Applied Spectroscopy Reviews, 46:251–260, 2011Copyright © Taylor & Francis Group, LLCISSN: 0570-4928 print / 1520-569X onlineDOI: 10.1080/05704928.2011.565532

Recent Applications of Fourier Transform InfraredSpectrophotometry in Herbal Medicine Analysis

ANDREI A. BUNACIU,1 HASSAN Y. ABOUL-ENEIN,2

AND SERBAN FLESCHIN3

1SC Hofigal Import Export SRL, Research & Development Department,Bucharest, Romania2Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical andDrug Industries Research Division, Dokki, Cairo, Egypt3University of Bucharest, Faculty of Chemistry, Department of OrganicChemistry, Biochemistry and Catalysis, Bucharest, Romania

Abstract: Fourier transform infrared spectroscopy (FTIR) is a fast and nondestructiveanalytical method. Associated with chemometrics, it is a powerful tool for the pharma-ceutical industry. It is becoming a suitable technique for analysis of herbal medicine.This review focuses on the recent developments and updates for the qualitative andquantitative analysis of herbal medicine using FTIR. Moreover, it can be implementedduring herbal drug development, in production for process monitoring, or in qualitycontrol laboratories.

Keywords: FTIR analysis, herbal medicine analysis

Introduction

There is no doubt that herbal medicine is one of the oldest forms of health care. TheWorld Health Organization (WHO) estimates that 80% of the world’s population stillrelies primarily on botanical medicines [1]. Some botanicals have come to be used widelyin standard allopathic medicine, such as morphine, cocaine, digitalis, etc., and their useis regulated by the Food and Drug Administration with standards for purity, safety, andefficacy carefully upheld.

Infrared spectroscopy is certainly one of the most important analytical techniquesavailable to scientists. One of the great advantages of infrared spectroscopy is that virtuallyany sample can be studied. As a consequence of the improved instrumentation, a varietyof new sensitive applications have been developed in order to examine formerly intractablesamples [2–6].

Finally, the major advantage of infrared (IR) over other spectroscopic techniques isthat practically all compounds show absorption (emission) and can thus be analyzed bothqualitatively and quantitatively.

Address correspondence to Professor H.Y. Aboul-Enein, Pharmaceutical and Medicinal Chem-istry Department, Pharmaceutical and Drug Industries Research Division, Tahrir Street, Dokki, Cairo12311, Egypt. E-mail: enein@gawab.com

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Fourier transform infrared spectroscopy (FTIR) was originally a spectroscopic tech-nique used to identify the functional groups of the chemical constituents, but it has beenwidely used for the identification, quality control, and manufacturing process supervisionof herbal medicine in recent years.

The efficiencies of herbal medicines depend on the amount of active components inthem, which could vary significantly in contents. Therefore, the quality control of herbalmedicines is an essential issue.

The objective of this review is to present the new developments in the applications ofFTIR spectroscopy for herbal medicine analysis.

Fourier Transform Infrared Technique

The principle of IR spectroscopy is the measurement of the amount of IR radiation, which isabsorbed (or emitted) by a sample as a function of the wavelength [6]. The IR measurementcan be carried out in the modality of transmission or reflectance, with the first being themost popular.

IR spectroscopy has a high potential for the elucidation of molecular structures. TheIR spectrum of a poly-atomic molecules is based on molecular vibrations, each specificallydependent on atomic masses, bond strengths, and intra- or intermolecular interactions. As aconsequence, the entire IR spectrum of an organic compound provides a unique fingerprint,which can be readily distinguished from the IR absorption pattern of other compoundsincluding isomers. In other words, when reference spectra are available, most compoundscan be unambiguously identified on the basis of their IR spectra.

The IR spectrum contains abundant structural information. IR spectroscopy has beenthe classic analytical method for organic compound structure. Recently, FTIR spectroscopyhas developed quickly due to its low noise, rapid speed, high repeatability, easy operation,low expense, and so on. Associated with other sciences just like mathematics or computers,or with other techniques such as two-dimensional correlation analysis (2D-IR), FTIR hasbecome increasingly useful in the field of evaluating herbal qualities.

The vast majority of molecules exhibit infrared bands in the mid-infrared regionbetween 4000 and 400 cm−1. The position and intensity of a vibrational band are charac-teristic of the underlying molecular motion and consequently of the atoms participating inthe chemical bond, their conformation, and their immediate environment. Thus, a certainsubmolecular group produces bands in a characteristic spectral region. These characteris-tic bands form the empirical basis for the interpretation of vibrational spectra. Moreover,characteristic absorption bands can be used for compound-specific detection.

FTIR spectrometers have almost entirely replaced dispersive instruments because oftheir better performance in nearly all respects. The application of this technique has im-proved the acquisition of IR spectra dramatically.

Since early 1987 the fingerprint spectra (FPS) has been used in identification of herbalmedicines [7]. The components are extracted from the herb with certain solvents of differentpolarities on the order of petroleum ether (or cyclohexane), chloroform, ethanol, and waterand their UV/FTIR fingerprint spectra are measured.

Three methods are applied for collecting IR FPS for quality control of herbal medicines[3, 8]. In the first method, solvents of different polarities are used to extract components.After evaporating the solvent, the components are blended with KBr powder and pressuredinto a pellet, and then the IR FPS of samples are collected. The samples enable us todistinguish different herbal medicines effectively. In the second method, powders of herbal

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medicine are blended with KBr and pressured into a pellet and then the FTIR spectra ofthe samples are collected. In the last method, the reflectance spectra are obtained directlyfrom herbal materials. Although the last two methods are convenient, they provide lowerresolution ability in identification of herbal medicines as compared with first method.

Selected Applications of Herbal Medicine Analysis

The literature studied shows a great number of papers dedicated to herbal drug analysisusing FTIR. Most of the papers are related to qualitative assays of an active compound, butthere are also many papers dedicated to quantitative methods. The subjects can be divided intwo main categories: first and the most important are related to proposing new methods forqualitative and quantitative determinations and the second are concerned with establishingnew methods for analytical estimation of geographic origin.

Radix Aconiti kusnezoffii is an important plant for officinal noxiousness, which wasanalyzed from various processed products as well as from their ether extracts by usingFTIR spectroscopy and 2D-IR [9]. There are distinctive differences in the absorption peaksin the range of 1800–1500 cm−1 in the second derivative spectra of different processedproducts. The authors used the second derivative spectra because of their better resolution.With the advantages of high resolution, high speed, and convenience, FTIR can quicklyand precisely distinguish various processed products such as Radix A. kusnezoffii and canbe applied to predict the tendency of transformation of the complicated chemical mixturesystems under heat perturbation.

The root of Angelica sinensis (Oliv.) Diels (Umbellifeae) is a well-known traditionalChinese medicine in common use [10]. Modern pharmacological research indicates thatit could be used to treat anemia, apoplexy, hypertension, coronary heart disease, throm-boangiitis obliterans, superficial thrombosed phlebitis, etc. FTIR associated with secondderivative infrared spectroscopy and 2D-IR was used [11] to study the main constituentsin traditional Chinese medicine Angelica and its different extracts (extracted by petroleumether, ethanol, and water in turn). The use of the macroscopic fingerprint characters of FTIRand 2D-IR spectrum can not only identify the main chemical constituents in medicinal ma-terials and their different extracts but can also compare the components differences amongthe similar samples. This analytical method is highly rapid, effective, visual, and accuratefor pharmaceutical research.

The quality of buchu oil obtained from two South African species, Agathosma be-tulina and Agathosma crenulata, belonging to the Rutaceae family were analyzed usingFTIR spectroscopy [12]. Samples of A. betulina and A. crenulata were collected fromdifferent natural localities and cultivation sites in South Africa. The essential oil was ob-tained by hydrodistillation and scanned using three spectroscopy techniques, such as nearinfrared (NIR), mid-infrared (MIR), and Raman. A comparison of the three spectroscopytechniques showed that MIR together with PLS algorithms produced the best model for thequantification of six of the seven major oil constituents.

FTIR was used to simultaneously analyze the main chemical constituents in differentsolvent extracts of several kinds of Chrysanthemum samples of different regions [13].The findings indicate that different Chrysanthemum samples have dissimilar fingerprintcharacters in FTIR spectra. Such spectral techniques can provide substance structuralinformation for complicated test samples. Liu et al. [13] reported that they could identifythe main components of different extracts and distinguish the origins of the Chrysanthemumsamples from different regions easily.

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Multistep infrared spectroscopic methods, including conventional FTIR, second deriva-tive spectroscopy, and 2D-IR correlation spectroscopy, have been proved to be effectivemethods to examine complicated mixture system such as the investigation on the effectof flowering on the pharmaceutical components of Cistanche tubulosa [14]. The resultsprovide a scientific explanation for the traditional experience that flowering consumes thepharmaceutical components in stem and the seeds absorb some nutrients of stem afterflowering.

A new method using single reflection Fourier transform infrared spectroscopy wasproposed for direct and fast determination of Corydalis yanhusuo W. T. Wang of traditionalChinese herbal medicines and its confusable varieties [15]. By converting FTIR spectra ofthe samples into second derivative spectra by derivative spectra software, it is possible toidentify Corydalis yanhusuo W. T. Wang from the confusable varieties using statistics.

FTIR spectroscopy and 2D-IR were employed to propose a new method for analysis ofCordyceps [16]. Their secondary derivative spectra can amplify the differences and confirmthe potential characteristic IR absorption bands. The different fingerprints display differentchemical constitutes.

Chestnut (Castanea sativa) shell and eucalyptus (Eucalyptus globulus) bark, wasteproducts of the food and wood industries, respectively, were analyzed as potential sourcesof antioxidant compounds [17]. The antioxidant activity and the total phenols content of theextracts had positive linear correlations. FTIR spectroscopy confirmed the higher contentof phenolic compounds in chestnut shell extracts compared to eucalyptus bark extracts.

The determination of sweet cherry anthocyanins in crude material of three varietiesusing diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method has been described [18]. A linear relationship between thesweet anthocyanins content and the peak area at 1640–1630 cm−1 was established witha high correlation coefficient (0.990). The deconvolution analysis using the curve-fittingmethod allowed the elimination of spectral interferences from other cell wall components.The proposed method is simple, rapid, and nondestructive and could be applicable to anycherry varieties.

A systematic study of the effect of the commercially available, ultradiluted drug, Digi-talis purpurea (extract of foxglove leaves) with aqueous ethanol was conducted to monitorchanges in its chemical structure/functional group arrangements using vibrational (FTIRand Raman) spectroscopy [19]. These changes suggest a significant effect of ultradilution(microvolumes) in the spectrum profile of Digitalis bands in the fingerprint region. The tech-nique is useful in the detection and identification of compounds/chemical groups presentat levels lower than microvolume in drugs used in alternative/complementary medicine.

The composition of fenugreek seeds in the form of powder, ash, and oil was investigatedthrough FTIR and Raman spectra measurements [20]. The results indicated that fenugreekseeds (powder) are rich in proteins. Fats (lipids) and starch are present in small amountsin the seeds. The fenugreek oil FTIR absorbance ratios A3009/A2924, A3009/A2854, andA3009/A1740 were considered for measuring the iodine values. These ratios revealed that theiodine value of fenugreek oil is higher than that of other oils. On the other hand, the ashof fenugreek is very rich in phosphate compounds. The spectra showed some absorptionbands that are due to phosphate compounds. It could be concluded that the inorganic partof fenugreek consists mainly of phosphate compounds.

One of the most important herbals analyzed using FTIR spectroscopy is ginseng[21–25]. Ginseng is an expensive herb, and adulteration with other cheaper products mayoccur. Quality assurance of ginseng is required because many of its commercial productsnow come in various formulations such as capsules, powder, soft gels, and teas. A review

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was published by Angelova et al. [21], who discussed the most recent developments inginseng analysis. Yap et al. [22] discussed a rapid means of distinguishing American andAsian ginsengs from two morphological fakes, namely, sawdust and Platycodon grandi-florum, via pattern differences and principal component analysis of their infrared spectra.The results showed that ginseng can be distinguished from both sawdust and Platycodongrandiflorum; hence there is a potential for the use of infrared spectroscopy as a novelanalytical technique in the authentication of ginseng. A simple and rapid protocol (2–6PC)based on infrared wavelengths and principal component analysis for the identification andcategorization of ginseng was elaborated [23]. The advantage of this protocol is that it isable to provide rapid identification of natural products because it avoids tedious extractionor purification procedures. The results showed that this protocol was not only able to dis-criminate raw ginseng roots but also different types of ginseng in three commercial ginsengproducts. Another report was made regarding the case of a misidentification of ginsengbased on traditional methods of authentication via morphology and the ability of infraredspectroscopy and principal component analysis as a rapid form of quality surveillancefor discrimination [24]. Molecular spectroscopy (including near-infrared diffuse reflectionspectroscopy, Raman spectroscopy, and infrared spectroscopy) with OPUS/Ident software(Thermo Scientific-Nicolet, Waltham, MA) was applied to clustering ginseng according tospecies and processing methods [25]. The results demonstrate that molecular spectroscopicanalysis could provide a rapid, nondestructive, and reliable method for identification ofChinese traditional medicine. Compared with traditional methods, which are laborious andtime consuming, molecular spectroscopic analysis is more effective. The herbal materi-als of Asian ginseng (the root of Panax ginseng), American ginseng (the root of Panaxquinquefolius), and Notoginseng (the root of Panax notoginseng) were differentiated byconventional Fourier transform infrared spectroscopy (1D-FTIR) as shown in Figure 1and 2D correlation FTIR applying a thermal perturbation [26]. Large differences in sec-ond derivative FTIR patterns among the three species also provided information for easydifferentiation. These species of herbs were further identified based on the positions andintensities of relatively strong auto-peaks and positive or negative cross-peaks in their2D-FTIR spectra. The findings provide a rapid and new operational procedure for thedifferentiation of these notable herbs.

Huanglongbing (HLB), also known as citrus greening, has greatly affected citrusorchards in Florida and was studied for the detection using FTIR [27]. Leaf samples ofhealthy, nutrient-deficient, and HLB-infected trees were processed in two ways (process1 and process 2) and analyzed using a rugged, portable mid-infrared spectrometer. Thespectral peak in the region of 952–1112 cm−1 was found to be distinctly different betweenthe healthy and HLB-infected leaf samples. This carbohydrate peak could be attributed tothe starch accumulation in the HLB-infected citrus leaves. Thus, this study demonstratedthe applicability of mid-infrared spectroscopy for HLB detection in citrus.

FTIR spectroscopy has been emphasized as a widespread technique in the quick assessof food components. In one such work, procyanidins were extracted with methanol and ace-tone/water from the seeds of white and red grape varieties [28]. FTIR spectroscopy allowedthe creation of a partial least squares (PLS1) regression model with eight latent variables(LVs) for the estimation of the degree of polymerization (DPn), giving a root mean squareerror of cross-validation (RMSECV) of 11.7%. The application of orthogonal projectionto latent structures (O-PLS1) clarifies the interpretation of the regression model vectors.Moreover, the O-PLS procedure removed 88% of noncorrelated variations with the DPn.

Kava has been used as a folk medicine and well-known traditional beverage. A previousstudy focused on quantitative analyses of kavalactones in kava samples using FTIR [29]

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Figure 1. FTIR spectra for different species for (a) Asian ginseng, (b) American ginseng, and(c) notoginseng

integrated with an attenuated total reflectance (ATR) accessory and multivariate calibrationanalysis. Derivative transformations (first and second), mathematical enhancements such asmean centering and variance scaling, and multivariate regression by PLS were implementedto develop and enhance the calibration model. Results showed that the FTIR-predictedvalues were similar to gas chromatography (GC)-determined values (R2 = 0.75–0.98),indicating that the FTIR method is suitable for determination of the contents of the sixkavalactones in kava samples and thereby the chemotypes.

Many deciduous perennial fruit crops require winter chilling for adequate bud breakand flowering. Recent research has shown that changes in sugar and amino acid profiles areassociated with the release of buds from dormancy. Judd et al. [30] applied FTIR spectrom-etry to provide an alternative mechanism for tracking metabolic changes in the meristemsof kiwifruit buds during winter dormancy. The results suggested that the application ofmultivariate analysis to FTIR spectra has the potential to be a reliable and fast method fordetecting structural and compositional changes in fruit crops. These wavenumbers appearto be associated with carbohydrate, pectin, and cellulose levels in the meristems. It is ex-pected that this FTIR signature can be used to advance our understanding of the influenceof the various environmental and physiological factors on the breaking of bud dormancyand shoot outgrowth, including the optimum timing and concentrations of applications ofbud break regulators, such as hydrogen cyanamide.

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Figure 2. Conventional IR spectra (Panel I) and second derivative IR spectra (Panel II) of fourpatchouli samples of different geographical origins at room temperature. (A) Nanxiang, (B) Paixiang,(C) Zhanxiang, and (D) Zhaoxiang.

Other important applications of FTIR for herbal analysis are related to geographicorigin determination [31–36]. In addition to the applications already mentioned, related toAgathosma betulina and Agathosma crenulata [12] and ginseng analysis [22, 26], thereare other reports where FTIR was used as discrimination technique for Epimedium ko-rean Nakai [31] from Jilin province, China, and patchoulis (Pogostemon cablin Blanco)from different Chinese provinces [32] (Nanxiang, Paixiang, Zhanxiang, and Zhaoxiang; seeFigure 2). A study on the identification of four species of Mongolian herbal medicine byultraviolet (UV)/fluorescence/IR spectroscopy was carried out [33]. Ultrasonic methano-lic extracts of Mongolian herbal medicine Rubus sachalinensis Leveille, its substitutematerials Sambucus williamsii Hance and Uncaria rhynchophylla (Miq.) Jacks., and itsadulterant Cinnamomum cassia Presl showed clear differences in the IR spectra. IRspectroscopy provided more information through the fingerprints region of herbal

Figure 3. FTIR spectrum of Basella rubra leaf sample at room temperature.

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medicines, rendering the technique direct and simple. Lakshimi Prasuna et al. [34] studiedthe FTIR spectra of Basella rubra and Moringa oliefera leaves and showed evidence for theprotein matrix bands and those corresponding to carboxylic C-O bonds (Figure 3). Also,Phyllagathis praetermissa [35] collected from Pasoh Forest Reserve (Negeri Sembilan),Ampang Forest Reserve (Selangor), and Bukit Lagong (Selangor) in Peninsular Malaysiacould be differentiated based on their chemical constituents by using multistep infraredmacrofingerprinting. Conventional infrared spectra showed limited differences but the sec-ond derivative infrared spectra could amplify the differences of P. praetermissa collectedfrom different localities. Zhao et al. [36] investigated the FTIR fingerprint of 53 soil samplesused in Chinese herbal cultivation from various geographical locations. These data provideduseful information for the best choice of soil, geographical location and transplantation ofChinese herb culture.

Conclusions

According to these spectral fingerprint features, we cannot only identify the main compo-nents of different herbal plants and extracts but can also distinguish the origins of samplesfrom different regions easily, which is troublesome using existing analytical methods.Compared with traditional methods, which are laborious and time consuming, molecularspectroscopic analysis is more effective and efficient.

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