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Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin (4-(#-D-glucopyranosyloxy)-2,6,6trimethyl–1–cyclohexane–1-carboxaldehyde) Anastasia Kyriakoudi, Yvonne C. O'Callaghan, Karen Galvin, Maria Z. Tsimidou, and Nora M. O'Brien J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03363 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 5, 2015

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Journal of Agricultural and Food Chemistry

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Cellular Transport and Bioactivity of a Major Saffron

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Apocarotenoid, Picrocrocin (4-(β-D-glucopyranosyloxy)-2,6,6-

3

trimethyl–1–cyclohexane–1-carboxaldehyde)

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Running Title: Cellular Transport and Bioactivity of the Saffron Apocarotenoid,

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Picrocrocin

7 8 9 10

Anastasia Kyriakoudia, Yvonne C. O’Callaghanb, Karen Galvinb, Maria Z. Tsimidoua Nora M. O’Brien*b

11 12

a

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University of Thessaloniki (AUTh), 54124, Thessaloniki, Greece

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b

Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle

School of Food and Nutritional Sciences, University College Cork, Ireland

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*To whom correspondence should be sent: Professor Nora M. O’Brien, School of

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Food and Nutritional Sciences, University College Cork, Cork, Ireland. E-mail:

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[email protected]. Fax: +353 21 4270244. Tel: +353 21 4902884

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ACS Paragon Plus Environment


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ABSTRACT: The cellular transport and bioactivity of the second major saffron

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apocarotenoid, picrocrocin, was examined in parallel to that of the major group,

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crocetin sugar esters, in aqueous extracts. The transport of pure picrocrocin was

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investigated in comparison to that of other saffron apocarotenoids, trans-crocetin (di-

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β-D-gentiobiosyl) ester and crocetin using the Caco-2 cell model coupled with an in

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vitro digestion procedure. RP-HPLC-DAD was employed to quantify the

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bioaccessible and bioavailable amounts of individual apocarotenoids. Picrocrocin and

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crocetin sugar esters though highly bioaccessible (75% and 60%, respectively), were

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transported at minute quantities (0.2% and 0.5%, respectively; 10 fold lower than

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crocetin). Picrocrocin did not protect against oxidant-induced DNA damage in U937,

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human monocytic blood cells at the concentration investigated however, it reduced

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the proliferation of human adenocarcinoma and hepatocarcinoma cells. Our findings

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may be useful for the requirements of food legislation regarding saffron preparations,

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in which both apocarotenoid groups co-exist.

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KEYWORDS: picrocrocin, saffron apocarotenoids, crocetin, cellular transport,

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bioactivity

41 42 43 44 45 46 47 48 49 2


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INTRODUCTION

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Saffron, the dehydrated red stigmas of the plant Crocus sativus L. that

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comprise the most expensive spice in the world, is highly appreciated for the

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appealing bright yellow hues, the characteristic bitter taste and distinctive aroma

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which it imparts to certain dishes and beverages. Its coloring properties are attributed

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to a group of water-soluble apocarotenoids rarely found in nature, the sugar esters of

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crocetin (8,8′-diapocarotene-8,8′-dioic acid), known as crocins. The bitter taste is

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principally assigned to the colorless monoterpene glucoside picrocrocin (4-(β-D-

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glucopyranosyloxy)-2,6,6-trimethyl–1–cyclohexane–1-carboxaldehyde).

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secondary metabolite groups originate from the same precursor, zeaxanthin.1,2

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Crocetin sugar esters account for up to 30% of dehydrated stigmas weight.

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Picrocrocin is the second most abundant apocarotenoid of saffron (up to 20%, w/w).3

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Aroma is the result of the presence of many volatiles among which safranal (2,6,6-

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trimethyl-1,3-cyclohexadiene-1-carboxaldehyde),

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picrocrocin is most abundant.1 The chemical structures of the major saffron

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apocarotenoids are illustrated in Figure 1.

a

degradation

Both

product

of

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Apart from its known applications in the food industry, saffron can be

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considered as a functional spice4 because of the many biological activities assigned to

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its polar extracts or individual apocarotenoids. So far, attention has been focused on

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the properties of crocetin sugar esters as well as to the parent molecule, crocetin.

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Previous data has assigned protective effects against various types of cancer,

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atherosclerosis, hepatotoxicity and antiradical properties to these apocarotenoids (e.g.

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5,6

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present in saffron extracts at high concentrations.

). Less is known about the bioactivity of picrocrocin, which is concomitantly

3



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Studies on the bioactivity of saffron apocarotenoids are numerous but

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available data on their absorption and metabolism are rather limited. The first data on

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the bioaccessibility of crocetin sugar esters and picrocrocin from aqueous saffron

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extracts, reported by Kyriakoudi et al. (2013)7, indicated that ~50% and ~70% of

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them, respectively, were bioaccessible upon in vitro gastrointestinal digestion

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conditions. The authors did not detect free crocetin in the saffron extract digestate, a

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finding verified a few years later by Lautenscläger et al. (2015).8 In vivo studies,

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however, point out that crocetin is the form detected in biological fluids9-11 suggesting

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thus, that this is the most biologically relevant form. In addition, Lautenscläger et al.

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(2015)8 detected free crocetin in the digestate only when they added a freshly

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prepared tissue homogenate from purged small mouse intestine to their in vitro model.

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However, no information is available in the literature for the intestinal absorption and

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metabolism of picrocrocin.

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The present study aimed at investigating the cellular transport of the second

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major saffron apocarotenoid picrocrocin as this glucoside is co-extracted with crocetin

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sugar esters using water or alcoholic mixtures. A Caco-2 cell model coupled with an

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in vitro digestion procedure was employed. Caco-2 cells are human colon

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adenocarcinoma cells, which can be differentiated to resemble small intestinal

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epithelial cells.12 This cell line is a well-established tool for the in vitro assessment of

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cellular transport and prediction of the bioavailability of nutrients and non-nutrient

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bioactive compounds (e.g. carotenoids and phenols). Using the same model, the

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cellular transport of pure picrocrocin was also investigated in comparison to that of

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the other two major saffron apocarotenoids, i.e. trans-crocetin (di-β-D-gentiobiosyl)

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ester (trans-4-GG crocetin ester) and crocetin. Moreover, the potential biological

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activity of picrocrocin in terms of protection against oxidant-induced DNA damage in 4


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U937 cells and anti-proliferative effects in Caco-2 and HepG2 cells were also

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investigated using the Comet and MTT assays, respectively. The data of the present

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study will add to existing knowledge on the degree of cellular transport of polar

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apocarotenoids and also to the limited information regarding the bioactivity of

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picrocrocin. Our findings may also be useful to the requirements of food legislation

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regarding saffron preparations.

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MATERIALS AND METHODS

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Chemicals. Authentic Greek saffron (harvest year 2013, Category I according

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to ISO 3632-1 specifications13) was donated by the Saffron Cooperative of Kozani

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(Greece).

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All of the chemicals were of the highest purity needed for each assay. In particular,

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the enzymes (pepsin, pancreatin) and bile salts used were from Sigma Chemical Co.

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(Dublin, Ireland). Fetal bovine serum (FBS) was purchased from Invitrogen (Paisley,

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Scotland, UK). Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt

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solution (HBSS) and RPMI-1640 medium were from Sigma Chemical Co. (Dublin,

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Ireland). Cell culture plastics were supplied by Cruinn Diagnostics (Dublin, Ireland).

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Corning 24 mm transwell plates with collagen coated PTFE membrane and 0.4 µm

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pore size (product code: CLS3491) were supplied by Sigma Chemical Co. (Dublin,

119

Ireland). All solvents used were of HPLC grade. Ultra-high purity water was

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produced in the laboratory using a SG Ultra Clear Basic UV system (SG

121

Wasseraufbereitung und Regenerierstation GmbH, Barsbüttel, Germany).

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Apocarotenoid Isolation. Individual apocarotenoids were isolated and characterized as follows:

5



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Picrocrocin isolation. Picrocrocin was isolated according to Sánchez et al.

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(2009).14 Purity (91%) of the isolated compound was checked chromatographically by

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RP-HPLC-DAD (HPLC system I) and calculated as the percentage of the total peak

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area at 250 nm. The HPLC system I consisted of a pump, model P4000 (Thermo

128

Separation Products, San Jose, CA, USA), a Midas autosampler (Spark, Emmen, The

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Netherlands), and a UV 6000 LP diode array detector (DAD) (Thermo Separation

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Products, San Jose, CA, USA). Separation was carried out on a LiChroCART

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Superspher 100 C18 (125×4 mm i.d.; 4 µm) column (Merck, Darmstadt, Germany).

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The elution system used consisted of a mixture of water:acetic acid (1 %, v/v) (A) and

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acetonitrile (B). The linear gradient was 20 to 100 % B in 20 min. The flow rate was

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0.5 mL/min. Chromatographic data were processed using the ChromQuest Version

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3.0 software (Thermo Separation Products, San Jose, CA, USA). Monitoring was in

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the range of 200 – 550 nm. Its identity was confirmed by LC-ESI-MS analysis (model

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2010 EV, Shimadzu, Kyoto, Japan) and by Nuclear Magnetic Resonance (NMR)

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spectroscopy recording the 1H 1D spectra at 300 MHz on a Brucker 300AM

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spectrometer (Rheinstetten, Germany) (see Supporting Information I).

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Trans-4-GG crocetin ester isolation. The procedure described below was

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according to Kyriakoudi et al. (2012).3 In particular, saffron stigmas were carefully

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ground with an agate pestle and mortar and passed through a 0.4 mm sieve just prior

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to further analysis. Finely ground saffron (0.36 g) was extracted with ultra-high purity

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water (20 mL) with the aid of ultrasounds for 30 min. Sample temperature was kept at

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15 ± 0.5 °C in a thermostated water bath. The extract was centrifuged (4,100 rpm, 15

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min, 4 oC) and the supernatant was lyophilized and stored at -22 oC until further use.

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Trans-4-GG crocetin ester was then isolated by semi-preparative RP-HPLC (HPLC

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system II). The system consisted of two Marathon IV series HPLC pumps (Rigas 6


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Labs, Thessaloniki, Greece), a Rheodyne injection valve (model 7125) with a 250 µL

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fixed loop (Rheodyne, Cotati, CA) and a diode array linear UVIS-206 multiple

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wavelength detector (Linear Instruments, Fermont, CA). Separation was carried out

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on a Nucleosil 100 C18 (250×10 mm i.d.; 7 µm) chromatographic column (Macherey-

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Nagel, Düren, Germany). The gradient elution system used consisted of water (A) and

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methanol (B). The gradient was: 0 min, 30 % (B), 0–10 min, 45 % (B), 10–20 min, 70

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% (B), 20–30 min, 100 % (B), 30– 40 min, 100 % (B), 40–50 min, 30 % (B) with a

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flow rate of 3.0 mL/min. Monitoring was at 440 nm. Purity (97%) of isolated trans-4-

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GG crocetin ester was checked chromatographically by RP-HPLC-DAD (HPLC

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system I) in the range 200 – 550 nm and calculated as the percentage of the total peak

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area at 440 nm. The identity of the isolated trans-4-GG crocetin ester was confirmed

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by LC-ESI-MS analysis and by NMR spectroscopy, recording the 1H 1D spectra at

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300 MHz on a Brucker 300AM spectrometer (Rheinstetten, Germany) (see

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Supporting Information II).

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Crocetin preparation. Crocetin was prepared from an aqueous saffron extract

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by acid hydrolysis according to a protocol described by Ordoudi et al. (2009).15 Purity

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(99%) of isolated crocetin was checked chromatographically by RP-HPLC-DAD

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(HPLC system I) in the range of 200 – 550 nm and calculated as the percentage of the

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total peak area at 440 nm. Its identity was confirmed by LC-ESI-MS analysis and by

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NMR spectroscopy recording the

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spectrometer (California, United States) (see Supporting Information III).

1

H 1D spectra at 500 MHz on a Agilent

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Preparation and Characterization of Saffron Aqueous Extract. Saffron

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was carefully ground with a pestle and mortar just prior to analysis. The finely ground

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plant material (0.05 g) was extracted with ultra-high purity water (100 mL) by

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rigorous agitation (~ 500 rpm) at ambient temperature for 1 h. All manipulations were 7



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performed under subdued (yellow) light to minimize photo-decomposition of crocetin

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esters. The characterization was achieved using RP-HPLC. The HPLC system (HPLC

176

system III) (Finnigan Spectra SYSTEM; Thermo Scientific, Philadelphia, PA)

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consisted of a P2000 pump, a AS300 autosampler and a diode array detector (DAD;

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Finnigan Spectra SYSTEM; Thermo Scientific, Philadelphia, PA). Separation was

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carried out on the same chromatographic column [LiChroCART Superspher 100 C18

180

(125×4 mm i.d.; 4 µm) column (Merck, Darmstadt, Germany)]. The elution system

181

consisted of a mixture of water:acetic acid (1 %, v/v) (A) and acetonitrile (B). The

182

linear gradient was 20 to 100 % B in 20 min. The flow rate was 0.5 mL/min and the

183

injection volume was 20 µL. Chromatographic data were processed using

184

ChromQuest software (version 4.2, Thermo Fisher Scientific). Monitoring was in the

185

range of 200 – 550 nm. Quantification of picrocrocin was accomplished with the aid

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of a proper calibration curve (y = 12830x – 2950; R2 = 1.00; 9.8 – 570 ng/20 µL

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injected volume; n = 6). The total and major individual crocetin esters content of

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saffron was also determined by constructing a calibration curve of pure trans-4-GG

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crocetin ester within the range (y = 35561x – 381548; R2 = 0.99; 12 – 600 ng/20 µL

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injected volume; n = 6).

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In Vitro Gastrointestinal Digestion Procedure. The in vitro digestion

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procedure was carried out as previously described by Kyriakoudi et al. (2013).7

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Briefly, aliquots (2 mL) of the saffron aqueous extract were transferred into amber

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bottles and HBSS was added to a final volume of 20 mL. To each bottle, 1 mL of

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freshly prepared pepsin (0.04 g pepsin/0.1 mol/L HCl) was added and the pH was

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acidified to 2.0 using 1 mol/L HCl. The samples were overlaid with nitrogen gas and

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incubated at 37 oC for 1 h in a shaking water bath (Grant, OLS200; Keison Products,

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Chelmsford, Essex, UK) at 95 rpm to mimic the gastric phase of human digestion. 8


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The intestinal phase involved increasing the pH to 5.3 with 0.9 mol/L sodium

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bicarbonate followed by the addition of 200 µL of bile salts glycodeoxycholate (0.8

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mmol/L), taurodeoxycholate (0.45 mmol/L) and taurocholate (0.75 mmol/L) and 100

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µL of porcine pancreatin (0.08 g/mL HBSS). The final pH was adjusted to 7.4 using 1

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mol/L NaOH. Samples were overlaid with a layer of nitrogen gas and incubated at 37

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o

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phase, the digestate was centrifuged at 14,000 g using a Sigma 4K15 centrifuge

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(SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany) for 60 min at 4 oC.

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After centrifugation, the supernatants were collected and filtered through a 0.45 µm

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membrane filter. The resulting filtrates were stored at -80 oC until further analysis.

C for 2.5 h to mimic the duodenal phase of human digestion. After the intestinal

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Cellular Transport of Picrocrocin. Human colon adenocarcinoma Caco-2

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cells were purchased from the European Collection of Animal Cell Cultures

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(Salisbury, Wilts, UK). Cells were maintained in the DMEM supplemented with 10%

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(v/v) fetal bovine serum and 1% (v/v) nonessential amino acids. Cells were grown at

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37 oC in a humidified incubator with 5% CO2 in the absence of antiobiotics. Culture

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medium was replaced three times a week. Caco-2 cells were seeded at a density of 1.0

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x 105 cells/mL on transwell inserts in 6-well transwell plates. Cells were maintained

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in DMEM (10% FBS) for 21 to 25 days to obtain a differentiated cell monolayer;

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media was changed every 2-3 days. Transepithelial electrical resistance (TEER)

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measurements were taken before each transport experiment by a transepithelial

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electrical resistance voltohmmeter (Millicel-ERS, Merck Millipore, Merck KgaA,

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Darmstadt, Germany) to ensure that the monolayer was intact. Only inserts with

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TEER values > 250 Ω ⋅ cm2 in culture medium were selected for transport

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experiments. At the beginning of each experiment, the inserts were washed twice with

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2 mL HBSS. 9



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Saffron Extract Digestate. The apical (mucosal) compartment received

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increasing volumes of the saffron extract digestate diluted with HBSS up to a final

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volume of 2 mL. The basolateral (serosal) compartment received 1.5 mL of HBSS.

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Pure compounds. The cellular transport of pure picrocrocin was determined

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after its dissolution in HBSS at a concentration range between 8 and 24 µM. Pure

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trans-4-GG crocetin ester and crocetin were also examined at equimolar

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concentrations for comparison.

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The incubation time of the cells with the tested samples was 3 hours for all

232

experiments. Following incubation, the TEER values were determined again and

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wells with TEER values < 250 Ω ⋅ cm2 were discounted. Media from both the apical

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and basolateral chambers was isolated and stored at -80 oC until further analysis.

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Samples were analyzed by RP-HPLC (HPLC system III) as detailed above. The

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injection volume was 90 µL. Quantification of picrocrocin, trans-4-GG crocetin ester

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and crocetin was accomplished with the aid of proper calibration curves. Cellular

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transport was the quantity measured in the basolateral chamber expressed as a

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percentage of the concentration of the compound that was initially added to the Caco-

240

2 cells.

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In Vitro Bioactivity Studies of Picrocrocin and Other Major Saffron

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Apocarotenoids. Cell cultures. Caco-2 cells were maintained in the DMEM and

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supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) nonessential

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amino acids. HepG2 cells were maintained in DMEM and supplemented with 10%

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(v/v) FBS. U937 cells were maintained in RPMI-1640 and supplemented with 10%

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(v/v) FBS. The cells were grown at 37 oC and 5% (v/v) CO2 in a humidified incubator.

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Cells were cultured in the absence of antibiotics and were sub-cultured every two to

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three days. 10


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Sample Preparation for Bioactivity Assays. A 5000 µM stock solution of

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picrocrocin and trans-4-GG crocetin ester in water as well as crocetin in DMSO:PBS

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(10:90, v/v) were prepared. Samples were filtered through a 0.45 µm membrane filter.

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The resulting filtrates were stored at -20 oC until further analysis.

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Determination of DNA damage (Comet Assay). U937 cells (1 × 105 cells/mL)

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were supplemented with increasing non toxic concentrations of individual

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apocarotenoids, i.e. picrocrocin, trans-4-GG crocetin ester, crocetin (25 µM) in 6-well

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plates with a final volume of 2 mL and incubated for 24 h at 37 °C. The cytotoxicity

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of the tested compounds against this cell line was determined by the fluorescein

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diacetate/ethidium bromide (FDA/EtBr) assay as previously detailed.7 Following

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incubation, cells were treated with 75 µM H2O2 for 30 min. Cells were harvested,

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suspended in a low melting point (LMP) agarose, placed on microscope slides, and

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allowed to solidify. Slides were placed in cold lysis solution [2.5 M NaCl, 100 mM

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EDTA, 10 mM Tris, pH = 10, 1% (w/v) sodium sarcosinate], with 1% (v/v) Triton X-

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100 and 10% (v/v) DMSO, added freshly before each use, for 1.5 h. Slides were then

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placed in a horizontal gel electrophoresis tank containing fresh electrophoresis

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solution (1 mM EDTA, 300 mM NaOH) for 30 min. Subsequently, electrophoresis

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was carried out for 25 min at 4 °C with a current of 25 V (300 mA) using a compact

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power supply. After electrophoresis, the slides were washed three times with

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neutralizing buffer (0.4 M Tris, pH = 7.5) at 4 °C for 5 min each. Slides were stained

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with ethidium bromide (20 µg/mL) and covered with coverslips. Komet 5.5 image

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analysis software (Kinetic Imaging Ltd., Bromborough, UK) was utilized to measure

271

the level of DNA damage, which was expressed as percentage tail DNA.

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Cell proliferation. The anti-proliferative effects of individual apocarotenoids

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(picrocrocin, trans-4-GG crocetin ester, crocetin) were examined in the Caco-2 and 11



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HepG2 cell lines using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) assay (MTT I proliferation kit, Roche Diagnostics, West Sussex,

276

United Kingdom). Caco-2 and HepG2 cells were seeded at a density of 1 × 105

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cells/mL in 96-well plates and allowed to adhere overnight. Cells were then exposed

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to increasing concentrations of individual apocarotenoids. After 24 h incubation at 37

279

°C, 5 µL of MTT solution was added to each of the samples and incubated for 4 h at

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37 °C. Following incubation, 100 µL of the solubilisation solution (10% SDS in 0.01

281

M HCl) was added. A further incubation period of 24 h at 37 °C followed.

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Absorbance was read using a Tecan Spectrafluor Plus microplate reader at a

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wavelength of 570 nm with a reference wavelength of 690 nm. Cell proliferation was

284

expressed as a percentage of the control, untreated cells. GraphPad Prism version 4.00

285

software (San Diego, USA) was used to determine IC50 values for each of the samples

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in Caco-2 and HepG2 cells.

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Statistical analysis. Statistical comparisons of the mean values were

288

performed by one-way ANOVA, followed by the multiple comparison Duncan test (p

289

< 0.05 confidence level) using the SPSS 14.0 software (SPSS Inc., Chicago, IL,

290

USA).

291 292

RESULTS AND DISCUSSION

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Chemical Characterization of Saffron Aqueous Extract. The RP-HPLC-

294

DAD profile of the saffron aqueous extract at 250 nm and 440 nm indicated the

295

presence of picrocrocin and crocetin sugar esters, respectively, as expected. The

296

picrocrocin content (14.4 ± 0.7 g/100 g dry stigmas) and that of total crocetin sugar

297

esters (29.3 ± 0.5 g/100 g dry stigmas) and major individual ones, i.e. trans-4-GG

298

crocetin ester (15.6 ± 0.3 g/100 g dry stigmas) and trans-crocetin (β-D-glucosyl)-(β12


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D-gentiobiosyl) ester (trans-3-Gg crocetin ester) (7.4 ± 0.2 g/100 g dry stigmas) were

300

representative of those of high quality saffron and of similar size to reported

301

data.3,14,16

302

Bioaccessibility of Picrocrocin in Saffron Aqueous Extract. Treatment of

303

the saffron aqueous extract under the simulated digestion conditions showed that

304

~75% of the initial amount of picrocrocin survived the harsh gastrointestinal

305

conditions, in agreement with the results of our previous study.7 Picrocrocin was

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found to be more stable under these conditions than the total crocetin sugar esters of

307

the saffron extract (~40% loss) in agreement with previous results.7,8 Picrocrocin is

308

reported to be resistant to thermal treatment,17 however there is no information

309

available about the effect of pH and/or digestive enzymes to its degradation rate.

310

Cellular

Transport

of

Picrocrocin

and

Other

Major

Saffron

311

Apocarotenoids. Saffron Extract Digestate. The cellular transport of picrocrocin was

312

then examined by applying the saffron digestate to the Caco-2 cell monolayer. As the

313

digestate itself can be toxic to the Caco-2 cells due to the presence of proteolytic and

314

lipolytic enzymes, it was first diluted with medium (HBSS), at least 1:2 (v/v), and

315

then added to the cell monolayer. Blank digestates that contained all the digestive

316

enzymes except for the saffron extract were also examined. Dilutions that resulted in

317

TEER values < 250 Ω ⋅ cm2 were not examined further. After a 3 h incubation period

318

in the Caco-2 cells with three increasing concentrations of the saffron digestate

319

corresponding to 2.3, 2.6 and 2.8 mg picrocrocin/L digestate; no picrocrocin was

320

detected in the basolateral compartment even though a high fraction of its initial

321

amount was bioaccessible. This observation could be attributed to the presence of the

322

sugar moiety that results in lack of affinity towards the cellular membranes and does

323

not allow passive diffusion.18 Numerous studies have demonstrated that hydrolysis by 13



324

digestive enzymes or intestinal microflora may be necessary for the absorption of

325

glucosides of phenolic compounds due to their structural complexicity. Two

326

metabolic pathways have been reported for the absorption of glucosides.19 Glucosides

327

may be hydrolyzed by lactase phloridizin hydrolase (LPH) which is located on the

328

brush border of small intestinal epithelial cells into the aglycone or alternatively, they

329

may be absorbed by means of the active sodium-dependent glucose transporter

330

(SGLT-1) and then they can be hydrolyzed within the intestinal cells by cytosolic β-

331

glucosidase. In the present study, however, no sign of picrocrocin hydrolysis and

332

release of the aglycone 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde

333

(HTCC)1 was evidenced on the chromatogram at 250 nm [Relative Retention Time

334

(RRT) towards picrocrocin = 1.53]. It has also been observed in the literature that

335

other glucosides such as quercetin-4′-glucoside,20 trans-piceid,21 genistin and

336

daidzein22 are not transported across the Caco-2 cell monolayer possibly due to an

337

absence of β-glucosidase activity in this in vitro model. The crocetin sugar esters of

338

the saffron extract digestate were found to be barely transported across the Caco-2

339

cell monolayer (data not shown). In particular, only 0.4% of the bioaccessible fraction

340

of total crocetin sugar esters was found to be transported to the basolateral

341

compartment, i.e. was bioavailable. Similarly, the major crocetin ester, trans-4-GG,

342

which accounts for ~60% of the total crocetin esters content of saffron was

343

transported by ~0.4% - 0.5%. Trans-3-Gg crocetin ester was detected in the

344

basolateral chamber but at concentrations below the limit of quantification (LOQ) of

345

the specific analytical method employed. No evidence of the hydrolysis of crocetin

346

sugar esters to release the aglycone crocetin was evidenced during cellular transport,

347

experiments, probably due to the reasons detailed above. Lautenschläger et al. (2015)8

348

also found that only 0.34% of pure trans-4-GG crocetin ester was transported across 14


Page 14 of 33

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349

the Caco-2 cells monolayer with no concomitant generation of free crocetin during

350

absorption. However, the presence of free crocetin has been observed in rodent

351

plasma after the oral administration of either a mixture of crocetin esters or pure

352

trans-4-GG crocetin ester, derived from the dried fruits of Gardenia jasminoides

353

Ellis.9,10 Moreover, when human volunteers consumed a cup of saffron tea, no

354

crocetin esters but only crocetin was detected in their blood stream.11 Compounds that

355

are not absorbed in the small intestine are expected to reach the colon, where they

356

may exert their biological effects.

357

Pure compounds. The cellular transport of pure picrocrocin versus that of the

358

other two major saffron apocarotenoids, i.e. trans-4-GG crocetin ester and crocetin

359

was then examined at equimolar concentrations (8 – 24 µM) using the same in vitro

360

model. The pure compounds were applied directly onto the Caco-2 cell monolayer. In

361

this case the cellular transport of picrocrocin was found to be ~0.2% (Figure 2). The

362

observed cellular transport in comparison to that found for picrocrocin in the saffron

363

extract digestate could be attributed to antagonistic interactions with other co-present

364

constituents (e.g. crocetin sugar esters) that may have similar mechanisms of

365

absorption.23 Data in Figure 2 showed a 10-fold higher cellular transport of the

366

aglycone crocetin (~5% in the range 8 – 24 µΜ) compared to that of picrocrocin and

367

also its natural sugar ester forms. Lautenschläger et al. (2015)8 measured the

368

absorption of trans-crocetin in Caco-2 cells and found an average permeation rate of

369

32%; they also found that the absorption was similar for each concentration in the

370

range of 10 - 114 µM and was not dose dependent. In addition, there was no specific

371

transporter associated with the transport of trans-crocetin and the authors concluded

372

that transport was via the paracellular route. Several experimental factors such as the

373

pH of the transport buffer, the solvent used to deliver the test compound24 and the 15



374

TEER values of the Caco-2 monolayers can affect transport across Caco-2 cells.25

375

Theoretically, crocetin is expected to be efficiently absorbed after oral administration

376

as it meets the Lipinski’s requirements for optimal oral bioavailability.26 According to

377

Lipinski’s rule27 a compound is expected to display optimum gastrointestinal

378

absorption if «it has a molecular mass of equal or less than 500 g/mol, less than five

379

hydrogen bond donors and less than ten hydrogen bond acceptors and a calculated

380

partition coefficient (logP) less than five”. Therefore, crocetin, with a molecular mass

381

of 328.40 g/mol, four hydrogen bond acceptors, two hydrogen bond donors and a

382

value of logP equal to 4.7228 may fulfill better these requirements in comparison to

383

the more polar apocarotenoids.

384

Once carotenoids are absorbed by the intestinal cells, metabolism becomes the

385

major barrier to their bioavailability.29 The metabolism of apocarotenoids derived

386

from saffron has not been fully eludidated however, β-carotene 9', 10'-oxygenase

387

(BOC2), which is expressed in mammalian tissues, has a broad substrate specificity

388

and has been shown to metabolise xanthophylls.30 In the present study, however,

389

peaks at retention times earlier than those of picrocrocin and trans-4-GG crocetin

390

ester were not detected on the chromatograms of the basolateral medium. Similar

391

observations have been also made in the case of quercetin from red onions31 and

392

apples32 using the same cell line. It is well documented that conjugation via

393

glucuronidation occurs to a greater extent in the liver where the expression of

394

glucuronosyltranferases (UGTs) is much higher compared to that in the small

395

intestine.33

396

DNA Protective Effects of Picrocrocin and Other Major Saffron

397

Apocarotenoids. To determine the protective effects of the individual major saffron

398

apocarotenoids against oxidant-induced DNA damage, the Comet assay was 16


Page 16 of 33

Page 17 of 33


399

employed. The Comet assay is a sensitive tool for measuring DNA single-strand

400

breaks in individual cells.34 In the present study, the addition of 75 µM Η2Ο2 to U937

401

cells caused an increase in DNA damage to 52.6% tail DNA compared to the

402

unchallenged control (4% tail DNA) (Figure 3). Cell supplementation with a non-

403

toxic concentration (data not shown) of pure picrocrocin and trans-4-GG crocetin

404

ester (25 µM) did not exhibit a significant (p < 0.05) DNA protective effect under

405

these experimental conditions. When the aglycone crocetin was examined at an

406

equimolar concentration (25 µM), it caused a significant (p < 0.05) decrease in DNA

407

damage (~43% tail DNA). It has been shown that saffron compounds including

408

crocetin may form a complex with DNA and scavenge free radicals which can protect

409

DNA from oxidant induced damage.35 Literature data focus on the protective effects

410

of saffron extracts and pure trans-4-GG crocetin ester against oxidant-induced DNA

411

damage. In particular, Kyriakoudi et al. (2013)7 reported a significant decrease

412

(13.2% tail DNA) in DNA damage induced by hydrogen peroxide after

413

supplementation of U937 cells with a saffron aqueous extract. Trans-4-GG crocetin

414

ester has previously demonstrated a dose dependent decrease in H2O2-induced DNA

415

damage, as determined by the Comet assay, in a mouse lymphoma (L5178Y) cell line

416

however, it did not protect against methyl methanesulfonate (MMS)-induced DNA

417

damage in the same study.36 The protective effect of a saffron extract and pure trans-

418

4-GG crocetin ester has been also reported in vivo against methyl methanesulfonate–

419

induced DNA damage in mice organs using the Comet assay.37

420

Anti-proliferative effects of Picrocrocin and Other Major Saffron

421

Apocarotenoids in human cancer cell lines. Several constituents of saffron have

422

shown anti-tumor effects through an inhibition of cancer cell growth. The anti-

423

proliferative effects of saffron extracts have been demonstrated in breast cancer cells 17



424

(MCF-7), hepatacarcinoma cells (HepG2), colorectal cancer cells (HCT-116, SW-

425

480, HT-29) and lung cancer cells (NSCLC), amongst others.38 Additionally,

426

ethanolic extracts of saffron have demonstrated selective toxicity to cancer cells and

427

did not induce toxicity in normal, non-cancerous cells.39 The effect of picrocrocin,

428

trans-4-GG crocetin ester and crocetin on the in vitro proliferation of human tumoral

429

cells was studied using the human adenocarcinoma, Caco-2, and the human

430

hepatocellular carcinoma, HepG2, cell lines. As illustrated in Figure 4A, picrocrocin

431

and trans-4-GG crocetin ester reduced Caco-2 cell proliferation in a dose-dependent

432

manner. Crocetin was found to stimulate cell growth at the lower concentrations (125

433

and 250 µM) but reduced cell proliferation at higher ones. The IC50 value for each

434

compound is presented in Table 1. These values were found to range from 840 to

435

1,700 µM with crocetin exhibiting the highest anti-proliferative effect followed by

436

picrocrocin and trans-4-GG crocetin ester. Picrocrocin had an IC50 value of 3,000 µM

437

in TC-1, malignant murine, cells following a 48 hour incubation and trans-4-GG

438

crocetin ester had an IC50 value of 1.5 µM.40 The differences in IC50 values for the

439

various saffron constituents could result from differences in the uptake, transport and

440

retention of these compounds. In the HepG2 cell line, it was found that picrocrocin

441

triggered a dose-dependent decrease in cell proliferation (Figure 4B) similar to the

442

trend observed in the Caco-2 cell line. In this cell line, trans-4-GG crocetin ester and

443

crocetin were found to increase cell viability at the lower concentrations examined,

444

125 µM and 250 µM, but decreased cell proliferation at higher concentrations. The

445

IC50 values for the tested apocarotenoids were found to be similar in both the HepG2

446

and the Caco-2 cell line (Table 1). These data indicate that picrocrocin, trans-4-GG

447

crocetin ester and crocetin may have chemopreventative effects. The ability of

448

crocetin to impede cancer cell growth has been related to an enhancement of the 18


Page 18 of 33

Page 19 of 33


449

cellular antioxidant systems, the inhibition of nucleic acid synthesis and the induction

450

of apoptosis.41

451

In conclusion, even though picrocrocin is the most bioaccesible among the

452

major saffron apocarotenoids, it was found to be transported across the Caco-2 cell

453

monolayer at very low levels. When pure picrocrocin was examined at equimolar

454

concentrations with other major saffron apocarotenoids, i.e. trans-4-GG crocetin ester

455

and crocetin using the same in vitro model, its cellular transport was found to be

456

approximately similar to that of trans-4-GG crocetin ester and ~10 fold lower than

457

that of the aglycone crocetin. To the best of our knowledge, this is the first time that

458

the data on the cellular transport of picrocrocin itself or in the presence of other

459

saffron apocarotenoids are presented. Picrocrocin also demonstrated anti-proliferative

460

activity in human cancer cell lines. This study highlights the relevance of picrocrocin

461

to investigations of the bioavailability and bioactivity of saffron extracts.

462 463

ABBREVIATIONS USED

464

trans-4-GG crocetin ester: trans-crocetin (di-β-D-gentiobiosyl) ester; trans-3-Gg

465

crocetin ester, trans-crocetin (β-D-glucosyl)-(β-D-gentiobiosyl) ester; TEER,

466

transepithelial electrical resistance; FBS, fetal bovine serum; HBSS, Hank’s balanced

467

salt solution; DMEM, Dulbecco’s modified Eagle’s medium, MTT, 4,5-

468

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

469

diacetate/ethidium bromide; LOQ, limit of quantification; H2O2, hydrogen peroxide;

470

RP-HPLC-DAD, reversed phase-high performance liquid chromatography-diode

471

array detector; RRT, relative retention time; NMR, nuclear magnetic resonance;

472

ANOVA, analysis of variance.

bromide;

473 19


FDA-EtBr,

fluorescein


474

ACKNOWLEDGMENT

475

A.K. thanks the Foundation of State Scholarships (IKY, Athens, Greece) for financial

476

support for doctoral studies.

477 478

SUPPORTING INFORMATION DESCRIPTION

479

LC-ESI-MS and NMR data for the identification of picrocrocin (I), trans-4-GG

480

crocetin ester (II) and crocetin (III).

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498

20


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499

REFERENCES

500

(1) Carmona, M.; Zalacain, A.; Alonso, G. L. The chemical composition of saffron:

501

color, taste and aroma. 1st ed., Editorial Bomarzo S.L. Albacete, Spain, 2006;

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Chapt. 4, pp 77-104.

503

(2) Frusciante, S., Diretto, G., Bruno, M., Ferrante, P., Pietrella, M., Prado-Cabrero,

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A., Rubio-Moraga, A., Beyer, P., Gomez-Gomez, L., Salim Al-Babili, Giovanni

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Giuliano. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step

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in saffron crocin biosynthesis. Proc Natl Acad Sci U S A. 2014, 111, 12246-

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12251.

508

(3) Kyriakoudi, A.; Chrysanthou, A.; Mantzouridou, F.; Tsimidou, M. Z. Revisiting

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(saffron). Anal. Chim. Acta 2012, 755, 77-85.

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(4) Kyriakoudi, A.; Ordoudi, S. A.; Roldán-Medina, M.; Tsimidou, M. Z. Saffron, a functional spice. Austin J. Nutr. Food Sci. 2015, 3, 1059-1063. (5) Giaccio, M. Crocetin from Saffron: An Active Component of an Ancient Spice, Crit. Rev. Food Sci. Nutr. 2004, 44, 155-172. (6) Alavizadeh, S. H.; Hosseinzadeh, H. Bioactivity assessment and toxicity of crocin: A comprehensive review. Food Chem. Toxicol. 2014, 64, 65-80.

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digestion of saffron aqueous extracts. J. Agric. Food Chem. 2013, 61, 5318-5327.

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Riehemann, K.; Hensel, A. Intestinal formation of trans- crocetin from saffron

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Xi, L.; Qian, Z.; Du, P.; Fu, J. Pharmacokinetic properties of crocin (crocetin

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Chryssanthi, D. G.; Lamari, F. N.; Georgakopoulos, C. D.; Cordopatis, P. A

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Application after saffron tea consumption. J. Pharm.Biomed. Anal. 2011, 55, 563-

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Ordoudi, S. A.; Befani, C. D.; Nenadis, N.; Koliakos, G. G.; Tsimidou, M. Z.

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Williamson, G.; Swallow, D. M.; Kroon, P. A. Deglycosylation by small intestinal

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dietary flavonoid glycosides in humans. Eur J Nutr 2003, 42, 29-42.

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Walgren R. A.; U. Walle, K.; Walle, T. Transport of quercetin and its

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Henry, C.; Vitrac, X.; Decendit, A.; Ennamany, R.; Krisa, S.; Mérillon, J. M.

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Cellular uptake and efflux of trans-piceid and its aglycone trans-resveratrol on the

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Steensma, A.; Noteborn, P. J. M.; van der Jagt, R. C. M.; Polman, T. H.G.;

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Mengelers, M. J. B.; Kuiper, H. A. Bioavailability of genistein, daidzein, and

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A.; Polissiou, M. G. An overview of structural features of DNA and RNA

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Choi, H. Y.; Kim, Y. J.; Ryu, J. C. Study of genotoxicity of crocin, a

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Hosseinzadeh, H.; Abootorabi, A.; Sadeghnia, H. R. Protective effect of

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Crocus sativus stigma extract and crocins (trans-crocin 4) on methyl

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Bolhassani, A.; Khavari, A.; Bathaie, S. Z. Saffron and natural carotenoids:

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Biochemical activities and anti-tumor effects. Biochimica et Biophysica Acta,

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(39) Li, C. Y.; Huang, W. F.; Wang, Q. L.; Cai, F. W.; Hu, B.; Du, J. C.;

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Wang, J.; Chen, R.; Cai, X. J.; Feng, J.; Li, H. H. Crocetin induces cytotoxicity in

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Khavari, A.; Bolhassani, A.; Alizadeh, F.; Bathaie, S. Z.; Balaram, P.; Agi, E.;

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Vahabpour, R. Chemo-immunotherapy using saffron and its ingredients followed

621

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622

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499-508.

624

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Gutheil, W.G.; Reed, G.; Ray, A.; Anant, S.; Dhar, A. Crocetin: an agent

625

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626

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627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 26


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647

FIGURE CAPTIONS

648 649

Figure 1. Chemical structure of the major saffron apocarotenoids.

650 651

Figure 2. Cellular transport (%) of pure picrocrocin, trans-4-GG crocetin ester and

652

crocetin across the Caco-2 cell monolayer. Cells were incubated with increasing

653

concentrations (8 - 24 µM) of the compounds for 3 h. Data are mean values ± SD of

654

three independent experiments (n = 3) and were analyzed by one-way ANOVA.

655

Different lowercase letters for each compound indicate significant difference

656

according to Duncan’s test at p < 0.05.

657 658

Figure 3. DNA damage in U937 cells following pretreatment with or without pure

659

picrocrocin, trans-4-GG crocetin ester and crocetin (25 µM) for 24 h followed by

660

exposure to 75 µM H2O2 for 30 min at 37 °C. DNA damage was assessed by the

661

comet assay. Data are the mean values of six independent experiments, with standard

662

errors represented by vertical bars. Statistical comparisons of the mean values were

663

performed by one-way ANOVA followed by Duncan’s test.

664

significantly different from that for H2O2-challenged cells (p < 0.05).

a,b,c

Mean value was

665 666

Figure 4. Cell proliferation in Caco-2 (A) and HepG2 (B) cell lines following 24 h

667

exposure to picrocrocin, trans-4-GG crocetin ester and crocetin. Cell proliferation was

668

assessed using the MTT assay. Data are the mean values ± SD of six independent

669

experiments (n = 6).

27



TABLES

Table 1. IC50 Values for Picrocrocin, Trans-4-GG Crocetin Ester and Crocetin in Human Colon Adenocarcinoma Caco-2 Cells and Human Heptacarcinoma HepG2 Cells. IC50 values (µM)a

a

Caco-2 Cells

HepG2 Cells

Picrocrocin

1160 ± 0.88

1100 ± 0.13

Trans-4-GG crocetin ester

1700 ± 0.11

1250 ± 0.03

Crocetin

840 ± 0.21

750 ± 0.25

Each value is the mean of triplicate determinations ± SD.

28


Page 28 of 33

Page 29 of 33


FIGURES

Figure 1

O OR2

R1O O

trans R1, R2 = glucose or gentiobiose (major crocetin esters) R1 = R2 = H (crocetin) H3C CH3O H CH3

Safranal

Picrocrocin

29



Figure 2

30


Page 30 of 33

Page 31 of 33


Figure 3

31



Figure 4

32


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Graphic for table of contents (TOC)

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Cellular Transport and Bioactivity of a Major ... - ACS Publications

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