FITOTE-03139; No of Pages 12 Fitoterapia xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Fitoterapia

Isoxanthohumol — Biologically active hop flavonoid

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journal homepage: www.elsevier.com/locate/fitote

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Anna Katarzyna Żołnierczyk, Wanda Krystyna Mączka, Małgorzata Grabarczyk, Katarzyna Wińska, Edyta Woźniak, Mirosław Anioł ⁎

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Department of Chemistry, The Faculty of Food Science, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, C. K. Norwida 25, Poland

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Article history: Received 14 January 2015 Accepted in revised form 2 March 2015 Accepted 6 March 2015 Available online xxxx

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Keywords: Angiogenesis Antiproliferative activity Antitumor activity Isoxanthohumol Prenylflavonoids

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Isoxanthohumol (IXN), apart from xanthohumol (XN) and 8-prenylnaringenin (8PN), is one of the most important prenylflavonoids found in hops. Another natural source of this compound is a shrub Sophora flavescens, used in traditional Chinese medicine. Main dietary source of IXN is beer, and the compound is produced from XN during wort boiling. In the human body, the compound is O-demethylated to 8PN, the strongest known phytoestrogen. This process takes place in the liver and in the intestine, where it is mediated by local microflora. It has been reported in some studies that even though beer contains small amounts of hops and its preparations, these compounds may affect the functioning of the human body. IXN exhibits an antiproliferative activity against human cell lines typical for breast cancer (MCF-7), ovarian cancer (A-2780), prostate cancer (DU145 and PC-3), and colon cancer (HT-29 and SW620) cells. It strongly inhibits the activation of the following carcinogens: 2-amino-3-methylimidazol-[4,5-f]quinoline and aflatoxin B1 (AFB1) via human cytochrome P450 (CYP1A2). It also inhibits the production of prostate specific antigen (PSA). IXN significantly reduces the expression of transforming growth factor-β (TGF-β) in the case of invasive breast cancer MDA-MB-231. It interferes with JAK/STAT signaling pathway and inhibits the expression of proinflammatory genes in the monoblastic leukemia cell line (MonoMac6). It activates apoptosis in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMCs). In addition, IXN shows an antiviral activity towards herpes viruses (HSV1 and HSV2) and bovine viral diarrhea virus (BVDV). © 2015 Published by Elsevier B.V.

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1. Introduction

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There has been a visible trend in industry to look for plant metabolites exhibiting interesting biological properties [1]. Some of the most exciting ones are phenolic compounds, including e.g. flavonoids, chalcones, xanthones, coumarins, tannins and lignins. These compounds are classified according to the number of phenolic rings and they all have at least one hydroxyl group directly bonded to the aromatic ring [2]. Flavonoids are characterized by particularly broad spectrum of interesting properties. They may be present in all plant organs, often contributing to their distinctive color. They are also consumed with food. These compounds are not

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⁎ Corresponding author. Tel.: +48 71 3205468; fax: +48 71 3284124. E-mail address: [email protected] (M.ł Anioł).

necessary for the proper functioning of the human body, but they improve resistance to various diseases, infections, or ailments and help to maintain our body in a good shape. Flavonoids represent about two-thirds of all the phenolic compounds present in our diet [3]. In plants, they are mainly found as glycosides or esters [2,4]. There are 6 basic subclasses of these compounds: flavonols, flavanones, isoflavones, flavan3-ols, flavons and anthocyanins, with basic structure modified by different substituents [5]. The group of phenolics also includes chalcones, serving as precursors of flavonoids. Considering their beneficial effects on human health, special attention should be given to prenylated flavonoids that are particularly interesting as active substances for the production of drugs and functional foods. So far, about 1000 of prenylated flavonoids have been identified [1]. These compounds can be found in only a few plant families, e.g., Leguminosae (Fabaceae),

http://dx.doi.org/10.1016/j.fitote.2015.03.007 0367-326X/© 2015 Published by Elsevier B.V.

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

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2. Structure, physical properties and methods of analysis

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(the maximum at 282.6 nm) is applied in HPLC analysis. 1H NMR, 13C NMR, HMQC and IR spectra are presented and discussed in [22]. Chemistry of isoxanthohumol derivatives and preparation of acyl- and alkoxy-8-prenylnaringenins from corresponding isoxanthohumols via demethylation process is described in [23]. Over the years many methods of IXN and other hop flavonoid analysis have been developed. The samples included many beers, hops, hop products as extracts, granules and pellets, biofluids, tissues and extracts obtained after biotransformation. The analyzed beers were degassed in an ultrasonic bath, the liquid samples were dissolved mainly in MeOH, and the solid samples were homogenized and next diluted in the solvent. To concentrate and purify the samples solid phase extraction (SPE) was used frequently using mainly C-18 sorbent. Some researchers for the release of conjugated hop flavonoids applied an enzymatic hydrolysis by Helix pomatia glucuronidase. HPLC analysis was usually performed using a C-18 column and a three component eluent system: organic solvent (MeCN or MeOH)–H2O–acid (HCOOH or less frequently CF3COOH and H3PO4). The detection methods were based on DA, MS and the most advanced and convenient MS/MS detectors. The experimental data are summarized in Table 1.

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Moraceae, Cannabaceae, Guttiferae (Clusiaceae), Umbelliferae, Rutaceae and individual species of Euphorbiaceae and Asteraceae. Plants probably use them in defense against pathogens and other pests and in prevention of environmentally induced abiotic stress [6]. The important type and currently involved in a lot of attention are prenylated flavonoids [7–10]. They exert powerful biological effects and many of them have been identified in the plants used in alternative medicine, e.g. licorice used in traditional Chinese medicine as an anti-inflammatory agent [11,12]. One of the prenylated flavonoids is kurarinone, isolated from Sophora flavescens (Leguminosae) [13], and able to inhibit the activity of tyrosinase and glycosidase [12–14]. Another prenylated flavonoid, involving considerable interest in the recent years, is isoxanthohumol (IXN). It is found, together with xanthohumol (XN) and 8-prenylnaringenin (8PN), in hops and hop waste left after an extraction with supercritical carbon dioxide. The extract obtained in this process is used for hopping in the brewing industry [15]. Mentioned above compounds are also present in the beer and in recent years have received much attention for their biological effects, especially cancer chemopreventive and estrogen activity. 8-Prenylnaringenin is the most potent phytoestrogen isolated until now. These compounds and other constituents of beer have been investigated and described many times [7,16–18]. Much attention has been paid to XN and 8PN but in the literature we have not found the comprehensive review on IXN.

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Isoxanthohumol (5-O-methyl-8-prenylnaringenin) belongs to prenylated flavanones. A core part of the molecule is a 97 heterocyclic flavone. Its two hydroxyl groups are located at C-7 98 and C-4′, and the prenyl group is at C-8 (Fig. 1). 99 In its pure form isoxanthohumol is a crystalline pale yellow 100 Q10 solid with the melting points of 196–208 °C [19] and 178– 101 180 °C (MeOH) [20]. It is well soluble in tetrahydrofuran, 102 acetone, ethyl acetate, dimethyl sulfoxide, methanol, and 103 ethanol. It is poorly soluble in water (5.0 mg/L, 23 °C), but 104 still to a greater extent than xanthohumol (1.3 mg/L, 23 °C) 105 from which it is obtained [21]. The UV–VIS spectra of 106 methanolic solutions of IXN have two major maximum 107 absorbance bands at 225.0 nm (ε = 26,407 L/(mol·cm)) and 108 282.6 nm (ε = 25,495 L/(mol·cm)). Usually the second band

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3. Occurrence

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The main natural source of isoxanthohumol is the roots of S. flavescens, a shrub growing in the northern part of China and Mongolia. Local communities call this shrub “Shen Ku”, which means “bitter root”. This name is probably due to the presence of many flavonoids and alkaloids in the shrub roots. S. flavescens is used in traditional Chinese folk medicine for the treatment of various conditions, such as fever, inflammation, ulcers and skin burns [41]. Another source of isoxanthohumol is a hop (Humulus lupulus L.), and more precisely its female flowers. They contain very small amounts of this compound. The percentage content of xanthohumol and isoxanthohumol in the dry weight of the flowers is 0.48% and 0.008%, respectively [42]. Isoxanthohumol content in hop products (granulate, ethanol extract, and the extract resulting from an extraction with supercritical carbon dioxide) is also negligible and below the detection limit [32]. It is worth noticing that isoxanthohumol, xanthohumol and 8-prenylnaringenin are present only in female flowers of hop. Hop leaves and bines are devoided by the presence of these compounds [43,44]. Isoxanthohumol is much more abundant in beer, as during wort boiling thermal isomerization of xanthohumol to isoxanthohumol occurs [32]. A research involving twelve beer types from different countries showed that various types of beer contain from 0.04 to 3.44 mg/L of IXN, 0.001 to 0.11 mg/L of 8PN and 0.002 to 0.69 mg/L of XN. Isoxanthohumol was also identified in a non-alcoholic beer in an amount of 0.11 mg/L (Table 2) [45].

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4. Preparation methods

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Xanthohumol, precursor of isoxanthohumol, can be prepared in a process of a six-step chemical synthesis starting from chloroacetophenone [46]. However, this method is hardly effective, with a yield about 10%. For this reason, it is better to

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Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

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Phytoestrogens are a group of naturally occurring compounds with a structure similar to 17β-estradiol. They have an estrogenic or antiestrogenic activity. They belong to one of four groups of compounds, i.e. flavonoids, coumestans, lignans and stilbene derivatives [62]. Prenylflavonoids, the category of compounds comprising isoxanthohumol, are third of the largest group of phytoestrogens, after isoflavones and lignans [63]. To determine the effects of hop flavonoids on the human endocrine system, the effect of 8-prenylnaringenin and isoxanthohumol on the process of human chorionic gonadotropin (hCG)-activated steroidogenesis was examined in a culture of primary rat Leydig cells at different stages of their development. Leydig cells synthesize androgens from cholesterol. Luteinizing hormone (LH) and chorionic gonadotropin (hCG) stimulate steroidogenesis in the Leydig cells during testosterone formation from pregnenolone [64]. The study demonstrated that both isoxanthohumol and 8prenylnaringenin modify the comprehensive developmentdepending effects during steroidogenesis and inhibit hCGinduced androgen production in the Leydig cells [62]. Although 8-prenylnaringenin is one of the most potent dietary phytoestrogens [16,65,66], isoxanthohumol and xanthohumol showed only a weak estrogenic activity.

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5. Biological activity

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extracted with methanol and then subjected to column chromatography with methanol and acetic acid. Recrystallization from acetic acid yielded 0.63 g of xanthohumol and 0.34 g of isoxanthohumol from 8 g of the resin derived from 700 g of hop waste [56] (Table 3). Isoxanthohumol can also be isolated from dried and pulverized roots of S. flavescens by extraction with methanol at room temperature. Following solvent evaporation, the extract is subjected to further extraction with chloroform and purified on a chromatographic column using a mixture of hexane and ethyl acetate as an eluent. The process yields, depending on a report, 0.21 g [14], 0.088 g [57], 0.011 g of [58] or 0.02 g [59] of isoxanthohumol per 1 kg of dried roots. In the search for an alternative method of more efficient isolation of isoxanthohumol, some researchers extracted 3 kg of dried roots of S. flavescens with hot ethanol or methanol (heated using a reflux column). Main differences concerned further processing of the resulting extract. In the first approach, the extract was dissolved in 2% citric acid and extracted with hexane, and then with ethyl acetate. Separation and purification of individual fractions yielded 0.12 g of isoxanthohumol [41]. In the second approach, the extract was dissolved in water and extracted with e.g. methylene chloride. Purification of this fraction yielded 0.05 g of isoxanthohumol [13]. In another method, instead of whole roots, 3 kg of S. flavescens bark was used. It was extracted with hot methanol, and then the extract was dissolved in water and fractionated using methylene chloride and ethyl acetate. The fraction extracted with ethyl acetate was purified yielding 0.01 g of isoxanthohumol [60] (Table 4). Efforts have also been made to obtain isoxanthohumol by means of tsugafolin prenylation using prenyltransferase gene from S. flavescens. Efficiency of this process was also unsatisfactory [61].

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isolate xanthohumol from natural sources, such as hops or granules, where it may constitute up to 1% of dry weight (dw) [42], and then isomerize it to isoxanthohumol. Isoxanthohumol (Fig. 2) can be readily prepared by acid or base-catalyzed cyclization of xanthohumol. The highest yield, up to 100%, can be obtained by means of xanthohumol isomerization with 1% NaOH at 0 °C, and next in a solution acidified with 50% H2SO4 [47,48]. The compound can be also obtained in reactions involving acid catalysts or chiral basic catalysts, but with lower productivity [48] (Fig. 2). The most common methods of xanthohumol isolation involve its extraction from hops using hot ethanol [49,50], extraction with diethyl ether [51] or methanol [52] from hop granules, or high-pressure extraction with supercritical CO2 [32,53]. The last method is especially interesting, from both industrial and environmental perspectives (it does not involve environmentally damaging solvents), as it yields high purity product, without various admixtures resulting from ethanol extraction (plant pigments, waxes, water-soluble compounds). As hop flavonoids are usually extracted using methanol or ethanol, attempts were made at extraction of xanthohumol and isoxanthohumol with different solvents, from commercially available products such as granules, ethanol extract and extract obtained with supercritical CO2. Out of five tested solvents involving methanol, acetonitrile, ethyl acetate, hexane and water, the most effective was methanol, providing a recovery rate of 90–92% for xanthohumol and 93–94% for isoxanthohumol. The content of both flavonoids in commercial products containing hops was assessed by means of HPLCDAD. The largest amounts of these compounds were found in ethanol extract, i.e. 3.75 g/100 g of xanthohumol and 0.17 g/100 g of isoxanthohumol. Very small amounts of xanthohumol were extracted from granules (0.62 g/100 g) and CO2 extract (0.089 g/100 g), and isoxanthohumol content was below the detection limit in both products. This is due to the fact that the flavonoids are readily soluble in polar solvents such as ethanol, and very poorly soluble in non-polar CO2 at low pressure [32]. While looking for a still better method, other researchers employed a pressurized liquid extraction to obtain xanthohumol and isoxanthohumol from hop pellets. The best results were achieved when the solvent was water heated to 150 °C under a pressure of 1500 psi (10.68 MPa). The resulting extract contained 2.34 mg of isoxanthohumol and 0.11 mg of xanthohumol in 1 g of dry weight. When using ethanol, a traditional solvent used for the extraction of hop flavonoids, the yield was 0.76 mg and 1.89 mg of xanthohumol and isoxanthohumol, respectively, in 1 g of dry matter. Such huge differences in isoxanthohumol and xanthohumol ratio (21 and 0.35) may be explained by xanthohumol isomerization to isoxanthohumol under high temperature and high pressure [54], which is also observed during beer brewing, as mentioned before. One of the most recent methods of isolating xanthohumol from ethanol hop extract is the use of high speed countercurrent chromatography (HSCCC). It employs a biphasic solvent system composed of hexane, ethyl acetate, methanol and water 5:5:4:3 (v/v) that allows for obtaining xanthohumol of purity greater than 95% in a single-step purification process [55]. Hops and its products serve not only as a source of xanthohumol, but also of its isomer. Hard hop resins were

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Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

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t1:1 Table 1 t1:2 Q1 Procedures for quantitative analysis of isoxanthohumol.

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Sample

The highest level of IXN Other analytes in the matrix

The highest level of 8PN; XN

Solvent (V/V)

Method of isolation details

Analytical Ref. separation–detection

t1:4

Czech beers and others

2.50 mg/L

XN, DMX, BA

–; 0.50 mg/L

MeOH:H3PO4 (100:0.2)

HPLC-DAD

[24]

t1:5

Czech beers

1.28 (MS) mg/L

8PN, XN, 6PN, DMX, DPN, PXN, BA

0.02; 0.09 mg/L

SPE on Strata C-18 E adsorbent. Interfering substance elution: MeOH:H2O:H3PO4 (50:50:0.2). a Beer was degassed and filtered, hop extracts were dissolved in solvent.

HPLC–MS

[25]

t1:6

Hop extracts

t1:7

HPLC-DAD HPLC–MS HPLC-DAD HPLC-DAD

[26]

Beer was directly used for analysis. Beer was degassed and MeCN was added. MEPS using automated analytical syringe (500 μL) containing 4 mg of the C2, C8, C18, SIL or M1 sorbent. Interfering substances elution: H2O-HCOOH (99.9:0.1).a SPE on Strata C-18 E adsorbent. Interfering substance elution: MeOH:H2O:H3PO4 (20:80:0.2). a Samples were extracted with solvent.

LC–MS/MS UPLC–MS UPLC-DAD

[27] [28] [29]

HPLC-DAD

[30]

HPLC-DAD–MS

[31]

MeOH:HCOOH (99:1)

Samples were extracted with solvent.

HPLC-DAD

[32]

MeOH:H2O (80:20) MeOH

Samples were extracted with solvent.

HPLC-DAD

[33]

Samples were enzymatically hydrolyzed and HPLC-DAD underwent SPE analysis on Bond Elut® C18

[34]

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Hop extracts

1.79 (DAD) mg/L 20.0 mg/L 17.3 mg/L 15.1 mg/g

8PN, XN, 6PN,

ND; ND 0.25; 125 mg/L ND; 107 mg/L 1.9; 38.8 mg/g

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American beers Spanish and other beers Portuguese beers

1.82 mg/L Detected 0.200 mg/L

XN XN, BA XN

–, 12.7 mg/L –; Detected –; 0.091 mg/L

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Beer

0.15 mg/L

8PN, XN, 6PN, others 0.013; 0.061 mg/L

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Beer lee Capsules (229 mg of hop extract) Ethanolic extract

36.2 μg/g, 11.5 mg/g 1.7 mg/g

8PN, XN, 6PN, BA

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CO2 extract Dietary supplements for breast enhancement Human urine

ND 81.1 μg/g

–; 0.9 mg/g XN, 8PN, 6PN, others 10.9; 323.0 μg/g

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XN

7.84; 29.6 μg/g 1.4; 29.4 mg/g –; 37.5 mg/g

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MeOH-H3PO4 (100:0.2) EtOH

Samples were dissolved with MeOH

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Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

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Capsules 1 (200 mg of hop extract) Capsules 2 (200 mg of hop extract) Human urine after drinking 330 mL of beer

t1:23 t1:24 t1:25

Above tested beer Human plasma after a single oral dose of max. 180 mg XN

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Rat plasma after 6 weeks treatment 100 (M) 181 (F) nM using max. dose of 16.9 mg (free and conjugated) XN/kg BW.

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Liver tissue

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Human liver microsomes

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Human serum extract 2 h after consuming a hop extract standardized to 1 mg 8PN.

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Microbial transformation broth

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1.2 mg/g 6.0 mg/g 3.06 μg/g creatinine

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460.7 μg/L 25 μg/L (free and conjugated)

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8PN, XN

0.50; 6.9 mg/g 0.50; 10.2 mg/g ND; ND

MeOH MeOH MeOH

ND; 55.4 μg/L NDW; 120 μg/L (free and conjugated)

MeOH:HCOOH (99.9:0.1)

364; 389 (M) 211; 555 MeOH:HCOOH (F) nM (free and conjugated) (99.9:0.1)

LC–MS/MS

[35]

Samples were enzymatically hydrolyzed and LC–MS/MS extracted using a paper strip extraction with solvent. Samples were enzymatically hydrolyzed LC–MS/MS with Helix pomatia sulfatase/glucuronidase and extracted using Et2O. After Et2O evaporation extracted with solvent. Samples after homogenization were extracted with solvent.

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0.22; 1.1 (M) 0.11; 1.9 (F) nM (free and conjugated)

MeOH:H2O (90:10)

8PN, XN and others

D; D

32.0 μg/L

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7.32; 24.4 μg/L

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MeOH:0.05% Pre-treated samples were extracted with HCOOH (80:55) HPLC solvent. Methyl-t-butyl ether LLE (compared to SPE) Samples were extracted with solvent. After evaporation to dryness reconstituted in 100 μL 70% MeOH. AcOEt Supernatant was extracted with AcOEt concentrated and redissolved in MeOH

0.21 (M) 0.39 (F) nM (free and conjugated) D

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HPLC–MS

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UPLC–MS/MS

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HPLC-DAD

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Abbreviations: 6PN — 6-prenylnaringenin; AcOEt — ethyl acetate; BA — bitter acids; D — detected; DAD — diode-array detector; DMX — desmethylxanthohumol; DPN — 6,8-diprenylnaringenin; EtOH — ethanol; F — female; HPLC — high pressure liquid chromatography; LC — liquid chromatography; LLE — Liquid–liquid extraction; M — male; MeCN — acetonitrile; MeOH — methanol; MEP — semi-automatic microextraction by packed sorbents; ND — not detected; NDW — not detected or very low level was noticed; PXN — 5′-prenylxanthohumol; SPE — solid phase extraction; UPLC — ultra-high pressure liquid chromatography. a Analytes were desorbed with solvent.

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A.K. Żołnierczyk et al. / Fitoterapia xxx (2015) xxx–xxx

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

t1:19 Q2 t1:20 Q3 t1:21 t1:22

adsorbent. Interfering substances elution: MeOH:H2O (2:3).a Samples were dissolved with MeOH. Samples were dissolved with MeOH. SPE on Oasis MCX 96-well plates. Interfering substances elution: MeOH:5 mM ammonium acetate buffer pH = 5 (1:1). a

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Table 2 Quantities of xanthohumol, isoxanthohumol and other flavonoids in different types of beer [45].

297 298 299 300 301 302 303 304 305 306 307 308 309 310

Hard hop resins

0.17 – – 0.234 0.515 0.067 0.007 0.014 0.049

78.4 93.0 83.8 96.4

12.7 4.7 4.1 1.1

8.9 2.3 12.1 2.5

91.7

2.5

5.8

Northwest/US microbrews Am. porter 1.33 Am. hefeweizen 0.30 Strong ale 3.44 India pale ale 0.80

45.9 90.9 86.0 69.0

Imported beers Imported stout Imported lager Imported pilsner 1 Imported pilsner 2

2.10 0.04 0.57 1.06

Other Non-alcohol beer

0.11

C

Therefore, these compounds have not been in the center of attention for many years. However, it was interesting that a dose of 8PN, consumed with one beer, was 500–1000 times lower that the concentration necessary to induce a noticeable estrogenic activity in vivo (≈ 100 mg/L) [67]. Studies on IXN were resumed when it was found that it could be readily O-demethylated by microorganisms inhabiting the gastrointestinal tract or, to a lesser extent, converted by enzymes associated with cytochrome P450 in the liver. The concentration of isoxanthohumol in beer is 10–30 times higher than 8PN [68]. Thus, it seems that most of 8PN present in the human body results from IXN bioactivation. Studies based on Simulator of the Human Intestinal Microbial Ecosystem (SIME) showed that IXN and other prenylflavonoids remain unchanged after passing through the stomach and small intestine. Transformation occurs in the colon, with a maximum yield of 80% [68]. In addition, an analysis of 51 fecal samples revealed high interindividual variability in IXN conversion by intestinal bacteria [34]. It is estimated that only one third of the population has high or medium ability of converting IXN to 8PN [59,68], which is not surprising given the fact that over 160 strains of bacteria were isolated from the human gastrointestinal tract [69]. Works are underway to find strains that may inhabit the human

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t3:9 t3:10

[56]

gastrointestinal tract and efficiently convert IXN to 8PN. One of such strains is Eubacterium limosum — anaerobic Grampositive bacteria, which could be included into dietary probiotic supplements [63,67]. IXN metabolism is shown in Fig. 3. Most of IXN is first absorbed into the blood, and then secreted by the liver as glucuronides back into the intestines, similarly as in the case of other flavonoids. About 30% of IXN and 46% of 8PN are excreted with urine in the form of aglycone [63]. It is worth noticing that, due to enterohepatic recirculation, the half-life of these compounds reaches 20 h [70]. Apart from glucuronidation, other processes that take place in the liver include demethylation of IXN to 8PN and hydroxylation of prenyl groups resulting in the formation of cis and trans alcohols [71]. The last two transformations are mediated by enzymes associated with cytochrome P450, hydroxylation is mediated by CYP2C19 and CYP2C8 isoforms, and demethylation by CYP1A2 isoform [72]. It is worth noticing that the enzymes of the CYP2C subfamily account for about 25% of the total pool of cytochrome P450 enzymes in the human liver [70]. These enzymes also show polymorphism in humans. Furthermore, their activity can be regulated by both environmental and genetic factors. Environmental factors that can induce or inhibit P450 enzymes include numerous prescription drugs, diet, alcohol consumption and smoking [70]. For example, there are reports on the inhibitory effect of alcohol on the activity of CYP1A2 in mammals [34]. Isoxanthohumol was found to be the most potent inhibitor of CYP2C8 in comparison to XN and 8PN with IC50 = 0.2 μM. 8-Prenylnaringenin was the most active in the case of CYP1A2 (IC50 = 0.1 μM), CYP2C9 (IC50 = 0.1 μM), and CYP2C19 (IC50 = 0.4 μM) [34]. Ames Salmonella assay revealed that isoxanthohumol strongly inhibited the activation of mutagenic carcinogen 2-

HO

OH O

O

HO

O

OH XN

[54]

F

5.8 1.2 3.0

E

295 296

30.3 7.6 8.0 17.2

3.75 0.62 0.089 0.011 0.257 0.189 0.035 0.001 0.09

t3:5 t3:6 t3:7 t3:8

23.8 1.5 6.0 13.8

MeOH MeOH MeOH Hot H2O EtOH–hexane EtOH Hexane EtOH–H2O MeOH

84.7 90.7 87.0

R

293 294

Ethanol extract Granules CO2 extract Pellets

[32]

9.5 8.1 10.0

0.50 0.68 0.40

R

291 292

t3:3 t3:4

O

289 290

References

N C

287 288

Amount of IXN (g/100 g)

Other flavonoids (%)

U

286

Amount of XN (g/100 g)

XN (%)

R O O

US major brands Lager/pilsner 1 Lager/pilsner 2 Lager/pilsner 3

Solvent used for extraction

IXN (%)

P

t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23

t3:1 t3:2

Hop products

IXN (mg/L)

D

Type of beer

E

t2:4

Table 3 Amount of xanthohumol and isoxanthohumol isolated from hop products.

T

t2:1 t2:2 t2:3

O OH

OH IXN

8-PN

Fig. 2. Synthesis and biological transformation of isoxanthohumol (IXN) and 8-prenylnaringenin (8PN) from xanthohumol (XN).

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342

A.K. Żołnierczyk et al. / Fitoterapia xxx (2015) xxx–xxx Table 4 Preparation of isoxanthohumol from dried, pulverized roots of Sophora flavescens per 1 kg of raw material. Raw material

Extraction method

Amount of IXN

References

t4:5

Roots

MeOH at r.t.

t4:6

Bark

0.21 g 0.088 g 0.011 g 0.02 g 0.04 g 0.017 g 0.003 g

[14] [57] [58] [59] [41] [13] [60]

MeOH or hot EtOH

F

t4:4

360 361 362 363 364 365 366 367 368

D

E T

C

358 359

E

356 357

R

354 355

R

353

O

351 352

N C

349 350

U

347 348

Therefore, it may substantially modulate the activity of the enzymes involved in carcinogen metabolism and detoxification processes [73]. Efficiency of the detoxification system is linked to the susceptibility to tumor development. Antiproliferative properties of 6 flavonoids, xanthohumol and its derivatives were tested in the cell lines of human ovarian cancer (A-2780), breast cancer (MCF-7), and colon cancer (HT-29). The compounds were administered at a concentration range of 0.1–100 μM and their activity was strongly dose-dependent. The most active compound in all the lines was XN. The cells most sensitive to IXN were those of MCF-7 line, as their IC50 after 2 days was 15.3 μM, and after 4 days 4.69 μM. The cells of HT-29 line were resistant to the compound. Sensitivity of A-2780 cells was lower after 2 days than after 4 days (IC50 18.0 and 25.7 μM, respectively) [73,74]. Isoxanthohumol antiproliferative effect against prostate cancer cells was examined in adenocarcinoma cells from mixed cultures (co-cultures), incubated in specially created conditions, and in the presence of potentially active hop compounds [75]. Prostate cancer may be derived from the epithelium lining the prostatic urethra. It involves an enhanced production of prostate specific antigen (PSA) and its increased

P

343 Q12 amino-3-methyloimidazo-[4,5-f]quinoline (IQ) and aflatoxin 344 (AFB1), catalyzed by human cytochrome P450 (CYP1A2). 345 346

blood concentration (PSA tests facilitate a diagnosis of prostatitis and prostate cancer). All investigated compounds inhibited PSA production by epithelial cells to a different degree, in a dose-dependent manner. Isoxanthohumol activity was noticeable at a concentration of 10 μM [76]. Compounds isolated from hops were also studied in classical cell lines of human prostate cancer cells DU145 and PC-3, obtained from the American Type Culture Collection (ATCC). Xanthohumol most actively inhibited the cancer cell proliferation among all the examined compounds, and isoxanthohumol showed a slightly lower activity [75]. Furthermore, the studies revealed that the positive effect of the compound was dose dependent. The dose of 200 μM of the compound in 100 μL of a growth medium reduced viability of DU145 and PC3 cells by at least 50% in 2 h [77]. No direct evidence of apoptosis induction in the prostate cancer cells PC3 and DU145 was found. However, it was confirmed that hop flavonoids (isoxanthohumol, 8-prenylnaringenin and 6prenylnaringenin) induced cellular vacuole formation. This can cause autophagocytosis i.e. the process of degradation of the cellular components by the cell, leading to the cell death [77]. Another interesting phenomenon is a reduced risk of prostate cancer observed in men consuming beer, as compared to those who do not drink this beverage [78]. Hop extract and xanthohumol and isoxanthohumol alone were also tested for their antiproliferative properties in the cell lines of colorectal carcinoma HT-29 and SW620 and nonmalignant IEC-6 line. Both compounds inhibited cell differentiation even at micromolar concentrations. IC50 for IXN and HT-29 cell line was 16.9 ± 0.9 μmol·dm−3, and for SW620 line it was 37.3 ± 3.2 μmol·dm−3. No impact of the compound on IEC-6 cell line was observed. However, in both lines the effects of IXN were weaker than those of xanthohumol (HT-29: 3.1 ± 0.2 μmol·dm−3, SW620: 1 ± 0.2 μmol·dm−3, IEC-6: 65.5 ± 11.3 μmol·dm−3) [79]. Apoptosis strongly interferes with the process of carcinogenesis. The studies involving murine 3T3-L1 adipocytes showed that isoxanthohumol and xanthohumol decreased cell viability and induced apoptosis. Concentration range of the investigated

R O O

t4:1 t4:2 t4:3 Q4

7

Fig. 3. Isoxanthohumol metabolism.

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

369 370 371 372 373 374 375 Q13 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407

A.K. Żołnierczyk et al. / Fitoterapia xxx (2015) xxx–xxx

429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

F

R O O

427 428

P

425 426

E

423 424

R

421 422

R

419 420

O

417 418

N C

415 416

U

413 414

D

Table 5 Antiproliferative properties of isoxanthohumol tested in cell lines.

412

vasculogenic mimicry of the cancer cells. In the studies involving MonoMac6 monoblastic leukemia cell line (DSMZ ACC124, Braunschweig, Germany), the same research team found that isoxanthohumol disrupted JAK/STAT signaling pathway and inhibited the expression of proinflammatory genes [83]. It is important to indicate that angiogenesis is associated with chronic inflammation. Inflammatory mediators produced by immune cells induce a production of angiogenic factors by the endothelial cells (ECs). These cells can directly release large amounts of various angiogenic factors. On the other hand, angiogenesis sustains inflammation by facilitating the transport of oxygen, nutrients and immune cells [85]. Isoxanthohumol was in a large group of compounds tested in vitro as potential inhibitors of angiogenesis. The studies confirmed its antiproliferative properties in these conditions and inhibition of formation of tubular microcapillaries, corresponding to vascular capillaries [86]. Other studies, involving the lines of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) showed that both xanthohumol and isoxanthohumol activated the mechanism of programmed cell death and prevented the formation of capillary-like structures. Since these polyphenols affected differentiation and migration of smooth muscle cells (SMCs), they can be also used in the treatment of diseases causing their proliferation, such as atherosclerosis and restenosis [85]. It is intriguing that despite such a strong anti-tumor activity isoxanthohumol exhibits only weak anti-microbial properties. Only antiviral activity was showed and it was investigated using hop extracts containing a mixture of compounds and purified components. These tests were cell-based assays designed to assess inhibition of cytopathic effects (CPEs). The tests involved herpes viruses type I and II (HSV1 and HSV2), bovine viral diarrhea virus (BVDV), cytomegalovirus (CMV) causing cytomegaly and rhinovirus (Rhino). Among the tested compounds present in hops (xanthohumol, isoxanthohumol, α- and β-bitter acids, and hop oils) isoxanthohumol exhibited the highest activity against rhinovirus (Rhino) and cytomegalovirus (CMV) [87]. The antioxidant properties of isoxanthohumol (IXN), as determined by DPPH (Fig. 3), are represented by EC50 = 8.4 mM, i.e. they are 450 times weaker than the standard ascorbic acid and 2.7 times stronger than its structural analogue naringenin (N) lacking isoprenyl moiety and O-methyl group. The same study showed that a conversion of isoxanthohumol into its oxime derivative increased its antioxidant activity by about 200 times [22] (Fig. 4).

E

t5:1 t5:2 Q5

410 411

C

454 455

compounds was 25–100 μM and the experiment lasted for 24 and 48 h. The best results were obtained for the maximum concentration of the inhibitor and the longest time. It was found that apoptosis was induced by oxidative stress. Additionally, the mitochondrial membrane potential was decreased and greater amounts of cytochrome c were released. These compounds also inhibited lipid accumulation in 3T3-L1 cells [80] (Table 5). Phosphoinositide-dependent kinase 1 (PDK1) is involved in the development and progression of melanoma. However, it is a constitutive enzyme and it is essential for the transmission of signals stimulating normal growth and development in mammals, as it mediates a number of physiological processes such as cell proliferation and survival, cell cycle regulation or insulin secretion [81]. The protein kinase C (PKC) plays an important role in many signal transduction cascades. Isoxanthohumol is active, but non-selective towards PKC-α and θ (Protein Kinase C α), which is due to binding mechanism based on hydrophobic interactions, and it is moderately active towards PDK1, to a much lower extent than xanthohumol. Investigation of the molecular interactions of isoxanthohumol and PDK1 indicated that its binding sites were located more externally than in xanthohumol [82]. Another important factor associated with cancer development is a transforming growth factor-β (TGF-β), a polypeptide from the group of cytokines involved in the regulation of cell proliferation and apoptosis [83]. Although produced mainly in platelets, it is expressed in most body tissues. It occurs in three isoforms. It inhibits growth processes by blocking the cell cycle in the middle and late G1 phase. Many reports indicate a role of this cytokine not only in tumor development, but also in serious degenerative diseases such as osteoporosis, diabetic retinopathy, Alzheimer's disease and myocardial infarction, or auto-aggressive diseases [84]. TGF-β has a dual effect on tumor growth. On the one hand, in tumors characterized by rapid growth, and in the initial phase of development, this cytokine activates the transcription of the cyclin-dependent kinase inhibitors — p15Ink4B and p21Waf1/Cip1. On the other hand, it stops cell cycle progression by inhibiting protein phosphorylation. In the late stage of tumor development, TGF-β promotes angiogenesis by increasing the expression of matrix metalloproteinases and proangiogenic factors such as VEGF and PDGF, thus leading to infiltration and metastases [83]. Studies conducted in the cell line of extremely invasive breast cancer MDA-MB-231 (ATCC HTB-26) showed that isoxanthohumol strongly reduced the expression of TGF-β. It was also demonstrated for the first time that it antagonized the cellular effects of TGF-β and blocked

408 409

T

8

t5:3

Cell line

Method

Activity

References

t5:4 t5:5

LAPC-4 prostatic adenocarcinoma PC-3 (prostate cancer)

Induced PSA secretion in LAPC-4 cells WST1 assay

[76] [77]

t5:6

DU145 (prostate cancer)

t5:7 t5:8 t5:9 t5:10

HT-29 (colorectal carcinoma) SW620 (colorectal carcinoma) IEC-6 (colorectal carcinoma, non-malignant) 3T3-L1 (murine adipocytes)

N100 μM 80% inhibition (100 μM) after 2 h 95% inhibition (200 μM) after 2 h 22% inhibition (100 μM) after 2 h 65% inhibition (100 μM) after 2 h IC50 = 16.9 ± 0.9 μmol dm−3 IC50 = 37.3 ± 3.2 μmol dm−3 No impact 60% inhibition (100 μM) after 48 h

XTT assay

MTS assay

[79]

[80]

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

456 457 458 459 460 461 462 463 464 465 Q14 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 Q15 481 482 483 484 485 486 487 488 Q16 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503

A.K. Żołnierczyk et al. / Fitoterapia xxx (2015) xxx–xxx

HO

O

O

O

O

HO

O

O

O

HO

OH

N

OH

O

OH

N: ED50 = 22.9 mM

OH

F

H

9

IXNOX: ED50 = 0.041 mM

R O O

IXN: ED50 = 8.4 mM

Fig. 4. The antioxidant activity of isoxanthohumol and its selected structural analogues determined by DPPH.

518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548

E

516 517

R

514 515

R

512 513

O

510 511

N C

P

508 509

U

507

D

Isoxanthohumol is one of the three major flavonoids in hops with the highest content in beer. Due to its extensive and interesting biological properties, easy synthesis from xanthohumol and the ability to convert into 8prenylnaringenin inside the human body, isoxanthohumol has recently enjoyed considerable attention of scientists. Its biological properties include antitumor, antioxidant, antimicrobial and proestrogenic activity, thus offering a possibility of using isoxanthohumol and its derivatives in medicine. An additional advantage of isoxanthohumol is the fact that it is a natural compound of plant origin. But there are some questions to take into consideration. The only source of IXN, XN and 8PN in the human diet is beer. This does not apply to beers based on the supercritical carbon dioxide hop extracts used in brewing due to trace amounts of flavonoids. Due to the large conversion of XN to IXN during wort boiling [45,88] and next to 8PN in human body, including its microbiota [89], it is possible that the dose of phytoestrogen present in the beer is significant and might have an influence on human health [67]. This is because the IXN content in beer can be 30 times higher than 8PN [45] due to thermal isomerization, especially in the case of highly hopped beers. On the other hand, in vitro and in vivo studies have shown no significant estrogenic activity of beer. The effective oral dose in Low-Dose Hormone Therapy (LDHT) in postmenopausal hormone therapy is 0.5 mg/day of 17β-estradiol [90]. A number of in vitro studies indicated that 8PN is a 100 times weaker estrogen than 17β-estradiol [91]. This allows estimation of quantity of 8PN recalculated on 17β-estradiol, assuming that the entire amount of IXN (c = 3.44 mg/L [45]) is converted into 8PN and that 3 L of beer is consumed, what is possible. The simple calculation is as follows: (3.44 mg/L × 3 L) / 100 = 0.1 mg. It is about 20% of dose used in LDHT. Xanthohumol, the second hop flavonoid in terms of content in beer, can also be a source of 8PN via microbial demethylation to desmethylxanthohumol and spontaneous cyclization to 8PN and 6PN [36]. It should make an estrogenic activity of beer greater than it would result from the content of IXN and 8PN. On the other hand, the subtle latent health effects of estrogens is still considered and beer is a significant source of estrogens with the highest possible content of estrogen with the exception of soy-based foods [92]. What is more the study examined on 67 menopausal women showed significant reduction of Kupperman index after

E

505 506

6 and 12 weeks of treatment of extract standardized on 0.1 and 0.25 mg/day of 8PN against placebo [93]. The content of IXN was not investigated but probably was about up to 10 times higher than the content of 8PN. The amount of estrogen (8PN) delivered to the human body is difficult to determine due to the fact that the quantity of this compound depends not only on production technology of beer, but also on the variety of hops, the climatic conditions of cultivation, and on the ability of the organism to transform IXN into 8PN, including the strains of the human bacterial flora. Estrogenic activity of 8PN in vivo trials involving rats was much weaker than in vitro by factors 3 × 103 and 2 × 105 in comparison to 17β-estradiol and according to researchers can be omitted [91,94]. The investigations on XN showed that metabolism of this compound between rodents and humans can be different because the production of 8PN by rats via IXN is strong, but humans are very weak producers of 8PN [36]. Promberger et al. after conducting the Yeast Estrogen Screen assay (YES) concluded also that the human health hazard of beer drinking is negligible [95]. According to Sauerwein and Meyer [96] the daily equivalent of dose of 2 mg of 17β-estradiol could be delivered by 103 L of beer (0.5 mg by 250 L). The investigations were carried out using estrogen receptor assay on extracted beer samples. The content of IXN, XN and 8PN in beer was not investigated. On the market there are many oral formulations based on the hop extracts which alleviate the symptoms of menopause which contain active estrogen 8PN [93] and its precursors IXN and XN in concentrations 10–100 times higher, respectively. In addition to the above information, XN (the highest content in hop extract) can be also converted into IXN through acidcatalyzed cyclization in the stomach [38]. Thus, when determining their estrogenic activity, the IXN and XN contents should be taken into account because their impact can be higher than 8PN. Due to the influence of many factors, the effective estrogen dose is difficult to determine. Nowadays the hydrogenated hop extracts are very common in beer production. Taking into consideration that XN can be hydrogenated to tetrahydro-XN (H2, Pd/C) [97] and that IXN or 8PN can be also hydrogenated (H2/PtO2) to dihydro-IXN or dihydro-8PN, respectively [56], it is possible that such compounds are present in larger quantities in hydrogenated hops. According to the available author's knowledge, such studies have not been yet conducted despite the fact that the biological activity of these compounds was considered.

T

6. Summary and open questions

C

504

Please cite this article as: Żołnierczyk AK, et al, Isoxanthohumol — Biologically active hop flavonoid, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.03.007

549 550 551 552 553 554 555 556 557 558 559 560 Q17 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594

A.K. Żołnierczyk et al. / Fitoterapia xxx (2015) xxx–xxx

603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626

R

627

Conflict of interest

629 630

The authors confirm that this article content has no conflicts of interest.

631

Acknowledgments

632 633

This work was supported by grant from the National Science Centre (Poland), grant No. 2011/01/B/NZ9/02890.

634

References

N C

O

R

628

[1] Smejkal K. Cytotoxic potential of C-prenylated flavonoids. Phytochem Rev 2014;13:245–75. [2] Haminiuk CWI, Maciel GM, Plata-Oviedo MSV, Peralta RM. Phenolic compounds in fruits — an overview. Int J Food Sci Technol 2012;47:2023–44. [3] Robbins RJ. Phenolic acids in foods: an overview of analytical methodology. J Agric Food Chem 2003;51:2866–87. [4] Belitz HD, Grosch W, Schieberle P. Fruits and fruit products. Food chem. Berlin Heidelberg: Springer; 2009 807–61. [5] Peterson J, Dwyer J. Flavonoids: dietary occurrence and biochemical. Nutr Res 1998;18:1995–2018. [6] Yazaki K, Sasaki K, Tsurumaru Y. Prenylation of aromatic compounds, a key diversification of plant secondary metabolites. Phytochemistry 2009; 70:1739–45. [7] Chen X, Mukwaya E, Wong MS, Zhang Y. A systematic review on biological activities of prenylated flavonoids. Pharm Biol 2014;52:655–60. [8] Yu L, Zhang F, Hu Z, Ding H, Tang H, Ma Z, et al. Novel prenylated bichalcone and chalcone from Humulus lupulus and their quinone reductase induction activities. Fitoterapia 2014;93:115–20.

U

635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652

F

601 602

R O O

599 600

E

598

P

6PN 8-Prenylnaringenin 8PN 8-Prenylnaringenin A-2780 Ovarian carcinoma A-2780 AFB1 Aflatoxin B1 BVDV Bovine virus diarrhea CMV Cytomegalovirus CPE Cytopathic effect CYP1A2 Cytochrome P450 1A2 CYP2C19 Cytochrome P450 2C19 CYP2C8 Cytochrome P450 2C8 DPPH 2,2-Diphenyl-1-picrylhydrazyl ECs Endothelial cells HASMCs Human aortic smooth muscle cells hCG Human chorionic gonadotropin HPLC-DAD High-performance liquid chromatography with diode-array detection HSCCC High-speed countercurrent chromatography HSV Herpes simplex virus HUVECs Human umbilical vein endothelial cells IXN Isoxanthohumol LH Luteinizing hormone PDGF Platelet-derived growth factor PDK1 Phosphoinositide-dependent kinase 1 PKC Protein kinase C PSA Prostate-specific antigen SIME Simulator of the human intestinal microbial ecosystem SMCs Smooth muscle cells TGF-β Transforming growth factor-β VEGF Vascular endothelial growth factor XN Xanthohumol

D

596 597

[9] Luo G, Yang Y, Zhou M, Ye Q, Liu Y, Gu J, et al. Novel 2-arylbenzofuran dimers and polyisoprenylated flavanones from Sophora tonkinensis. Fitoterapia 2014;99:21–7. [10] Dat NT, Binh PTX, Quynh LTP, Minh CV, Huong HT, Lee JJ. Cytotoxic prenylated flavonoids from Morus alba. Fitoterapia 2010;81:1224–7. [11] Shin EM, Zhou HY, Guo LY, Kim JA, Lee SH, Merfort I, et al. Anti-inflammatory effects of glycyrol isolated from Glycyrrhiza uralensis in LPS-stimulated RAW264.7 macrophages. Int Immunopharmacol 2008;8:1524–32. [12] Kim SJ, Son KH, Chang HW, Kang SS, Kim HP. Tyrosinase inhibitory prenylated flavonoids from Sophora flavescens. Biol Pharm Bull 2003;26: 1348–50. [13] Jung HA, Yokozawa T, Kim BW, Jung JH, Choi JS. Selective inhibition of prenylated flavonoids from Sophora flavescens against BACE1 and cholinesterases. Am J Chin Med 2010;38:415–29. [14] Kim JH, Ryu YB, Kang NS, Lee BW, Heo JS, Jeong IY, et al. Glycosidase inhibitory flavonoids from Sophora flavescens. Biol Pharm Bull 2006;29: 302–5. [15] Stevens JF, Ivancic M, Hsu VL, Deinzer ML. Prenylflavonoids from Humulus lupulus. Phytochemistry 1997;44:1575–85. [16] Zanoli P, Zavatti M. Pharmacognostic and pharmacological profile of Humulus lupulus L. Ethnopharmacology 2008;116:383–96. [17] Karabin M, Hudcová T, Jelinek L, Dostálek P. Wyznam chmielowych prenylflavonoidu pro lidske zdravi (The importance of hop prenylflavonoids for human health). Chem Listy 2012;106:1095–103. [18] Steenackers B, De Cooman L, De Vos D. Chemical transformations of characteristic hop secondary metabolites in relation to beer properties and the brewing process: a review. Food Chem 2015;172:742–56. [19] Hwang EM, Ryu YB, Kim HY, Kim DG, Hong SG, Lee JH, et al. BACE1 inhibitory effects of lavandulyl flavanones from Sophora flavescens. Bioorg Med Chem 2008;16:6669–74. [20] Haensel R, Schulz J. Desmethylxanthohumol: Isolierung aus Hopfen und Cyclisierung zu Flavanonen. Arch Pharm (Weinheim) 1988;321:37–40. [21] Stevens JF, Taylor AW, Clawson JE, Deinzer ML. Fate of xanthohumol and related prenylflavonoids from hops to beer. J Agric Food Chem 1999;4: 2421–8. [22] Potaniec B, Grabarczyk M, Stompor M, Szumny A, Zieliński P, Żołnierczyk AK, et al. Antioxidant activity and spectroscopic data of isoxanthohomol oxime and related compounds. Spectrochim Acta A 2014;118:716–23. [23] Anioł M, Świderska A, Stompor M, Żołnierczyk AK. Antiproliferative activity and synthesis of 8-prenylnaringenin derivatives by demethylation of 7-Oand 4-O-substituted Isoxanthohumols. Med Chem Res 2012;21:4230–8. [24] Krofta K. The content of hop prenylflavonoids in Czech and foreign beers. Kvasny Prum 2010;56:2–9. [25] Česlova L, Holčapek M, Fidler M, Drštičkovă J, Lisa M. Characterization of prenylflavonoids and hop bitter acids in various classes of Czech beers and hop extracts using high-performance liquid chromatography–mass spectrometry. J Chromatogr A 2009;1216:7249–57. [26] Dhooghea L, Naessensa T, Heyerick A, De Keukeleire D, Vlietinck AJ, Pieters L, et al. Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary Standards. Talanta 2010;83:448–56. [27] Henley T, Reddivari L, Broeckling CD, Bunning M, Miller J, Avens JS, et al. American India Pale Ale matrix rich in xanthohumol is potent in suppressing proliferation and elevating apoptosis of human colon cancer cells. Int J Food Sci Technol 2014;49:2464–71. [28] Andres-Iglesias C, Blanco CA, Blanco J, Montero O. Mass spectrometrybased metabolomics approach to determine differential metabolites between regular and non-alcohol beers. Food Chem 2014;157:205–12. [29] Goncalves JL, Alves VL, Rodrigues FP, Figueira JA, Câmara JS. A semiautomatic microextraction in packed sorbent, using a digitally controlled syringe, combined with ultra-high pressure liquid chromatography as a new and ultra-fast approach for the determination of prenylflavonoids in beers. J Chromatogr A 2013;1304:42–51. [30] Jurkova M, Čejka P, Houška M, Mikyška A. Simultaneous determination of prenylflavonoids and isoflavonoids in hops and beer by HPLC-DAD method: study of green hops homogenate application in the brewing process. Kvasny Prum 2013;59:41–9. [31] Kao TH, Wu GY. Simultaneous determination of prenylflavonoid and hop bitter acid in beer lee by HPLC-DAD–MS. Food Chem 2013;141:1218–26. [32] Magalhaes PJ, Guido LF, Cruz JM, Barros AA. Analysis of xanthohumol and isoxanthohumol in different hop products by liquid chromatographydiode array detection–electrospray ionization tandem mass spectrometry. J Chromatogr A 2007;1150:295–301. [33] Coldham NG, Sauer MJ. Identification, quantitation and biological activity of phytoestrogens in a dietary supplement for breast enhancement. Food Chem Toxicol 2001;39:1211–24. [34] Bolca S, Possemiers S, Maevoet V, Huybrechts I, Heyerick A, Vervarcke S, et al. Microbial an dietary factors associated with the 8-prenylnaringenin producer phenotype: a dietary intervention trial with fifty healthy postmenopausal Caucasian women. Br J Nutr 2007;98:950–9.

E

Abbreviations

C

595

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[59] Olennikov DN, Tankhaeva LM, Pankrushina NA, Sandanov DV. Phenolic compounds of Sophora flavescens Soland of Russian Origin. Russ J Bioorg Chem 2013;39:755–60. [60] Quang TH, Ngan NTT, Minh CV, Kiem PV, Tai BH, Thao NP, et al. αGlucosidase inhibitors from the roots of Sophora flavescens. Bull Korean Chem Soc 2012;33:1791–3. [61] Chen R, Liu X, Zou J, Yin Y, Ou B, Li J, et al. Regio- and stereospecific prenylation of flavonoids by Sophora flavescens Prenyltransferase. Adv Synth Catal 2013;355:1817–28. [62] Izzo G, Söder O, Svechnikov K. The prenylflavonoid phytoestrogens 8prenylnaringenin and isoxanthohumol differentially suppress steroidogenesis in rat Leydig cells in ontogenesis. J Appl Toxicol 2011;31:589–94. [63] Possemiers S, Rabot S, Espin JC, Bruneau A, Philippe C, Gonzalez-Sarrıas A, Heyerick A, Tomas-Barberan FA, Keukeleire DD, Verstraete W. Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine. J Nutr 2008;138:1310–6. [64] Keiler AM, Zierau O, Kretzschmar G. Hop extracts and hop substances in treatment of menopausal complaints. Planta Med 2013;79:576–9. [65] Oszukowska E, Słowikowska-Hilczer J, Lipiński M, Kula K. Zmiany wydzielania estradiolu i testosteronu przy leydigioma i po usunięciu jądra z guzem. Urol Pol 2003;56:57–60. [66] Chen W, Becker T, Qian F, Ring J. Beer and beer compounds: physiological effects on skin health. J Eur Acad Dermatol Venereol 2014;28:142–50. [67] Possemiers S, Heyerick A, Robbens V, Keukeleire DD, Verstraete W. Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; conversion of isoxanthohumol into 8-prenylnaringenin. J Agric Food Chem 2005;53:6281–8. [68] Possemiers S, Bolca S, Grootaert C, Heyerick A, Decroos K, Dhooge W, et al. The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) Is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine. J Nutr 2006;13:1862–7. [69] Bolca S, Van de Wiele T, Possemiers S. Gut metabotypes govern health effects of dietary polyphenols. Curr Opin Biotechnol 2013;24:220–5. [70] Yuan Y, Qiu X, Nikolic D, Chen SN, Huang K, Li G, et al. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols. Eur J Pharm Sci 2014;53:55–61. [71] Bolca S, Li J, Nikolic D, Roche N, Blondeel P, Possemiers S, et al. Disposition of hop prenylflavonoids in human breast tissue. Mol Nutr Food Res 2010; 54:284–94. [72] Guo J, Nikolic D, Chadwick LR, Pauli GF, van Breemen RB. Identification of human hepatic cytochrome P450 enzymes Involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus Lupulus L.). Drug Metab Dispos 2006;34:1152–9. [73] Botta B, Vitali A, Menendez P, Misiti D, Monache GD. Prenylated flavonoids: pharmacology and biotechnology. Curr Med Chem 2005;1: 713–39. [74] Miranda CL, Stevens JF, Helmrich A, Henderson MC, Rodriguez RJ, Yang YH, et al. Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines. Food Chem Toxicol 1999;37:271–85. [75] Delmulle L, Bellahcène A, Dhooge W, Comhaire F, Roelens F, Huvaere K, et al. Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines. Phytomedicine 2006;13:732–4. [76] Vollmer G, Amri H, Arnold J. Assessment of polyphenols on PSA expression in a human co-culture model of reactive prostate stroma cells and LAPC4 prostate adenocarcinoma cells. BMC Complement Altern Med 2012; 12(Suppl. 1):27. http://dx.doi.org/10.1186/1472-6882-12-S1-P27. [77] Delmulle L, Vanden Berghe T, Keukeleire DD, Vandenabeele P. Treatment of PC-3 and DU145 prostate cancer cells by prenylflavonoids from hop (Humulus lupulus L.) induces a caspase-independent form of cell death. Phytother Res 2008;22:197–203. [78] Gerhäuser C. Beer constituents as potential cancer chemopreventive agents. Eur J Cancer 2005;41:1941–54. [79] Hudcová T, Bryndová J, Fialová K, Fiala J, Karabín M, Jelínek L, et al. Antiproliferative effects of prenylflavonoids from hops on human colon cancer cell lines. J Inst Brew 2014;120:225–30. [80] Yang JY, Della-Ferra MA, Rayalam S, Baile CA. Effect of xanthohumol and isoxanthohumol on 3T3-L1 cell apoptosis and adipogenesis. Apoptosis 2007;12:1953–63. [81] Michel JJC, Townley IK, Dodge-Kafka KL, Zhang F, Kapiloff MS, Scott JD. Spatial restriction of PDK1 activation cascades by anchoring to mAKAPα. Mol Cell 2005;20:661–72. [82] Lauro G, Masullo M, Piacente S, Riccio R, Bifulco G. Inverse virtual screening allows the discovery of the biological activity of natural compounds. Bioorg Med Chem 2012;20:3596–602. [83] Serwe A, Rudolph K, Anke T, Erkel G. Inhibition of TGF-β signaling, vasculogenic mimicry and proinflammatory gene expression by isoxanthohumol. Invest New Drugs 2012;30:898–915.

T

C

[35] Quifer-Rada P, Martínez-Huélamo M, Jáuregui O, Chiva-Blanch G, Estruch R, Lamuela-Raventós RM. Analytical condition setting a crucial step in the quantification of unstable polyphenols in acidic conditions: analyzing prenylflavanoids in biological samples by liquid chromatography–electrospray ionization triple quadruple mass spectrometry. Anal Chem 2013;85:5547–54. [36] Legette LC, Karnpracha C, Reed CR, Choi J, Bobe G, Christensen JM, et al. Human pharmacokinetics of xanthohumol, an antihyperglycemic flavonoid from hops. Mol Nutr Food Res 2014;58:248–55. [37] Legette LCL, Moreno Luna AY, Reed RL, Miranda CL, Bobe G, Proteau RR, et al. Xanthohumol lowers body weight and fasting plasma glucose in obese male Zucker fa/fa rats. Phytochemistry 2013;91:236–41. [38] Nikolic D, Li Y, Chadwick LR, Pauli GF, Breemen RB. Metabolism of xanthohumol and isoxanthohumol, prenylated flavonoids from hops (Humulus lupulus L.), by human liver microsomes. J Mass Spectrom 2005; 40:289–99. [39] Yuan Y, Qiu X, Nikolic D, Dahl JH, van Breemen RB. Method development and validation for ultra-high-pressure LC/MS/MS determination of hop prenylflavonoids in human serum. J AOAC Int 2012;95:1744–9. [40] Fu M, Wang W, Chen F, Dong Y, Liu X, Ni H, et al. Production of 8prenylnaringenin from isoxanthohumol through biotransformation by fungi cells. J Agric Food Chem 2011;59:7419–26. [41] Jin JH, Kim JS, Kang SS, Son KH, Chang HW, Kim HP. Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens. J Ethnopharmacol 2010;127:589–95. [42] Stevens JF, Page JE. Xanthohumol and related prenylflavonoids from hops and beer: to your good health! Phytochemistry 2004;65: 1317–30. [43] Tanaka Y, Yanagida A, Komeya S, Kawana M, Honma D, Tagashira M, et al. Comprehensive separation and structural analyses of polyphenols and related compounds from bracts of hops (Humulus lupulus L). J Agric Food Chem 2014;62:2198–206. [44] Rong H, Zhao Y, Lazou K, De Keukeleire D, Milligan SR, Sandra P. Quantitation of 8-prenylnaringenin, a novel phytoestrogen in hops (Humulus lupulus L.), hop products, and beers, by benchtop HPLC–MS using electrospray ionization. Chromatographia 2000;51:545–52. [45] Stevens JF, Taylor AW, Deinzer ML. Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography– tandem mass spectrometry. J Chromatogr A 1999;832:97–107. [46] Khupse RS, Erhardt PW. Total synthesis of xanthohumol. J Nat Prod 2007; 70:1507–9. [47] Anioł M, Żołnierczyk A, Szymańska K. An efficient synthesis of the phytoestrogen 8-prenylnaringenin from isoxanthohumol with magnesium iodide etherate. Tetrahedron 2008;64:9544–7. [48] Wilhelm H, Wessjohann LA. An efficient synthesis of the phytoestrogen 8prenylnaringenin from xanthohumol by a novel demethylation process. Tetrahedron 2006;62:6961–6. [49] Olas B, Kołodziejczyk-Czepas J, Wachowicz B, Jędrejek D, Stochmal A, Oleszek W. The extract from hop cones in plasma protects against changes following exposure to peroxynitrite. Cent Eur J Biol 2011;6:990–6. [50] Rozalski M, Micota B, Sadowska B, Stochmal A, Jedrejek D, WieckowskaSzakiel M, et al. Antiadherent and antibiofilm activity of Humulus lupulus L. derived products: new pharmacological properties. BioMed Res Int 2013; 2013. http://dx.doi.org/10.1155/2013/101089 (Article ID 101089, 7 pages). [51] Kac J, Plazar J, Mlinaric A, Zegura B, Lah TT, Filipic M. Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol. Phytomedicine 2008;15:216–20. [52] Arráez-Roman D, Cortacero-Ramirez S, Segura-Carretero A, Martin-Lagos Contreras JA, Fernandez-Gutierrez A. Characterization of the methanolic extract of hops using capillary electrophoresis electrospray ionizationmass spectrometry. Electrophoresis 2006;27:2197–207. [53] Faltermeier A, Massinger S, Schulmeyr J. Process for preparing high-purity xanthohumol-containing powder and use thereof. Patent U.S. 20070254086 A1 2007. [54] Gil-Ramirez A, Mendiola JA, Arranz E, Ruiz-Rodriguez A, Reglero G, Ibanez E, et al. Highly isoxanthohumol enriched hop extract obtained by pressurized hot water extraction (PHWE). Chemical and functional characterization. Innovative Food Sci Emerg Technol 2012;16:54–60. [55] Chen QH, Fu ML, Chen MM, Liu J, Liu XJ, He GQ, et al. Preparative isolation and purification of xanthohumol from hops (Humulus lupulus L.) by high-speed counter-current chromatography. Food Chem 2012; 132:619–23. [56] Mizobuchi S, Sato Y. A new flavanone with antifungal activity isolated from hops. Agric Biol Chem Tokyo 1984;48:2771–5. [57] Jeong TS, Ryu YB, Kim HY, Curtis-Long MJ, An SJ, Lee JH, et al. low density lipoprotein (LDL)-antioxidant flavonoids from roots of Sophora flavescens. Biol Pharm Bull 2008;31:2097–102. [58] Jeong TS, An S, Ryu YB, Kim HY, Cho MH, Park JS, et al. Human ACAT inhibitory effects of flavonoids from Sophora flavescens. Bull Korean Chem Soc 2008;29:2287–90.

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[91] Biendl M. Estrogenic activity of hop components. , 1Brauwelt; 2004 24–8. [92] Stanfors BD, Snyder SA, Trenholm RA, Holady JC, Vanderford BJ. Estrogenic activity of US drinking waters: a relative exposure comparison. J Am Water Works Assoc 2010;102:1–11. [93] Heyerick A, Vervarcke S, Depypere H, Bracke M, De Keukeleire D. A first prospective, randomized, double-blind, placebo-controlled study on the use of a standardized hop extract to alleviate menopausal discomforts. Maturitas 2006;54:164–75. [94] Miyamoto M, Matsushita Y, Kiyokawa A, Fukuda C, Iijima Y, Sugano M, et al. Prenylflavonoids: a new class of non-steroidal phytoestrogen (part 2): estrogenic effects of 8-isopentenylnaringenin on bone metabolism. Planta Med 1998;64:516–9. [95] Promberger A, Dornstauder E, Frűhwirth C, Schmid ER, Jungbauer A. Determination of estrogenic activity in beer by biological and chemical means. J Agric Food Chem 2001;49:633–40. [96] Sauerwein H, Meyer HHD. Erfassung östrogenwirksamer Substanzen in Bier und in dessen Rohstoffen. Monatsschr Brauwiss 1997;50:142–6. [97] Miranda CL, Stevens JF, Ivanov V, McCall M, Frei B, Deinzer ML, et al. Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. J Agric Food Chem 2000;48:3876–84.

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O

R

R

E

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E

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[84] Stępień-Wyrobiec O, Hrycek A, Wyrobiec G. Transformujący czynnik wzrostu beta (TGF-beta) — budowa, mechanizmy oddziaływania oraz jego rola w patogenezie tocznia rumieniowatego układowego. Postepy Hig Med Dosw 2008;62:688–93. [85] Negrao R, Costa R, Duarte D, Gomes TT, Mendanha M, Moura L, et al. Angiogenesis and inflammation signaling are targets of beer polyphenols on vascular cells. J Cell Biochem 2010;111:1270–9. [86] Bertl E, Becker H, Eicher T, Herhaus C, Kapadia G, Bartsch H, et al. Inhibition of endothelial cell functions by novel potential cancer chemopreventive agents. Biochem Biophys Res Commun 2004;325:287–95. [87] Buckwold VE, Wilson RJ, Nalca A, Beer BB, Voss TG, Turpin JA, et al. Antiviral activity of hop constituents against a series of DNA and RNA viruses. Antivir Res 2004;61:57–62. [88] Magalhães PJ, Dostalek P, Cruz JM, Guido LF, Barros AA. The impact of a xanthohumol-enriched hop product on the behavior of xanthohumol and isoxanthohumol in pale and dark beers: a pilot scale approach. J Inst Brew 2008;114:246–56. [89] Possemiers S, Bolca S, Verstraete W, Heyerick A. The intestinal microbiome: a separate organ inside the body with the metabolic potential to influence the bioactivity of botanicals. Fitoterapia 2011;82:53–66. [90] Langer RD. Efficacy, safety, and tolerability of low-dose hormone therapy in managing menopausal symptoms. J Am Board Fam Med 2009;22: 563–73.

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