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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1244–1249

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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Fourier transform infrared spectroscopy combined with chemometrics
for discrimination of Curcuma longa, Curcuma xanthorrhiza and Zingiber
cassumunar
Eti Rohaeti a, Mohamad Rafi a,b,⇑, Utami Dyah Syafitri c, Rudi Heryanto a,b
a

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia
Biopharmaca Research Center – Research and Community Empowerment Institute, Bogor Agricultural University, Jalan Taman Kencana No. 3 Kampus IPB Taman Kencana,
Bogor 16128, Indonesia
c
Department of Statistics, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Meranti Kampus IPB Dramaga, Bogor 16680, Indonesia
b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t 

Discrimination of C. longa, C.

xanthorrhiza and Z. cassumunar by
FTIR. 
Principal component analysis and
canonical variate analysis are used for
discrimination. 
Canonical variate analysis gave
clearer discrimination between the
three species.

a r t i c l e

i n f o

Article history:
Received 22 March 2014
Received in revised form 14 July 2014
Accepted 31 August 2014

Keywords:
C. longa
C. xanthorrhiza
Z. cassumunar
FTIR
Chemometrics
Discrimination

a b s t r a c t
Turmeric (Curcuma longa), java turmeric (Curcuma xanthorrhiza) and cassumunar ginger (Zingiber
cassumunar) are widely used in traditional Indonesian medicines (jamu). They have similar color for their
rhizome and possess some similar uses, so it is possible to substitute one for the other. The identification
and discrimination of these closely-related plants is a crucial task to ensure the quality of the raw materials. Therefore, an analytical method which is rapid, simple and accurate for discriminating these species
using Fourier transform infrared spectroscopy (FTIR) combined with some chemometrics methods was
developed. FTIR spectra were acquired in the mid-IR region (4000–400 cm 1). Standard normal variate,
first and second order derivative spectra were compared for the spectral data. Principal component
analysis (PCA) and canonical variate analysis (CVA) were used for the classification of the three species.
Samples could be discriminated by visual analysis of the FTIR spectra by using their marker bands.
Discrimination of the three species was also possible through the combination of the pre-processed FTIR
spectra with PCA and CVA, in which CVA gave clearer discrimination. Subsequently, the developed
method could be used for the identification and discrimination of the three closely-related plant species.
Ó 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis Kampus IPB Dramaga, Bogor
16680, Indonesia. Tel./fax: +62 251 8624567.
E-mail address: mra@ipb.ac.id (M. Rafi).
http://dx.doi.org/10.1016/j.saa.2014.08.139
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

xanthorrhiza [4. Phenolic compounds (diarylheptanoid. they may have some similar chemical components.02 (Addinsoft. diabetes. Kulonprogo. While in Z. sesquiterpenoid. FTIR has been widely used and is a well-established tool for quality control in various industries. xanthorrhiza and Z. These ethanol extracts were then used for FTIR measurement. xanthorrhiza and 29 samples of Z. java turmeric (Curcuma xanthorrhiza) and cassumunar ginger (Zingiber cassumunar) cultivated mostly in the Java Island are widely used in traditional Indonesian medicines (jamu). In this case. NMR. Indonesia: Bogor. Cassumunar.E. hepatitis. diarylheptanoids (curcumin). The color of their rhizome is yellow to orange for C. About 5 mg of ethanol extract of each sample was mixed with 95 mg of KBr and then pressed to form a tablet. and Kediri (Table 1). although the composition of a chemical compound is certainly not known [8]. Diarylheptanoids/curcuminoids (curcumin. [10]. Analytical-grade ethanol for solvent extraction was obtained from Merck (Darmstadt. zingiberene. and are used to treat stomachic. Obvious advantages of FTIR application to discriminate different medicinal plants are not only effective and specific. longa and C. were compared. Longa. Changes in the position and intensity of bands in the FTIR spectra would be associated with the changes in the chemical composition of a sample. turmerone. diarrhea. Sample preparation Ninety-nine samples consisting of 35 samples of C. Material and methods Chemicals Potassium bromide (KBr) for spectroscopy was purchased from Sigma–Aldrich (St Louis. baseline variation. Although medicinal plants will contain a large variety of chemical components.3]. the aid from chemometrics methods is needed [9]. In this experiment. and first and second order derivative spectra. including herbal industry [8]. and triterpenoid) have been identified in C. fast. Voucher specimens were deposited at Biopharmaca Research Center. Ethanol was used to extract the samples and after the extraction process. xanthorrhiza and Z. etc. and GC) have been used in the development of method for identification and discrimination of medicinal plants. muscle and joint pain [2. longa and some of them also present in C. Several analytical techniques such as spectroscopy (UV–Vis. This combined method was successfully applied for the identification and discrimination of the three species. the ethanol was evaporated by rotary evaporator. and pale yellow to yellow for Z.) and sesquiterpenoids (curcumene. Fourier transform infrared (FTIR) spectroscopy may be an attractive option because it can meet the criteria of efficient analysis. safety and efficacy of the raw materials before they are converted to final products. stomachic. cassumunar is usually used to treat various diseases such as asthma. [12] in all FTIR spectra of the samples. Because of the advantages 1245 of FTIR spectroscopy. Germany). Ponorogo. Due to the similarity of the color of their rhizomes which possess some similar uses. their FTIR spectra sometimes are found to differ even in the same species [7]. etc. we used this techniques in combination with chemometrics methods for the first time to develop a qualitative method for discrimination of C. etc. substitution for each other could be possible.) were found as a major group of compounds in C. Bogor Agricultural University. Discrimination by visual inspection in the FTIR spectra is not easy because the FTIR spectra pattern is very complex. .2 software (Bruker. cassumunar were collected during 2008–2010 from 11 regencies (2–5 samples from each regency) in Java Island. xanthorrhiza are usually aromatic and carminative. C. cassumunar and these analyses were performed in XLSTAT software version 2012. HPLC. systematic noise. There are several methods to obtained IR spectra for the purpose of identification and discrimination as described by Zou et al. phenylbutanoids ((E)-1(3. USA). and to address this concern. xanthorrhiza is much higher than C. Purworejo. Karanganyar. Rohaeti et al. FTIR spectral data pre-treatment Spectral data pre-treatment is an important step before subjecting the FTIR spectra data for multivariate analysis. the identification and discrimination of these closely-related plants are crucial in order to ensure the quality. cassumunar. a rapid. FTIR spectral analysis alone or in combination with chemometrics methods has been extensively used for species identification and discrimination of some closely-related species [11–22]. USA). Pacitan. colic. New York. xanthorrhiza. FTIR spectra contain complex data information describing the overall chemical signal in a sample. i. easy to use. and pulverized prior to use. simple and accurate analytical method is essentially required for the discrimination of these species. xanthorrhiza. the extracts were blended with KBr. It is necessary to perform this step in order to minimize the effect of light scattering. FTIR spectra could be used to discriminate closely-related species. but also rapid and non-separative. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1244–1249 Introduction Turmeric (Curcuma longa). In this study. especially if in the powdered form and if the price of C.e. Therefore.5]. monoterpenoids (sabinene) and sesquiterpenoids (zingiberene) were identified [2]. The advantage of using chemometrics for the interpretation of FTIR is the ability to link the spectral pattern with hidden information contained in a sample [10]. diarylpentanoid. The sample tablet was placed in the sample compartment and FTIR spectra were recorded in the region of 4000–400 cm 1 with 32 scans/min and resolution of 4 cm 1. Chemometrics analysis Principal component analysis (PCA) and canonical variate analysis (CVA) were used to build a model for discrimination of C. C. FTIR. atherosclerosis and bacterial infections [1]. C. demethoxycurcumin. while Z. and MS) and chromatography (TLC. Germany). the samples were extracted with a solvent and after evaporating the solvent. Semarang. Sumedang. cassumunar. data from three different pre-treatment methods. diterpenoid. it could be a serious problem because of inconsistency in their biological properties. If the substitution occured. longa. longa and C. and inexpensive [6]. These three plants are belongs to the Zingiberaceae family and therefore. This method will provide better resolution in the identification and discrimination of closely related plants [10].4-dimethylphenyl) butadiene). All samples were sieved. Curcuminoids are responsible for the color of the three plants used in this study. phenylpropene derivative) and terpenoids (monoterpenoid. longa. Among these techniques. jaundice. Indonesia.2. namely standard normal variate (SNV). FTIR spectroscopy measurement FTIR spectra were obtained using a Bruker Tensor 37 FTIR spectrophotometer equipped with deuterated triglycine-sulphate (DTGS) as detector and controlled by OPUS 4. carminative. longa or Z. 35 samples of C. Wonogiri. Therefore. Sukabumi. bisdemethoxycurcumin. longa and C. dried. cassumunar.

Semarang. Ponorogo. Sukabumi. Central Java Tawangmangu. Central Java Tembalang. West Java Rancakalong. Rohaeti et al. However. West Java Tanjungkerta. province) CX-32 CX-33 CX-34 CX-35 Bandar. East Java Tanjungkerta. longa CL-1 CL-2 CL-3 CL-4 CL-5 CL-6 CL-7 CL-8 CL-9 CL-10 CL-11 CL-12 CL-13 CL-14 CL-15 CL-16 CL-17 CL-18 CL-19 CL-20 CL-21 CL-22 CL-23 CL-24 CL-25 CL-26 CL-27 CL-28 CL-29 Wonogiri. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1244–1249 Table 1 Sources of samples. Wonogiri. East Java Slahung. Purworejo. Central Java Ngadirejo. Semarang. Kediri. Kulonprogo. East Java Semen. Central Java Tawangmangu. Sumedang. Kulonprogo. Central Java Kalibawang. Karanganyar. Semarang. Purworejo. East Java Bandar. Central Java Tembalang. Table 1 (continued) Species Species Sample code Sources (subdistrict. Kediri. xanthorrhiza were compared to the FTIR spectra of Z. Central Java Tembalang. Central Java Tawangmangu. Semarang. Bogor. Semarang. Central Java Ngadirejo. Pacitan. Central Java Tembalang. West Java Gunung Putri. Central Java Tembalang. Special Region of Yogyakarta Ponorogo. Pacitan. Sukabumi. East Java Slahung. Karanganyar. East Java Tanjungkerta. FTIR spectra could be used for the purposes of identification and discrimination of some closely-related medicinal plants. Central Java Ngadirejo. Central Java Tembalang. Semarang. East Java Bandar. East Java Semen. longa samples while the C. Purworejo. Two peaks at 833 cm 1 and 816 cm 1 were only found in C. Semarang. Comparison of the FTIR spectra of C. East Java Tanjungkerta. Central Java Tembalang. West Java Tanjungkerta. Purworejo. West Java Cikembar. East Java Tegalombo. Central Java Kalibawang. Karanganyar. West Java Cikembar. Pacitan. Karanganyar. West Java Cikembar. Central Java Tawangmangu. Kulonprogo. Semarang. West Java Ciampea. Ponorogo. East Java Slahung. Sukabumi. Central Java Kalibawang. Wonogiri. at 2800–3000 cm 1 to methyl (ACH3) and methylene (ACH2) symmetric and asymmetric stretching vibration. East Java ZC-27 ZC-28 ZC-29 Result and discussions Discrimination by FTIR spectral analysis Medicinal plants are complex mixture of chemicals. Central Java Wonogiri. West Java Gunung Guruh. Wonogiri. cassumunar could be discriminated from C. Sumedang. Purworejo. West Java Ciampea. Sumedang. West Java Cikembar. Central Java Ngadirejo. Wonogiri. Kulonprogo. cassumunar samples. Purworejo. C. so their FTIR spectra will show overlapping of some characteristic absorption bands of the functional groups in the sample [11]. province) C. Central Java Purworejo. longa. West Java Pituruh. Bogor. West Java Rancakalong. Central Java Karangpandan. Central Java Tawangmangu. Central Java Tembalang. Sukabumi. Ponorogo. longa and C. cassumunar obvious differences were observed. Special Region of Yogyakarta Z. xanthorrhiza and Z. Central Java Tembalang. Sumedang. Sumedang. at 1740–1680 cm 1 are attributed to C@O absorption. Wonogiri. Pacitan. Karanganyar. Pacitan. Karanganyar. Kediri. Purworejo. Central Java Tembalang. Special Region of Yogyakarta Bandar. Central Java Purworejo. Kulonprogo. West Java Purworejo. West Java Gunung Putri. Ponorogo. Central Java Semen. East Java Semen. Kediri. Semarang. the FTIR spectra of each species from different locations showed high similarities. xanthorrhiza showed slight differences because they are from the same genus that they may have similar chemical components. Z. Bogor. Central Java Tegalombo. Central Java Ngadirejo. Special Region of Yogyakarta Pengasih. Sukabumi. Central Java Semen. Special Region of Yogyakarta Wates. 1. Bogor. Kulonprogo. East Java CL-30 CL-31 CL-32 CL-33 CL-34 CL-35 C. Wonogiri. East Java Tegalombo. Central Java Tembalang. cassumunar are shown in Fig. longa and C. East Java Ponorogo. and at 1030 cm 1 are due to CAOH stretching vibration. regency. East Java Slahung. East Java Ngrayun. West Java Cibungbulang. West Java Cikembar. Kulonprogo. Central Java Purworejo. Wonogiri. Special Region of Yogyakarta Pengasih. xanthorrhiza by using a peak at 1737 cm 1 for the peak only appeared in Z. Karanganyar. cassumunar samples . Ponorogo. Sukabumi. Central Java Wonogiri. Kediri. Sukabumi. Ponorogo. Sumedang. Ponorogo. Bogor. Semarang. when the FTIR spectra of C. Central Java Wonogiri. Special Region of Yogyakarta Pengasih. Bogor. The FTIR spectra of C. Under the same experimental conditions. East Java Semen. Special Region of Yogyakarta Wates. Wonogiri. Bogor. East Java Ponorogo. East Java Ngrayun. regency. Wonogiri. Semarang. Ponorogo. Sumedang. Pacitan. Kulonprogo. Central Java Purworejo. West Java Rancakalong. it is recognized that there are variations in their peak positions and intensities. East Java ZC-1 ZC-2 ZC-3 ZC-4 ZC-5 ZC-6 ZC-7 ZC-8 ZC-9 ZC-10 ZC-11 ZC-12 ZC-13 ZC-14 ZC-15 ZC-16 ZC-17 ZC-18 ZC-19 ZC-20 ZC-21 ZC-22 ZC-23 ZC-24 ZC-25 ZC-26 Wonogiri. West Java Cibadak. West Java Rajapolah. Sukabumi. Wonogiri. Kediri. at 1510 cm 1 are assigned to aromatic skeletal stretching vibration. xanthorrhiza and Z. West Java Dramaga. West Java Gunung Putri. longa and C. East Java Slahung. xanthorrhiza CX-1 CX-2 CX-3 CX-4 CX-5 CX-6 CX-7 CX-8 CX-9 CX-10 CX-11 CX-12 CX-13 CX-14 CX-15 CX-16 CX-17 CX-18 CX-19 CX-20 CX-21 CX-22 CX-23 CX-24 CX-25 CX-26 CX-27 CX-28 CX-29 CX-30 CX-31 Wonogiri. West Java Dramaga.1246 E. Special Region of Yogyakarta Wates. Ponorogo. Bogor. Ponorogo. East Java Slahung. By comparing the FTIR spectra of the three species. Sumedang. Central Java Tembalang. Semarang. Kulonprogo. West Java Dramaga. Ponorogo. Pacitan. As can be seen in Fig. 1. the characteristic peaks in the FTIR spectra of the three samples appeared at 3400 cm 1 corresponds to OAH absorption. East Java Tegalombo. Central Java Tawangmangu. cassumunar Sample code Sources (subdistrict. West Java Purworejo. West Java Cikembar. Tasikmalaya. Sukabumi. Special Region of Yogyakarta Wates. Kulonprogo. Pacitan. Wonogiri. West Java Gunung Guruh. Bogor.

longa. In this study. longa (A). C. longa could be discriminated from the other two species in this study. Representative FTIR spectra of C. Pre-treatment of FTIR spectra is a standard procedure before using the spectra in chemometrics analysis. SNV and first and second order derivative spectra were applied and compared. cassumunar could be observed from the intensities of OH absorption near 3400 cm 1. xanthorrhiza. 2. are used. xanthorrhiza. xanthorrhiza and Z. longa and Z. cassumunar from the other two plants could be obtained by using two positive peaks at 1452 and 1430 cm cassumunar sample. thus giving the FTIR spectra a unit standard deviation. cassumunar (C). only showed one peak at 816 cm 1. 1 that were only found in Z. Negative peak near 1738 cm 1 could be used for the identification of Z. With second derivative spectra. Representative second derivative FTIR spectra of C. and Z. Beside the negative peak. such as PCA and CVA. by using this rationale. SNV removes slope variation and also the scatter effects [23. longa sample. only appeared in C. Discrimination of C. xanthorrhiza and Z. xanthorrhiza from C. 2 shows the second derivative FTIR spectra of the samples in the region 1800–400 cm 1 and it is seen that there are some differences in the spectra features. As illustrated in Fig. some overlapping bands could also be resolved. Fig. some techniques in chemometrics. longa (A). xanthorrhiza samples than C. cassumunar To confirm the results obtained from the visual inspection of the FTIR spectra for the discrimination of C. cassumunar. SNV works by calculating the standard deviation of all data points in a given FTIR spectra and then the entire FTIR spectra is normalized by this value.1247 E. longa. a combination of FTIR spectra and chemometrics methods was used. discrimination of Z. longa and C. The first and second order derivative spectra are usually . C. xanthorrhiza (B). C. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1244–1249 Fig. respectively. C. Enhancement of the spectral resolution and amplification of small differences in the ordinary spectra could be obtained using second derivative spectra. and finding the similarities or dissimilarities between observations and variables. the peak of Z. xanthorrhiza (B). cassumunar because in this sample. Chemometrics is widely used to analyze a huge amount of data such as in FTIR spectra with an aim to resume information contained in the data matrix by reducing dimensions of the data.24]. cassumunar (C). xanthorrhiza from the other samples could be obtained by using negative peaks at 989 cm 1 that were only observed in C. Fig. it was clearly observed that typical positive and negative peaks at 1590 and 1579 cm 1. cassumunar samples. longa and Z. C. 2. Discrimination of C. 1. The intensities of the FTIR spectra of this functional group were found much greater in all C. and Z. Rohaeti et al. cassumunar gave higher intensities compared to those of C. Combination of FTIR spectra and chemometrics method for discrimination of C.

The main objective of the PCA is to reduce the data and extract the information in order to find a combination of variables or factors for describing major trends in a data set. Although C. Table 2 provides the information regarding the eigenvalues and cumulative percentage of total variance from the 10 initial PCs. ZC-10 and ZC-29) were detected in the C. 4. The full 99 objects  831 variables data matrices were submitted to PCA. From the result of CVA. This result indicates that the model gives satisfactory prediction of the samples tested. Canonical variate analysis CVA is one of the supervised pattern recognition and widely used for multiple groups discrimination. CVA was used to discriminate the three herbs more clearly. PCA plot for the first two PCs is used and being the most useful in the analysis because both PCs contain the most variations in the data.145 3. xanthorrhiza ( ).443 41. In this study.3%). the distance between them was so close. indicating that the CVs obtained could clearly discriminate the three species. This criterion will retain PCs with eigenvalues greater than 1 because these PCs explain as much variance as are observed in the variables [27]. The criterion proposed by Kaiser was used to determine the number of PCs to be retained and used them in the CVA model.7%).915 99. Only three Z. pre-treatment of the FTIR spectra using SNV gave the best result for optimum groups separation for the three species.61% of total variance were explained by this 10 initial PCs.023 14. the tested samples were clustered into three different groups. longa and C. longa ( ).911 3.714 97. PCA plot of samples: C. xanthorrhiza cluster. Principal component analysis PCA is a well-known unsupervised pattern recognition. From the PCA plot. it could be obtained pattern of a sample. Fig. therefore. Fig. cassumunar samples (ZC-3. and Z. The closer the PCs values.536 46. cassumunar ( ).7% and CV2 = 13. Most of the Z.262 1. xanthorrhiza ( ). PCA was employed in the FTIR spectra data of samples to get PCs before building a predictive model using CVA. 4). The CVA predictive model was built based on eight initial PCs (eigenvalues greater than 1) as input variables with the cumulative percentage of total variance greater than 99%. the total variance from the two CVs was 100% (CV1 = 86. CVA plot of samples: C. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1244–1249 Table 2 Eigenvalues and cumulative percentage of total variance for each PCs. CVA will work effectively when the number of samples is more than the number of variables. Fig.3% and PC2 = 16.949 90.465 99. PCA will transform the original variables into new uncorrelated variables called principal components (PCs) that maximize the explained variance in the data on each successive component under the constraint of being orthogonal to the previous PCs [25]. 3. In this case.760 1. cassumunar samples were separated from the C. Typically. one of sample is removed at a time and the rest of the samples are used to build a model.309 75.614 used to eliminate the baseline drifts and for the enhancement of the small spectral features [12]. Principal component Eigenvalue Cumulative percentage of total variance 1 2 3 4 5 6 7 8 9 10 165. SNV was selected to normalize the FTIR spectra before subjecting the spectra to PCA and CVA. longa ( ). Leave-one-out cross-validation (LOOCV) method was used for evaluation of the predictive ability of this model.285 98.470 0. PCA was employed to discriminate the samples according to the species based on the FTIR spectra in the region of 2000–400 cm 1.297 99. longa and C. Canonical variates (CVs) are the name for these linear combination [26]. PCA could discriminate the three species.647 95. about 98% of all samples used in this study were correctly classified and only 1 sample (CL-4 and CL-7) was misclassified.116 98. This region was selected because it is complex and full of information with many vibrations attributed to the chemical components in all samples. Separate covariance matrices were used in this classification because there are differences in the within-class covariance matrices from the Box test.415 59. groupings. C. cassumunar ( ). C. LOOCV works by a single training set. This situation occurred due to the fact that the chemical profile of the two plants was nearly similar as can be seen in their FTIR spectra (Fig. similarities and differences [16].1248 E. xanthorrhiza samples. xanthorrhiza could be discriminated. The goal in CVA is to find linear combination of variables that exhibit maximum amonggroups variations to within-groups variations. In this work. As a result from the LOOCV. . First. In general.066 0. About 99. Rohaeti et al. 1). As can be seen in Fig. the greater the similarities among the samples. This means that it is clearly showed that 100% of the original groups were correctly classified into its own group (Fig. 3 showed the score plot derived from PCA using the first two PCs which accounted for 76% of the total variance (PC1 = 59. 3. In this work. Then the removed sample is treated as an unknown and its class membership is predicted [28]. and Z.

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