american india pale ale matrix rich in xanthohumol is potent in suppressing proliferation and...

Original article

American India Pale Ale matrix rich in xanthohumol is potent in

suppressing proliferation and elevating apoptosis of human colon

cancer cells

Twila Henley,1† Lavanya Reddivari,2† Corey D. Broeckling,3 Marisa Bunning,1 Jeff Miller,1 John S. Avens,1

Martha Stone,1 Jessica E. Prenni3 & Jairam Vanamala4*

1 Department of Food Science and Human Nutrition, Colorado State University, 1571 Campus mail, Fort Collins, CO 80523, USA

2 Department of Plant Science, Pennsylvania State University, University Park, PA 16802, USA

3 Proteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO 80523, USA

4 Department of Food Science, Pennsylvania State University, University Park, PA 16802, USA

(Received 22 March 2014; Accepted in revised form 3 April 2014)

Summary American India Pale Ales (IPAs) with and without addition of dark/roasted malts (DRM) and dry hop-

ping (DP) were analysed to determine whether these processes will increase total phenolic content (TP),

antioxidant capacity (AA) and the levels of bioactive compounds (xanthohumol, XN and isoxanthohu-

mol, IX). In addition, bioactivity of whole beer matrices, that is, the ‘phytochemical team approach,’ was

compared to isolated compounds, the ‘silver bullet approach,’ by measuring antiproliferative and pro-

apoptotic properties using HCT 116 human colon cancer cells. DP and addition of DRM elevated the

XN, IX, TP and AA. Dark malts reduced losses in XN and TP due to filtration. Xanthohumol content

positively correlated with phenolic content (r = 0.88, P = 0.0002), indicating that the processes which

increased xanthohumol content also elevated other bioactive compounds in beer. However, whole extract

from IPAs were more potent in suppressing proliferation and elevating apoptosis in colon cancer cells

compared with xanthohumol alone.

Keywords Antioxidant activity, apoptosis, beer extracts, cell proliferation, UPLC/MS/MS, xanthohumol.

Introduction

Beer contains multiple compounds beneficial to healthincluding silicone, benzoic and cinnamic acid deriva-tives, ferulic acid, vitamin B6, betaine glycine, catechinsand proanthocyanidins. A hop-derived prenylated chal-cone in beer, xanthohumol (XN), has received muchattention (Magalhaes et al., 2008) because of its puta-tive health-beneficial properties including antioxidant,antiproliferative, pro-apoptotic, anti-inflammatory,antimicrobial and antimalarial activities (Bamforth,2002; Gerhauser, 2005; Festa et al., 2011; Yen et al.,2012). Recently researchers showed that XN (2.5–20 lM) suppressed cancer cell proliferation and cancercell invasion and increased apoptosis in various cancercell lines (Pan et al., 2005; Wang et al., 2012; Kimet al., 2013). Because of the wide spectrum of beneficialproperties XN possesses, XAN technology, whichincludes modifying brewing ingredients (using dark,

roasted malts and XN-enriched hop products) andbrewing procedures (wort boiling, fermentation, matu-ration/storage and filtration/stabilisation) has beendeveloped to increase the amount of XN in beers (Wun-derlich et al., 2005; Wunderlich & Back, 2007; Karab�ınet al., 2013). XN is reported to be slightly bitter tasting(Haseleu et al., 2010), and the amount in commercialbeers is low: 0.2 mg L�1 or less (Wunderlich et al.,2005). However, addition of dark malts that containseveral unidentified soluble substances with a moleculesize range of 300 000–600 000 Da form complexes withbeer compounds and might protect XN during the boil-ing process, thus reducing its isomerisation to iso-xanthohumol (IX), a compound which is not asbiologically active as XN (Wunderlich et al., 2005;Wunderlich & Back, 2007). By utilising the combinationof dark malts and XN-enriched hop products, a beerwith a XN content of 17.2 mg L�1 was developedWunderlich et al., 2005).One example of a beer style that may inherently con-

tain more XN is the India Pale Ale (IPA). Specifically,*Correspondent: Fax: 814 863 6132; e-mail: [email protected]†These authors contributed equally for this work.

International Journal of Food Science and Technology 2014, 49, 2464–2471

doi:10.1111/ijfs.12570

© 2014 Institute of Food Science and Technology

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American IPA is a style of beer containing Americanhop varieties which often utilises dry hopping wherehops are added after the wort has been cooled and whilethe beer ferments (Guinard et al., 1990). Dry hoppingcontributes to hop aroma but not to bitterness as alphaacids need to be isomerised during wort boiling to formthe bitter tasting isoalpha acids (De Keukeleire, 2000).Information on bioactive compounds in American IPAstyled beers is limited. Researchers have analysed pren-ylflavonoids in different beer styles and reported XNcontent of 0.16 mg L�1 in IPAs (Stevens et al., 1999),but this amount is expected to be higher in beers thatare dry-hopped.

We hypothesised that (i) American IPAs will havehigher amounts of XN, total phenolics (TP) and antioxi-dant activity (AA) compared with non-IPA style beersand the addition of dark malts and dry hopping willincrease the XN content, TP and AA; (ii) the beermatrix will have greater antiproliferative and pro-apop-totic properties in vitro compared with isolated XN and/or IX. To test these hypotheses, isolated compoundsXN and/or IX, as well as different brands of whole IPAmatrices were analysed for TP, AA, XN and IX content,and pro-apoptotic and antiproliferative properties inHCT 116 (P53+/+) colon cancer cells. Results indicatedthat the amount of XN, TP and antioxidant capacity ofIPAs vary widely depending on the ingredients and pro-cessing methods used. However, certain IPAs containedmuch higher amounts of XN than previously reported(Stevens et al., 1999). Finally, beer matrices with a mix-ture of bioactive compounds along with XN and IXshowed potent antiproliferative and pro-apoptoticproperties compared with isolated XN and/or IX atconcentrations found in the beer extracts.

Materials and methods

Materials

Folin-Ciocalteu reagent, Trolox and 2,20-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicalwere purchased from Sigma (St. Louis, MO, USA).Na2CO3 was purchased from Mallinckrodt (Paris, KY,USA), and gallic acid was purchased from AcrosOrganics (North Hampton, NH, USA). XN and IXstandards were purchased from Enzo Life Sciences(Plymouth Meeting, PA, USA), and chlorogenic acidwas procured from Indofine (Hillsborough, NJ, USA).For the cell culture assays, McCoy’s 5A modified med-ium, Dulbecco’s modified Eagle’s medium F-12,bovine serum albumin and sodium bicarbonate werepurchased from Sigma. Foetal bovine serum, strepto-mycin/penicillin mix and 5% activated charcoal wereobtained from Fisher Scientific (North Hampton, NH,USA). Caspase-Glo 3/7 was purchased from PromegaCorporation (Madison, WI, USA).

Beer preparation

A total of ten different beer samples were collectedfrom four different breweries (Odell Brewing, New Bel-gium Brewing, Equinox Brewing and CooperSmith’sBrewing) in Fort Collins, Colorado, and the originalbrand names were substituted with letters brand A, B,C, etc. Beers were made in batches from the originalbrand A, B, C and D IPA recipes and from alteringthe Brand B recipe to contain 8.7% more darker maltsto assess the effect of addition of dark malts on xan-thohumol (XN) content and bioactivity. The effect ofDE filtration, a process to clarify the beer, on finalXN content in American IPAs was analysed by col-lecting samples before and after filtration for threebeer types (Brand A, Brand B and BD). Two differentversions of Brand C IPA, one dry-hopped and theother non-dry-hopped, were tested to determine theeffect of dry hopping on the final XN content. Infor-mation on beer preparation of each of these IPAs ispresented in Table 1. A German-style Pilsner beer(Brand E; EGP) made with Pilsner malts, German hopvarieties and German lager yeasts (4.9% alcohol byvolume), served as the control beer. Compared withthe other beers, it was lightest in colour and highest incarbonation.

Sample preparation for analytical assays

Beer samples collected at breweries from differentbatches were transported on ice (0 °C) to the labora-tory. All samples were stored at �80 °C in 50-mLtubes. The samples were thawed at 4 °C for 6 h andcentrifuged at 515 g for 20 min, and the supernatantwas collected for analysis. Although solid-phaseextraction was used in some studies to extract bioac-tive compounds from beers before determining thecontent of XN (Magalhaes et al., 2008), a reductionhas been shown in the XN recovery after C18 solid-phase extraction of dark beers (Wunderlich et al.,2009; Haseleu et al., 2010). Thus, in this study, thesupernatant after centrifugation of beer was directlyused for antioxidant activity, total phenolic contentand LC-MS analysis. Beer supernatants were filteredand evaporated under nitrogen gas to remove ethanolprior to cell culture assays.

Quantification of XN and IX Using LC-MS/MS

A Waters Acquity UPLC system with BEH C18 col-umn (1.7 lM, 1.0 9 100 mm) was used for chromato-graphic separation. Samples were eluted using thefollowing linear gradient at a constant flow rate of140 lL min�1: 0% B for 1 min, 0–100% B for 10 min,100% B for 2 min, 0% B for 3 min (total runtime 16 min). The column and autosampler were

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© 2014 Institute of Food Science and Technology International Journal of Food Science and Technology 2014

Bioactivity of American IPAs T. Henley et al.

maintained at 50 °C and 5 °C, respectively. Solvent Acontained 5% acetonitrile, 95% water and 0.1% for-mic acid. Solvent B consisted of 95% acetonitrile, 5%water and 0.1% formic acid.

Column eluent was directly coupled to a WatersQ-Tof Micro mass spectrometer fitted with an electro-spray source. Data were collected in a negative mode,scanning from 50 to 1000 m/z with a 0.5-s scan timeand a 0.1-s interscan delay. Calibration was performedprior to sample analysis via infusion of sodium for-mate solution, with mass accuracy within 3 ppm. Thecapillary voltage was held at 2200V, the source tempat 130 °C and the desolvation temperature at 300 °Cwith nitrogen desolvation gas flow rate of 300 L h�1.The quadrupole was held at collision energy of 7 eVfor parent ion detection and 18 eV for product ionformation. For both internal standards and target ana-lytes, quantification was based on product ions toensure specificity. Chlorogenic acid, an internal stan-dard eluting at 2.7 min, was isolated using a parentmass 353.08 m/z, and the product ion 191.06 m/zserved as the target for quantitation. Both IX and XNwere detected using the parent ion 353.14 m/z andquantified using the fragment ion 233.08 m/z at reten-tion times 6.01 and 7.84 min, respectively. Each beertype was tested in quadruplicate with two technicalreplications for each sample, for a total of eight repli-cations. All sample areas were normalised againstchlorogenic acid.

Total phenolics assay

The Folin-Ciocalteu reagent (FCR) assay was used tomeasure the total phenolic content of beer samples.Gallic acid was used to set the standard curve

(R2 > 0.99). Samples were diluted and tested in four rep-lications according to the published protocols (Madi-wale et al., 2011). In brief, 150 lL of 0.2 M Folin-Ciocalteu reagent was added to 35 lL of sample andvortexed. After 5 min, 115 lL of 7.5% Na2CO3 wasadded to each sample and held at 25 °C for 1 h beforemeasuring the phenolic content using spectrophotome-ter (Synergy 2; BioTek, Winooski, VT, USA) at a wave-length of 765 nm. Values were expressed as milligramsgallic acid equivalent (GAE) per litre of sample � SE.

Antioxidant capacity assay

The 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) Trolox equivalent antioxidant capacityassay (TEAC) was used to measure total antioxidantactivity of the beer samples. A stock solution of ABTSwas prepared by mixing 8 mM ABTS solution and 3 mM

of potassium persulphate, followed by incubation in thedark for 12 h. A working solution was freshly preparedevery time by adding 5 mL of the stock solution to45 mL of phosphate buffer. Samples were diluted sothat the absorbance reading was between 0.1 and 1.5.Ten microlitres of diluted samples and 290 lL of pre-pared ABTS working solution were mixed in a 96-wellplate and measured in the spectrophotometer at734 nm. Each beer type was tested in quadruplicate withfour technical replications (sixteen values in total).Results were expressed in milligrams TEAC per litre ofsample � SE.

Cell proliferation and apoptosis

The human colon carcinoma cell line HCT-116 p53+/+,from Bert Vogelstein (affiliation), was maintained in

Table 1 Description and specifications of India Pale Ales (IPA) samples and preparation method

IPA Malts Hops Filtration

Brand A Pre-filtered (AP) Two-row Pale and Crystal 120 T-90 pellets and CO2 extract (Cascade Chinook and Simcoe)

Brand A Filtered (AF) Two-row Pale and Crystal 120 T-90 pellets and CO2 extract (Cascade Chinook and Simcoe) DE filtration

Brand B Pre-filtered (BP) Pale, Munich, Crystal and Cara Pelleted Columbus, Chinook, Perle and Centennial,

0.055 oz per 0.3 L beer

Brand B Filtered (BF) Pale, Munich, Crystal and Cara Pelleted Columbus, Chinook, Perle and Centennial,


DE filtration

Brand B Dark

Pre-filtered (BDP)

Pale, Munich, Crystal, Cara, ESB,

Carafa and Amber

Pelleted Columbus, Chinook, Perle and Centennial,


Brand B Dark

Filtered (BDF)

Pale, Munich, Crystal, Cara, ESB,

Carafa and Amber

Pelleted Columbus, Chinook, Perle and Centennial,


DE filtration

Brand C Non-Dry

Hopped (CNH)

Pale, Aromatic, Munich, Carapils,

Crystal/Caramel and Debittered black

Pelleted Columbus, Golding, Amarillo, Simcoe

and Centennial added

at beginning of whirlpool

Biofine clear

treatment

Brand C Dry

Hopped (CDH)

Pale, Aromatic, Munich, Carapils,

Crystal/Caramel and Debittered black

Pelleted Columbus, Golding, Amarillo, Simcoe

and Centennial, 0.016

oz per 0.3 L beer

Biofine clear

treatment

Brand D Dry

Hopped (DDH)

Pale, light and medium Crystal Pelleted Centennial, Columbus, Cascade and Glacier,


DE filtration

© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014

Bioactivity of American IPAs T. Henley et al.2466

McCoy’s 5A modified medium supplemented with2.0 g L�1 sodium bicarbonate, 0.11 g L�1 sodium pyru-vate, 4.5 g L�1 glucose, 100 mL L�1 foetal bovineserum and 10 mL L�1 antibiotic antimycotic solution.Four beers were selected based on their content of XNor IX. Brand B Pre-filtered (BP), Brand B Dark Pre-fil-tered (BDP) (highest XN content), Brand C DryHopped (CDH) (highest IX content) and EGP (lowestXN content) were used for in vitro assays. Centrifugedbeer samples were filtered using a 0.2-lm nylon filtermembrane and evaporated to completion under nitro-gen gas. Cells were plated at a density of 5.0 9 104 perwell in a 12-well plate. After 24 h, the growth mediumwas replaced with 2.5% serum stripped Dulbecco’smodified Eagle’s medium F-12 containing either fourbeer samples in three concentrations (5, 10, 15 lg mL�1

GAE) or XN/IX at highest beer concentra-tion (12.69 ng mL�1) (2 ng mL�1), combination ofXN + IX (12.69 ng mL�1 + 1 ng mL�1), and XN at aconcentration that had previously shown antiprolifera-tive activity (3.5 lg mL�1) (Hadjiolov & Frank, 2009).Ethanol (3%) was used as the control, as isolated com-pounds were dissolved in 80% ethanol. After 24 h ofincubation with the treatments, cell proliferation (cellnumber) was measured using a Cellometer Auto T4counter (Nexcelom Bioscience, Lawrence, MA, USA).Apoptosis was determined by measuring luminescenceafter addition of 100 lL of Caspase-Glo 3/7 accordingto the manufacturer’s protocol. Results were expressedas means � SEs.

Statistical analysis

Differences among means were calculated using Tu-key’s honestly significant difference (HSD) test usingSPSS Software, v 18.0 (SPSS Inc., Chicago, IL, USA).

For all comparisons, significant differences at P ≤ 0.05were indicated. ANOVA was performed using SPSS. Pear-son correlation coefficients were determined using theSAS PROC CORR procedure, v.9.2 (SAS Institute Inc.,Cary, NC, USA).

Results and discussion

Effect of addition of roasted malts, filtration and dryhopping on XN and IX content

Ten different beers (Table 1) were analysed for xan-thohumol (XN), isoxanthohumol (IX), total phenoliccontent (TP) and antioxidant activity (AA). Of the tenbeers tested, eight contained detectible amounts of XN(Fig. 1a). Most IPAs tested in this study showedhigher levels of XN compared with the earlier reportsof 0.16 mg L�1 XN (Stevens et al., 1999). Higher lev-els of XN observed in this study may be due the useof larger amounts of dry hops in the IPAs. Beers withadditional dark malts, BDP and Brand B DarkFiltered (BDF) had significantly greater levels of XN,12.69 and 12.62 mg L�1, respectively. XN content inthe original IPA recipes BP (11.18 mg L�1) and BF(9.94 mg L�1) was lower than the dark IPAs. Sub-stances (300 000–600 000 Da) in roasted malts mightform complexes with XN to protect it from isomerisa-tion to IX during wort boiling resulting in increasedXN content in beers with additional roasted malts(Wunderlich et al., 2005, 2009; Wunderlich & Back,2007). The amount of XN in prefiltered and filteredbeers (Fig. 1a) was dependent on the type of IPA. Fil-tration resulted in significant decrease in XN content.DE filtration removed 34.3% of the XN content in theBrand A beer and 11.1% in the Brand B IPA. Theaddition of dark malts reduced the removal of XN

(a) (b)

Figure 1 (a) Xanthohumol (XN) content and (b) isoxanthohumol (IX) of beer samples using UPLC/MS/MS. Abbreviations of beer samples

are explained in Table 1. EGP is the control Brand E German pilsner. Data expressed as means � SE. Different letters on the bars represent

significant (P ≤ 0.05) differences in XN or IX content among the beer samples.

2467



upon filtration, and only a 0.55% reduction in XNcontent was observed in the Brand B Dark IPA sam-ple. The data support the claim that the use of darkmalts decreases the removal of XN during filtration, aswas observed in PVPP (polyvinylpyrrolidone) filtration(Wunderlich & Back, 2007).

In addition to the use of dark malts, both the BrandB Dark IPA and the Brand B IPA were dry-hopped.This ratio of dry hops to beer was among the highest of

all beers tested in this study. Low levels of XN werefound in the other four beer samples: AP (0.67 mg L�1),AF (0.44 mg L�1), CNH (0.15 mg L�1) and CDH(0.26 mg L�1). The low amount of XN in AP and AF,0.67 and 0.44 mg L�1, respectively, may be due to theuse of a smaller amount of dry hops (Biendl et al.,2004).Brand C Dry Hopped (CDH) was prepared with

1 oz. of dry hops, while the CNH contained the same

(a) (b)

Figure 2 (a) Total phenolic (TP) content as analysed by the Folin-Ciocalteu reagent assay and (b) antioxidant capacity (AA) as analysed by

the ABTS Trolox equivalent antioxidant capacity assay. Abbreviations of beer samples are explained in Table 1. EGP is the control Brand E

German pilsner. Data expressed as the mean � SE. Different letters on the bars represent significant (P ≤ 0.05) differences in TP content and

AA among the beer samples.

(a) (b)

Figure 3 Effect of beer bioactives on (a) HCT 116 p53+/+ cancer cell proliferation and (b) apoptosis. Solvent control (ethanol); XN at highest

beer concentration (12.69 ng mL�1); IX at highest beer concentration (2 ng mL�1); XN and IX together at concentrations 12.69 ng mL�1 and

1 ng mL�1, respectively; XN at 3.5 lg mL�1; and beer samples (abbreviations of beer samples are explained in Table 1). EGP is the control

Brand E German pilsner) at gallic acid equivalent (GAE) concentrations of 5, 10 and 15 lg lL�1. Data expressed as the mean of three replica-

tions �SE. Different letters on the bars represent significant (P ≤ 0.05) differences in cell proliferation and apoptosis among the beer samples.



amount of hops, but all hops were added before thewort was cooled. This change in hop addition did notresult in a significant difference in XN content ofCDH (0.26 mg L�1) and CNH (0.15 mg L�1). It issurprising that both Brand C beers were low in XNalthough these were prepared with debittered blackmalt. These beers were both treated with Biofine Clear,a product which removes yeast and other haze formingparticles based on colloidal silicon dioxide. BiofineClear treatment might be associated with removal ofXN also. Other explanations for varying amounts ofXN in dry-hopped beers analysed in this study includedifferences in the temperature and time, and durationof hop addition during fermentation.

The IX content was the lowest in AF (0.30 mg L�1)and the highest in CDH (1.82 mg L�1) (Fig. 1b). Theuse of roasted malts resulted in an increased amountof IX. BP contained 0.92 mg IX L�1 and BDP con-tained 1.08 mg IX L�1. Filtration did not reduce theamount of IX in dark IPAs. In BDP, the IX contentincreased from 1.08 to 1.27 mg L�1 with filtration.However, a slight decrease was seen in the other beersafter filtration (AP: 0.49; AF: 0.30; BP: 0.92; BF:0.86). Differences in IX content before and after filtra-tion of beers were not significant (P ≤ 0.05).

The amount of IX was expected to be higher in cer-tain beers because of the small amount of XNdetected. For example, Brand D Dry Hopped (DDH)contained low levels of XN and IX even though it wasdry-hopped. Low amounts of these compounds in thisbeer might be due to poor extraction from the pelletedhops or lower amount of XN in the pelleted hopsthemselves, although XN is not typically lost in thepelletising process (Biendl et al., 2004b). For otherbeers, such as CNH and CDH, the low amount of XNcould be the result of the isomerisation of XN to IXas amounts were higher than in all other beers. Theamount of IX was lower in CNH than in CDH. Thereverse was expected as all hops in CNH were exposedto heat with the exception of CDH where 1 oz. ofhops was not exposed to heat.

Variation in total phenolic content and antioxidantactivity

The TP content and antioxidant activity of all IPAswere greater (P ≤ 0.05) than that of EGP, theGerman-style Pilsner (190.5 mg L�1 GAE and837 mg L�1 TEAC, respectively Fig. 2). This is likelydue to IPAs containing more hops and a largervariation in malts than Pilsner style beer. The highestTP content and antioxidant activity in the IPAswere observed in BDP (504.4 mg L�1 GAE and1327 mg L�1 TEAC, respectively) and could be attrib-uted to high content of dark malts, dry hopping andnonfiltration. BDF had a slight reduction in TP con-

tent (482.9 mg L�1 GAE) indicating DE filtration didresult in a reduction of total phenolics in beer. Uponfiltration, the Brand B IPA contained lower total phe-nolic content (392.5 mg GAE L�1) compared withnonfiltered Brand B IPA (480.7 mg GAE L�1). Theseresults indicate that the addition of roasted maltscould also minimise the removal of other phenoliccompounds besides XN from the beer matrix uponDE filtration. CNH contained less XN and IX com-pared with CDH, but this did not correlate with totalphenolic content, 360.81 and 413.22 mg GAE L�1,respectively. Even though some of the IPAs did notcontain increased levels of XN as was expected, thesebeers still showed significant TP content. This may bedue to the presence of other hop-derived compounds.For example, AP and AF were made with both CO2

hop extracts and pelleted hops. While the CO2 extractrecovers <5% XN in the original hop cone (Biendlet al. 2004b), other hop-derived compounds such asalpha and beta acids are captured and preserved inthis extract (Hopsteiner 2014). Given that hop-derivedcompounds besides XN are present in the supercriticalCO2 hop extracts, it is not surprising the Brand Abeers had similar total phenolic content as that ofCNH, CDH and DDH.Antioxidant capacity of AP and AF was 1324.0 and

1115.5 mg L�1 TEAC, respectively. These results indi-cate that compounds other than phenolic compoundscontributed to antioxidant capacity in beer samplessuch as certain hop-derived compounds like alphaacids, trans-and cis-iso-alpha acids, trans-isohumulone,cis-isohumulone and others upon exposure to heat andstorage (Miranda et al., 2000). These compoundsmight not have been accounted for in the total pheno-lics assay, but could play a role in antioxidant capac-ity. Thus, this could contribute to AP and AF havinga higher antioxidant capacity than total phenolic con-tent compared with other beers.

Cell proliferation and apoptosis

Xanthohumol (XN) is well known for its biologicalbenefits and has been found to inhibit colon cancer

Table 2 Correlation coefficients between antioxidant activity

(ABTS), total phenolics (TP), xanthohumol (XN), isoxanthohumol

(IX), colon cancer cell proliferation (P) and apoptosis (A)

TP XN IX P A

ABTS 0.852* 0.857* 0.008 �0.796* 0.876*

TP 0.878* 0.276 �0.901* 0.953*

XN 0.172 �0.785* 0.970*

IX �0.207 0.033

P �0.863*

A

*Significance at P ≤ 0.05.

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cell proliferation at 10 mM concentrations in HCT-116cell lines (3.5 lg mL�1) (Hadjiolov & Frank, 2009).However, no studies have focused on the synergisticeffect of beer compounds in relation to colon cancercell proliferation and cell death. In this study, fourbeer samples were tested at three different concentra-tions on colon cancer lines HCT 116 p53+/+ as well aspure XN and/or IX. Brand B and Brand C wereselected because of higher XN or IX content andBrand E served as control. IPAs differed in their sup-pression of cancer cell proliferation (Fig. 3a) and ele-vation of apoptosis (Fig. 3b). IPAs with greateramounts of XN contained greater antiproliferative andpro-apoptotic properties, and the effect was dosedependent. BDP and BP were the most potent insuppressing cell proliferation and elevating apoptosis.The highest amounts of XN in these beer matriceswere only 12.7 and 11.2 ng mL�1, respectively, andboth showed reduced cell proliferation and elevatedapoptosis compared with XN and/or IX at the similarconcentrations. However, isolated XN reduced prolif-eration and increased apoptosis at higher concentra-tion. These results indicate that XN in combinationwith other inherent beer compounds was more potentthan isolated XN in suppressing cancer cell prolifera-tion. Earlier results suggest that XN elevated apoptosiscan be attributed to activation of Caspase 3/7 enzyme,inhibition of DNA synthesis and cell cycle arrest in S-phase (Miranda et al., 2000; Hadjiolov & Frank,2009).

Correlation coefficients among different parameters

Significant positive correlations were observed betweenXN vs. TP (r = 0.88, P = 0.0002), XN vs. antioxidantactivity (r = 0.86, P = 0.0004) and XN vs. apoptosis(r = 0.970, P = 0.0001) (Table 2). Significant negativecorrelations were observed between XN content andcell proliferation (r = �0.79, P = 0.0025). These corre-lations further confirm that increased XN content inbeers increases the antioxidant, pro-apoptotic and an-tiproliferative properties. However, no significant cor-relations were seen between IX and any of theparameters analysed.

Conclusion

Brewing processes such as addition of dark malts, dryhopping and filtration altered xanthohumol content ofthe IPAs. Intensely dry-hopped (0.06 oz. of hops per0.3 L) beers contained an increased amount of XNcompared with the Pilsner beer. Beer made withincreased amounts of dark malts contained greaterXN levels, and prefiltered beer contained more XNthan filtered beer. Addition of dark malts amelioratedthe negative effects of filtration on XN content. The

dry-hopped formulation had greater XN content thanthe method which did not include adding dry hops.Cell culture assays confirmed beer matrices withgreater levels of XN suppressed cell proliferation andelevated apoptosis compared with isolated componentsalone or together. The results substantiated both ourhypotheses concerning the characteristics of IPAs andbeer matrices. It is possible that elevated amounts ofXN in combination with other inherent beer com-pounds could provide beneficial effects to the humanbody, such as antioxidant, antiproliferative and anti-carcinogenic activities.

Acknowledgment

This work was supported by College of Health andHuman Sciences challenge grant, CSU.

Conflict of interest

Authors of this manuscript declare that they have noconflict of interest.

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american india pale ale matrix rich in xanthohumol is potent in suppressing proliferation and...

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