/w) and bisdemethoxycurcumin (12.84 ± 0.57% w/w) were
found in the samples from the south while the highest average content of demethoxycurcumin (22.63 ±
1.33% w/w) was found in the samples from the north-east. In contrast, the lowest average contents of
curcumin (53.65 ± 8.31% w/w) and demethoxycurcumin (16.23 ± 5.23% w/w) were found in the samples
from the north, while the lowest content of bisdemethoxycurcumin (7.83 ± 0.28% w/w) was found in the
samples from the central area. Total curcuminoid content in the crude curcuminoid extract was in the range
of 67.13-96.25% w/w. This is the first report of each curcuminoid content in crude curcuminoid extract of
C. longa from various locations of Thailand and will provide a useful guidance for further standardization
of curcuminoid extracts used in pharmaceutical products and cosmetics. All right reserved.
Keywords: bisdemethoxycurcumin, Curcuma longa, curcumin, curcuminoid extract, demethoxycurcumin

INTRODUCTION agent in foods, household medicine and

insect repellent.2 Recently, C. longa is widely
Thailand is a country endowed with a variety used as a nutritional supplement and coloring
of medicinal plants with strong potential for agent in drugs and cosmetics. It has been
therapeutic applications. Curcuma longa Linn. found to be a rich source of polyphenolic
or turmeric is one of the most popular curcuminoids, i.e. curcumin, demethoxycurcumin
medicinal herbs1, which is listed as one of the and bisdemethoxycurcumin3 (Figure 1). Several
product champions of Thailand. It has been biological activities of turmeric have been
used for thousand years as a spice, coloring studied both in vitro and in vivo. Curcuminoids
*
Corresponding author: Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Road, Bangkok 10400, Thailand.
Fax: (622) 6448701. Email: pywgs@mahidol.ac.th
24 W. Pothitirat and W. Gritsanapan

are well known for their antioxidant, anti- All chemicals and reagents used were
inflammatory, antitumour and cytotoxic analytical grade, except ethanol which was
properties.4-7 the commercial grade obtained from the
Excise Department, Bangkok, Thailand.
In Thailand, C. longa is cultivated throughout
the country, mostly in the south.8 Thai Herbal Plant Material
Pharmacopoeia and the Standard of ASEAN
The rhizomes of C. longa were collected
Herbal Medicine have recommended that
from 10 different locations in the north,
dried turmeric should contain not less than 5%
north-east, south and central area of Thailand
w/w of total curcuminoids.8,9 It has also been
(Figure 2) during January - April 2005. The
reported that turmeric from various regions in
samples were identified by comparison with
Thailand had a high variation of curcuminoids
the specimens at the Forest Herbarium,
and volatile oil contents,10-12 but there is no
Department of National Park, Wildlife and
report concerning the amount of each
Plant Conservation, Ministry of Natural
curcuminoid in the crude curcuminoid extracts.
Resources and Environment, Bangkok. The
At present, a variety of methods for voucher specimens (WCL0105-WCL1005)
quantitative analysis of curcuminoid content were deposited at the Department of
were reported. Most of them are spectro- Pharmacognosy, Faculty of Pharmacy, Mahidol
photometric methods.13-15 Disadvantages of University, Bangkok, Thailand.
this method are that it is not possible to
Fresh rhizomes were cleaned, cut into small
analyze individual curcuminoids and the
pieces and air-dried for 2 days. The samples
precision is not good due to some interferences
were then further dried in a hot air oven at
by other pigments presented in the extract. A
50°C for 24 hours, ground into powder and
rapid and simple TLC-densitometric method
passed through a sieve (20 mesh).
has been developed for the simultaneous
quantitation of curcumin, demethoxycurcumin Extraction and Isolation of Reference Standards
and bisdemethoxycurcumin in C. longa
powder. The accuracy and precision of this Dried powder of C. longa collected from
Surat Thani Province (10.0 g) was extracted
method were reported to be reliable.10,11,16
with 95% ethanol (600 ml) using a soxhlet
Thus, this study was undertaken to determine apparatus for 56 hours.17 The ethanol extract
the amount of each curcuminoid in the crude was dried using a rotary evaporator and
curcuminoid extracts of C. longa, collected yielded crude ethanol extract (2.56 g). The
from different locations in Thailand by TLC- dried ethanolic extract (1 g) was further
densitometric method. fractionated by silica gel 60 column
chromatography (17.5 x 2.5 cm) eluted with
MATERIALS AND METHODS hexane, hexane : dichloromethane and then
dichloromethane : ethyl acetate with increasing
Chemicals and Reagents polarity. Fractions containing curcumin
(fractions 10-50, 200 ml) were refractionated
Curcumin, demethoxycurcumin and bis- by the column chromatography eluted with
demethoxycurcumin were isolated in our
100% dichloromethane to yield pure curcumin
laboratory from the ethanolic extract of C. longa.

Compounds R1 R2
Curcumin OMe OMe
Demethoxycurcumin H OMe
Bisdemethoxycurcumin H H
Figure 1. Chemical structure of curcuminoids.
 Quantitative Analysis of Curcumin, Demethoxycurcumin and Bisdemethoxycurcumin in the Crude 25
Curcuminoid Extract from Curcuma longa in Thailand by TLC-Densitometry

1= Tambol Wang Tai, Amphoe Wang Nuea, Lampang North

2= Tambol Noen Kum, Amphoe Bangkrathum, Phitsanulok North
3= Tambol Wang Mi, Amphoe Wang Nam Khiao, Nakhon Ratchasima North-East
4= Tambol Kut Bak, Amphoe Kut Bak, Sakon Nakhon North-East
5= Tambol Tha Sao, Amphoe Sai Yok, Kanchanaburi Central
6= Tambol Mungtarod, Amphoe Muang, Nakhon Pathom Central
7= Tambol Ban-Tai, Amphoe Ban rai, Uthai Thani Central
8= Tambol Khao Hin Son, Amphoe Phanom Sarakham, Chachoengsao Central
9= Tambol Tham Thong Lang, Amphoe Thap Put, Phangnga South
10 = Tambol Khao Wong, Amphoe Ban Ta Khun, Surat Thani South

Figure 2. Ten different locations of Thailand where C. longa was collected.

Fractions containing demethoxycurcumin and of each sample was done in triplicate and the
bisdemethoxycurcumin were further eluted yield was reported as mean ± S.D.
with dichloromethane : ethyl acetate (95:5) in
a column chromatography. Curcumin, demethoxy- Preparation of Standard Solutions and the
curcumin and bisdemethoxycurcumin were Calibration Curves
recrystallized and yielded 103.2, 43.3 and
86.0 mg, respectively. Stock solutions of individual curcuminoid
standards were separately prepared in
Identification of the compounds was characterized methanol at 1.0 mg/ml. One milliliter of the
using 1H NMR and 13C NMR comparing to stock solution was transferred to a 10-ml
references.18 Melting point of each curcuminoid volumetric flask and adjusted to volume with
was also investigated and compared with the methanol.
reference.19
Calibration curves of curcumin, demethoxy-
Extraction of Crude Curcuminoids20 curcumin and bisdemethoxycurcumin were
derived from separately applying five
The dried powder (15.0 g) of each sample
concentrations of each curcuminoid on the
was extracted with hexane (300 ml) using a
TLC plate to obtain final amounts of 100-
soxhlet apparatus for 20 hours. The hexane
1,200, 150-800 and 200-1,000 ng/spot,
extract was evaporated at a reduced pressure
respectively.
to separate volatile oil. The marc was further
extracted with 95% ethanol using a soxhlet The amount of each curcuminoid presented in
apparatus for 10 hours, two times, and the the sample was calculated using peak area
ethanol extracts were combined and filtered. with linear regression. Linearity, reproducibility
The filtrate was concentrated under reduced and accuracy of each curcuminoid were
pressure at 50°C using a rotary vacuum determined.
evaporator. The concentrated extract was
evaporated on a boiling water bath to yield Preparation of Sample Solutions
crude curcuminoids. The crude extract was For sample preparation, five milligrams of
further purified by dissolving in 95% ethanol, each crude curcuminoid extract were transferred
1:6 w/v. The yellow curcuminoids were to a 10 ml volumetric flask. The sample was
precipitated, filtered and dried. The extraction dissolved in methanol and adjusted to a
concentration of 0.5 mg/ml.
26 W. Pothitirat and W. Gritsanapan

Instrumentation and Analytical Condition RESULTS AND DISCUSSION

Four microliters of each sample solution
Purified curcumin, demethoxycurcumin and
were spotted as a band width of 6.0 mm on a
bisdemethoxycurcumin showed single spots
precoated silica gel aluminium plate 60F254
on TLC (Si-60GF254, chloroform : benzene :
(20 × 10 cm; E. Merck, Germany) using a
methanol = 80:15:5) after detecting by UV
Camag Linomat 5 syringe. The following
366 (Figure 3). The Rf values of these
conditions were employed: application rate,
compounds were investigated as shown in
150 nl/s; space between each band, 13.0 mm;
Table 1. By NMR, Rf values and melting
slit dimension, 5.00 mm × 0.45 mm; and
points, the compounds were confirmed to be
scanning speed, 20 mm/s. The mobile phase
curcumin, demethoxycurcumin and bis-
consisting of chloroform : benzene : methanol
demethoxycurcumin.
(80:15:5) was used. Linear ascending
development was carried out in 20 × 10 cm Linearity of curcumin was found in the
twin trough glass chamber (Camag, Muttenz, concentration range of 100-1,200 ng/spot while
Switzerland) saturated with the mobile phase. linearity of demethoxycurcumin and bis-
The length of each chromatogram run was 8 demethoxycurcumin were found in the
cm. After developing, the TLC plate was concentration ranges of 150-800 and 200-
dried using an air dryer. Densitometric 1,000 ng/spot, respectively with high
scanning was performed on Camag TLC reproducibility and accuracy.
Scanner 3 in the reflectance-absorbance
mode at 420 nm, operated by CATS software The linear regression equations with correlation
coefficient (r2) of curcumin, demethoxy-
(V 1.2.6, Camag). The source of radiation
utilized was a tungsten lamp. Video densito- curcumin and bisdemethoxycurcumin were
metry of the TLC chromatogram was carried given in Table 1.
out with the help of Camag Reprostar 3 with From TLC-densitometric chromatogram (Figure
cabinet cover and mounted digital camera. 4), it was found that a major peak in the crude
Determination of Individual Curcuminoid curcuminoid extracts of all samples was
curcumin. The other two minor peaks were
Content
demethoxycurcumin and bisdemethoxy-
A volume of each sample solution was curcumin. The identification was done by
applied in a triplicate manner on TLC plate spiking with their standards and by
and analyzed by the proposed method. Each determination of the Rf values. Chloroform :
curcuminoid content was calculated using its benzene : methanol (80:15:5) was found to be
calibration curve. The contents of curcumin, a suitable mobile phase for separation of
demethoxycurcumin and bisdemethoxycurcumin curcumin, demethoxycurcumin and bis-
were expressed as the amount in gram per 100 demethoxycurcumin (Figure 3). Wavelength
grams of the extract. at λ max 420 nm which was used to analyze

Table 1. Linear regression equation, correlation coefficient (r2), Rf value and melting point (m.p.) of each
curcuminoid

Curcuminoids Equation r2 Rf (cm) m.p. (°C)

Curcumin Y = 31.233X-1910.30 0.9959 0.69 ± 0.02 180-181
Demethoxycurcumin Y = 32.716X+3204.10 0.9962 0.51 ± 0.02 160-161
Bisdemethoxycurcumin Y = 14.386X+7340.50 0.9953 0.39 ± 0.02 220-221
 Quantitative Analysis of Curcumin, Demethoxycurcumin and Bisdemethoxycurcumin in the Crude 27
Curcuminoid Extract from Curcuma longa in Thailand by TLC-Densitometry

each curcuminoid content in the samples is From the results, C. longa samples from the
the same wavelength recommended by Thai south of Thailand where it rains the whole
Herbal Pharmacopoeia 9 and a previous study.10 year and from the north-east where the weather
is warm in summer and cool in winter, should
Curcumin content in all extracts was found in
be selected for the extraction of curcuminoids
the range of 46.45 ± 3.21% to 67.31 ± 0.97%
due to high total curcuminoid contents
w/w while the content of demethoxycurcumin
(average 89.72 ± 4.81% and 93.02 ± 2.55%
and bisdemethoxycurcumin were found in the
w/w, respectively). These results support the
ranges of 11.47 ± 0.61% to 23.81 ± 0.28% w/w
former reports that C. longa grown in the
and 5.97 ± 0.41% to 13.88 ± 0.86% w/w,
southern part of Thailand contains high
respectively. The average contents of these
content of curcuminoids.12,21,22
curcuminoids in the extracts were found to be
58.12 ± 6.46%, 18.48 ± 3.94% and 10.29 ±
CONCLUSION
2.72% w/w for curcumin, demethoxycurcumin
and bisdemethoxycurcumin, respectively. The
highest average contents of curcumin (60.16 ± C. longa grown in different parts of Thailand
contains different amounts of various
3.23% w/w) and bisdemethoxycurcumin (12.84
± 0.57% w/w) were found in the samples curcuminoids. Therefore, for obtaining the
from the south while the highest average high yield of certain curcuminoid, the area for
plant collection should be considered. This is
content of demethoxycurcumin (22.63 ±
1.33% w/w) was found in the samples from the first report of each curcuminoid content in
the north-east. In contrast, the lowest average crude curcuminoid extract of C. longa from
various locations of Thailand and it will
contents of curcumin (53.65 ± 8.31% w/w)
and demethoxycurcumin (16.23 ± 5.23% w/w) provide a useful guidance for further
were found in samples from the north, while standardization of curcuminoid extracts used
in pharmaceutical products and cosmetics.
the lowest average content of bisdemethoxy-
curcumin (7.83 ± 0.28% w/w) was found in
samples from the central area (Table 2).

Figure 3. TLC fingerprints of curcuminoids in the extracts of C. longa collected from various locations.
Stationary phase: Si-60GF254; solvent system: CHCl3 : C6H6 : MeOH (80:15:5); detection: UV 366.
1= Tambol Wang Tai, Amphoe Wang Nuea, Lampang
2= Tambol Noen Kum, Amphoe Bangkrathum, Phitsanulok
3= Tambol Wang Mi, Amphoe Wang Nam Khiao, Nakhon Ratchasima
4= Tambol Kut Bak, Amphoe Kut Bak, Sakon Nakhon
5= Tambol Tha Sao, Amphoe Sai Yok, Kanchanaburi
6= Tambol Mungtarod, Amphoe Muang, Nakhon Pathom
7= Tambol BanTai, Amphoe Ban rai, Uthai Thani
8= Tambol Khao Hin Son, Amphoe Phanom Sarakham, Chachoengsao
9= Tambol Tham Thong Lang, Amphoe Thap Put, Phangnga
10 = Tambol Khao Wong, Amphoe Ban Ta Khun, Surat Thani
C = curcumin, DMC = demethoxycurcumin and BDMC = bisdemethoxycurcumin
 28 W. Pothitirat and W. Gritsanapan

Table 2. The content of each curcuminoid in crude curcuminoid extracts of C. longa from different locations of Thailand analyzed by TLC-densitometry

Yield of crude Each curcuminoid content (% w/w in extract)* Total curcuminoid

curcuminoids in (% w/w in extract)
Location Code
dried powder
(%)* C Average DMC Average BDMC Average Content Average
North 1 4.03 ± 0.25 60.84 ± 2.70 20.98 ± 0.33 13.18 ± 0.06 95.00 ± 3.07
53.65 ± 8.314 16.23 ± 5.234 11.20 ± 0.353 81.07 ± 15.544
(N) 2 3.05 ± 0.41 46.45 ± 3.21 11.47 ± 0.61 9.21 ± 0.64 67.13 ± 3.40
North-East 3 4.59 ± 0.57 55.46 ± 0.51 21.45 ± 0.39 13.88 ± 0.86 90.79 ± 0.78
58.60 ± 3.473 22.63 ± 1.331 11.79 ± 0.672 93.02 ± 2.551
(NE) 4 2.98 ± 0.42 61.74 ± 0.52 23.81 ± 0.28 9.69 ± 0.48 95.24 ± 0.86
5 2.74 ± 0.11 64.42 ± 0.73 21.66 ± 0.35 10.17 ± 0.04 96.25 ± 0.72
Central 6 3.10 ± 0.11 67.31 ± 0.97 21.51 ± 0.29 5.97 ± 0.41 94.78 ± 1.57
59.10 ± 7.192 18.40 ± 3.642 7.83 ± 0.284 85.32 ± 10.813
(C) 7 4.10 ± 0.10 52.27 ± 1.30 13.38 ± 1.25 7.76 ± 0.07 73.40 ± 2.30
8 3.17 ± 0.42 52.41 ± 0.65 17.06 ± 0.46 7.40 ± 0.59 76.86 ± 1.06
South 9 4.72 ± 0.88 57.63 ± 2.58 15.89 ± 0.75 12.23 ± 0.51 85.75 ± 3.16
60.16 ± 3.231 16.73 ± 1.083 12.84 ± 0.571 89.72 ± 4.812
(S) 10 4.13 ± 0.06 62.69 ± 0.38 17.57 ± 0.45 13.44 ± 0.63 93.70 ± 0.60
Average 3.66 ± 0.73 58.12 ± 6.46 18.48 ± 3.94 10.29 ± 2.72 86.89 ± 10.42

1= Tambol Wang Tai, Amphoe Wang Nuea, Lampang 6= Tambol Mungtarod, Amphoe Muang, Nakhon Pathom
2= Tambol Noen Kum, Amphoe Bangkrathum, Phitsanulok 7= Tambol Ban-Tai, Amphoe Ban rai, Uthai Thani
3= Tambol Wang Mi, Amphoe Wang Nam Khiao, Nakhon Ratchasima 8= Tambol Khao Hin Son, Amphoe Phanom Sarakham, Chachoengsao
4= Tambol Kut Bak, Amphoe Kut Bak, Sakon Nakhon 9= Tambol Tham Thong Lang, Amphoe Thap Put, Phangnga
5= Tambol Tha Sao, Amphoe Sai Yok, Kanchanaburi 10 = Tambol Khao Wong, Amphoe Ban Ta Khun, Surat Thani

C = curcumin, DMC = demethoxycurcumin and BDMC = bisdemethoxycurcumin

1…..4
= ordering, maximum to minimum content
*
Extraction and analysis of each sample were done in triplicate and % contents were expressed as mean ± S.D.
 Quantitative Analysis of Curcumin, Demethoxycurcumin and Bisdemethoxycurcumin in the Crude 29
Curcuminoid Extract from Curcuma longa in Thailand by TLC-Densitometry

Figure 4. Densitometric thin layer chromatograms of crude curcuminoid extract of C. longa from various
locations (at λ = 420 nm).
C = curcumin, DMC = demethoxycurcumin and BDMC = bisdemethoxycurcumin
30 W. Pothitirat and W. Gritsanapan

ACKNOWLEDGEMENTS 13. The medicine commission. British Pharmaco-

poeia A19. London: Her Majesty’s Stationary
This study was supported by Drug Program of Office, 1973.
National Research Council of Thailand (NRCT) 14. Ministry of Public Health. Manual of
and Mahidol University, Bangkok, Thailand. Medicinal Plant for Primary Health Care.
Bangkok: Text and Journal Corporation, 1990.
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