Food Chemistry 113 (2009) 640–644

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

High-performance thin layer chromatographic method for quantitative

determination of curcuminoids in Curcuma longa germplasm
M. Paramasivam a,*, R. Poi a, H. Banerjee a, A. Bandyopadhyay b
a
Department of Agricultural Chemicals, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia-741 252, India
b
Department of Spices and Plantation Crops, Faculty of Horticulture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia-741 252, India

a r t i c l e i n f o a b s t r a c t

Article history: Phytochemical investigation of the rhizomes of Curcuma longa leads to the isolation of pharmacologically
Received 25 February 2008 active curcuminoids viz Curcumin, demethoxycurcumin and bisdemethoxycurcumin. These were iso-
Received in revised form 4 July 2008 lated from turmeric rhizomes by soxhlet extraction followed by column chromatography, crystallisation
Accepted 19 July 2008
and identified by spectroscopic studies. The purity of the curcuminoids was analysed by HPTLC method.
The method employed TLC aluminium plates precoated with silica gel 60GF254 as the stationary phase.
The solvent system consisted of chloroform: methanol (48:2, v/v). This system was found to give compact
Keywords:
spots for curcumin, demethoxycurcumin and bisdemethoxycurcumin (RF value of 0.66 ± 0.02, 0.48 ± 0.02
Turmeric
Curcuminoid
and 0.30 ± 0.02), respectively. Densitometric analysis of curcuminoids was carried out in the absorption–
Curcuma longa reflection detection mode at 425 nm. Seven different germplasm of turmeric were analysed to detect the
Phytochemical percentage of these three curcuminoids.
HPTLC Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Chandrasekhara, 1992; Toda, Miyase, Arichi, Tanizawa, & Takino,

The rhizomes of turmeric (Curcuma longa L.), belongs to the
Zingiberaceae family and widely cultivated throughout tropical
and subtropical regions of the world, mainly in India and China.
Its rhizomes contain curcumins, the yellow pigment, belonging to
the diferuloyl methane group. Turmeric powder is extensively used
as a spice, food preservative and colouring material. It has been
used in traditional medicine as a household namely for various
diseases, including bilary disorders (Chattopadhyay, Biswas,
Bandyopadhyay, & Banerjee, 2004), anorexia, cough, diabetic
wounds and hepatic disorders. The main colouring substances in
the rhizomes of curcuma species are curcumin and two related
compounds, demethoxycurcumin and bisdemethoxycurcumin
shown in Fig. 1 (Jayaprakasha, Rao, & Sakariah, 2002).
Curcuminoids has been valued worldwide as a functional food
because of its health promoting properties (Jayaprakasha, Rao, &
Sakariah, 2002). There are several reports in the literature human
immunodeficiency virus, antimicrobial, antioxidant, antiparasitic,
antimutagenic and anticancer (Ahsan, Parveen, Khan, & Hadi,
1999; Kim, Park, & Kim, 2001; Reddy & Chandrakasan, 1989; Simon
et al., 1998; Jayaprakasha, Rao, & Sakariah, 2005). It is also efficient
in the treatment of circulatory problems, liver diseases, and derma-
tological disorders (Osawa, Sugiyama, Inayoshi, & Kawakishi, 1995;
Semwal, Sharma, & Arya, 1997; Srinivasan, Sambaiah, &
* Corresponding author. Tel.: +91 9903380372.
E-mail address: sivam25@gmail.com (M. Paramasivam).
1985). Curcumin (diferuloyl methane), the natural yellow pigment
in turmeric, is isolated from the rhizomes of the plant C. longa.
The pharmacological activities of turmeric have been attribu-
ted to its ethanol extracts, which contain three different curcumi-
noid pigments, Curcumin (Cur-I) 1,7-bis (4-hydroxy-3-methoxy-
phenyl)-1,6-heptadiene-3,5-dione], Demethoxy curcumin (Cur-II)
[1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxy phenyl)-1,6-hept-
adiene-3,5-dione], Bisdemethoxycurcumin (Cur-III) [1,7-bis
(4-hydroxy phenyl)-1, 6-heptadiene-3, 5-dione] (Bong, 2000;
Jayaprakasha, Rao, & Sakariah, 2002; Verghese, 1993).
A variety of methods for the quantification of the curcuminoids
have been reported. Most of them are spectrophotometric
methods, expressing the total colour content of the sample. Tur-
meric (rhizome) products contain mixtures of curcumin, deme-
thoxycurcumin, and bisdemethoxycurcumin. However, it is not
possible to quantify the individual curcuminoids with spectropho-
tometric methods. Usually, separation of the curcuminoids has
been achieved by silica gel thin layer chromatography (Barrero &
Carrero, 1999 and Chempakam, Leela, & John, 2000).
Several studies were undertaken to separate curcumin, deme-
thoxycurcumin, and bisdemethoxycurcumin by thin-layer chroma-
tography (TLC) and column chromatography (CC) (Govindarajan,
1980; Janaki & Bose, 1967; Janben & Gole, 1984; Krishnamurthy
et al., 1976; Osawa, Sugiyama, Inoyoshi & kawakishi, 1995 and
Srinivasan, Sambaiah & Chandrasekhara, 1992). However, poor
resolution limited the curcumin bands to 80% purity. Furthermore,
no separation was obtained for demethoxycurcumin and bisde-
methoxycurcumin.

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.07.051
 M. Paramasivam et al. / Food Chemistry 113 (2009) 640–644
O O
R1 R2
HO OH
Curcumin : R1 = R 2 = OCH3
Demethoxycurcumin : R 1 =H, R 2 = OCH3
Bisdemethoxycurcumin : R 1 = R 2 = H
Fig. 1. Chemical structure of curcuminoids.
A few methods are reported for quantification of curcuminoids
by HPLC (Chauhan, Singh, & Agrawala, 1999; Khurana & Ho, 1998;
Taylor & McDowell, 1992; Schieffer, 2002). Due to its accuracy and
simplicity but lack of precision may be due to interference by other
pigments present in the plant. Very little information is available
on the determination of curcuminoids in turmeric using high per-
formance thin layer chromatography (Gupta, Gupta, & Kumar,
1999). It should be the first choice in medicinal and aromatic plant
research programmes as most of the phytochemical are complex
biological materials. It has advantages in terms of flexibility, paral-
lel analysis of a large number of samples and different modes of
development (Nyiredy & Glowniak, 2001). HPTLC is now utilized
frequently in the crop improvement programs involving rapid
plant screening.
Commercially available curcumin consists of a mixture of natu-
rally occurring curcuminoids with curcumin as the main constitu-
ent (Jayaprakasha, Rao, & Sakariah, 2002). Since the curcuminoid
pigments vary in chemical structures, it is possible that the chem-
ical and color characteristics, as well as the functional properties,
will vary among the pigments. Pure Curcumin is scarce and expen-
sive, whereas Cur-II and Cur-III are not commercially available.
Therefore, it is important to obtain pure pigments and to character-
ize them individually to provide subsidies for the determination of
each curcuminoid pigment.
This study was undertaken to provide information on the phys-
ico-chemical characteristics of individual curcuminoid pigments to
facilitate their identification in the mixture. The specific objectives
were (i) to compare the efficiency of different solvent systems to
separate curcumin, demethoxycurcumin, and bisdemethoxycurcu-
min by TLC; (ii) to develop an easy, effective and efficient method
for determination of curcumin, demethoxy curcumin, and bisde-
methoxy curcumin by high performance thin layer chromatogra-
phy (HPTLC).
Here, we have developed a simple and rapid High Performance
Thin Layer Chromatography method for the rapid analysis of major
curcuminoids in C. longa germplasm. The method was found suit-
able for rapid screening of plant materials for their genotypic
assessment, crop improvement, export quality and can be per-
formed without any special sample pretreatment.
2. Experimental methods
(a) Plant materials: The plant rhizomes of Curcuma longa
(Nimbarg, Kalimpong, Armoor, PCT-13, PTS-43, Sugandham and
RS-5) were obtained from the Experimental Farm of Medicinal
and Aromatic Plants at B.C.K.V., Mohanpur, West Bengal, India.
(b) Curcuminoids: Curcumin, demethoxycurcumin, and bisde-
methoxycurcumin have been isolated by column chromatography
of the benzene extract of Curcuma longa. Compounds were identi-
fied by spectral analysis.
(c) Reagents: All solvents/chemicals (AR/HPLC grade) were ob-
tained from E-Merck, Mumbai, India and the HPTLC precoated alu-
minum plates silica gel 60 GF254, 20  10 cm, layer thickness 0.2
mm used were from Camag, Multenz, Switzerland.
641
2.1. Isolation of curcuminoid standards
The rhizomes (1 kg) of Curcuma longa were air dried and about
100 g of rhizomes were extracted using hexane (500 mL) in a soxh-
let apparatus (Torres, Bonifacio, Herrera, & Lanto, 1998) to remove
volatile oil. The defatted rhizome was further extracted with ben-
zene (500 mL) for separation curcuminoids (Chatterjee, Desai,
Thomas, & Chatterjee, 1999). The benzene extract was concen-
trated to dryness using rotary vacuum evaporator at 40 °C. The
concentrated dry material (8 g) was impregnated with 10 g of silica
gel, loaded onto a column of silica gel [60–120 mesh] (100 g), and
eluted with 500 mL of hexane. Then subjected to column chroma-
tography over silica gel and eluted with solvents of increasing
polarity for isolation of different phytoactive compounds.
2.2. Efficiency of different solvents system for separation of
curcuminoids by TLC
The analytical separation of curcuminoid pigments by TLC was
investigated using silica gel 60G plates 10  10 cm developed with
different solvent system as indicated in Table 1. The method was
selected according to the RF values for each pigment. The different
solvents were tested for TLC including the elution system benzene,
hexane, ethyl acetate, chloroform and methanol.
2.3. Curcuminoid crystallization
Crystallization of the curcuminoid was performed by dissolving
a curcuminoid in 50 mL of methanol at 40 °C. After dissolution,
20 mL of chloroform was added, and the mixture was kept at
5 °C for 2 h. The curcumin crystals were separated from the mother
liquor by vacuum filtration. The melting points were determined in
open capillary tubes on a sulfuric acid bath and are uncorrected.
Curcumin (Cur-I) was obtained from fractions 31–48 of with
benzene: hexane (9:1 v/v) furnished a yellow solid which was
purified by repeated and crystallized in Chloroform: MeOH, as
bright yellow needle shaped crystals and melting point 185–
186 °C, 1H NMR (CD3)2CO d: 5.82 (1 H, s,1-H),8.97(s,2-OH),6.48 (2
H, d, J = 16 Hz, 3, 30 -H2), 7.62 (2 H, d, J = 16 Hz, 4, 40 -H2), 7.28 (2
H, brs, 6, 60 -H2), 8.2 (2H, s, 8, 80 OH), 6.84 (2 H, d, J = 8 Hz, 9, 90 -
H2), 7.22 (2 H, brd, J = 8 Hz, 10, 100 -H2), 3.95 (6 H, s, 2  OMe).
Demethoxycurcumin (Cur-II), isolated from fractions 67–92 of
Benzene:ethylacetate (7:3) as the brown color residue which was
purified by repeated crystallization from chloroform: methanol
as light yellow crystals and melting point 175–176 °C, 1H NMR
(CD3)2CO d: 5.96 (1 H, s, 1-H), 8.95 (s,2-OH), 6.62 (2 H, d, J = 16
Hz, 3, 30 -H2), 7.60 (2 H, d, J = 16 Hz, 4, 40 -H2), 7.52 (2 H, d, J = 8
Hz, 6,60 -H2), 6.88 (1 H, d, J = 8 Hz, 7-H), 6.85 (2H, d, J = 8Hz, 9, 90 -
H2), 7.15 (1 H, brd, J = 8 Hz, 10 H), 7.30 (1 H, brs, 100 H), 3.95 (3
H, s, OMe).
Bisdemethoxycurcumin (Cur-III) was isolated by column chro-
matography fractions of 93–113 from benzene: ethylacetate (9:1).
Compound Cur-III was crystallized in chloroform: methanol, as
reddish brown shinning crystals and melting point 231–232 °C,
Table 1
Different solvent systems on the separation of curcuminoids by TLC
TLC mobile phase RF values
Cur-I Cur-II Cur-III
Benzene:hexane (9:1) 0.69 0.48 0.40
Chloroform:methanol (48:2) 0.66 0.48 0.30
Chloroform:methanol (20:5) 0.40 0.34 0.26
Benzene:ethyl acetate (9:1) 0.75 0.54 0.46
DCM:MeOH (48:2) 0.49 0..32 0.21
642 M. Paramasivam et al. / Food Chemistry 113 (2009) 640–644

1
H NMR (CD3)2 CO d: 5.90 (1 H, s, 1-H), 8.95 (s,2-OH),6.58 (2 H, d, Different solutions of varying polarities were used for extraction
J = 16 Hz, 3, 30 -H2), 7.60 (2 H, d, J = 16 Hz, 4, 40 -H2), 7.50 (2 H, d, of curcuminoids from rhizome. Methanol was found to be the most
J = 8 Hz, 6, 60 – H2), 6.82 (4 H, d, J = 8 Hz, 7, 70 -H2),), 8.2 (2H, s, 8, appropriate solvent for the maximum extraction of curcuminoids
80 OH), 6.82 (2 H, d, J = 8 Hz, 9, 90 -H2), 7.50 (4 H, d, J = 8 Hz, 10, as the recoveries of curcuminoids were optimum in this solvent.
100 -H2). All structures were confirmed by comparison with spec- The identification of curcuminoids in the rhizome was confirmed
tral analysis data reported in literature (Jayaprakasha, Rao, & by superimposable of UV–visible spectra of the extract with that
Sakariah, 2002). of the standard within the same RF window.
The percentage amounts of curcuminoids of seven different
germplasm of C. longa by HPTLC are given in Table 2. Curcumin
2.4. HPTLC Analytical procedures
was found to be the major compound in all of the tested germplasm
followed by bisdemethoxycurcumin and demethoxycurcumin, the
(a) Chromatographic condition:
ratio hardly changed with maturity (Cooray, Janse, Ranatunga, &
Chromatography was performed on a pre-activated silica gel
Wimalasena, 1988).The highest content of Curcumin is perhaps
HPTLC plate (60GF 254, 20  10 cm). Samples and standard were
due to its stability compared to other two compounds. It was found
applied on the plate as 6mm wide bands with an Automated Ca-
that Nimbarg (6.18%) followed by Kalimpong (5.37%) have higher
mag TLC applicator, Linomat 5 (Camag, Multenz, Switzerland) with
amounts of total curcuminoids content, where as germplasm RS-5
N2 flow @ 150 gl/sec, positioned 15 mm from the bottom of the
(1.65%) has lower amount of total curcuminoids content. Hence
plate and 20 mm from side of the plate. The application parameters
these two varieties Nimbarg and Kalimpong are high quality tur-
were identical for all the analyses performed.
meric and may be a good source for the isolation of curcuminoids.
(b) Detection of curcuminoids:
The curcuminoids were dissolved in methanol. The TLC plates
3.2. Purity of curcuminoids
were developed in a camag twin trough glass tank presaturated
with the mobile phase (CHCl3: MeOH, 48: 2, v/v). It was then
The purity of isolated Curcumin, demethoxycurcumin and bis-
poured in twin trough glass solvent development chamber well
demethoxycurcumin was confirmed by HPTLC analysis and melt-
in advance (30 min) to allow complete saturation which was fur-
ing point determination. The isolated curcuminoids showed
ther enhanced by keeping one filter paper along one wall of the
single spots on HPTLC plate (Figs. 2 and 3) and gave a single peak
chamber and each plate was developed to a height of about 8 cm
on scanning at kmax 425 nm.
run. The TLC runs were made under laboratory conditions of 25 ±
5 °C and 50% relative humidity. After development, the plate was
removed and dried and spots were visualized in camag UV cham-
ber (425 nm) and Curcuminoids were quantified with a camag TLC Table 2
scanner model 3 equipped with WINCATS software under the fol- Percentage (w/w) of composition of curcuminoids in seven different germplasm of
lowing conditions [Slit width – 5  0.45 mm, Wavelength – Curcuma longa by HPTLCa

425 nm, absorption/reflection detection mode. The HPTLC analysis S No. Germplasm % Content (w/w)
of Cur-I, II and III showed single peak at RF 0.66 ± 0.02, 0.48 ± 0.02 Cur – I Cur – II Cur – III Total
and 0.30 ± 0.02 respectively.
1. Nimbarg 2.43 1.79 1.96 6.18
(c) Extraction procedure: 2. Kalimpong 2.19 1.58 1.60 5.37
Air dried (35–50 °C) rhizomes of C. longa (1 g each) were ex- 3. Armoor 1.95 1.01 1.24 4.20
tracted separately in (20 mL  3) methanol for 30 min by ultrason- 4. PCT – 13 1.80 1.02 1.21 4.03
ication, filtered, and concentrated. The individual extracts of C. 5. PTS – 43 1.21 0.72 1.22 3.15
6. Sugandham 0.83 0.57 0.70 2.10
longa were re-dissolved in 10 mL of methanol for quantification.
7. RS – 5 0.76 0.38 0.51 1.65
(d) Calibration graphs:
a
Stock solutions of Cur-I, Cur-II and Cur-III (1 mg/mL) were pre- Average of three replications.
pared in methanol and different amounts (1–20 lL) of these were
analysed by HPTLC exactly as described above.

3. Results and discussion

3.1. Comparison of TLC methods for the separation of Cur-I, Cur-II and
Cur-III

Different compositions of mobile phase for HPTLC analysis of

Cur-1, Cur-II and Cur-III were tested in order to obtain high resolu-
tion, symmetrical and reproducible peaks (Table 1). The desired
resolution of compounds was achieved using Chloroform and
methanol 48:2 as the mobile phase. On this system separation be-
tween curcuminoids is larger and the spots are well defined. Spots
of three compounds showed florescence when observed under UV
light. The scanning wavelength of 425 nm was found to be optimal
for highest sensitivity of curcuminoids spots. Peaks corresponding
to curcuminoids, checked via addition of standard, were well re-
solved for identification. For examination of recovery rates, known
amount of stock solutions of curcuminoids were added in C. longa
rhizome extract and quantitative analysis replicated three times.
Recovery of Cur-1, Cur-II and Cur-III was 98.71, 96.29 and 97.18% Fig. 2. HPTLC separation of curcumin, demethoxy curcumin, and bisdemethoxy
respectively. curcumin in standard, Curcuma longa tracks at 425 nm absorption/reflection mode.
 M. Paramasivam et al. / Food Chemistry 113 (2009) 640–644
Fig. 3. HPTLC separation of curcumin, demethoxy curcumin, bisdemethoxy curcu-
min and 1 and 5 unidentified peaks in sample, Curcuma longa tracks at 425 nm
absorption/reflection mode.
3.3. Peak purity test
Peak purity test of curcuminoid was done by comparing UV–vi-
sual spectra of curcuminoid in standard and sample tracks. Peak
purity results (obtained by scanning at 425 nm) were satisfactory.
Correlations of the peak start spectrum with the peak centre spec-
trum were 0.9999, 0.9998 and 0.9998 and 0.9999, 0.9998 and
0.9998 for standard and sample tracks, respectively. Correlation
of the peak centre spectrum with the peak end spectrum were also
0.9999, 0.9998 and 0.9998 and 0.9999, 0.9998 and 0.9998 for stan-
dard and sample tracks, respectively.
3.4. Detection limits
Detection limit of Cur-I, Cur-II and Cur-III was determined by
estimating the minimal mass that could be quantified. It was
0.1 lg/s pot.
3.5. Linearity
For determination of the linearity curve, different amounts (1–
20 lg) of stock solution of Cur-I, Cur-II and Cur-III (1 mg/ mL) were
applied on HPTLC plate and plate was developed as above and
scanned at kmax 425 nm. The calibration plot of peak area versus
concentration was linear. The linear regression equation was Y =
4447.26 + 61.993X, Y = 1089.881 + 70.003X and 2611.84 + 51.565X
for Cur-I, Cur-II and Cur-III and with correlation coefficient (r)
0.9999, 0.9998 and 0.9998 respectively.
HPTLC technique was able to determine micro quantities of cur-
cumin and other curcuminoids from turmeric rhizome, while
maintaining a high level of purity. This method provides a reliable
fingerprint for turmeric, distinguishing it from other curcuma spe-
cies. It has advantageous due to its shorter analysis time, minimal
sample preparation and it can also be used in the quality standard-
ization of turmeric extracts for pharmaceutical production and
cosmetics.
The method will be useful for rapid screening purpose of plant
samples for finding of high quality turmeric under the plant
breeding program, genotypes assessment and export quality of
curcuma species for curcuminoids. The proposed method is
shown to have the selectivity, accuracy, precision and high sam-
ple throughput that make it useful for routine determination of
curcuminoids of a large number of commercial samples of
C. longa.
643
Acknowledgements
The authors are indebted to Department of Agricultural Chem-
icals for providing infrastructural facilities, Bidhan Chandra Krishi
Viswavidyalaya, Kalyani, India for conduct the experiment. We also
extend our appreciation to all colleagues who participated in scien-
tific discussions and provided useful suggestions.
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