Emerging Medicinal Uses of Hops

SESSION ON: NEW TECHNOLOGIES – NON BREWING USES OF HOPSEmerging MedicinalUses of HopsDr. Arne HeyerickCenter for Evidence‐based Development of Natural TherapeuticsLaboratory of Pharmacognosy and PhytochemistryFaculty of Pharmaceutical SciencesGhent University –UGentHarelbekestraat 72, 9000 Gent, BelgiumE‐mail: Arne.Heyerick@UGent.beAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

GHENT ‐ BELGIUMEstablished in 181730.000 studentsAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

BELGIUM – BEER CULTUREAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP (Humulus(lupulus L.) Cannabaceae contains two genera:Humulus and Cannabis Two main species: Humulus lupulus L.and Humulus japonicus Sieb. & Zucc. Perennial fast-growing vine (6-7 m) Dioecious (male vs female plants) Between 35° and 55° latitutde (US,Europe, Asia (China), Australia, NewZealand, South-Africa, Argentina, Chile) Economic relevance: female hop flowersAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP: TRADITIONAL MEDICINE Sedative, antistress, and sleep-inducing properties(1% of hops used for these purposes usually in combination withother plants including valerian and melissa) Estrogenic activity (induction of menstruation/gynecomastia) Bacteriostatic and antiinflammatory activities Stimulation of digestive tract (hops » ‘amara’ or‘bitters’) Diureticum Against bladder complaints – kidney stones ‘Blutreinigend’ (liver – spleen) AnafrodisiacumAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP: TRADITIONAL & CURRENT USAGENatural preservativeBREWERY:Bitter tasteHoppy flavorStable foam headMEDICINAL: Sedative Estrogenic AntiinflammatoryOTHER:DelicaciesCosmeticsOrnamentalAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP (Humulus(lupulus L.)FemaleHop ConeGlandularStructuresHop Glandular TrichomesAverage composition of dried hop conesLupulinHop(Humulus lupulus L.)American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP SECONDARY METABOLITESComplex mixture of >100 different phenolicsbelonging to different classesAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP ACIDS – SEDATIVEFig. 1∆ T [°C]0.40.2-0.0-0.2-0.4-0.6-0.8-1.00 60 120 180Time [min]controlMelatonin 25 mg/kgMelatonin 50 mg/kgFig. 2Effect of oral intake of CO 2-extractor α-acids on pentobarbital inducedsleeping time.β-acids have melatonin-like activity: reduction in body core temperatureincreases sleep propensity.∆ T [°C]0.40.2-0.0-0.2-0.4-0.6-0.8-1.00 60 120 180Time [min]controlHE 60 mg/kgHE 125 mg/kgHE 250 mg/kgHE 500 mg/kgResults by Prof. Zanoli and Prof. BrattströmAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP ACIDS – ANTIMICROBIALAntimicrobial activity is well-established β-acids fraction most activeGrowth kinetics of Listeria monocytogenesin the presence of hop beta acidsIMPROVEDWITH HOPSShen and Sofos (2008) J Food Sci, 73, M438-M442.American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP ACIDS – ANTI‐INFLAMMATORYINFLAMMATORYTETRA&RHOResults from Metaproteomics/Metagenics, Gig Harbor, WA, USAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP ACIDS – ANTI‐CANCER?β-acids induce significant reduction in number ofpreneoplastic lesions and size and number oftumors in colorectal cancer modelLamy et al. (2007) Carcinogenesis, 28, 1575-1581.American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP ACIDS ‐ METABOLIC SYNDROME?Iso-α-acids are modulating blood sugar and blood lipidsResults from Kirin Brewery Research LaboratoriesAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP POLYPHENOLSLeaf-associated Proanthocyanidins Flavanols Hydroxybenzoic andhydroxycinnamic acids Flavonol glycosides Flavonols Multifidols StilbenesLupulin-associated Prenylflavonoids Prenylchalcones:desmethylxanthohumol(DMX), xanthohumol (X) Prenylflavanones:isoxanthohumol (IX),8‐prenylnaringenin (8‐PN),6‐prenylnaringenin (6‐PN)American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

USE OF HOP POLYPHENOLS Anti-allergic preparations (developed by Sapporo) Cold water extract of hop tissue Fractionation using XAD resins fraction with flavonolglycosides (kaempferol + quercetin derivatives) Inhibition of histamine release in human basophilic KU812cells (Segawa et al., 2006, Biosci Biotechnol Biochem, 70, 2990.)American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

USE OF HOP POLYPHENOLS Anti-allergic preparations (developed by Sapporo) Significant inhibition of nasal rubbing and sneezing insensitized mice (Takubo et al., 2006, Biol Pharm Bull, 29, 689) Double-blind, placebo-controlled trial with HWE at 100 mgper day (Segawa et al, 2007, Biosci. Biotechnol. Biochem., 71, 1955) significant reduction in summed nasal symptoms scoreAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

USE OF HOP POLYPHENOLS Oral hygiene preparations (developed by Asahi) Hydroalcohilic extraction + removal of divalent cationsusing bentonite + column fractionation Hopsphenon ® = Hop Bract Polyphenols (HBP) enriched in proanthocyanidins + some flavonolglycosides Active materials: high-molecular proanthocyanidins(> 6 kDa polymer of > 20 monomers) HBP inhibits the cellular adherence of Streptococcusmutans + glucosyltransferase activity which isinvolved in water-insoluble glucan synthesis (Tagashira etal. 1997, Biosci Biotech Biochem, 61, 332) Placebo-controlled clinical trial with mouth rinsecontaining 0.1% HBP significantly less plaqueregrowth after 3 days + significantly less Streptococci(Shinada et al., J Dent Res, 86, 848)American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

USE OF HOP POLYPHENOLS Anti-oxidant preparations High in xanthohumol (5%-95%) Prepared by ethanolic extraction combined with CO 2extraction or high pressure CO 2 extraction and furthercolumn fractionations Applications:Dietary Supplements Drinks CosmeticsAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

HOP POLYPHENOLS FOR MENOPAUSE Phytoestrogens from hop Extraction procedure: CO 2 extraction + recuperation of spent hops water extraction to remove polar residuals hydroalcoholic extraction including alkalinization andneutralization spraydrying Standardized extract:IXXTotal Prenylflavonoids: > 5%• Isoxanthohumol (IX): > 1%• 8-Prenylnaringenin (8PN): > 0.12%• 6-Prenylnaringenin (6PN): > 0.1%• Xanthohumol (X): > 3%• 8PN > 6-PN8PN6PNAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 1As assessed by the gynaecologistKupperman IndexHot flash scoreSignificant changes after six weeks for the normal dosage group Indications of activity, mostly targeting vasomotor symptomsAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 1As assessed by the participantDistribution of responders to treatments(strong resonder: > 25% reduction, non-responder: < 5% reduction)Quality-of-life(in % improvement)Highest improvements in QoL In MenoHop groupsAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 2Evaluating the effect of oral intake ofMenoHop on menopausal discomfortsassessed by Kupperman Index,Menopause rating scale and VASRandomized,placebo-controlled,double-blind,cross-overAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 2Overal estimates of treatment effect(active treatment – placebo)Significant reductions in placebo and active treatment, butno significant effect of active treatment over placebo??? Detailed statistical analysis of the results American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 2P = 0.244P = 0.143P = 0.030ActivePlaceboP = 0.017 P = 0.066P = 0.021 P = 0.277P = 0.007 P = 0.306Baseline After 8 weeks After 16 weeksBaseline After 8 weeks After 16 weeksBaseline After 8 weeks After 16 weeksPlacebo ActiveP = 0.008P = 0.008 P = 0.408P = 0.019P = 0.002 P = 0.786P = 0.008P = 0.038 P = 0.448Baseline After 8 weeks After 16 weeksBaseline After 8 weeks After 16 weeksBaseline After 8 weeks After 16 weeksAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 2Time-specific estimates of treatment efficacy(active treatment – placebo) for each outcome measureSignificant effects on KI and VAS, and marginally significant for MRS…VAS: vasomotor + sleep both contributing for 50%American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 3Evaluating the safetyof the oral intake of astandardized hopextract on theendometriumRecruitmentAt time = 0Baseline measurements:•Transvaginal ultrasound•Pap smearRandomizedInterventiontrialIncludedAfter 8-10 weeksnoEndometrial thickness > 4 mmMeasurements:•Transvaginal ultrasound•Pap smear+ Subjects provide a urine sampleyesExcludedAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 3Effect of MenoHop (2/day) on Endometrial ThicknessNo significant differences(P=0.833)Correlation with starting value trend for reductionwhen high at start vs increase when low at startAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 3Effect of MenoHop (2/day) on Estrogen Maturation Index(EMI)Small, but significant increase in EMI (P=0.002)Indication of estrogenic effect on vaginal lining positive for QoL?Exposure does not stimulate endometrial lining but does enhance renewal of vaginal wall.American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

FURTHER RESEARCH & DEVELOPMENTMicrobial activation of IX to 8PN in the gastrointestinalsystemOHOHHOOHOOOMeOOHOHigh interindividual variability(Possemiers et al., 2006, J Nutr, 136, 1862)American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 4Evaluationof theexposure to8-PNusing aninterventionstudyRFecal samplein vitro productionIX 8-PN1 2 3 4 5 6 7 8 9WASHOUTProducersInclusionTREATMENT3/dayNon-producersExclusionIX: 1.34 ± 0.04 mg/dose8-PN: 90.5 ± 1.7 µg/dose6-PN: 56.5 ± 3.1 µg/doseX: 2.07 ± 0.08 mg/dosebreath methaneserum & urineELISALC-MSBMI, age, antibiotic use,...American Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 4SCREENING Fecal 8-PN production (8-PN/(IX+8-PN))(MenoHop vs Phytoestrogen mix: soy, hop, flax)100In vitroproduction of 8-PN is influencedby addition ofotherphytoestrogens8-prenylnaringenin (%)908070605040302010r= 0.358;P= 0.00101 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71HOPMIXAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 4Analysis of relative 8-PN concentrations in urine(8-PN/(IX+8-PN))100Relative levelsof 8-PN in urineapparently notaffected byintake of otherphytoestrogens8-prenylnaringenin (%)908070605040302010r= 0.277; P< 0.001The relative levels of 8-PN inurine appear to be low incomparison with findings fromfecal incubations!But original ratio is 6%!Thus, ratio in urine issignificantly affected.Furthermore, this may reflectthe different pharmacokineticbehaviour of IX vs 8-PN.01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20HOPMIXAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

MENOHOP: CLINICAL TRIAL 4Relative activities of phytoestrogens vs E 2Circulating total 17β-estradiol (E 2 )levels• ♂ ~0.02 - 0.10 nM• ♀ pre-menopausal ~0.15 - 1 nM• ♀ post-menopausal ~0.015-0.05 nMAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

FURTHER RESEARCH & DEVELOPMENTSpecies capable of transformation has been isolatedand identified (E. limosum) (Possemiers et al., 2005, J AgricFood Chem, 53, 6281)Development of fermented extractsDevelopment of probioticFeasibility of probiotic has been shown in in-vivoexperiments with axenic rats (germ-free)(Possemiers et al., 2008, J Nutr, 138, 1310)Hop + = microbiota of converterHop - = microbiota of weak converterExpt.1 = in absence of E. limosumExpt. 2 = addition of E. limosum via oral gavageAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009

Contact details: Dr. Arne HeyerickIOF-Technology DeveloperCenter for Evidence-based Development of Natural TherapeuticsGhent University-UGentHarelbekestraat 72, B-9000 GhentTel: +3292648058Mobile: +32473562165Fax: +3292648192E-mail: Arne.Heyerick@UGent.beAmerican Hop Convention 2009 and Hop Research Council Winter Meeting, 29 January 2009