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ARTICLE IN PRESSG ModelBA-10261; No. of Pages 4

Journal of Pharmaceutical and Biomedical Analysis xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur na l ho mepage: www.elsev ier .com/ locate / jpba

uantification of 4′-geranyloxyferulic acid (GOFA) in honey samplesf different origin by validated RP-HPLC-UV method

alvatore Genovese, Vito Alessandro Taddeo, Serena Fiorito, Francesco Epifano ∗

ipartimento di Farmacia, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo, CH, Italy

r t i c l e i n f o

rticle history:eceived 6 August 2015eceived in revised form4 September 2015ccepted 15 September 2015vailable online xxx

a b s t r a c t

Natural honey has been employed as a nutraceutical agent with benefits and therapeutic promises forhumans for many centuries. It has been largely used as food and medicine by all generations, traditions,and civilizations, both ancient and modern. Several chemicals having beneficial effects for human healthhave been reported as components of natural honey and these include sugars, organic acids, aminoacids,minerals, and vitamins. Also some important phytochemicals have been described and these comprisetannins, flavonoids, terpenes, saponins, and alkaloids. In this note it is described the successful application

eywords:ancer chemopreventionieteranyloxyferulic acidoney

of a RP HPLC-UV–vis method for the separation and quantification of 4′-geranyloxyferulic acid (GOFA)in four honey samples of different origin. Concentration values showed a great variation between thefour samples tested, being chestnut honey the one richest in GOFA (7.87 mg/g). The findings describedherein represent the first example reported in the literature of the characterization of an oxyprenylatedphenylpropanoid in honey.

xyprenylated secondary metabolitesrenyloxyphenylpropanoids

. Introduction

In recent years much attention has been dedicated to cancerhemoprevention adopting a correct and suitable lifestyle [1]. Inhis context food supply (e.g. consumption of fruits, vegetables, andn general of food rich in antioxidants) has been seen to play a piv-tal role [2]. Honey intake was found to be beneficial in dietaryeeding chemoprevention of gastrointestinal cancers [3], leukemia4], renal carcinoma [5], and Ehrlich ascites carcinoma [6]. The can-er chemopreventive effect by honey has been largely attributed tots high content of polyphenols [4,6,7].

As a continuation of studies aimed to reveal the presence ofaturally occurring dietary feeding cancer chemopreventive phyto-hemicals in widely consumed food, it is reported herein a validatedPLC/UV–vis method for the quantification of 4′-geranyloxyferuliccid (designated with the acronym GOFA) (1), in four honey sam-les of different origin, namely forest honey, acacia honey, orangeoney, and chestnut honey.

Please cite this article in press as: S. Genovese, et al., Quantification oorigin by validated RP-HPLC-UV method, J. Pharm. Biomed. Anal. (201

The title oxyprenylated secondary metabolite has recentlyemonstrated great potentialities as a pharmacologically activegent in terms of anti-inflammatory, cancer chemopreventive, and

∗ Corresponding author. Fax: +39 8713554912.E-mail address: francesco.epifano@unich.it (F. Epifano).

ttp://dx.doi.org/10.1016/j.jpba.2015.09.018731-7085/© 2015 Elsevier B.V. All rights reserved.

© 2015 Elsevier B.V. All rights reserved.

neuroprotective activities. The phytochemical and pharmacologi-cal properties of GOFA have been recently reviewed [8,9].

2. Materials and methods

Honey samples were purchased from beekeepers of Marcheregion (Italy). GOFA has been synthesized as already reported andits purity (>98.6%) assessed by GC/MS and 1H and 13C NMR [10].Methanol (HPLC-grade) was purchased from Carlo Erba (Milan,Italy) and used without further purification. Double-distilled waterwas obtained by a Millipore Milli-Q Plus Waters treatment system(Millipore Bedford Corp., Bedford, MA, USA). Solid phase extrac-tions were carried out by Sep-Pak Vac 1 cc (100 mg) C18 cartridges(Waters, Milford, MA, USA) using the same experimental conditionsas previously reported [11]. HPLC analyses were performed using aWaters liquid chromatograph equipped with a model 600 solventpump and a 2996 photodiode array detector. Empower v.2 Software(Waters Spa, Milford Massachusetts, USA) was used for data acqui-sition. A C18 reversed-phase packing column (GraceSmart RP18,4.6 × 150 mm, 5 �m; Grace, Deerfield, IL, USA) was employed forthe separation. The column was thermostatated at 25 ± 2 ◦C using

f 4′-geranyloxyferulic acid (GOFA) in honey samples of different5), http://dx.doi.org/10.1016/j.jpba.2015.09.018

a Jetstream2 Plus column oven. The UV–vis acquisition wavelengthwas set in the range of 200–600 nm. Analogue output channel Awas set at wavelength 310 nm with a bandwidth of 9.6 nm. Thequalitative analyses were achieved at a wavelength of 288 nm.The injection volume was 20 �L. The mobile phase was directly

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Table 1Gradient elution profile for HPLC analyses of honey samples.

Time (min) % A % B

0 90 101 90 10

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3

3

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It is noteworthy to underline how analysis of the selected fourhoney samples in all cases under the same experimental condi-tions as above provided no significant results in terms of content ofother common and widespread oxyprenylated phenylpropanoids,

Table 2GOFA concentration values in chestnut, forest, acacia, and orange honeys expressedas mg/g ± SD.

Honey sample GOFA (mg/g ± SD)a R.S.D. (%)

30 10 9033 0 10040 90 10

n-line degassed by using a Degassex, mod. DG-4400 apparatusPhenomenex, Torrance, CA, USA). Mobile phase composition con-isted in double distilled water + trifluoroacetic acid 0.04% (solvent) and acetonitrile + trifluoroacetic acid 0.04% (solvent B) at a flowate of 1.0 mL/min following a gradient elution program as reportedn Table 1. Column re-equilibration was achieved in 7.0 min usinghe initial composition of the mobile phase used for the analyteeparation.

Sample solutions were centrifuged and the supernatant wasirectly injected into the HPLC-UV–vis system. Stock solutions ofOFA were prepared by dissolving the powder in acetonitrile at r.t.

o provide an initial concentration of 1 mM and stored in aliquots at20 ◦C in amber glass tubes. Working standard solutions were thenbtained by appropriately diluting the stock solutions to obtainoncentration values in the range 0.5–50 �g/mL. These latter werelso stored at −20 ◦C in amber glass tubes for a period not longerhan 4 weeks. Separate solutions were used to prepare calibrationtandards and quality control (QC) samples. Calibration curves haveeen obtained from the mean values of n = 3 measures in 13 points.he same statistical test as previously described was used to inves-igate the significance of the linear fit employed for calibration [12].PLC solutions from food matrices were prepared by adding 1 mgf each natural honey sample in 1 mL of acetonitrile. The resultingixtures were let to stand overnight at room temperature, thenere centrifuged and the supernatant separated. Aliquots of 20 �L

rom these solutions were then used for the HPLC analysis.

. Results and discussion

.1. HPLC parameters

Under the experimental analytical conditions described below,he mean retention time recorded for GOFA was 25.8 ± 0.2 minR.S.D.% = 2.8) The HPLC profile and LC/MS structural assignmentf peak related to the external standard obtained by chemicalynthesis was found to fully match those previously reported forhe same compound obtained using the same experimental condi-ions [10]. Conditions set for column purge and re-equilibrationnsured stability for column pressure and chromatogram back-round. The calculated capacity factor (k′) for GOFA was 3.96. Theead retention time, calculated with uracile, was 1.83 min. Limitf detection (LOD) was calculated by measuring standard to noiseS/N) values obtained in the mobile phase spiked at 0.2 �g/mLevel and extrapolation of the corresponding values to S/N = 3. Thealibration curves showed a good linearity in the concentrationange 0.5–50.0 �g/mL (r2 = 0.9977). The back-calculated calibra-ion standard points showed R.S.D. percentage values ranging from.9% to 6.0%. The differences in percentage between the stan-ard concentrations calculated from the calibration curves andhe theoretical ones ranged from −12.2% to −2.2%. The accuracynd precision results for GOFA have been obtained analyzing QCamples prepared at three different concentration levels (2.5, 20,

Please cite this article in press as: S. Genovese, et al., Quantification oorigin by validated RP-HPLC-UV method, J. Pharm. Biomed. Anal. (201

nd 45 �g/mL). Bias values ranged from 3.0% to 4.4%. Limit ofuantification (LOQ) was evaluated according to the guidance for

ndustry on the validation of bioanalytical methods [13], as the low-st analyte concentration corresponding to a response at least 10

Fig. 1. Structure of GOFA.

times higher than blank response and that can be determined with80–120% accuracy and 20% precision. The back-calculated concen-tration value, obtained from calibration curves, allowed to assess0.5 �g/mL as the validated LOQ. All weighting factors consisted in1/x2 values. The mean imprecision imprecisions (CVs) of the reten-tion times were 0.5% with no statistical differences compared withthe imprecision for the normalized retention times of the standard.As an explicative example, we report in Fig. 1 the HPLC profile of theextract solution obtained from chestnut honey (in which the peakof GOFA has a retention time of 25.52 min). The structural assign-ment of this diagnostic peak was carried out by LC–MS as depictedabove (Fig. 2).

3.2. Quantification of GOFA in natural honey samples

Honey represents a very complex natural matrix from an analyt-ical point of view. It has been seen to contain sugars, aminoacids,and minerals as the most abundant component, but also a widerange of minor compounds many of which are represented bypolyphenols (e.g. phenolic acids and flavonoids). To this concernseveral analytical methodologies (mainly HPLC and electrophore-sis) have been developed for the qualitative and quantitativedetermination of such natural products. A survey of the literaturein this context have been made by Pyrzynska and Biesaga in 2009[14].

The one described herein represents the first example reportedin the literature about the characterization of an oxyprenylatedphenylpropanoid in honey samples. GOFA has been isolated for thefirst time in 1966 from the bark extracts of Acronychia baueri Schott,an Australian tree (Rutaceae) [15]. In very recent years the presenceof GOFA was also disclosed in Citrus fruits [11,16]. Such a ferulicacid derivative exhibited anti-inflammatory and dietary feedingcolon cancer chemopreventive effects in rats and other effectsclosely related to cancer pathogenesis [17–19]. Results about theconcentration of GOFA in the four natural honey samples underinvestigation are reported in Table 2 and expressed as mg/g ofhoney ± SD.

Data reported in Table 2 show that chestnut honey contains thehighest concentrations of GOFA (7.87 ± 0.24 mg/g of honey) whileacacia honey has the less one (0.013 ± 0.002 mg/g). A total recoveryof the standard analyte strictly more than 100% was recorded foreach honey sample.

f 4′-geranyloxyferulic acid (GOFA) in honey samples of different5), http://dx.doi.org/10.1016/j.jpba.2015.09.018

Chestnut 7.87 ± 0.24 3.0Forest 5.36 ± 0.12 3.9Acacia 0.013 ± 0.002 2.9Orange 1.29 ± 0.05 2.1

a Data are reported as mean ± SD (n = 10).

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S. Genovese et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2015) xxx–xxx 3

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Fig. 2. HPLC profile of chestnut honey extr

ike coumarins, other ferulic acid derivatives (e.g. boropinic acidaving a 3,3-dimethylallyl side chain instead of a geranyl one inosition 4), or cinnamaldehydes, being all not detected or havingoncentration values under the LOQ.

The presence of a prenylated metabolite in chestnut and orangeoneys may reflect the fact that prenylation of phenylpropanoidsores is a typical enzymatic activity of Castanea [20] and Citrus gen-ra [11], while the very low concentration in acacia honey may behe consequence of a relatively low content of GOFA precursor fer-lic acid in robinia plants, Robinia pseudoacacia L., the pollen ofhich is the main natural source to obtain acacia honey [21].

Natural honeys are nowadays greatly taken into considerationor their commercial, feed, and nutraceutical values. As statedbove, they do represent a valuable source of several macro- andicronutrients, as well as of a wide range of phytochemicals, all

ontributing to a healthy diet. Witnessing its importance as food,roduction, and consumption of natural honeys largely increaseduring the last decade. As a parallelism in the same period a hugeumbers of studies and reports about beneficial properties of honey

or humans and animals have been cited in the literature [22], espe-ially in terms of cancer chemoprevention. In this context veryecently chestnut honey has been found to potentiate the cytotoxicffects of extracts of several medicinal plants as a results of a syner-ism of action [23], and forest honey was seen to be effective on oralquamous cell carcinoma and osteosarcoma cell line [24]. Resultsescribed in the present investigation may largely contribute tonhance the consideration of natural honeys as effective epidemi-logical means for a valuable prevention strategy of socially andconomically severe diseases like cancer, inflammatory-based syn-romes and associated disorders. The insofar observed beneficialffects of honey feeding have been mainly attributed to its highontent of carbohydrates, aminoacids, vitamins, minerals and tohytochemicals like flavonoids, phenolic acids and their esters,nd related secondary metabolites. The findings described in theresent study indicates that GOFA may be effectively regardeds an additional component of the nutraceutical pool of naturaloneys. For what concerns the observed cancer chemopreventivend related beneficial effects of honey feeding, GOFA may act in

synergistic way with already reported nutraceuticals and phy-ochemicals, like flavonoids and phenolic acids and their esters.uch potential synergies surely deserve to be investigated in moreetails in the next future, as well as the search for other prenylatedecondary metabolites and their effects. In this context it is note-orthy also to underline how prenylation of phenylpropanoids and

olyphenols in general has been reported to largely ameliorate theharmacological effectiveness respect to parent unprenylated sec-ndary metabolites [25].

. Conclusions

In this article it has been described an easy and effective HPLC

Please cite this article in press as: S. Genovese, et al., Quantification oorigin by validated RP-HPLC-UV method, J. Pharm. Biomed. Anal. (201

ethodology for the qualitative and quantitative analysis in fourelected natural honey samples of the naturally occurring bio-ogically active 4′-geranyloxyferulic acid. The findings describederein represent the first examples reported in the literature ofhe characterization of an oxyprenylated secondary metabolite in

[

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owing the presence of GOFA at Rt = 25.52.

honey. Adopting slight experimental modifications respect to pre-viously reported HPLC methods [11], the methodology describedin the present study is effective in selectively allow to qualitativelyand quantitatively determine GOFA in a real complex matriceslike honey samples. It may be represent a valuable basis for theinvestigation of the presence of other oxyprenylated secondarymetabolites in the same samples. On the basis of the already inso far reported pharmacological properties of such a secondarymetabolite, data reported herein may be a stimulus for future inves-tigations having as the scope the correlation of the presence ofGOFA with a potential dietary chemoprevention protocol basedon feeding with honey and other foods rich in natural productswith proven cancer chemopreventive agents like Citrus fruits [11].In view of the well-documented pharmacological effects so farascribed to GOFA both in vitro and in vivo, the analytical proce-dure set-up in the present study will be of sure help to evaluatethe presence of this secondary metabolite in natural honeys oforigins different than those described herein and but also in honey-containing preparations.

Acknowledgement

Financial support to this research from the University “G.D’Annunzio” of Chieti-Pescara was gratefully acknowledged.

References

[1] D. Serrano, M. Lazzeroni, B. Bonanni, Cancer chemoprevention: much hasbeen done, but there is still much to do. State of the art and possible newapproaches, Mol. Oncol. 9 (2015) 1008–1017.

[2] A.C. Tan, I. Konczak, D.M. Sze, I. Ramzan, Molecular pathways for cancerchemoprevention by dietary phytochemicals, Nutr. Cancer 63 (2011)495–505.

[3] M.M.M. Abdel Atif, Chemoprevention of gastrointestinal cancers by naturalhoney, World J. Pharmacol. 4 (2015) 160–167.

[4] M.B. Abubakar, W.Z. Abdullah, S.A. Sulaiman, A.B. Suen, A review of molecularmechanisms of the anti-leukemic effects of phenolic compounds in honey,Int. Mol. Sci. 13 (2012) 15054–15073.

[5] S. Samarghandian, J. Tavakkol Afshari, S. Davoodi, Honey induces apoptosis inrenal cell carcinoma, Pharmacogn. Mag. 7 (2011) 46–52.

[6] S.K. Jaganathan, M. Mandal, Effect of honey and eugenol on Ehrlich ascites andsolid carcinoma, J. Biomed. Biotechnol. (2010), http://dx.doi.org/10.1155/2009/830616, Article ID 830616.

[7] E.R. Kasala, L.N. Bodduluru, R.M. Madana, V. Ak, R. Gogol, C.C. Barua,Chemopreventive and therapeutic potential of chrysin in cancer: mechanisticperspectives, Toxicol. Lett. 233 (2015) 214–225.

[8] F. Epifano, S. Fiorito, V.A. Taddeo, S. Genovese, 4′-Geranyloxyferulic acid: anoverview of its potentialities as an anti-cancer and anti-inflammatory agent,Phytochem. Rev. 14 (2015) 607–612.

[9] S. Genovese, F. Epifano, Recent developments in the pharmacologicalproperties of 4′-geranyloxyferulic acid, a colon cancer chemopreventive agentof natural origin, Curr. Drug Targets 13 (2012) 1083–1088.

10] C. Bruyere, S. Genovese, B. Lallemand, A. Ionescu-Motatu, M. Curini, R. Kiss, F.Epifano, Growth inhibitory activities of oxyprenylated and non-prenylatednaturally occurring phenylpropanoids in cancer cell lines, Bioorg. Med. Chem.Lett. 21 (2011) 4173–4178.

11] S. Genovese, S. Fiorito, M. Locatelli, G. Carlucci, F. Epifano, Analysis ofbiologically active ferulic acid derivatives in Citrus fruits, Plant Foods Hum.Nutr. 69 (2014) 255–260, and references reported herein.

f 4′-geranyloxyferulic acid (GOFA) in honey samples of different5), http://dx.doi.org/10.1016/j.jpba.2015.09.018

12] T. Klein, R. Longhini, J.C. Palazzo de Mello, Development of an analyticalmethod using reversed-phase HPLC-PDA for a semipurified extract ofPaullinia cupana var. sorbilis (guaraná), Talanta 88 (2012) 502–506.

13] http://www.fda.gov/downloads/Drugs/./Guidances/ucm070107.pdf.14] K. Pyrzynska, M. Biesaga, Analysis of phenolic acids and flavonoids in honey,

Trends Anal. Sci. 28 (2009) 893–902.

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4 cal an

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[Antiproliferative effect of Tualang honey on oral squamous cell carcinomaand osteosarcoma cell lines, BMC Complement. Altern. Med. 10 (2010), http://dx.doi.org/10.1186/1472-6882-10-49.

[25] A.M. Alhassan, M.I. Abdullahi, A. Uba, A. Umar, Prenylation of aromatic

ARTICLEG ModelBA-10261; No. of Pages 4

S. Genovese et al. / Journal of Pharmaceuti

15] R.H. Prager, H.M. Thregold, Some neutral constituents of Acronychia baueri,Aust. J. Chem. 3 (1966) 451–456.

16] V.A. Taddeo, F. Epifano, S. Fiorito, S. Genovese, A novel oxyprenylatedmetabolite in Citrus paradisi Macfad. seeds extracts, Biochem. Syst. Ecol. 2(2015) 72–74.

17] S. Genovese, M. Curini, P. Gresele, T. Corazzi, F. Epifano, Inhibition of COX-1activity and COX-2 expression by3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid and itssemi-synthetic derivatives, Bioorg. Med. Chem. Lett. 21 (2011) 5995–5998.

18] M. Shimizu, T. Kochi, Y. Shirakami, S. Genovese, F. Epifano, S. Fiorito, T. Mori,T. Tanaka, H. Moriwaki, A newly synthesized compound, 4′-geranyloxyferulicacid-N(omega)-nitro-l-arginine methyl ester suppresses inflammationassociated colorectal carcinogenesis in male mice, Int. J. Cancer 135 (2014)774–784.

19] S. Genovese, S. Fiorito, F. Epifano, V.A. Taddeo, A novel class of emerging

Please cite this article in press as: S. Genovese, et al., Quantification oorigin by validated RP-HPLC-UV method, J. Pharm. Biomed. Anal. (201

anticancer compounds: oxyprenylated secondary metabolites from plantsand fungi, Curr. Med. Chem. (2015), http://dx.doi.org/10.2174/0929867322666150716114758 (in press).

20] S. Silici, M. Unlu, G. Vardar-Unlu, Antibacterial activity and phytochemicalevidence for the plant origin of Turkish propolis from different regions, WorldJ. Microbiol. Biotechnol. 23 (2007) 1797–1803.

PRESSd Biomedical Analysis xxx (2015) xxx–xxx

21] L.J. Potman, P.E. Laks, M.S. Pruner, Chemical cosntitutents of black locust barkand their biocidal activity, Holzforschung 4 (1989) 219–224.

22] A. Ajibola, J.P. Chamunorwa, K.H. Erlwanger, Nutraceutical values of naturalhoney and its contribution to human health and wealth, Nutr. Metab. 9 (2012)61–73.

23] C. Pereira, J.C.M. Barreira, R.C. Calhelha, M. Lopes, M.J.R.P. Queiroz, M.Vilas-Boas, L. Barros, I.C.F.R. Ferreira, Is honey able to potentiate theantioxidant and cytotoxic properties of medicinal plants consumed asinfusions for hepatoprotective effects? Food Funct. (2015), http://dx.doi.org/10.1039/c4fo01206b (in press).

24] A.A. Ghashm, N.H. Othman, M.N. Khattak, N.M. Ismail, R. Saini,

f 4′-geranyloxyferulic acid (GOFA) in honey samples of different5), http://dx.doi.org/10.1016/j.jpba.2015.09.018

secondary metabolites: a new frontier for development of novel drugs, Trop. J.Pharm. Res. 2 (2014) 307–314.