Botanicals and Their Bioactive Phytochemicals for Women’s Health

1. Vitex agnus-castus (Chasteberry).

Chasteberry is obtained from the dried ripe fruit of the chaste tree and has been used as a women’s health botanical since ancient Greece (Wuttke et al., 2003). One mechanism of action could be through the dopamine receptors, which decrease thyrotropin releasing hormone and prolactin levels, alleviating PMS symptoms (Meier et al., 2000; Tamagno, 2009). Chasteberry has also been shown to have hormone modulating properties in vitro that may play a role in regulating PMS symptoms (Girman et al., 2003; Lloyd and Hornsby, 2009). Weak binding affinity for the estrogen receptors and modulation of hormone sensitive genes have been reported in vitro (Liu et al., 2001, 2004; Hajirahimkhan et al., 2013a) as well as estrogenic effects on the uterus in rat models (Ibrahim et al., 2008). An alternative mechanism as an opiate receptor agonist has also been reported (Webster et al., 2006, 2011; Chen et al., 2011). A number of phytochemicals have been isolated from chasteberry including several flavonoids and linoleic acid, which could be responsible for the biological activities (Fig. 13) (Chen et al., 2011; Webster et al., 2011). Apigenin and penduletin in particular have been identified as weak ERβ-selective ligands (Fig. 13) (Jarry et al., 2003; Hu et al., 2007b), although apigenin is present in numerous plants. A small randomized, double-blind, placebo-controlled trial (67 Chinese women) did show significant efficacy of chasteberry compared with placebo in the treatment of moderate to severe PMS (Ma et al., 2010). Other small placebo controlled trials also suggested benefit; however, the placebo effect was large, which is often seen in clinical trials with effects on the CNS (Berger et al., 2000; Schellenberg, 2001, 2012; He et al., 2009; Zamani et al., 2012). These trials were of short duration and safety data were lacking. The German Commission E approved the use of chasteberry for PMS and chasteberry has the most convincing clinical data out of all of the botanicals for efficacy in the treatment of PMS (Freeman, 2010).

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Fig. 13.

Phytochemicals in chasteberry.

2. Angelica sinensis (Dong Quai).

Dong quai or danggui is the dried root of Angelica sinensis, which is commonly used in traditional Chinese medicine to promote blood circulation and treat menstrual disorders such as dysmenorrhea as well as other women’s health issues (Wu and Hsieh, 2011; Chen et al., 2013). It has been shown that a bioactive component of dong quai, Z-ligustilide (Fig. 10), inhibits the contraction of isolated rat uterus in a dose-dependent manner and improves microcirculation, suggesting it might be responsible for the antispasmodic effects of dong quai (Du et al., 2006). Ligustilide also has anti-inflammatory effects, which could contribute to the mechanisms of relief of menstrual symptoms (Su et al., 2011). Whether dong quai has estrogenic activity is controversial, and no estrogenic compounds have been isolated to date (Piersen, 2003; Hajirahimkhan et al., 2013a). Finally, there are no randomized, placebo-controlled, clinical trials evaluating the efficacy of dong quai for PMS.

3. Viburnum opulus and Viburnum prunifolium (Cramp Bark and Black Haw).

Cramp bark and black haw are two species of viburnum that are often used as uterine relaxants and antispasmodics. Animal studies have suggested that both species have antispasmodic effects on the uterus and experiments with human uterine tissue also showed a relaxant effect (Jarboe et al., 1966; Nicholson et al., 1972; Cometa et al., 2009). Scopoletin could be responsible for the smooth muscle antispasmodic activity of viburnum (Fig. 14) (Jarboe et al., 1967; Nicholson et al., 1972). There are no randomized, placebo-controlled, clinical trials evaluating the efficacy of viburnum for PMS.

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Fig. 14.

Phytochemical in cramp bark and black haw.

4. Zingiber officinale (Ginger).

The rhizome of the ginger plant has been used for centuries as a spice and a condiment as well as for medicinal properties, particularly for anti-inflammatory and immune modulating properties (Ali et al., 2008). Ginger may also be effective at reducing PMS symptoms. More than 60 active compounds have been identified in ginger, including gingerols, shogaols, and zingerone (Fig. 15) (Baliga et al., 2011). Ginger and its bioactive compound zingerone have reported antispasmodic activities (Fig. 15) (van Andel et al., 2014; Ahmad et al., 2015). There are some reports of estrogenic activities for ginger, although other studies have shown no effect (Kim et al., 2008; Kumar et al., 2015). A small trial suggested that ginger (250 mg, 4 times daily, for three days from the start of the menstrual period) was as effective as NSAIDs at reducing period pain (Ozgoli et al., 2009). A few other small trials in young women have also shown positive effects for PMS (Kashefi et al., 2014, 2015).

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Fig. 15.

Phytochemicals in ginger and their metabolites.

5. Valeriana officinalis (Valerian).

The roots and rhizomes of valerian have been used for centuries primarily for tranquilizing/sedative properties as well as for PMS (Jarema, 2008). Over 150 phytochemicals have been identified in valerian, including pyridine alkaloids and organic acids such as valerenic acid (Fig. 16) (Patocka and Jaki, 2010). The antispasmodic effects of valerian have been demonstrated in vitro and in vivo (Occhiuto et al., 2009). A double-blind, placebo-controlled, randomized clinical trial with 100 young women receiving 255 mg three times daily for 3 days at the beginning of the menstrual cycle for two consecutive cycles showed a significant reduction in the severity of pain (Mirabi et al., 2011). No adverse effects were reported in the valerian-treated group, which supports literature evaluations of safety (Kelber et al., 2014).

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Fig. 16.

Phytochemical in valerian.

6. Oenothera biennis (Evening Primrose).

The oil from the seeds of evening primrose are high in fatty acids, including γ-linolenic acid (Montserrat-de la Paz et al., 2014) (Fig. 17). Polyphenolic compounds are also present, including gallic acid and chatechin, which could be responsible for antioxidant effects (Wettasinghe et al., 2002) (Fig. 17). A double-blind randomized clinical trial with 40 young women receiving either femicomfort (evening primrose, vitamins B6 and E) or placebo for two menstrual cycles concluded that evening primrose was effective in relieving PMS symptoms (Kashani et al., 2010). Another small randomized crossover study with 38 women showed no effect (Budeiri et al., 1996).

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Fig. 17.

Phytochemicals in evening primrose.

1. Vaccinium macrocarpon (Cranberry).

Cranberry, which is the second most popular botanical sold in the US (Table 1), has a well-recognized medicinal use primarily in preventing UTIs (Yarnell, 2002; Rossi et al., 2010). The mechanism is generally thought to involve inhibition of adhesion of P-fimbriated (proteinaceous appendages) E. coli that firmly attach to uroepithelial cells by the type A proanthocyanidins in cranberry juice (Fig. 18) (Foo et al., 2000; Guay, 2009; Vasileiou et al., 2013). Three species of cranberry (V. macrocarpon, V. oxycoccus, V. vitis-idaea) have different amounts and types of proanthocyanidins, which may explain the variable clinical data and further emphasizes the need for full chemical and biological standardization of cranberry extracts (Davidson et al., 2014). The Cochrane Collaboration previously recommended cranberry products for the prevention of UTIs in young and middle-aged women; however, questions of efficacy remain because of the heterogeneity in the clinical trial design and the lack of consensus on dosage, formulation, and time of use (Jepson et al., 2012). The most recent update suggests that cranberry juice is less effective than previously indicated, and they do not currently recommend cranberry juice for the prevention of UTIs. In addition, other cranberry preparations such as powders need to be fully standardized both chemically and biologically to the active compounds to ensure potency before being evaluated in clinical studies or recommended for use. The existing clinical trials suggest that cranberry is more effective at preventing rather than treating UTIs (Vasileiou et al., 2013); however, it is quite possible that the studies with V. macrocarpon in particular were under-dosed in the bioactive proanthocyanidins and placebo-controlled clinical trials that use standardized cranberry products are needed to establish efficacy (Davidson et al., 2014).

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Fig. 18.

Phytochemicals in cranberry.

2. Arctostaphylos uva-ursi (Bearberry).

Bearberry is a North American shrub, the leaves of which have ethnobotanical use for treating lower urinary tract infections (Yarnell, 2002). Bearberry contains tannins, which have astringent effects and can help to shrink and tighten mucous membranes, reducing inflammation and fighting infection. The antimicrobial compound could be arbutin, which is hydrolyzed in the gut to produce glucose and hydroquinone that can be further oxidized to para-quinone (Fig. 19) (de Arriba et al., 2013). Because hydroquinone/quinone is carcinogenic and tannins can cause nausea and vomiting, low doses of bearberry (800 mg arbutin/day) are recommended for less than 1 week at a time. Currently there are no clinical trials on bearberry leaf alone to support the efficacy claims for treating UTIs.

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Fig. 19.

Phytochemical in bearberry and its metabolites.

1. Zingiber officinale (Ginger).

Ginger might be an effective treatment of nausea associated with pregnancy, although the clinical data have been mixed (Kennedy et al., 2013; Giacosa et al., 2015; Marx et al., 2015). In general, ginger extracts have been shown to accelerate gastric emptying (promotility, prokinetic) and stimulate gastric antral contractions (Micklefield et al., 1999; Wu et al., 2008b). In addition, four randomized controlled clinical trials on the use of ginger for pregnancy-induced nausea and vomiting showed that orally administered ginger was significantly more effective than placebo at reducing frequency and intensity of nausea (Ding et al., 2013). The current literature suggests that ginger is safe in pregnant women; however, it is still not clear what the dose and length of treatment should be as well as the potential for drug/botanical interactions. GraviFrisk Ferrosan A/S (Søborg, Denmark), which contained a daily dose of 6 g dried ground ginger, was withdrawn from the Danish market in February 2008 because of inadequate scientific documentation of safety (Jacobsgaard, 2008). It is believed that the active compounds responsible for these biological effects are gingerols and shogaols (Fig. 15), which are cholinergic M3 and serotonergic 5-HT₃ receptor antagonists (Abdel-Aziz et al., 2006; Pertz et al., 2011). Gingerols, shogaols, and zingerone are metabolically unstable and are readily oxidized to phenoxy radicals and potentially quinones or quinone methides (Fig. 15), which could play a role in their chemopreventive activities (Gan et al., 2013; Mason and Thompson, 2014). The instability of these compounds could explain some of the varied activities reported in clinical trials. In general, the popular use of ginger for the relief of nausea during pregnancy is substantiated by a number of clinical trials that generally report efficacy relative to placebo and little toxicity (Dante et al., 2014). Similar results have been reported for ginger clinical trials for chemotherapy-induced nausea, motion sickness, and postoperative nausea (Dante et al., 2014).

1. Trigonella foenum-graecum (Fenugreek).

Fenugreek, which is a seed spice used to enhance flavor, color, and texture of food, also has several medicinal properties including galactagogue (lactation inducer) effects (Yadav and Baquer, 2014). It is the most commonly used herbal galactagogue, although there is conflicting reports of its efficacy (Mortel and Mehta, 2013). Fenugreek is thought to stimulate sweat production and because the breast is a modified sweat gland, it may stimulate breast milk production through this mechanism (Gabay, 2002). It has also been reported that fenugreek contains diosgenin (Fig. 20), which could modify the endocrine system enhancing milk production (Sreeja et al., 2010). Sugars of the flavonoids apigenin (Fig. 13) and luteolin (Fig. 20) have also been reported. In a well-designed clinical trial of 66 mother-infant pairs randomly assigned to three groups; fenugreek tea, apple tea, and control, maximum weight loss was significantly lower in the infants and milk volume was higher in the mothers receiving fenugreek tea (Turkyılmazet al., 2011). The research on the health benefits of fenugreek including its use as a galactagogue and its lack of side effects suggest potential as a dietary supplement (Yadav and Baquer, 2014).

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Fig. 20.

Phytochemicals in fenugreek.

2. Silybum marianum (Milk Thistle).

The most common use of milk thistle supplements is for liver detoxification (Greenlee et al., 2007). Galactagogue activity of milk thistle has also been reported and its use is increasing (Forinash et al., 2012; Zapantis et al., 2012). It has been described in an in vivo study that milk thistle and specifically, silymarin (Fig. 21), a mixture of flavonolignans and some precursor flavonoids contained in milk thistle, increased prolactin levels in female rats and that this might lead to a galactagogue effect (Forinash et al., 2012). In an in vivo study with gestational pigs, feeding of 4 g silymarin twice daily for 20 days resulted in significant increase of circulating prolactin levels (Farmer et al., 2014). In addition, some parameters of mammary gland development changed through silymarin treatment; however, the increase in prolactin was not sufficient to have beneficial effects on mammary gland development in late gestation. The influence of silymarin on the amount of milk formation was not analyzed in this study. One placebo-controlled clinical trial analyzed silymarin’s effectiveness as galactagogue in women (Forinash et al., 2012). Although an increase in milk formation was observed in this small clinical trial, this study had several limitations, which warrants further examination of milk thistle as a galactagogue in larger clinical investigations. The estrogenic activity of milk thistle and of silymarin was analyzed in different studies with varying results. Some studies describe weak estrogenic activities for silymarin and especially for its major flavonolignan, silybin B, and for quercetin and taxifolin (Pliskova et al., 2005) (Fig. 21). One 12-week in vivo study showed that silymarin exerted estrogenic effects, such as increase of uterine weight and reduction of bone loss in ovariectomized rats (El-Shitany et al., 2010). A 1-week in vivo study using a lower dose of silymarin also showed an influence on osteoblast parameters but did not observe a uterotrophic effect for silymarin (Seidlova-Wuttke et al., 2003). Silymarin is reported to mainly exert estrogenic activity through the ERβ receptor (Seidlova-Wuttke et al., 2003); however, it is controversial whether silibin B (El-Shitany et al., 2010) or other compounds contained in silymarin, such as taxifolin and quercetin, are responsible for the described ERβ-selective effect (Powers and Setzer, 2015). In general, the safety and efficacy of milk thistle as a galactagogue has not been analyzed in detail and further studies are warranted.

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Fig. 21.

Phytochemicals in milk thistle.

1. Serotonergic Mechanism.

Estrogen withdrawal during menopause results in a decline in the release of neurotransmitters, primarily norepinephrine and serotonin (5-hydroxytryptamine; 5-HT), leading to a change in thermoregulation in the hypothalamus (Shanafelt et al., 2002) resulting in hot flashes and night sweats. Increase in the amount of serotonin and activating certain 5-HT receptors as well as inhibition of serotonin reuptake in synapses through blocking of serotonin transporters with prescription antidepressant SSRIs are viable approaches in preventing hot flashes (Fig. 22). However, there are also a number of undesirable outcomes such as nausea, sexual dysfunction, weight gain, and sleep disturbances associated with these remedies that have led women to consider natural botanicals with potential serotonergic efficacy (Nachtigall, 2010; Kintscher, 2012; Hajirahimkhan et al., 2013a).

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Fig. 22.

Serotonergic mechanism. In synapses, botanicals can directly act on the serotonin receptors (5-HT receptors) or reduce serotonin reuptake through inhibiting serotonin transporters (SERTs) to elicit a reduction in menopausal symptoms such as hot flashes.

2. Estrogenic Mechanism.

Botanicals with estrogenic activity can mimic the effects of endogenous estrogens lost during menopause (Hajirahimkhan et al., 2013a). Estrogens often activate biological responses through binding to estrogen receptors (ERs) including ERα and ERβ, followed by dimerization of ERs and interaction with estrogen responsive elements (EREs) at the promoter of the estrogen responsive genes, thus activating transcription and generating estrogenic effects that are crucial for normal physiological functions (Fig. 23) (Nilsson et al., 2001; Bjornstrom and Sjoberg, 2005; Ellmann et al., 2009; Jia et al., 2015). It is believed that ERα induction is associated with the proliferative effects of estrogens, whereas ERβ opposes the ERα-dependent proliferation (Deroo and Buensuceso, 2010; Shanle and Xu, 2010; Thomas and Gustafsson, 2011; Jia et al., 2015). Botanicals with estrogenic effects, particularly the ones with ERβ selectivity, may offer safer alternatives to conventional prescription HT regimens.

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Fig. 23.

Hormonal pathway. ERα and ERβ are activated by estrogens or phytoestrogens that cause dimerization of these receptors and translocation to the nucleus. Activation of ERE-responsive genes by ERα increases proliferation yet reduces menopausal symptoms and osteoporosis. On the other hand, ERβ decreases proliferation, while also decreasing menopausal symptoms and osteoporosis. Additionally, many botanicals reportedly decrease aromatase expression/activity to reduce synthesis of estrogen. This figure was simplified to represent only the classic estrogen signaling pathway.

a. Isoflavones.

i. Glycine max (Soy).

Soy is one of the major sources of protein in Asian diets in particular (Low Dog, 2005; Taylor, 2015). Isoflavones in soy foods are mainly present as glycosides (i.e., genistin, daidzin), which are hydrolyzed by β-glucosidases in the intestine forming the estrogenic ERβ-selective aglycones, genistein and daidzein (Fig. 24) (Lin and Giusti, 2005; Lopez-Gutierrez et al., 2014; Taylor, 2015). It has also been reported that daidzein metabolism by the intestinal microflora results in the formation of S-equol, which has better estrogenic activity and a higher affinity for ERβ than daidzein (Setchell et al., 2005; Jiang et al., 2013) (Fig. 24). Different anaerobic bacteria can reduce daidzein to S-equol through three different reductase enzymes and one dihydrodaidzein racemase (Shimada et al., 2011; Lee et al., 2016). Genetic polymorphism in equol production has been demonstrated, which in part may explain the variability in relieving menopausal symptoms (Hong et al., 2012).

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Fig. 24.

Isoflavones in red clover, soy, and kudzu. Genistein and daidzein are both found in soy and red clover; however, they are significantly more abundant in soy. Daidzein is metabolized by the intestinal microflora to S-equol.

Most clinical trials have used dosages ranging from 50 to 100 mg/day isoflavones (Low Dog, 2005). The North American Menopause society has concluded that soy-based isoflavones are modestly effective in relieving menopausal symptoms (North American Menopause Society, 2011). A meta-analysis of the use of soy dietary supplements for hot flashes showed a statistically significant decrease in hot flashes, although the clinical efficacy is questionable (Bolanos et al., 2010). A review published by the Cochrane Collaborative on 43 randomized controlled trials concluded that there was no conclusive evidence that soy supplements reduced menopausal hot flashes (Lethaby et al., 2013). Fortunately, no estrogenic effects on the endometrium or vagina were reported in these studies with up to 2 years of use. The poor bioavailability of genistein and daidzein from soy may explain the variable and relatively modest if any effects on menopausal symptoms (Yang et al., 2012; Rodriguez-Morato et al., 2015).

ii. Trifolium pratense (Red Clover).

Unlike soy, the major isoflavones in red clover are the proestrogens biochanin A and formononetin (Fig. 24) (Booth et al., 2006). These methoxy precursors are demethylated by cytochrome P450 isozymes in the intestine and liver to the estrogenic ERβ-selective genistein and daidzein, respectively (Tolleson et al., 2002). These data suggest that red clover isoflavones might have higher bioavailability compared with soy and could explain why the red clover clinical trials tend to show more efficacy for menopausal symptoms compared with those evaluating soy products (Piersen et al., 2004; Thomas et al., 2014). A meta-analysis of four clinical trials using the red clover extract marketed as Promensil (Novogen Ltd, Hornsy, Australia) showed a slight but statistically significant reduction in hot flashes (Lethaby et al., 2013). In addition, there was no increase in endometrial thickness or breast density, suggesting a lack of estrogenic activity in hormone-sensitive tissues. A recent trial of 72 women randomly divided between placebo and 40 mg dried red clover daily for 12 weeks showed a significant reduction in menopausal symptoms as measured by the menopause rating scale (Shakeri et al., 2015). Other clinical trials have not shown a significant difference from placebo, particularly for hot flash relief (Geller et al., 2009; Hall et al., 2011).

iii. Pueraria lobata (Kudzu).

Kudzu is used in traditional Chinese medicine for the treatment of menopausal symptoms (Hajirahimkhan et al., 2013a). The major isoflavone in kudzu is puerarin, which is the C8-glucoside of daidzein (Guerra et al., 2000; Delmonte et al., 2006) (Fig. 24). Puerarin is metabolized to daidzein by intestinal bacteria (Park et al., 2006; Nakamura et al., 2011). The same isoflavonoids found in red clover and soy are also found in kudzu in lower amounts (Delmonte et al., 2006; Zhang et al., 2009). A randomized controlled clinical trial (127 women, 50–65 years old, 3 months) receiving kudzu standardized to 100 mg isoflavones, HT, or no treatment showed no significant changes compared with control (Woo et al., 2003; Ulbricht et al., 2015).

iv. Eriosema laurentii (Guinea-Bissau).

Guinea-bissau is a botanical found in Cameroon traditionally used for infertility and menopausal symptoms (Ntie-Kang et al., 2013). Phytoestrogens isolated from guinea-bissau include ERα-selective lupinalbin A (Fig. 25) and ERβ-selective genistein (Fig. 24) (Ateba et al., 2014). No clinical trials have been reported on the use of guinea-bissau for menopausal symptoms.

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Fig. 25.

Isoflavone in guinea-bissau.

b. Humulus lupulus (hops).

The strobiles of hops are often added to beer to produce bitter, tart, hoppy flavors. Hop extracts are also a popular botanical for sleep-inducing effects, especially in Europe (Blumenthal and German Federal Institute for Drugs and Medical Devices Commission, 2000; Zanoli and Zavatti, 2008; Salter and Brownie, 2010). Hop extracts are also present in some dietary supplements for managing menopausal symptoms (Taylor, 2012). The most potent phytoestrogen in hops is the ERα-selective agonist 8-prenylnaringenin (8-PN), which is in part formed from the chemopreventive isoflavone xanthohumol through cyclization and P450 catalyzed demethylation as shown in Fig. 26 as well as through metabolism of isoxanthohumol by microbiota (Nikolic et al., 2005; Chadwick et al., 2006; Possemiers et al., 2006). A few clinical trials have been published evaluating the efficacy of hops extracts on menopausal symptoms (Taylor, 2015). A randomized placebo controlled trial with 67 menopausal women over 12 weeks receiving hops extract standardized to 100 or 250 μg 8-PN or placebo showed significant effects at 6 weeks for the lower dose. However, no effect was observed at higher doses, and the lower dose was not significantly different from placebo after 12 weeks as measured by the Kupperman index and quality of life questionnaire (Heyerick et al., 2006). The authors noted the relatively low effective dose of ERα-selective 8-PN, which is 100-fold more potent than the ERβ-selective isoflavones genistein and daidzein (Milligan et al., 1999; Overk et al., 2008). However, given the potent ERα agonistic activity of 8-PN in hops extracts, safety with respect to the effect on the endometrium and other hormone sensitive tissues needs further study. In a smaller study (randomized, double-blind, placebo-controlled, crossover study, 36 menopausal women), women receiving either placebo or hops extract standardized to 8-PN for 8 weeks and then switching treatments for 8 weeks showed weak efficacy of the extract over placebo when the extract was taken after the placebo treatment until week 16 (Erkkola et al., 2010). However, these results might not be robust, considering the small sample size and large number of variables included in the extensive statistical analysis (Taylor, 2015). A more recent trial using a daily dose of 500 mg hops in tablet form versus placebo (120 women, 90 days) showed a statistically significant reduction in menopausal symptoms and a dramatic reduction in hot flashes (Aghamiri et al., 2016). However, there are several problems with this trial including, the lack of standardization of the extract, the recruitment of women from multiple sites, and the lack of a placebo effect. No adverse effects were reported in any of these trials. More clinical trials are needed before the safety and efficacy of hops extracts for menopausal symptom relief can be established.

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Fig. 26.

Phytochemicals in hops.

c. Glycyrrhiza species (licorice).

Licorice is a widely used plant, mainly as a sweetening agent in tobacco, in food and beverages, and in toothpastes. It consists of more than 30 species, from which a few have been studied for several biological effects (Asl and Hosseinzadeh, 2008; Hajirahimkhan et al., 2013b). Licorice is commonly present in menopausal botanical supplements in the United States (Taylor, 2012). There are three pharmacopeial licorice species, Glycyrrhiza uralensis, G. glabra, and G. inflata; however, the specific species used is often not identified or mislabeled in supplements. The estrogenic activities of different licorice species are variable and likely depend on the type and amounts of bioactive compounds, emphasizing the importance of fully standardizing extracts using chemical, genetic, and biological methods (Liu et al., 2001; Hajirahimkhan et al., 2013a). After evaluating a large number of licorice samples that were local to certain regions of the world Kondo et al. (2007a) reported that G. uralensis and G. glabra had the highest and the lowest amounts of the phytoestrogen liquiritigenin (Fig. 27), respectively. Liquiritigenin was reported to be a selective ERβ agonist in the ER binding assay and in ERβ-ERE-luciferase activity assay in U2OS cells (Mersereau et al., 2008). In vivo liquiritigenin did not enhance proliferation of MCF-7 (ERα+) xenograft or induce uterine weight in nude mice, suggesting liquiritigenin is unlikely to affect hormone sensitive tissues (Mersereau et al., 2008). Isoliquiritigenin, the precursor chalcone of liquiritigenin was also shown to exhibit estrogenic effects (Fig. 27) (Maggiolini et al., 2002). However, the conversion of isoliquiritigenin to liquiritigenin could play a significant role in exerting the observed activity (Hajirahimkhan et al., 2013b). There have been very few clinical studies evaluating licorice for the treatment of menopausal symptoms. In a double-blind placebo-controlled trial, a significant drop in hot flashes was reported with 90 women receiving licorice 330 mg/day or placebo for 8 weeks (Nahidi et al., 2012). However, no mention about the species of licorice extract was available and no placebo effect was observed. Another recent trial with six women compared the efficacy of licorice extracts (1140 mg/day) for 90 days with traditional HT for hot flash relief claiming similar efficacy (Menati et al., 2014). Both trials have significant problems ranging from small size, lack of extract standardization, self-reporting surveys, lack of placebo group, and lack of placebo effect, which can be as high as 50% in menopause trials. More in-depth studies are needed to define the efficacy and safety of licorice for menopausal symptoms.

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Fig. 27.

Phytochemicals in licorice.

d. Rheum rhaponticum (rhubarb).

Rhubarb is also a common herb for menopausal symptom relief, particularly in Germany (Vollmer et al., 2010; Taylor, 2012; 2015). A special extract prepared from the roots of Rheum rhaponticum called ERr731 [marketed under the trade name Phytoestrol N (Mueller Goeppingen, Goeppingen, Germany)] has been in use in Germany for decades without any significant side effects such as endometrial hyperplasia, spotting, or breakthrough breeding (Wober et al., 2007). This extract mainly consists of rhaponticin and desoxyrhaponticin, which are converted to the resveratrol-like aglycones rhapontigenin and desoxyrhapontigenin by the microbiome and glycosylases (Fig. 28) (Kim et al., 2000; Wober et al., 2007). Weak ERβ activity has been measured for both the extract and the aglycones rhapontigenin and desoxyrhapontigenin in transfected HEC-1B adenocarcinoma cells and U2OS cells (Moller et al., 2007; Wober et al., 2007). Rhapontigenin is more active than desoxyrhapontigenin and it is tempting to suggest that P450-catalyzed O-demethylation giving the resveratrol catechol piceatannol might be responsible for the estrogenic activity (Wober et al., 2007). It has been shown that piceatannol binds estrogen receptors and stimulates the growth of estrogen-dependent cancer cells (Piotrowska et al., 2012). The efficacy and safety of ERr731 for menopausal symptoms has been studied in a number of double-blind placebo-controlled trials with positive results (Heger et al., 2006; Kaszkin-Bettag et al., 2008; Hasper et al., 2009). All these trials show that the rhubarb extract was well tolerated both for short duration (12 weeks) as well as long term (up to 2 years), successfully decreased the Menopause Rating Scale, and increased quality of life.

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Fig. 28.

Phytochemicals in rhubarb and their metabolites.

f. Linum usitatissimum (flaxseed).

Flaxseed is a major source of lignans that are metabolized by the intestinal flora into the weakly active phytoestrogens enterolactone and enterodiol (Fig. 29) (Dew and Williamson, 2013). Lignans reside in cell walls and are not bioavailable without crushing, and as a result the best sources for women are flaxseed flour and flaxseed meal. Flaxseed oil is a good source of α-linolenic acid but does not contain lignans (Taylor, 2015). In a randomized placebo-controlled clinical trial (90 women, 1 g/day flaxseed extract), modest but significant effects were observed in self-reporting relief of menopausal symptoms (Colli et al., 2012). No adverse effects related to flaxseed treatment were reported. A meta-analysis of recent randomized clinical trials examining the efficacy of flaxseed for menopausal symptom relief concluded that there is little evidence to support the use of this dietary supplement for menopause or for bone health (Dew and Williamson, 2013).

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Fig. 29.

Phytochemicals in flaxseed.

g. Epimedium species (horny goat weed).

Horny goat weed originating from different Epimedium species has been used in traditional Chinese medicine for over 2000 years to enhance libido in men and women as well as for symptoms of menopause and PMS (Ma et al., 2011). The flavonoid icariin can be metabolized to icaritin and desmethylicaritin, which is likely responsible for the mostly ERα-selective estrogenic bioactivities (Fig. 30) (Wang and Lou, 2004; Cao et al., 2012; Xiao et al., 2014). These prenylated flavonoids are structurally similar to the potent ERα estrogen 8-PN in hops (Fig. 26, see above). One clinical trial showed that an extract of horny goat weed could increase estrogen levels and improve lipid profiles in postmenopausal women without any significant side effects (Yan et al., 2008). No clinical trials using horny goat weed for menopausal symptoms have been reported.

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Fig. 30.

Phytochemicals in horny goat weed.

h. Lepidium meyenii (maca).

Maca has been used in South America for centuries for infertility and female hormone balance, especially for menopausal symptoms (Lee et al., 2011b; Gonzales, 2012; Taylor, 2015). Maca extracts have been shown to have estrogenic activity by increasing MCF-7 cell proliferation (Valentova et al., 2006), although the active phytoestrogens are not known. Maca extracts contain a number of phytosterols including beta-sitosterol and campesterol (Fig. 31) as well as free fatty acids including linolenic acid (Fig. 17) (Dini et al., 1994). A systematic review of clinical trials concluded that there was limited evidence for the effectiveness of maca for relief of menopausal symptoms; however, the sample size, the number of trials, and the quality of the trials were too limited to establish firm conclusions on efficacy and safety (Lee et al., 2011b).

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Fig. 31.

Phytochemicals in maca.

j. Medicago sativa (alfalfa).

Alfalfa is another botanical that is a good source of isoflavones. The coumestan derivative coumestrol (Fig. 32) is the major estrogenic phytochemical, although the licorice flavonoids liquiritigenin and isoliquirtigenin have also been detected (Fig. 27) (Hong et al., 2011). No clinical trials for menopause using alfalfa alone have been reported.

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Fig. 32.

Phytochemical in alfalfa.

3. Unknown Mechanism.

1. Isoflavones.

Because most isoflavones are ERβ-selective ligands, it should be possible to exploit this selectivity and target mainly bone cells without adversely affecting hormone-sensitive tissues such as the uterus and the breast. Genistein (Fig. 24) has been shown to stimulate bone formation, inhibit bone resorption, and prevent bone loss in ovariectomized rat models (Taylor et al., 2009; Fu et al., 2014). It has been demonstrated in a few randomized, double-blind placebo-controlled studies that genistein (54 mg/day for 1 or 2 years) was effective in preventing bone loss in postmenopausal women (Morabito et al., 2002; Marini et al., 2007). Favorable effects have been demonstrated in other clinical studies measuring bone mass density and bone turnover markers; however, the long-term effects on prevention of fractures have not been established (Lagari and Levis, 2014; Poluzzi et al., 2014). The data suggest that diets rich in isoflavones are beneficial for bone health in the long term, although the magnitude of the effects and the exact mechanism of how isoflavones could protect bones have not been determined. There is also evidence to suggest that the positive effects of isoflavones on bone health could vary as a function of age. Isoflavones could be more effective in younger women before loss of estrogen receptors observed in postmenopause (Reinwald and Weaver, 2006). Most clinical trials with soy proteins and enriched soy isoflavones were not effective in decreasing bone loss, in particular at common fracture sites in healthy perimenopausal and postmenopausal Western women (Alekel et al., 2010). Different studies have also analyzed red clover products for prevention of osteoporosis. A patented aqueous and fermented red clover extract standardized to 37.1 mg isoflavones per day or a placebo solution were administered to women suffering from menopausal symptoms in a 3-month, randomized, double-blind and parallel designed trial (Thorup et al., 2015). A significant decline in the bone mineral density of the lumbar spine (lower back) in the placebo group but not in the red clover group was observed, suggesting a positive effect on bone health by the red clover extract. However, it has to be noted that the actual changes were small and that the differences could also be due to the variability in bone density scanning. Studies with bisphosphonate therapy showed that reliable treatment effects can be best observed after 3 years, because the within-person variations differ widely with dual-energy X-ray absorptiometry measurement (Sharma and Stevermer, 2009). Therefore, longer clinical studies with red clover would be necessary to evaluate the efficacy as a treatment of osteoporosis. Until more rigorous clinical trials are completed, soy foods and/or soy or red clover supplements cannot be recommended as alternatives for antiosteoporotic medications (Messina et al., 2004; Nieves, 2013; Poluzzi et al., 2014).

2. Others.

The evidence supporting the use of other botanicals for prevention of osteoporosis is very limited. Maca prevents bone loss in rat models of estrogen-deficient bone loss (Zhang et al., 2006). Wild yam and fenugreek have effects on bone mineral density in ovariectomized rats (Chiang et al., 2011; Folwarczna et al., 2014). Silymarin has also been studied as remedy against osteoporosis in animal models (Milic et al., 2013). Horny goat weed has a long history in treating osteoporosis in traditional Chinese medicine, which was recently reviewed (Ma et al., 2011; Indran et al., 2016).

1. Hormonal Pathway.

In vitro studies revealed that isoflavones, such as genistein (IC₅₀ = 3.6 μM) and biochanin A (IC₅₀ = 25 μM), are moderate inhibitors of the estrogen synthesizing enzyme aromatase (van Meeuwen et al., 2007). More potent aromatase inhibitors have been designed from the isoflavone scaffold (Bonfield et al., 2012). Genistein and daidzein (Fig. 24) have also been reported to inhibit other steroidogenic enzymes in the micromolar range, such as 3β-hydroxysteroid dehydrogenase (CYP21), which might ultimately result in lower estradiol levels (Hasegawa et al., 2013). However, other investigations describe genistein as an inducer of aromatase expression/activity in extragonadal cells, which might lead to enhanced local estrogen concentrations (Edmunds et al., 2005; Ye et al., 2009a; van Duursen et al., 2013). Genistein, daidzein, and S-equol are preferential ERβ agonists with nanomolar potency, but at higher concentrations, they have ERα activities (Jiang et al., 2013). Because ERβ reduces ERα-induced cell proliferation, the safety of these isoflavones likely depends on their concentrations. In the micromolar range, genistein is a selective protein tyrosine kinase inhibitor, which might lead to reduction of cell growth through inhibition of core signaling tyrosine kinases, such as epidermal growth factor receptor, insulin receptor, and others (Russo et al., 2016). For example, in an ERβ-positive breast cancer cell line, genistein inhibited human epidermal growth factor 2 (HER2) overexpression and HER2 phosphorylation activity, likely through inhibition of its tyrosine kinase activity at concentrations >1 μM (Sakla et al., 2007; Russo et al., 2016). A recent study illustrated the distinct proliferative effects of genistein on different breast cancer cell types (Pons et al., 2016). In cell lines mainly expressing ERα, genistein lead to cell proliferation, whereas in ERβ-expressing cells, genistein treatment had no effect. These studies illustrate that genistein has different effects on the hormonal pathway depending on concentration, ER status, and breast cell type. This might explain some of the conflicting results obtained in breast cancer cells or in animal experiments studying estrogen sensitive tissues.

Several animal and clinical studies analyzed the effect of isoflavones on endometrial and mammary tissue with controversial outcomes (Dietz and Bolton, 2013). Some in vivo experiments suggest that genistein suppresses mammary carcinogenesis (Constantinou et al., 2001; Sahin et al., 2011); however, many recent in vivo investigations found that it promotes carcinogenesis in breast tissue and enhances metastasis formation (Ju et al., 2006; Yang et al., 2015). Three animal studies demonstrated that genistein stimulates mammary gland growth and enhances the growth of breast cancer cells (MCF-7) in animals (Hsieh et al., 1998; Allred et al., 2004; Ju et al., 2006). These reports show that isoflavones of red clover/soy have the potential to reduce and/or enhance the hormonal-related carcinogenic effects of endogenous estrogens. However, the activity of the whole red clover extract or soy supplementation might be distinct from the activity of the isolated isoflavones. For example, uterotrophic activities have been described for genistein (Diel et al., 2004a) but not for the red clover extract (Overk et al., 2008).

Most clinical trials suggest that isoflavones in dietary supplements are safe and do not show estrogenic effects on breast or endometrial tissues. For example, in one clinical trial that used a standardized (26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein) red clover extract in women (age 49–65 years), the extract did not increase breast density over 1 year (Atkinson et al., 2004). A clinical study (up to 3 years) with postmenopausal women suggested that purified genistein (54 mg/day) is safe with respect to breast and endometrial proliferation (Taylor et al., 2009). In contrast to the placebo group, the genistein arm showed a statistically significant decrease in endometrial thickness at the 36-month follow up. Genistein also significantly decreased sister chromatid exchanges in peripheral blood lymphocytes isolated from postmenopausal study participants after administration of 54 mg/day genistein for 1 year in a randomized placebo-controlled study (Atteritano et al., 2008). Soy isoflavone dietary intervention studies with premenopausal women showed no toxicity over a period of 2 years with no effect on breast density as analyzed by mammography (Taylor et al., 2009). Similarly, a randomized, double-blind, placebo-controlled study revealed that consumption of 250 mg of standardized soy extract (100 mg/day isoflavones) by postmenopausal women for 10 months did not influence breast density (Delmanto et al., 2013). The correlation of plasma genistein and daidzein concentrations with breast cancer or benign breast conditions was analyzed in a breast self-examination trial in Chinese women. The subjects included women diagnosed with breast cancer or benign breast conditions and age-matched controls with no known breast disease (Lampe et al., 2007). The results revealed that plasma daidzein and genistein concentrations were significantly higher in controls than in patients with breast cancer or benign breast conditions. These data may indicate selective uptake of the phytoestrogens by ER-positive tumors; however the overall chemopreventive effect of isoflavones on breast cancer is unknown.

However, some clinical studies depict an estrogenic or proliferative effect after consumption of isoflavone-containing dietary supplements. For example, a 2-week dietary supplementation study with soy (45 mg/day) in premenopausal women revealed an estrogenic effect as measured by modulation of estrogen-sensitive genes in nipple aspirate fluid (Taylor et al., 2009). A similar study with soy protein supplementation in premenopausal women suggested an estrogenic effect of soy due to an increase in proliferation of breast epithelium and an enhanced expression of progesterone receptors (Taylor et al., 2009).

A 6-month intervention study using a preparation of mixed soy isoflavones (150 mg genistein, 74 mg daidzein) was performed in healthy, high-risk adult Western women to examine the effect of these isoflavones on breast epithelial proliferation and other biomarkers in the breast tissue (Khan et al., 2012b). The study demonstrated that the isoflavones did not reduce breast epithelial proliferation, suggesting a lack of efficacy for breast cancer prevention. However, among treated premenopausal women, there was a statistically significant increase in the postintervention Ki-67 labeling index after soy supplementation. Gene expression studies demonstrated a mixed pattern, with more genes enhanced in the isoflavone supplementation group that suggested estrogenic activity. Further clinical studies with different concentrations are warranted to obtain a better picture of the hormonal and proliferative activities of isoflavone containing dietary supplements on breast tissue.

2. Chemical Pathway.

Isoflavones have been shown to modulate different estrogen metabolism enzymes through a variety of pathways in in vitro and in vivo studies (Mense et al., 2008; Snelten et al., 2012). They modulate the P450 enzymes, P450 1A1 and P450 1B1, through inhibition of enzyme activity and through induction or suppression of their transcription (Wagner et al., 2008; Takemura et al., 2013). Genistein has been demonstrated to decrease P450 1A1/1B1 activity in part through direct inhibition of the enzyme in several assays in the low micromolar range (Roberts et al., 2004; Mense et al., 2008; Scott et al., 2008; Takemura et al., 2013). Similarly, daidzein and equol have been reported to reduce P450 1A1 and 1B1 activity in different cell assays (Roberts et al., 2004; Scott et al., 2008).

CYP1B1 and CYP1A1 are both regulated through the AhR (Fig. 34) (Murray et al., 2014). Two AhR transactivation assays revealed biochanin A and formononetin as good AhR ligands (nM–μM range), whereas genistein, daidzein, and equol had no activity (Medjakovic and Jungbauer, 2008; Bialesova et al., 2015). Biochanin A was confirmed as an AhR ligand in a TCDD competitive AhR binding assay (Han et al., 2006). Biochanin A also induced the xenobiotic response element (XRE) activity in the breast cancer cell line MCF-7 (Chan et al., 2003), and at higher concentrations (10–50 μM), it caused a significant induction of CYP1A1 mRNA expression and CYP1 activity (Han et al., 2006), suggesting that biochanin A can increase oxidative estrogen metabolism. In the presence of a strong AhR agonist DMBA, biochanin A (≥5 μM) inhibited DMBA-induced XRE activity, CYP1A1 and CYP1B1 mRNA expression, as well as DMBA-induced P450 1 EROD activity in MCF-7 cells suggesting that biochanin A can act as an AhR agonist (Chan et al., 2003; Han et al., 2006).

Interestingly, genistein and daidzein reduced CYP1A1 mRNA levels and P450 1 activity at low concentrations in MCF-7 cells (Wagner et al., 2008). Because estradiol has been reported to downregulate CYP1A1 (L'Heritier et al., 2014), the effect of genistein and daidzein on CYP1A1 might be explained by their estrogenic effects. At the tested concentrations, CYP1B1 was not influenced by genistein and daidzein. Higher genistein concentrations (5–25 μM) were shown by Wei et al. (2015) to induce CYP1B1 mRNA levels. Genistein also inhibited DMBA-induced XRE-luciferase activity, P450 1 activity, CYP1A1 and 1B1 mRNA expression, and DNA adducts in the micromolar range in MCF-7 and MCF-10A cells (Chan and Leung, 2003; Leung et al., 2009). Only a limited number of in vivo studies have analyzed the influence of isoflavones on estrogen metabolism enzymes with different outcomes. One study analyzing the influence of the mixture of isoflavones on CYP1B1 and CYP1A1 in primate mammary glands and liver tissue did not show any significant effect (Scott et al., 2008). However, significant induction of CYP1A1 was observed after treatment of male Wistar rats with a standardized soybean extract containing 37% isoflavones (100 mg/kg) for 10 days (Mrozikiewicz et al., 2010).

Isoflavones have also been shown to modulate detoxification enzymes that facilitate the elimination of estradiol and its metabolites (Fig. 35) (Dietz and Bolton, 2013). Experiments in human colon cancer cells revealed that genistein (≥0.1 μM) caused significant induction of NQO1 activity, whereas biochanin A was less effective and daidzein and formononetin were inactive (Wang et al., 1998). Genistein induced NQO1 in various cell lines as well as in different tissues in animal studies (Wang et al., 1998; Bianco et al., 2005; Froyen et al., 2009; Wiegand et al., 2009). Mechanism of action studies in ER-positive cell lines demonstrated that genistein might influence NQO1 through ER-dependent and -independent pathways (Bianco et al., 2005; Wagner et al., 2008; Kaspar and Jaiswal, 2010). It has been shown that genistein (1 μM) and daidzein (10 μM) reduced NQO1 mRNA levels and catechol-O-methyltransferase mRNA and activity similar to estradiol in MCF-7 cells likely through an ERα-Nrf2-mediated mechanism (Wagner et al., 2008). However, in ERβ-positive cells, the isoflavones upregulated NQO1 mRNA through an ERβ-Nrf2-mediated mechanism (Bianco et al., 2005; Froyen and Steinberg, 2011). These variable results suggest that the NQO1 induction potential of genistein may be dependent on the cell type and ER status.

The influence of isoflavones on other detoxification enzymes might be less pronounced. Although genistein induced hepatic NQO1 activity, it did not induce the activity of other detoxification enzymes (glutathione-S-transferase, catalase, glutathione peroxidase, superoxide dismutase) (Wiegand et al., 2009). However, in a topical inflammation model, in which 1 or 2 μg of a soy isoflavone product was administered, the activity of the detoxification enzymes catalase and superoxide dismutase were induced, as well as glutathione levels (Khan et al., 2012a). An in vivo study in Sprague-Dawley rats (7 days) analyzed the effect of biochanin A on the Phase II enzyme sulfotransferase that is involved in the metabolism of estradiol and detoxification of various carcinogens (Raftogianis et al., 2000; Chen et al., 2010b). The study revealed that biochanin A (10 mg/kg/day) induced the expression and activity of sulfotransferase 1A1 and 2A1 in female rat liver and intestine.

In conclusion, the isoflavones have the potential to modulate estrogen metabolism. Biochanin A and formononetin seem to be AhR agonists and have the highest potential to induce P450 1A1/1B1 (Han et al., 2006; Bialesova et al., 2015). However, in vivo, biochanin A and formononetin are rapidly metabolized to genistein and daidzein, respectively (Fig. 24), and their concentration is likely relatively low (Piersen et al., 2004). Soy supplements contain much less biochanin A and formononetin than red clover extracts (Burdette and Marcus, 2013) and might therefore have a better safety profile with respect to estrogen metabolism. In general, the influence of genistein on the chemical pathway is probably cell and ER-status dependent. Therefore, more in vivo studies are warranted to analyze whether soy and red clover would reduce or enhance genotoxic estrogen metabolism.

3. Inflammatory Pathway.

Anti-inflammatory activities have been described for soy, red clover, and their isoflavones in in vitro and in vivo studies (Banerjee et al., 2008; Seibel et al., 2009; Masilamani et al., 2012; Nagaraju et al., 2013; Ganai et al., 2015). For example, a red clover extract (40% isoflavones) and isolated isoflavones reduced the proinflammatory cytokines IL-6 and TNF-α in lipopolycaccharide (LPS)-induced macrophages (Mueller et al., 2010). Biochanin A exhibited the strongest TNF-α inhibition (IC₅₀ = 6 nM), followed by genistein (IC₅₀ = 40 nM), whereas IL-6 was most efficiently inhibited by genistein and equol (IC₅₀ = 8 and 6 nM, respectively). Other studies reported the reduction of inflammatory cytokines by genistein, such as IL-4, IL-5, interferon-γ, in a murine model of asthma (Gao et al., 2012).

Various studies demonstrated the ability of isoflavones to reduce NF-κB pathways in different cell lines in the micromolar range (Gong et al., 2003; Kole et al., 2011; Masilamani et al., 2012; Wu et al., 2015b). Further analysis revealed that one mechanism through which genistein might reduce nuclear translocation of NF-κB is likely the inhibition of kinase pathways leading to a reduced phosphorylation of the NF-κB inhibitory protein IκBα (Fig. 36) (Gong et al., 2003; Masilamani et al., 2012; Ganai et al., 2015) or to a decreased phosphorylation of the NF-κB subunit p65 (Chung et al., 2014). Similarly, biochanin A has been shown to inhibit LPS-induced NO, IL-6, IL-1β, and TNF-α production in macrophages through prevention of phosphorylation and degradation of IκBα (Kole et al., 2011). In certain cell lines, a role for the ER in inhibiting NF-κB pathways has been postulated (Sas et al., 2012) and that isoflavones, such as genistein, might reduce inflammatory responses through their ER binding activity (Dijsselbloem et al., 2004).

Various in vitro and in vivo studies demonstrated that isoflavones repress or inhibit COX-2 activity in the micromolar range (Lam et al., 2004; Horia and Watkins, 2007; Seibel et al., 2009; Mueller et al., 2010; Chung et al., 2014). Comparison of the COX-2 inhibiting potential of the four isoflavones (Fig. 24) in murine macrophages showed genistein had the highest activity at 1 μM compared with 10 μM for biochanin A and 40 µM for formononetin and daidzein (Lam et al., 2004). Genistein (1 μM) also significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced prostaglandin E2 (PGE2) synthesis in ER negative breast cancer cells (MDA-MB-231) (Horia and Watkins, 2007). In addition, it significantly reduced the invasion capacity of this cell line at 10 μM, suggesting a chemopreventive potential of genistein in ER negative breast cancer cells. Pretreatment of human nontumorigenic breast epithelial cells (MCF-10A) with genistein (≈46 μM) reduced TPA-induced COX-2 expression and PGE2 synthesis through reduction of TPA-induced transcriptional activity of NF-κB (Chung et al., 2014). Genistein also reduced COX-2 expression in animal models of inflammation (Hwang et al., 2009; Willenberg et al., 2015). Because COX-2 is reported to be upregulated in breast cancer and epidemiological studies suggest a protective effect of COX inhibitory drugs on breast cancer (Howe, 2007), further in vivo studies are needed to determine if long-term treatment with isoflavone containing dietary supplements might modulate COX-2 in the breast tissue.

In a 2-year soy intervention study, 183 premenopausal women consumed either two daily soy servings containing 50 mg of isoflavones (aglycone equivalents) or their regular diet (Maskarinec et al., 2009). Serum samples were obtained in regular intervals and were analyzed for the inflammatory markers IL-6, C-reactive protein, leptin, and adiponectin by enzyme-linked immunosorbent assay. No significant effect of the soy food was determined; however, the leptin value increased in the control but not in the soy group. In a single-blind, randomized, controlled trial, 31 postmenopausal women received either three servings of soy milk or reduced fat dairy milk for 4 weeks (Beavers et al., 2009). Plasma markers of inflammation and oxidative stress (TNF-α, IL-6, IL-1β, COX-2, superoxide dismutase, and glutathione peroxidase) were determined before and after supplementation; however, no significant effects of the soy milk intervention were observed for any of these markers.

In conclusion, although there are clear anti-inflammatory activities reported for isoflavones from in vitro studies, the in vivo experiments and clinical trials analyzing the inflammatory pathways are less conclusive (Beavers et al., 2009; Maskarinec et al., 2009; Willenberg et al., 2015). More clinical trials will be necessary to evaluate the anti-inflammatory potential of isoflavones, soy, and red clover dietary supplements for their potential clinical application.

4. Epigenetic Pathway.

Recent studies indicated that isoflavones might exert cancer chemopreventive effects by targeting epigenetic mechanisms (Guo et al., 2015a; Hwang and Choi, 2015). Various in vitro studies demonstrated that genistein suppresses DNA methylation in breast cancer cells, possibly leading to increased mRNA expression of tumor suppressor genes (Guo et al., 2015a). For example, genistein (Fig. 24) has been shown to decrease mRNA levels and protein expression of DNA methyltransferase 1 (DNMT) 1 in MCF-7 cells. DNMT 1 has been reported to be upregulated in breast cancer tissue (Mirza et al., 2013). Also, genistein, has been reported to reactivate ERα expression and resensitize ERα-dependent cellular responses to E₂ in ERα-negative breast cancer cells, MDA-MB 231, in the micromolar range through epigenetic effects (Li et al., 2013c). In addition, genistein and daidzein in the micromolar range decreased hypermethylation of the oncosuppressor genes, tumor suppressor gene 1 and tumor suppressor gene 2, resulting in restored activity of these genes in different breast cell lines (Bosviel et al., 2012). Treatment with a genistein-enriched diet (250 mg/kg genistein) inhibited breast cancer development in a breast cancer xenograft model perhaps due to epigenetic regulation of the tumor suppressor genes p21 and p16 (Li et al., 2013a).

Isoflavones might have protective effects toward breast carcinogenesis when a diet rich in soy products is consumed early in life (Warri et al., 2008). This is in part based on the protective effect of a lifelong high-soy diet on breast cancer in Asian women. On the basis of this information, an in vivo study analyzed the influence of lifelong exposure to an isoflavone-rich diet on mammary gland proliferation, other estrogen sensitive endpoints, and on epigenetic markers (Blei et al., 2015). The authors concluded that isoflavones might alter the morphology of the mammary gland during pubertal breast growth and epigenetic modulation might play an important role. These effects might ultimately have an influence on the likelihood of breast carcinogenesis later in life; however, further studies are necessary to support this theory. In general, epidemiological studies suggest that soy consumption is associated with less breast cancer risk in Japanese women with an enriched soy diet (Yamamoto et al., 2003); however, the correlation between isoflavone intake and breast cancer risk is less clear in American women. The association between dietary isoflavone intake with breast cancer risk was analyzed using population-based, observational data from the Multiethnic Cohort Study (MEC) in 84,450 mostly postmenopausal American women, who were categorized based on their isoflavone intake (highest consumed amount: 20.3–178.7 mg/day) (Morimoto et al., 2014). After a mean follow up of 13 years, no statistical association between dietary isoflavone intake and overall breast cancer risk was observed. This study is in agreement with other epidemiological prospective studies suggesting that high soy intake in Asian women (around 25 and 50 mg of isoflavones) starting earlier in life shows a significant trend of breast cancer risk reduction; however, studies with Western women consuming a low soy diet do not show a significant association between dietary isoflavone exposure and breast cancer risk (Wu et al., 2008a; Dong and Qin, 2011).

In conclusion, numerous studies have shown that isoflavones, in particular genistein, might modulate estrogen carcinogenesis through hormonal, chemical, inflammatory, and epigenetic pathways. The data analyzing the influence of isoflavones on cell proliferation (hormonal) and on estrogen metabolism enzymes (chemical) reveal conflicting results, suggesting that isoflavones might enhance or decrease estrogen carcinogenesis through both hormonal and chemical pathways. The concentrations and cell type may play an important role whether isoflavones reduce or increase estrogen carcinogenesis. There is clear evidence mostly from in vitro studies that isoflavones, in particular genistein, have anti-inflammatory properties that might decrease estrogen carcinogenesis in the breast. However, dietary in vivo studies show conflicting results and it is not clear whether the bioavailability of genistein and of the other isoflavones in dietary supplements are sufficient to exert significant clinical anti-inflammatory activity. Epigenetic effects with subsequent reactivation of tumor suppressor genes and other genes have been shown (e.g., genistein). Some in vivo studies propose that lifelong exposure to isoflavones might modulate mammary gland development during puberty, potentially leading to breast cancer prevention. Overall, clinical studies often indicate no breast cancer preventive effect with low isoflavone intake in the diet during adulthood in Western cultures. However, some studies show a protective effect when isoflavones are enriched in the diet throughout life as in the case of Asian populations. Future animal and clinical studies are needed to determine if isoflavones are safe and whether they have the ability to elicit chemopreventive properties at clinically relevant concentrations.

1. Hormonal Pathway.

As outlined in section IV.A.2.b, 8-PN (Fig. 26) is a potent nanomolar ERα agonist (Overk et al., 2005; Helle et al., 2014). Various in vitro and in vivo studies demonstrate proliferative activities on breast cells/tissue or uterotrophic activities (Diel et al., 2004b; Overk et al., 2008). These proliferative activities might enhance hormonal estrogen carcinogenesis and may interfere with concurrent breast cancer treatments (van Duursen et al., 2013). However, XH has been reported to have antiproliferative activities in the micromolar range in several cancer cell lines (Vanhoecke et al., 2005; Yoshimaru et al., 2014). For example, in ERα-positive cells, XH suppressed E₂ signaling through reactivation of the tumor suppressor protein prohibitin 2 (Yoshimaru et al., 2014). XH has also been reported to inhibit cell invasion through upregulation of the epithelial cadherin/catenin complex in MCF-7 cells (Vanhoecke et al., 2005). Because the concentration of XH in hops extracts is usually much higher than that of 8-PN (van Breemen et al., 2014), XH might reduce the proliferative activities of 8-PN in the whole hop extract. For example, a hops extract showed estrogenic activities in vitro; however, in vivo studies did not reveal uterotrophic activities, suggesting that either the bioavailable 8-PN concentrations were not high enough or that the extract may contain various counteracting phytoconstituents such as XH or phytoprogestins (Overk et al., 2008). In general, 8-PN is a minor compound in hops, but it can accumulate in vivo through metabolism of IX by P450s or intestinal bacteria (Fig. 26) (Possemiers et al., 2006; van Breemen et al., 2014). The overall estrogenic properties are therefore highly dependent on the composition of the hops extract and the individual metabolism and duration of treatment in vivo.

Aromatase inhibition has also been reported for 8-PN (300 nM) in microsomes of human placental tissue or fibroblasts from healthy human mammary tissue (van Meeuwen et al., 2008). Similarly, in a human adrenal gland cell line, H295R, 8-PN was a potent aromatase inhibitor with an IC₅₀ of 0.1 μM (van Duursen et al., 2013). Interestingly, coculture of H295R cells with MCF-7 cells led to decreased estradiol concentrations and predominantly to an increased progesterone and 17α-OH-progesterone level. In breast cancer cells, 8-PN showed the highest aromatase inhibiting activity compared with IX and XH with an IC₅₀ of 0.08 μM (Monteiro et al., 2007). XH inhibited aromatase with a lower IC₅₀ of 3.2 μM, and IX showed the weakest activity (IC₅₀ = 25.4 μM).

2. Chemical Pathway.

Several hop polyphenols (Fig. 26) have been analyzed for their potential to inhibit P450 1A1 and 1B1, which are involved in estrogen metabolism (Henderson et al., 2000). Several of these compounds showed significant inhibitory potential for these P450 enzymes at 10 μM. However, 8-PN was also able to moderately induce CYP1A2 mRNA levels (Gross-Steinmeyer et al., 2009). Recently, P450 1A1 and 1B1 inhibiting potential has been confirmed for IX, 8-PN, XH, and 6-PN with IC₅₀ values in the high nanomolar to low micromolar range (Wang et al., 2016). 8-PN and 6-PN have also been shown to be agonists for the AhR receptor, resulting in an increase in AhR-mediated XRE-luciferase activity in HepG2 and MCF-7 cells. 6-PN also gave a significant increase in CYP1A1 gene expression, P450 1 activity, and estrogen 2-hydroxylation in MCF-10A and MCF-7 cells (Fig. 34). These data suggest that hops favors induction of the nongenotoxic 2-hydroxylation pathway in breast cells (Wang et al., 2016). Another study reported that a hop extract inhibited estrogen-induced malignant cell transformation of MCF-10A cells, suggesting that hops might have chemopreventive activity potentially through attenuation of the genotoxic estrogen metabolism pathway (Hemachandra et al., 2012).

Hops and its major Michael acceptor, XH, also influence detoxification enzymes. They have been shown to induce NQO1 activity in murine hepatoma cells, leading to prevention of menadione-induced DNA damage at low micromolar concentrations (Dietz et al., 2005a). XH has also been shown to increase the levels of Nrf2, GSTA, and GSTP in human hepatocarcinoma HepG2 cells, whereas in normal hepatocytes it increased the levels of Nrf2, GSTs, HO-1, and NQO1 in addition to p53 (Krajka-Kuzniak et al., 2013). XH also upregulated the transcription of NQO1 and HO-1 in mouse microglial cells via the Nrf2 pathway (Lee et al., 2011a). The in vivo NQO1-inducing potential by hops and XH has been confirmed in liver tissue of Sprague-Dawley rats (Dietz et al., 2013). These data suggest the potential of hops and its abundant Michael acceptor, XH, in modulating cytoprotective enzymes leading to chemoprevention.

3. Inflammatory Pathway.

Various in vitro and in vivo studies describe anti-inflammatory activities for hop extracts (Saugspier et al., 2012; Starkel et al., 2015) and XH (Hougee et al., 2006; Lupinacci et al., 2009; Dorn et al., 2012). Lupinacci et al. (2009) reported that a hop extract (0.1 μg/ml) significantly inhibited the production of the proinflammatory chemokine monocyte chemoattractant protein-1 in LPS-activated RAW 264.7 mouse macrophages. Bioassay guided fractionation of the extract revealed XH (2.5 μg/ml) as the major anti-inflammatory compound (Lupinacci et al., 2009; Dietz and Bolton, 2013). XH (micromolar range) inhibited the activation or binding of the transcription factor NF-κB in macrophages, glia cells, hepatic stellate cells, or breast cancer cells, thus reducing the induction of proinflammatory genes and cytokines (Harikumar et al., 2009; Cho et al., 2010; Lee et al., 2011a; Dorn et al., 2012). Studies in mouse microglial cells showed that the anti-inflammatory activity of XH depended on the transcription factor Nrf2, in addition to NF-κB (Lee et al., 2011a). Other mechanism of action studies showed that XH might inhibit TNF-induced IkBα phosphorylation and degradation, likely through interacting with cysteine residues of IKK and p65, thus leading to suppression of nuclear translocation of p65 and inhibition of NF-κB pathways (Harikumar et al., 2009). In vivo anti-inflammatory activities have also been reported for XH (Dietz and Bolton, 2013; Gupta et al., 2014). In a chronic dermatitis model in the mouse ear, topical XH treatment reduced the degree of ear thickening induced by oxazolone, suggesting that XH reduces skin inflammation (Cho et al., 2010). In two animal models of liver injury and inflammation, feeding of XH reduced mRNA levels of hepatic inflammatory markers (e.g., IL-1α, monocyte chemoattractant protein-1) (Dorn et al., 2012, 2013). The mechanism seemed at least in part through decrease of NF-κB activity (Dorn et al., 2012). Interestingly, oral administration of XH (100 μM) to nude mice inoculated with MCF-7 cells resulted in a significant decrease in NF-κB activity, reduced phosphorylated IκBα staining, and a decrease in the inflammatory interleukin IL-1β, which is often increased in breast cancer (Monteiro et al., 2008). Although the tumor weights between the control and XH group were not significantly different, XH did significantly increase the amount of apoptotic cells and decreased microvessel density in the tumors (Monteiro et al., 2008). A spent hops extract, supplemented to the diet of 5-week-old pigs for 4 weeks, also led to a significant decrease of the inflammation marker IL-1β in the duodenum, ileum, and colon (Fiesel et al., 2014).

COX-2 is another inflammatory marker in breast cancer that has been shown to be inhibited by many phytochemicals. COX-2 inhibition has been demonstrated for a hop extract and XH (Gerhäuser et al., 2002; Hougee et al., 2006). A hops CO₂ extract inhibited PGE2 production in LPS-stimulated peripheral blood monocytes (IC₅₀ = 3.6 μg/ml) likely through inhibition of COX-2 (IC₅₀ = 20.4 μg/ml) (Hougee et al., 2006). Mice treated with this hop extract showed decreased production of PGE2 in a zymosan-induced acute arthritis model; however, the extract did not reduce joint swelling (Hougee et al., 2006). XH, but not IX, moderately inhibited COX-1 and COX-2 activity in microsomal fractions (IC₅₀ = 16.6 μM and 41.5 μM, respectively) (Gerhäuser et al., 2002), suggesting that other hop compounds might add to the COX inhibiting activity of hops extracts.

4. Epigenetic Pathway.

The influence of hops or its compounds on epigenetic pathways has not been examined in detail. One study analyzed protein targets of the electrophile XH in the breast cancer cell line MCF-7/6 (Wyns et al., 2012). Immunoprecipitation experiments using a mouse-XH-antibody revealed that XH binds to proteins of the histone H2A family. Therefore, XH may exert epigenetic effects through modification of histones that could influence gene expression, potentially leading to chemopreventive effects.

In summary, the in vitro and in vivo studies suggest that hops may modulate estrogen carcinogenesis. 8-PN elicits estrogenic, proliferative, and aromatase inhibiting activities in the nanomolar range; however, other hops phytochemicals might counteract these proliferative activities. The CYP inhibiting activity of 8-PN might also influence estrogen metabolism, although much higher concentrations are needed (micromolar) for these effects, which may not be clinically relevant. 6-PN has been shown to be a potent inducer of P450 1A1, which catalyzes the benign estrogen 2-hydroxylation pathway leading to lower estrogen levels. Hops and XH have been shown to exert chemopreventive activity in vitro and in vivo through the Keap1-Nrf2 pathway, resulting in the induction of detoxification enzymes such as NQO1 and HO-1. Anti-inflammatory activities of hops and XH have been reported in vitro and in vivo through modulation of NF-κB, COX-2, and prostaglandins. Hops and XH might have the potential to exhibit chemoprevention via epigenetic mechanisms, which needs further studies. Although a human pharmacokinetic study demonstrated the bioavailability of the hops compounds 8-PN and XH (van Breemen et al., 2014), clinical studies are needed to analyze the chemopreventive activities of hops. It is also important to define whether the concentrations of the bioactive compounds in standardized hop dietary supplements are sufficient to achieve chemopreventive effects in women.

1. Hormonal Pathway.

It has been shown that extracts of Glycyrrhiza glabra and G. uralensis stimulated MCF-7 (ERα) cell growth at low concentrations (Dong et al., 2007; Hu et al., 2009). In contrast, G. uralensis was reported to have antiproliferative effects in MCF-7 breast cancer cells at higher concentrations (Jo et al., 2004, 2005). The estrogenic compound liquiritigenin did not enhance proliferation of MCF-7 (ERα) xenograft or induce uterine weight in nude mice (Mersereau et al., 2008), likely because of its ERβ selectivity. The marker compound in G. glabra, glabridin (Fig. 27), was shown to stimulate ER-dependent cell growth at concentrations lower than 10 µM and to inhibit cell growth at concentrations higher than 15 µM (Tamir et al., 2000). Selective estrogen receptor modulator-like activities of isoliquiritigenin and liquiritigenin have also been reported in an array of cell based assays (Boonmuen et al., 2016). Finally, isoliquiritigenin inhibited aromatase in MCF-7 cells stably transfected with CYP19 (MCF-7aro) and deterred the growth of MCF-7aro xenograft tumors in ovariectomized athymic mice when administered in the diet (Ye et al., 2009b).

2. Chemical Pathway.

Licorice species and its bioactive compounds have been shown to differentially modulate estrogen metabolism and thus may have varied effects on breast cancer prevention. It was shown that G. glabra and G. uralensis as well as isoliquiritigenin increased the genotoxic estrogen 4-hydroxylation pathway through enhanced AhR-mediated induction of P450 1B1 activity in MCF-10A cells (Dunlap et al., 2015). However, in the same study G. inflata and its specific compound licochalcone A (Fig. 27) inhibited estrogen metabolism by antagonizing AhR and blocking XRE at the promoter region of CYP1B1 (Dunlap et al., 2015). When DMBA or TCDD were used as the AhR agonists in MCF-7 cells, isoliquiritigenin reduced AhR translocation to the nucleus and diminished AhR interaction with XREs leading to downregulation of DMBA and TCDD-mediated induction of CYP1A1, 1A2, and 1B1 (Wong et al., 2014). Similarly, G. glabra extract suppressed the tumorigenic effects of TCDD by downregulating AhR, arylhydrocarbon receptor nuclear translocator, and CYP1A1 genes and inducing cell cycle arrest in MCF-7 cells (Chu et al., 2014). These data suggest that different species of licorice could have a variety of effects on estrogen metabolism, which further emphasizes the importance of authentication of material and standardization to bioactive compounds.

Various licorice species have also been shown to differentially modulate detoxification pathways. G. uralensis was reported to induce Nrf2-mediated genes in hepatoma cells and animal tissues (Wu et al., 2011). In a comparative analysis, G. glabra, G. uralensis, and G. inflata induced NQO1 dose dependently in murine hepatoma (hepa1c1c7) and breast epithelial (MCF-10A) cells (Hajirahimkhan et al., 2015). However, when G. glabra and G. uralensis were administered orally to female rats, no NQO1 induction was observed in liver, whereas G. glabra had a slight but significant NQO1 induction in mammary tissue (Hajirahimkhan et al., 2015). It was shown that the common Michael acceptor of licorice species, isoliquiritigenin activated NQO1 dose dependently in vitro but failed to induce this enzyme in liver and mammary glands of the female rats when administered orally (Hajirahimkhan et al., 2015). Other studies showed slight increase in NQO1 at very high doses of isoliquiritigenin, but the effect did not lead to the inhibition of mammary tumor growth (Cuendet et al., 2006, 2010). These observations with isoliquiritigenin could be associated with its conversion to the estrogenic compound liquiritigenin as discussed earlier and also to the low bioavailability of isoliquiritigenin in vivo (Hajirahimkhan et al., 2013b, 2015; Simmler et al., 2013). Licochalcone A in G. inflata is also a Michael acceptor and has been shown to induce NQO1 in hepa1c1c7 and MCF-10A cells (Kondo et al., 2007a; Hajirahimkhan et al., 2015; Simmler et al., 2015a). These data suggest that various species of licorice could differentially modulate the chemical estrogen carcinogenesis pathway.

3. Inflammatory Pathway.

Licorice has been traditionally used for several ailments such as coughs or ulcer that could be associated with inflammation (Asl and Hosseinzadeh, 2008). There are also numerous scientific reports about the anti-inflammatory effects of licorice and compounds in different organs such as lung, skin, nervous system, liver, and cardiovascular system to name a few (Chu et al., 2013; Dohil, 2013 Kim et al., 2013b;; Kang et al., 2015; Yu et al., 2015). The role of licorice in preventing breast cancer through modulating inflammatory pathways has also been studied. Comparative evaluation of three medicinally used licorice species showed that G. inflata followed by G. glabra and G. uralensis inhibited iNOS activity in macrophage cells. In this study, isoliquiritigenin and licochalcone A strongly blocked iNOS activity, suggesting the defining role of these two compounds in the anti-inflammatory effects of licorice species (Dunlap et al., 2015). Isoliquiritigenin was also shown to modulate COX-2 and iNOS and reduce PGE2 and nitric oxide levels and also exert antiproliferative chemopreventive effects in vitro and in vivo (Takahashi et al., 2004; Lau et al., 2010). Isoliquiritigenin has been shown to block colitis-associated tumorigenesis in animal models after 12 weeks of treatment through the suppression of PGE2 and interleukin-6 (IL-6) (Zhao et al., 2014). It was also reported that isoliquiritigenin exhibited growth inhibition and apoptosis in MCF-7 and MDA-MB-231 cells as well as in MDA-MB-231-inoculated nude mice through downregulating arachidonic acid metabolism enzymes such as COX-2 and deactivating phosphatidyl inositol 3 kinase/protein kinase B pathway (Li et al., 2013b). Additionally low concentrations of isoliquiritigenin might have breast cancer therapeutic benefits via inhibiting p38, phosphatidyl inositol 3 kinase/protein kinase B, and NF-κB pathways (Lorusso and Marech, 2013; Wang et al., 2013). Isoliquiritigenin inhibited phorbol ester-induced COX-2 expression in MCF-10A cells by modulating the ERK1/2 signaling pathway (Lau et al., 2010). Glabridin was reported to exert chemopreventive effects by attenuating angiogenesis via inhibiting NF-κB/IL-6/signal transducer and activator of transcription 3 signaling pathway in MDA-MB-231 and Hs-578T cells (Mu et al., 2015).

4. Epigenetic Pathway.

Licorice species and their related compounds have not been well studied for their potential to modulate epigenetic factors in breast cancer. It has been suggested that glabridin inhibits cancer stem cell-like properties of human breast cancer cells via modulation of micro RNA signaling (Jiang et al., 2016). In summary, licorice species have the potential to protect women from breast cancer; however, more rigorous studies with well-characterized/standardized licorice extracts are needed before recommendations of long-term use for breast cancer chemoprevention.

1. Hormonal Pathway.

Weak estrogenic activities have been shown for silymarin in in vitro and in vivo studies [Silybum marianum (Milk Thistle)] (Seidlova-Wuttke et al., 2003; Pliskova et al., 2005; El-Shitany et al., 2010). The estrogenic activities of silymarin are at least in part mediated through the ERβ-receptor; silibin B, taxifolin, and quercetin show some weak ERβ estrogenic activity (Pliskova et al., 2005; El-Shitany et al., 2010; Powers and Setzer, 2015), although other studies reported no estrogenic activities for taxifolin and quercetin (Jefferson et al., 2002; Resende et al., 2013). The effect of dietary silymarin against mammary carcinogenesis was tested in a N-methyl-N-nitrosourea-induced Sprague-Dawley rat model. In contrast to the expected protective effect, the authors reported a modest increase in the number of N-methyl-N-nitrosourea-induced mammary tumors, suggesting a cautionary note for breast cancer prevention (Malewicz et al., 2006). Finally, silymarin has also been reported to inhibit aromatase activity with an IC₅₀ of 6.7 μg/ml (Jeong et al., 1999); however, it is not known which constituents of silymarin are responsible for the aromatase inhibiting activity.

2. Chemical Pathway.

Several studies report the antioxidant properties of silymarin that prevent toxin-induced liver diseases and might also block estrogen-induced genotoxicity (Vargas-Mendoza et al., 2014). Strong inhibition of recombinant P450 1A1 has been shown for dehydrosilybin (Fig. 21) (IC₅₀ = 0.43 μM), whereas silibinin only moderately inhibited the P450 1A1 activity (IC₅₀ = 23 μM) (Dvorak et al., 2006). Silymarin also reduced benzo(a)pyrene (BaP)-induced hepatic P450 1A/1B activity in an in vivo toxicity model (Kiruthiga et al., 2015). In addition, silymarin enhanced NQO1, sulfotransferase activities, and glutathione levels, leading to a reduction of BaP-induced toxicity in this animal model. There have been no reports on the effects of milk thistle or its bioactive compounds on estrogen metabolism.

3. Inflammatory Pathway.

A number of studies support anti-inflammatory activity of silymarin and especially its major component, silibinin (Fig. 21) (Gazak et al., 2007; Deep and Agarwal, 2013). For example, silibinin treatment significantly reduced NF-κB activity, levels of IL-1β, and TNF-α in inflammatory colitis models (Esmaily et al., 2011). Silymarin also suppressed NF-κB activation by a variety of inflammatory agents in cell culture (Manna et al., 1999; Polyak et al., 2010; Kim et al., 2013a). Mechanism of action studies showed inhibition of phosphorylation of IκBα by silymarin, thus reducing NF-κB activation and nuclear translocation (Fig. 36) (Kim et al., 2013a). Downregulation of TPA-induced COX-2 by silibinin (around 50 μM) was also observed in two breast cancer cell lines (Kim et al., 2009). These results demonstrate that silibinin has several anti-inflammatory properties in cells, including breast cell lines as well as in vivo; however, further studies are necessary to determine if the high concentrations used in these studies will translate into clinical activity.

4. Epigenetic Pathway.

Silymarin has not been extensively studied for epigenetic effects and no studies in breast cancer models have been reported (Gerhauser, 2012; 2013). It has been reported that silibinin (80 or 160 mg/kg BW, gavage, for 5 weeks) reduced human hepatocellular xenograft growth, which was associated with increased histone acetylation of H3 and H4, indicating their potential role in hepatocellular carcinoma growth (Cui et al., 2009). In contrast, another study described silymarin (around 500 μM) as an activator of the deacetylase SIRT1 in human melanoma cells, (Li et al., 2007; Gerhauser, 2012). Because very high concentrations were used in these studies, their translation into clinical effects is questionable. In conclusion, several in vitro studies depict that silymarin and its constituents might influence breast cancer through the hormonal, chemical, inflammatory, and perhaps epigenetic pathways; however, it is not known yet whether these effects will translate into clinical effects. Also, more studies are needed to describe the exact bioactivities of individual silymarin compounds.

1. Hormonal Pathway.

As mentioned in section IV.A.3.a, whether dong quai has estrogenic activities is highly controversial (Liu et al., 2001; Lau et al., 2005; Circosta et al., 2006). Some studies describe proliferative activities of a dong quai extract in MCF-7 (ERα) cells (Amato et al., 2002; Lau et al., 2005) and in BT20 (ER-negative cells) (Lau et al., 2005) and an increase in uterine weight in female Wistar rats (Circosta et al., 2006). Chang et al. (2006) demonstrated that one of dong quai’s phytoconstituents, ferulic acid (100 nM) (Fig. 10), ER dependently stimulated MCF-7 breast cancer cell proliferation, likely through upregulation of ERα-mRNA and HER2 oncogene expression. However, other investigations did not observe estrogenic activities or even described antiestrogenic effects (Zhang et al., 2005; Godecke et al., 2012). Therefore, the proliferative or estrogenic activities of dong quai might be dependent on the kind of extract, and further studies are necessary to delineate the estrogenic properties of dong quai and its phytochemicals. Aromatase modulating effects have not been reported for dong quai.

2. Chemical Pathway.

Studies in breast cells are less known; however, phytochemicals of dong quai have been reported to modulate enzymes that are involved in estrogen metabolism. Z-ligustilide (5–50 μM) (Fig. 10) has been demonstrated to significantly inhibit BaP-induced CYP1A1 upregulation in human keratinocytes (Wu et al., 2014). Z-ligustilide (50 μM) did not activate or inhibit BaP-induced AhR translocation, but it induced Nrf2 translocation and transfection of si-Nrf2 abrogated the preventive effect of Z-ligustilide on BaP-induced CYP1A1. Therefore, the authors concluded that Z-ligustilide reduced BaP-induced CYP1A1 upregulation likely through a Nrf2-mediated mechanism (Wu et al., 2014). Z-ligustilide (5–10 μM) also induced Nrf2-dependent detoxification genes, such as heme-oxygenase and NQO1 in this cell line. Similarly, other studies demonstrated that lipophilic extracts of dong quai, such as the CO₂ supercritical fluid-extract, which is rich in Z-ligustilide, induced NQO1 protein and activity, ARE-luciferase activity, and Nrf2 mRNA in hepatoma cells (Saw et al., 2013). Z-ligustilide was the main compound responsible for this activity and doubled the NQO1 activity at a concentration of 6.9 ± 1.9 μM and had a chemopreventive index of 10 in hepatoma cells (Dietz et al., 2008). Mechanism of action studies showed that Z-ligustilide alkylated cysteine residues in Keap1, thus enhancing Nrf2 translocation and ARE-luciferase activity (Fig. 35). This suggests that dong quai extracts that are rich in Z-ligustilide have the potential to induce detoxification enzymes and therefore might increase the detoxification of genotoxic estrogen metabolites (Fig. 34).

3. Inflammatory Pathway.

Various in vitro and in vivo studies analyzed the inflammatory activity of dong quai and its phytochemicals. For example, the lipophilic supercritical CO₂ extract of dong quai (1.1–17 μg/ml), which is rich in Z-ligustilide, and Z-ligustilide itself (6.3–50 μM) suppressed LPS-induced NO production in macrophages (RAW 264.7 cells) (Saw et al., 2013). The extract (17 μg/ml) also significantly inhibited LPS-induced IL-1β in these macrophages. The volatile oil of dong quai (0.176 ml/kg BW) inhibited LPS-induced acute inflammation in a rat model (Li et al., 2016). Specifically, the oil decreased the levels of proinflammatory cytokines, such as TNF-α and IL-1β; inflammatory mediators, such as PGE2 and NO; the inflammation-related enzymes (iNOS and COX-2); and promoted the production of the anti-inflammatory cytokines IL-10. Z-ligustilide (5–25 μM), one of the most abundant ingredients in the volatile oil, also significantly suppressed the production of NO, iNOS, PGE2, and TNF-α in LPS-induced inflammation in macrophages (RAW 264.7) (Su et al., 2011). Mechanism of action studies demonstrated that Z-ligustilide decreased the phosphorylation of IKK, thus reducing the cytosolic degradation of IκBα and decreasing nuclear activation of NF-κB (Fig. 36). In addition to its anti-inflammatory effects, Z-ligustilide also significantly reduced reactive oxygen species in this cell line. Not only Z-ligustilide (IC₅₀ = 25 μM), but also another compound in dong quai, ferulic acid (IC₅₀ = 10 μM) (Fig. 10), inhibited NF-κB luciferase activity in LPS and interferon-γ-stimulated murine macrophages (Chao et al., 2010) as well as decreased hydrogen peroxide-induced IL-1β and TNF-α mRNA in porcine chondrocytes (Chen et al., 2010a). An ethyl acetate extract of dong quai, which contains both compounds as major constituents, significantly reduced inflammatory markers, such as TNF-α, in vitro (concentrations ≥5 μg/ml) and in vivo (1.56 mg/kg BW tube feeding for 1 week before LPS injection) inflammation models (Chao et al., 2010).

4. Epigenetic Pathway.

There are very few studies on the influence of dong quai and its constituents on epigenetic pathways. Z-ligustilide (50 μM) and the supercritical CO₂ fluid extract of dong quai (8.5 μg/ml) significantly decreased the relative amount of methylated DNA in the Nrf2 gene promoter region, thus leading to enhanced Nrf2 mRNA and protein expression as well as to upregulation of its downstream target genes in TRAMP C1 prostate cancer cells (Su et al., 2013; Guo et al., 2015b). In conclusion, several studies suggest that dong quai and its compounds, mainly Z-ligustilide and ferulic acid, might have the possibility to modulate estrogen carcinogenesis; however, conflicting data of dong quai on its effect of the hormonal pathway as well as limited data on its influence on estrogen metabolism and epigenetic pathways warrant further investigations before clinical studies can be performed.