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FTIR Spectrophotometric MethodsUsed for Antioxidant Activity Assay inMedicinal PlantsAndrei A. Bunaciu a , Hassan Y. Aboul-Enein b & Serban Fleschin ca SC HOFIGAL SA, Research & Development Department, Bucharest,Romaniab Pharmaceutical and Medicinal Chemistry Department,Pharmaceutical and Drug Industries Research Division, Dokki, Cairo,Egyptc Department of Organic Chemistry, Biochemistry and Catalysis,Faculty of Chemistry, University of Bucharest, Bucharest, Romania

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To cite this article: Andrei A. Bunaciu, Hassan Y. Aboul-Enein & Serban Fleschin (2012): FTIRSpectrophotometric Methods Used for Antioxidant Activity Assay in Medicinal Plants, AppliedSpectroscopy Reviews, 47:4, 245-255

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Applied Spectroscopy Reviews, 47:245–255, 2012Copyright © Taylor & Francis Group, LLCISSN: 0570-4928 print / 1520-569X onlineDOI: 10.1080/05704928.2011.645260

FTIR Spectrophotometric Methods Usedfor Antioxidant Activity Assay in Medicinal Plants

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

AND SERBAN FLESCHIN3

1SC HOFIGAL SA, Research & Development Department, Bucharest, Romania2Pharmaceutical and Medicinal Chemistry Department, Pharmaceuticaland Drug Industries Research Division, Dokki, Cairo, Egypt3Department of Organic Chemistry, Biochemistry and Catalysis, Facultyof Chemistry, University of Bucharest, Bucharest, Romania

Abstract: Fourier transform infrared spectroscopy (FTIR) is a fast and nondestructiveanalytical method. Associated with chemometrics, it is a powerful tool for research andindustry. The present review discusses the antioxidant activities assay of some plants(fruits, leaves, aerian part) with medicinal properties using an FTIR spectrophotometricmethod in comparison with other ultraviolet-visible (UV-Vis) spectrophotometric meth-ods. A good correlation was found between the different methods used for measuringthe antioxidant capacity of some of these herbs.

Keywords: Antioxidant activity, FTIR spectrophotometric methods, medicinal plants

Introduction

Medicinal plants constitute a major source of phytopharmaceutical products and dietarysupplements. A whole range of natural products derived from plants, phytochemicals, andprovitamins, which help to maintain health and fight disease, are now described as foodsupplements. The products derived from plants become more and more accepted and usedin the cosmetics industry.

In 1987, the World Health Organization (WHO) stated the importance of scientificresearch on herbal supplements, and there is sufficient evidence that such products mayhave beneficial effects. Without doubt, herbal drug use is one of the oldest forms of healthcare, so the WHO has estimated that 80% of the world population still relies on botanicmedication (1). National flora are remarkable for their diversity and richness; many speciesof plants are known for their therapeutic role. However, more research is still needed toexplore their utility in modern therapy.

In recent years, antioxidants have gained a lot of importance due to their potential asprophylactic and therapeutic agents in many diseases (2, 3). Traditionally, herbal medicines

This article is in memory of Magda Carmen Bunaciu (1955–2011).Address correspondence to Professor Hassan Y. Aboul-Enein, Pharmaceutical and Medicinal

Chemistry Department, Pharmaceutical and Drug Industries Research Division, Tahrir Street, Dokki,Cairo12311, Egypt. E-mail: haboulenein@yahoo.com

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with antioxidant properties have been used for various purposes, and epidemiologicaldata also point to widespread acceptance and use of these agents. Presently, the activeconstituents from these herbal sources are extracted, purified, and tested for their activities.

Infrared (IR) spectrometry provides a useful method for herbal analysis (4, 5) aswell as for quantitative analysis of drugs (6), and the help of Fourier transform infrared(FTIR) permits continuous monitoring of the spectral baseline and simultaneous analysisof different components of the same sample.

There are numerous studies related to evaluation of antioxidant capacity of plant mate-rials. Methods that measure the antioxidant’s radical scavenging ability include total peroxylradical trapping parameter (TRAP) (7), oxygen radical absorbance capacity (ORAC) (8),Trolox equivalent antioxidant capacity (TEAC) (9), 2,2-diphenyl-1-picrylhydrazyl radical(DPPH) (10), and N,N-dimethyl-p-phenylenediamine (DMPD) radical (11) assays. Thereducing ability of plasma in the presence of antioxidant was determined by the ferricion–reducing antioxidant parameter (FRAP) assay (12). Other commonly used antioxidantactivity methods include cupric reducing antioxidant capacity (CUPRAC) (13), cerium(IV) reduction (14), phosphomolybdenum complex (15), and inhibited oxygen uptake(IOU) (16).

The present review discusses the antioxidant activities of some plants (fruits, leaves,and/or aerial part) with medicinal properties using FTIR spectrophotometric methods incomparison with other methods. Prior to the review on this subject, it is useful to providea short introduction to the concept of the antioxidant activity and substances involved inthis mechanism. Furthermore, the quantitative and qualitative determinations of antioxidantactivity in different plants are presented.

Antioxidant Activity

Antioxidants are a class of compounds of great interest for the pharmaceutical industry andbiochemists and other health professionals because they are designed to reduce damagecaused by reactive oxygen species (ROS), nitrogen (RNS), or even chlorine (RCS). Thediscovery of the role of free radicals in cancer, diabetes, cardiovascular diseases, autoim-mune diseases, neurodegenerative disorders, aging, and other diseases has led to a medicalrevolution that is promising a new paradigm of health care. There is also great interest intrying to achieve partial or total replacement of synthetic antioxidants with natural ingredi-ents, in view of concerns about possible side effects—including cancer—of some syntheticantioxidants in food.

The importance of the mechanism of oxidation in the body as well as in food is widelyaccepted. Oxidative metabolism is an essential process for cell survival. A side effect of thismetabolism is that excess production of free radicals and other ROS can cause undesirableoxidative changes. There is increasing evidence for the involvement of high reactivity ofthese species in a variety of diseases.

Antioxidants are substances that counteract free radicals and prevent damage causedby them. These can greatly reduce the adverse damage due to oxidants by crumblingthem before they react with biologic targets, preventing chain reactions or preventing theactivation of oxygen to highly reactive products (17).

Antioxidants can be classified into two major groups; that is, enzymatic and nonen-zymatic antioxidants. Some of these antioxidants are endogenously produced, includingenzymes, low-molecular-weight molecules, and enzyme cofactors. Many nonenzymaticantioxidants are obtained from dietary sources. Dietary antioxidants can be classified into

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Figure 1. Classification of antioxidants.

various classes (18), of which polyphenols is the largest class. Polyphenols consist ofphenolic acids and flavonoids. The other classes of dietary antioxidants include vitamins,carotenoids, organosulfural compounds, and minerals (Figure 1).

Many methods reported for determination of antioxidant activity (19). Table 1 lists themajor methods involved in antioxidant activity assays.

Applications

A large number of publications are available in the literature that are dedicated to antioxidantactivity assays using FTIR spectrometry. Most of these papers are related to the qualitativeand quantitative assays of active compounds. In this section, new methods for quanti-tative determinations of antioxidant activity will be presented followed by examples forthe utilization of FTIR spectroscopy in studying the relationship between the chemicalstructure and antioxidant activity.

The antioxidant content of fruits has made them a desirable component of the humandiet. Several wet chemistry techniques, including the ORACFL assay, have been reportedfor measuring the antioxidant activities of fruit. Lam et al. (20) investigated the use ofFTIR to measure fruits’ antioxidant activity. This study shows that an FTIR technique issuitable for rapidly measuring fruit extract antioxidant activity. A good calibration model(R2 = 0.97) for antioxidant activity was obtained with satisfactory predictive ability (rootmean square error [RMSE] = 5.35) using the spectral region 2000 to 900 cm−1.

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Table 1Methods involved in antioxidant activity assays

Assays involving hydrogen atomtransfer reactions (HATs):ROO∗ + AH − ROOH + A∗

ROO∗ + LH − ROOH + L∗

TRAP (7)ORAC (8)Crocin methodIOU (16)Inhibition of linoleic acid oxidation

Assays involving electron transfer (ET):Men− + e− (din AH) − Me(n−1)− +AH∗−

TEAC (9)DPPH (10)FRAP (12)CUPRAC (13)Ce(IV) ions reduction (14)Mo(VI) ions reduction (15)Folin-Ciocalteu

Other methods Total oxidant scavenging capacity (TOSC)(54)

Briggs-Rauscher reaction (55)Chemiluminiscence (56)Electrochemiluminscence (57)FTIR

In other paper, Lam et al. (21) investigated the use of FTIR for measuring the changes inhuman low-density lipoproteins’ (LDL) primary and secondary lipid oxidation products andmodification of the apolipoprotein B-100’s (apoB-100) secondary structures during Cu2+-mediated oxidation in the presence of catechin, quercetin, and α-tocopherol at physiologicalconcentrations. Relationships between peroxide formation, carbonyl products, and LDLprotein denaturation were shown by the FTIR approach. The FTIR technique provided asimple new tool for a comprehensive evaluation of antioxidant performance in protectingLDL during in vitro oxidation.

An FTIR spectroscopic method was developed for the determination of α-tocopherolin refined, bleached, and deodorized (RBD) palm olein (22). The calibration and validationsamples were prepared by spiking a known amount of α-tocopherol to produce a wide rangeof α-tocopherol up to 2,000 ppm. The partial least squares (PLS) calibration models forpredicting α-tocopherol were developed using the FTIR spectral region at 3100–2750 cm−1.The accuracy of the method was comparable to that of high-performance liquid chromatog-raphy (HPLC) (23), with coefficients of determination (R2) from calibration samples of0.9922.

A simple, rapid, and direct FTIR spectroscopic method was developed for the deter-mination of butylated hydroxytoluene (BHT) content in RBD palm olein and RBD palmoil (24). The method used sodium chloride windows with a 50-mm transmission path.FTIR results for both oils correlated well with results obtained by the IUPAC HPLC-basedmethod. Due to the significant decrease in analysis time and reduction in solvent usage,this FTIR method for BHT is especially well suited for routine quality control applicationsin the palm oil industry.

Phycocolloids present in three brown seaweeds (Himanthalia elongata, Bifurcariabifurcata, Saccharina latissima) and five red edible seaweeds (Mastocarpus stellatus,

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Gigartina pistillata, Chondracanthus acicularis, Nemalion helminthoides, and Dumontiacontorta) were studied by Fourier transform infrared–attenuated total reflectance (FTIR-ATR) spectroscopy. Infrared spectra of polysaccharide standards (alginate, agar, and iota-,kappa-, and lambda-carrageenan) were obtained for comparison (25). Therefore, FTIR-ATR spectroscopy is proposed as a useful tool for the food, pharmaceutical, and cosmeticsindustries to check the phycocolloid quality of a raw seaweed material by a quick andnondestructive method.

Total phenolic content (TPC) and total antioxidant capacity (TAC) of four onion vari-eties (red, white, yellow, and sweet) and shallot from selected locations (Washington, Idaho,Oregon, Texas, and Georgia) were determined using FTIR spectroscopy (4000–400 cm−1)(26). The Folin-Ciocalteu (FC) assay was used to quantify TPC and three different methodswere used to determine TAC, including 2,2-diphenyl-picrylhydrazyl (DPPH) (10), TEAC(9) and FRAP (12) assays. A correlation of r > 0.95 was obtained between FTIR-predictedand reference values (by FC, DPPH, TEAC, and FRAP assay) with standard errors ofcalibration (SEC) and standard errors of cross-validation (SECV) less than 2.85, 0.35, and0.45 µmol Trolox/g fresh weight (FW) of extracts for TEAC, FRAP, and DPPH assays,respectively, and 0.36 mg gallic acid/g FW of extracts for the FC assay.

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 was described in Pappas et al. (27). A linear relationshipbetween the sweet anthocyanins content and the peak area at 1640–1630 cm−1 was es-tablished with a high correlation coefficient (0.990). The deconvolution analysis usingthe curve-fitting method allowed elimination of spectral interferences from other cell wallcomponents. The proposed methodology was compared with a reference ultraviolet-visible(UV-Vis) spectrophotometric method (28) and found equivalent in reproducibility andaccuracy.

FTIR spectroscopy provides rapid and nondestructive analysis of wine, with almost nosample preparation (29). Versari et al. (29) studied the use of FTIR measurement for theprediction of red wine TAC. PLS regression was chosen for evaluation of the FTIR spectra.A plot of the full cross-validated PLS-predicted TAC values showed a good correlation(r = 0.85), and the slope of 0.74 and the prediction error provided by the PLS model wereconsistent with the uncertainty derived from the reference method.

Rapid FTIR spectroscopy combined with ATR was applied for quantitative analysis ofvirgin coconut oil (VCO) in binary mixtures with olive oil (OO) and palm oil (PO) (30).The spectral bands correlated with VCO, OO, PO; blends of VCO and OO; and VCO andPO were scanned, interpreted, and identified. Two multivariate calibration methods, PLSand principal component regression (PCR), were used to construct calibration models thatcorrelate between actual and FTIR-predicted values of VCO contents in the mixtures at theFTIR spectral frequencies of 1120–1105 and 965–960 cm−1. In general, FTIR spectroscopyserves as a suitable technique for determination of VCO in mixtures with the other oils.

A rapid FTIR-ATR spectroscopic method was applied to the determination of wa-ter content (WC), total phenol amount (TP), and antioxidant activity (ABTS+) of virginolive oils (VOOs) and olive oils (31). Calibration models were constructed using partialleast squares regression. The FTIR-ATR method provided results that were comparableto conventional procedures. Moreover, the ATR-FTIR-PLS method developed herein wasfaster; the complete determination takes only a few minutes compared to the long timerequired for both the WC analysis (approximately 30 min) by titrimetric analysis and TPand ABTS+ spectrophotometric determinations (several hours taking into considerationphenol extraction, reactive preparation, and the long wait for the complete reaction).

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Vegetable oils (e.g., soybean oil) are the main biodegradable lubricant base stocks usedworldwide. Castro et al. (32) compared the oxidative and wear performance of oils to betterunderstand how the chemical composition of the base stock affects these properties. Oxi-dation products from the micro-oxidation test were analyzed by FTIR and gel permeationchromatography. All oils were evaluated neat, without additives.

Soybean oil (SBO) was blended with 10–40% palm kernel olein (PKO) to obtainSBO:PKO blends with different degrees of unsaturation. These blends can be determinedusing an FTIR method (33). Oil-in-water (O/W) emulsions were prepared with 70 wt%of SBO or SBO:PKO blends and monitored for their chemical destabilization after anaccelerated oxidation process up to 12 days at 60◦C. The formation rate of hydroperoxides,as demonstrated by peroxide value (PV) evolution, throughout the oxidation period wasrelatively high for a fully SBO-based emulsion compared to those with PKO incorporation.An FTIR spectroscopic method was also performed in parallel with PV determination,providing further information on structural changes of the functional groups due to lipidoxidation in the emulsions. By using a PLS chemometric method, a developed calibrationmodel that was based on the spectral region between 1800 and 1480 cm−1 was shown to beable to predict the PV in oxidized emulsions over the range of 4–45 meq/kg. The calibrationmodel provides a good coefficient of determination of 0.98 and a root mean standard errorof cross-validation of 2.09.

FTIR spectroscopy was investigated as a method for analysis of acesulfame-K contentafter a simple extraction procedure for certain commercial diet food samples (34). Partialleast squares models were developed for prediction of acesulfame-K using select spectralranges on the basis of relevant IR absorption bands associated with acesulfame-K. Theacesulfame-K content of test food samples was predicted accurately in the fingerprint regionbetween 1100 and 1300 cm−1 with a maximum prediction error of 9.82% when comparedwith conventional HPLC methods. The PLS was found to be a consistently better predictorwhen both PLS and PCR analyses were used for quantification of acesulfame-K.

Peridural scarring, or the excessive formation of scar tissue following spinal surgery, isone of the important contributing factors that result in persistent pain and disability in manyindividuals who have undergone elective back surgery. Treatment with anti-inflammatoryagents following surgery may reduce oxidative stress and scarring, leading to a reductionin postoperative pain. Wiens et al. (35) used a surgical rat model to test the hypothesis thatpostsurgical inflammation and oxidative stress following laminectomy can be reduced bysystemic administration of l-2-oxothiazolidine-4-carboxylate (OTC) and quercetin. OTCis a cysteine precursor required for the synthesis of glutathione, an important antioxi-dant. Quercetin is a flavonoid with antioxidant properties found in fruits and vegetables.Synchrotron FTIR microspectroscopy data have been collected on OTC, quercetin, andsaline (control)-treated postsurgery animals, sacrificed at 3 and 21 days (n = 6 per ageand treatment group). Preliminary IR results were presented, supported by immunocyto-chemistry, on the heterogenous distribution of biological components present in the healingtissue.

Thermal and mechanical degradation of natural rubber (NR) mixed with N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), polymerized 1,2-dihydro-2,2,4-trimethyl-quinoline (TMQ), and 50/50 weight basis mixture under high-temperature andshearing conditions were investigated using a moving die processability test and FTIRspectroscopy (36). The antioxidative capability of those antioxidants on NR was orderedbased on their effectiveness as follows: 6PPD > (6PPD mixed with TMQ) > TMQ. Itwas also found that the moving die processability test and FTIR spectroscopy are efficientroutes to estimate the oxidative degradation of NR molecules. Therefore, the techniques

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could be applied to assess or compare antioxidative capability of various types and amountsof antioxidants used in the rubber formulation within a reasonable testing time. The rela-tionship between dynamic properties in terms of tan δ value and chemical changes of NRmolecules during degradation were correlated.

A PLS regression model based on ATR-FTIR spectra of heated olive oil sampleshas been developed for determination of polymerized triacylglycerides (PTGs) generatedduring thermal treatment of oil (37). Three different approaches for selection of the spectralregions used to build the PLS model were tested and compared: (1) variable selection basedon expert knowledge, (2) uninformative variable elimination PLS, and (3) interval PLS.Each of the three variable selection methods provided PLS models from heated olive oilsamples with excellent performance for the prediction of PTGs in fried olive oils withcomparable model statistics. Furthermore, it was verified that the determination of PTGswas not influenced by the type of foodstuff fried in the olive oil.

A rapid FTIR-ATR spectroscopic method coupled with PLS was developed to estimatethe oxidation degree of extra virgin olive oil (EVOO) (38). The reference values of EVOOoxidation for the FTIR calibration were obtained by the specific absorptions at 232 and270 nm, due to the presence of conjugated diene (CD) and conjugated triene (CT) groups, asmonitored by the UV spectrophotometric determination. The results of the study indicatedthat a strong correlation existed between FTIR and UV-Vis peak intensities.

A fast, versatile, inexpensive, and environmentally safe analytical method was devel-oped to quantify simple sugars, malic acid, and total phenolic compounds in apple pomace,considering its potential use as a raw material with value instead of as an industrial waste(39). DRIFTS measurements of 26 samples of apple pomace were analyzed by PLS regres-sion using several signal preprocessing methods. Multivariate models developed with fourto five latent variables (LVs) and based in the mid-infrared (MIR) region had good predic-tion for the determination of sucrose, fructose, malic acid, and total phenolic compounds,with average errors between 3.9 and 6.6%. By contrast, glucose was better determined bymodels developed in the near-infrared (NIR) region and using six LVs, yielding an averageerror lower than 7.4%.

Red fruit (Pandanus conoideus Lam) is an endemic plant of Papua, Indonesia, andPapua New Guinea. The price of its oil (red fruit oil, RFO) is 10–15 times higher than that ofcommon vegetable oils; consequently, RFO is subjected to adulteration with lower price oils(40). Among common vegetable oils, canola oil (CaO) and rice bran oil (RBO) have similarfatty acid profiles to RFO as indicated by the score plot of principal component analysis;therefore, CaO and RBO are potential adulterants in RFO. An FTIR spectroscopy methodwas developed in combination with chemometrics of PLS regression and discriminantanalysis (DA) for authentication of RFO from CaO and RBO. FTIR spectroscopy combinedwith PLSR and DA can be successfully used for quantification and classification of oiladulterants in RFO.

Quality characteristics of wheat are determined by different physiochemical and rhe-ological analyses using different American Association of Clinical Chemists (AACC)methods (41). AACC methods are expensive, time consuming, and cause destruction ofsamples. FTIR spectroscopy is one of the most important and emerging tools used foranalyzing wheat for different quality parameters. This technique is rapid and sensitive, witha great variety of sampling techniques. Amir et al. (41) used different wheat varieties forquality assessment and were characterized using AACC methods and FTIR techniques.FTIR works on the basis of functional groups and provides information in the form ofpeaks. On the basis of the peaks the values of moisture, protein, fat, ash, carbohydrates,and hardness of grain were determined.

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FTIR spectrometry was used also in order to study the relationship between chemicalstructure and properties of modified substances versus antioxidant activity. κ-Carrageenanoligosaccharides prepared through mild hydrochloric acid hydrolysis of the polysaccharide(42), polysaccharides extracted from Ulva pertusa Kjellm (Chlorophyta) (43), melaninproduced by Hypoxylon archeri, a companion fungus of Tremella fuciformis, and purifiedfrom the submerged culture medium (44), chestnut (Castanea sativa) shell and eucalyptus(Eucalyptus globulus) bark, waste products of the food and wood industries, respectively(45), where FTIR spectroscopy confirmed the higher content of phenolic compounds inchestnut shell extracts compared to chestnut shell and eucalyptus bark and waste productsof the food and wood industries, respectively. A water-soluble polysaccharide from theseeds of Plantago asiatica L. was extracted with hot water and purified by gel filtrationchromatography (46), the nutrient and chemical contents of two Korean teas: traditionalChungtaejeon tea (CTJ) and green tea (GT) (47), fresh and stored maize (white and blue)tortillas (48), the structural change of the degraded κ-carrageenans, by an oxidative methodinvolving hydrogen peroxide (H2O2), was characterized (49), propolis extracts (50), thebioactive metabolites from a benthic, mat-forming strain dominating a polluted wastewatercanal in Egypt (51), the water and acetone extracts of raw and boiled for 10, 20, 40, and60 min Korean lotus roots (KLR) and Polish white onion (PWO) (52), as well was differentextra virgin olive oils (EVOOs) (53).

Conclusions

In conclusion, FTIR spectroscopy is a promising technique to rapidly provide informationon the TAC of products of interest and has a high potential to be implemented for therapid screening of several TAC methods concurrently. The use of MIR spectroscopy topredict the total antioxidant capacity of vegetables provides a rapid and precise alternativeto traditional wet chemistry analysis. With its speed and ease of data manipulation bycomputer software, FTIR spectroscopy is advantageous as a simple and rapid quantitativedetermining analytical tool for antioxidant activity assays in herbal medicine.

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