Estrogen-Like Activity of Licorice Root Extract and its Constituents Jacob Vaya1*, Snait Tamir1, Dalia Somjen2 1 Laboratory of Natural Compounds for Medicinal Use, Migal – Galilee Technological Center, Kiryat Shmona 10200, Israel 2 Institute of Endocrinology, Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Key Words: licorice, phytoestrogens, depression, SAR, whitening * Corresponding author: Dr. Jacob Vaya Laboratory of Natural Compounds for Medicinal Use Migal – Galilee Technological Center, Kiryat Shmona 10200, Israel Fax: 972-4-6944980 Tel.: 972-4-6953512 E-mail: firstname.lastname@example.org 1 Table of Contents 1. Introduction 1.1 Estrogens 1.2 Studies on Structure Activity Relationships 1.3 Phytoestrogens 2. Estrogen-like Activity of Licorice Root Extract 3. Estrogen-like Activity of Licorice Root Constituents 3.1 Glabridin A. Binding of glabridin to ERα B. Effect of glabridin on breast cancer cells C. Effect of glabridin on cardiovascular cells D. In vivo effects of glabridin on female rat tissues 3.2 Glabrene and other constituents from the licorice root 4. The effect of glabridin derivatives on their ERs biding 5. Effects of Licorice Constituents on Cell Proliferation 6. Differential Effects of Glabridin and Glabrene on ERα and ERβ Expressions 7. Licorice Constituents Inhibit Serotonin Re-uptake 8. Whitening Effect of Licorice Extract and its Constituents 9. Summary 10. References 2 1. Introduction 1.1 Estrogens Estrogens are steroid hormones that exhibit a broad range of physiological activities. 17β-hydroxy estradiol is the female sex hormone active in developing the mammary gland and the uterus, maintaining pregnancy, relieving menopausal symptoms and preventing cardiovascular and bone diseases (Korach 1994). An apparent consequence of estrogen is the increase in short-term menopausal symptoms including vasomotor hot flashes, urogenetal atrophy and psychological functioning. A hot flash is the classic sign of menopause and the primary clinical symptom experienced by women during this transitional stage (Barton, Loprinzi et al. 2001). Estrogen is beneficial in reducing the risk of cardiovascular disease (Seed 1991; Iafrati, Karas et al. 1997; Sourander, Rajala et al. 1998). The incidence of heart disease among pre-menopausal women is low compared with males, whereas the incidence among post-menopausal women approaches that of males. The administration of estrogen to post-menopausal women decreases the incidence of heart disease (Stampfer, Colditz et al. 1991). This protective effect of estrogen may partially be attributed to its influence in decreasing the ratio between LDL and HDL (Shewmon, Stock et al. 1994), to reduction of thrombus formation and improvement in vascular compliance. Estrogen is known to be involved in osteoporosis (Ettinger, Genant et al. 1985), which affects more than 25 million women, causing some 250,000 hip fractures annually. Osteoporosis is characterized by a reduction in bone mineral density to the extent that a fracture may occur after minimal trauma. However, estrogen can also stimulate malignant growths and in this way contributes to the development of 3 estrogen-dependent tumors such as breast and uterus cancer(Russo and Russo 1998). Breast cancer is the most common malignancy among women in Western society and is the leading cause of death among American women aged between 40 and 55 years. The mechanism of estrogen action includes binding to the estrogen receptor in the target cell. The estrogen receptor complex is then translocated from the cytosol to the cell nucleus, where it binds to the DNA, and modulates the transcription rate of certain specific genes in the nucleus of the target cells. At present two estrogen receptors (ERs) are known, ERα and ERβ, which have different structure and tissue distributions (Katzenellenbogen, Sun et al. 2001). The biological effect of an ER ligand in a specific tissue is determined by the expression of ERα and ERβ in that tissue. The two key events controlling the tissue selectivity of an estrogen are the receptor’s shape and the interaction with adaptor proteins (Horwitz, Jackson et al. 1996; Smith, Nawaz et al. 1997). Compounds known as selective estrogen receptor modulators (SERMS) function as estrogen antagonists in some tissues and as agonists in others. For example, tamoxifen, an antagonist in breast tissue, is used to treat breast cancer and acts as an estrogen agonist in bone(Pritchard 2001), whereas raloxifene functions as an agonist in the bone, breast, and cardiovascular system but not in the uterus (Somjen, Waisman et al. 1996; Yang, Bryant et al. 1996). This discrepancy led to a search for new therapeutic agents such as phytoestrogens that would mimic specific activities of estrogen. Thus, compounds that inhibit the estrogen receptor in breast tissue or function like estrogen in non-reproductive tissues (such as bone and cardiovascular tissues) may be of therapeutic use. 4 1.2 Studies on Structure Activity Relationships Several studies have been carried out on the relationship between the structure of compounds and their estrogen-like activity (Egner, Heinrich et al. 2001; Katzenellenbogen, Sun et al. 2001). Although it is expected that the binding affinity of the ligand to the ER does not accurately indicate the biological activity of the ligand in vivo, receptor binding is still a requisite for the stimulation of biological activity. Wiese et atl., (Wiese, Polin et al. 1997) evaluated 42 analogs of estradiol for their ER binding affinity and toxicity to breast cancer cell lines. They tried to correlate the structure of these compounds with the above activities by means of 3-dimensional quantitative structure activity (QSAR), employing a comparative molecular field analysis (CoMFA). They concluded that additional structural characteristics to those responsible for tight receptor binding must be present to induce an optimal mitogenic response, such as steric factor interference in specific zones and electronegative and electropositive properties near position 3. Sadler et al., (Sadler, Cho et al. 1998) used the CoMFA method, which can visualize the steric and electrostatic features of the ligands corresponding to ER binding affinity. Using the above technique, 30 compounds sharing the trans-stilbene structure were examined and compared to information from the ER binding affinities of substituted estradiol analogs. This study demonstrated the importance of hydroxy substituents in non-steroidal ligands that mimic the 3-OH and 17-OH of estradiol to obtain a high binding affinity. Grese et al., (Grese, Cho et al. 1997) examined a series of raloxifene analogs in vitro and in vivo in which the 2-arylbenzothiophene substructure had been modified, measuring the reduction of serum cholesterol, uterine weight gain and uterine eosinophil peroxidase activity in an ovariectomized (OVX) rat model. In this study, they showed the importance of highly electronegative 4’-substituents, such as hydroxy or fluoro 5 attached to the raloxifene molecule, in their ability to bind to the receptor. They also showed that increasing steric bulk at position 4’ led to increased uterine stimulation in vivo and that additional substitutions at the 4-, 5-, or 7-position of the benzothiophene moiety resulted in reduced biological activity, while an additional substitution of the 2-aryl moiety had little effect. Shiau et al., (Shiau, Barstad et al. 1998) investigated the crystal structure of the human LBD complex with an agonist (diethylstilbestrol), together with a peptide derived from an ERα coactivator and the crystal structure of LBD with an antagonist (4-hydroxy tamoxifen). They showed that the peptide binds as a short α helix to a hydrophobic groove on the LBD surface in the complex with the agonist, while the binding of the antagonist promotes a helix 12 conformation inhibiting the binding of a coactivator. They concluded that two effects occur when the antagonist binds to LBD: a change in the position of the helix 12 so that it occupies part of the coactivator- binding groove; and a change in LBD conformation resulting from the interaction with the antagonist that stabilizes this conformation. These data suggest that the ligand structure will have a direct effect on the complex ER-ligand structure, which dictates the specific biological activities. Thus a search for natural and synthetic ligands that form complexes leading to tissue-specific beneficial effects is desired. 1.3 Phytoestrogens Phytoestrogens are naturally occurring ligands for the estrogen receptor that are derived from plants. They are part of the human diet and exhibit estrogen-like activity (Cassidy, Bingham et al. 1993; Tham, Gardner et al. 1998). Phytoestrogens include the subclasses of lignans, coumestans, isoflavones and isoflavans (Fig. 1) that are widely distributed in oilseeds (flax, cereals), vegetables, soybeans and roots. The main 6 mammalian lignans are enterolactone (II) and enterodiol (III) (Liggins, Grimwood et al. 2000), and coumestan coumestrol. The major food active isoflavonoids are genistein (IV) and daidzein (V) (Wiseman 2000) while the major isoflavan is glabridin (Vaya, Belinky et al. 1997). Epidemiological evidence indicates that soy intake (rich in isoflavonoids) is associated with lower breast cancer risk in women (Lee, Gourley et al. 1991; Fournier, Erdman et al. 1998). Genistein is reported to prevent cancellous bone loss and to maintain or increase bone density in post- menopausal women (Valente, Bufalino et al. 1994; Arena, Rappa et al. 2002). The effects of different phytoestrogens in a wide range of concentrations on estrogen receptor binding, PS2 induction (estrogen regulated antigen) and cell proliferation rate in human breast cancer cells were compared to the effects of estradiol. Phytoestrogens were shown to have weak estrogenic activity, ranging from 500-15,000 times less than estradiol (Zava, Blen et al. 1997; Bingham, Atkinson et al. 1998). Insert Figure 1 here 2. Estrogen-like Activity of Licorice Root Extract The phytoestrogenic activity of licorice root extract (Glycyrrhiza glabra L.) was tested among 150 other herbal extracts exerting a high ER binding affinity (Zava, Dollbaum et al. 1998) while others reported that it showed a low binding affinity (Liu, Burdette et al. 2001). Licorice root extract in combination with a mixture of other herbal extracts was reported to exert potent estrogenic activity in vitro in animals and in patients with prostate cancer (Rafi, Rosen et al. 2000), which attributed to Licochalcone A present in the extract. The estrogen-like effects of licorice extract in 7 vivo was tested in our laboratory (Tamir, Eizenberg et al. 2001). Ovariectomized female rats fed with licorice extract (25 µg/day/rat) or estradiol (0.5 µg/day/rat) for four weeks showed a significant increase in creatine kinase (CK) activity in the epiphysis, diaphysis, left ventricle of the heart, aorta, uterus and pituitary gland (Table 1). CK activity is known to be induced by estrogens in vivo and in vitro (Malnick, Shaer et al. 1983; Somjen, Waisman et al. 1998), and can therefore be used as an ER response marker. These results showed that at 0.5 µg/day/rat, estradiol stimulated CK activity at the same level as licorice extract at 25 µg/day/rat only in diaphyseal bone and the pituitary gland. A histomorphometric analysis of the diaphysis and epiphysis of the femoral bone showed similar effects of licorice and estradiol on the bone’s tracular volume and trabecular width, but not on the cartilage width or the growth plate height (Table 2). These results suggest that licorice extract is as active as estradiol in some parameters and may be safer for use. Additional in vivo experiments exceeding one month may better clarify the licorice extract potential. Insert Tables 1 and 2 here 3. Estrogen-like Activity of Licorice Root Constituents The known constituents of licorice were isolated from the aqueous extract, and included glycyrrhizin and its aglycone, glycyrrhetinic acid, which are used in the treatment of hyperlipaemia, atherosclerosis, viral diseases and allergic inflammation (Kimura, Okuda et al. 1993). The organic extract of licorice root (acetone or ethanol) is known to contain isoflavans, isoflavene and chalcones (Fig. 2) such as Glabridin, Glabrol, Glabrene, 3-Hydroxyglabrol, 4’-O-Methylglabridin 8 (4’-OMeG), Hispaglabridin A (hisp A), Hispaglabridin B (hisp B), Isoprenylchalcone derivative (IPC), Isoliquitireginin chalcone (ILC) and Formononetin (Saitoh and Kinoshita 1976; Mitscher, Park et al. 1980; Vaya, Belinky et al. 1997). Licorice root is one of the richest sources of a unique subclass of the flavonoid family, the isoflavans. We recently showed that glabridin, the major compound of this class having diverse biologically activities (see Aviram et al., in this chapter) and is present in the extract in more than 10% w/w, also exhibits estrogen-like activity (Tamir, Eisenberg et al. 2000; Tamir, Eizenberg et al. 2001). The isoflavans contain ring A fused to ring C connected to ring B through carbon 3 (Fig. 2). Several functional groups, mainly hydroxyl, may be attached to this basic skeleton. The heterocyclic ring C of the isoflavans does not contain a double bond between carbon 2 and 3, nor a carbonyl group attached to carbon 4. This structure does not allow a conjugation of the double bonds between rings A and B. The similarity of the glabridin structure and lipophilicity to that of estradiol (Fig. 2) encouraged us to investigate the subclass of isoflavans as a possible candidate for mimicking estrogen activity. In vivo studies testing the effects of licorice extract suggested that there may be more compounds in the extract contributing to its estrogen-like activity. This led us to identify other active constituents, such as glabrene and chalcones. Insert Figure 2 here 3.1 Glabridin 9 Among the licorice constituents isolated and tested, the most active phytoestrogen in vitro and in vivo is glabridin (Tamir, Eisenberg et al. 2000; Tamir, Eizenberg et al. 2001). Several features are common to the structures of glabridin and estradiol (Fig. 2). Both have an aromatic ring substituted with a hydroxyl group at para (glabridin) or position 3 (estradiol), with three additional fused rings of a phenanthrenic shape. Both are relatively lipophilic, containing a second hydroxyl group, although not at the same position (17β in estradiol and 2’ in glabridin). i. Binding of glabridin to the ERα Glabridin binds the ER with IC50 of 5 µM (Tamir, Eisenberg et al. 2000) and with approximately the same affinity as genistein, the best known phytoestrogen (Zava, Blen et al. 1997), 104 times lower than estradiol (Wang and Kurzer 1997) (Fig. 3). Insert Figure 3 here ii. Effect of glabridin on breast cancer cells Glabridin stimulated growth over a range of 0.1 – 10 µM, reaching a maximum level at about 10 µM; at a higher level (15 µM), it inhibited cell growth (Tamir, Eisenberg et al. 2000) (Fig. 4). Growth stimulation of ER (+) cells by glabridin closely correlated to its binding affinity to ER. The concentrations at which the proliferative effects of glabridin were observed are well within the reported in vitro range of other phytoestrogens, such as genistein, daidzein and resveratrol from grapes (Gehm, McAndrews et al. 1997; Wang and Kurzer 1997; Breinholt and Larsen 1998; Hsieh, Santell et al. 1998). 10 Using human breast cancer cells that do not express active ERs (MDA-MB-468) and cells that express active ERs (T47D) confirmed that this cell growth inhibition at a high concentration exhibits ER-independent behavior. Insert Figure 4 here iii. Effect of glabridin on cardiovascular cells Animal and human studies indicate that estrogens are protective against coronary atherosclerosis (Iafrati, Karas et al. 1997). Since endothelial and vascular smooth muscle cells are involved in vascular injury and atherogenesis, the potential modulation of such processes by estrogen and estrogen-like compounds is of obvious interest. Glabridin as an estradiol-induced, dose-dependent increase of DNA synthesis of human endothelial cells (ECV304) had a biphasic effect on the smooth human primary vascular smooth muscle cells (VSMC) (Table 3) (Somjen, Kohen et al. 1998). The inhibition of VSMC proliferation and the induction of ECV304 cell proliferation by either estradiol or glabridin, which are estrogen-mimetic, are beneficial in preventing atherosclerosis. Insert Table 3 here iv. In vivo effects of glabridin on female rat tissues Ovariectomized female rats fed with estradiol or glabridin for four weeks (Table 1) showed that 0.5 µg/day/rat of estradiol stimulated CK activity at the same level as 25 µg/day/rat of glabridin in all tissues tested. The histomorphological analysis suggests that glabridin is slightly more active than the licorice extract and is similar to estradiol (Table 2). The above effects of glabridin on estrogen-responsive tissues 11 suggest that it has the potential to mimic the beneficial activities of estrogen in bone and cardiovascular tissues, but also has a hazardous influence on the uterus. 3.2 Glabrene and other constituents from the licorice root Glabrene, an isoflavene and ILC that was isolated from organic extract, binds to the human estrogen receptor with about the same affinity as glabridin and genistein. The hisp A and B, two additional isoflavans in the licorice root, were barely inactive, whereas IPC, another chalcones was totally inactive. Glabrene and ILC showed ER- regulated growth-promoting effects such as glabridin (Fig. 4) and genistein. Glabrene produced dose-dependent transcriptional activation with half-maximal induction at 1 µM, corresponding to the concentration required for the inhibition of estradiol binding, and showed a maximum induction level similar to that achieved by 10 nM of estradiol. The administration of 25 µg/day/rat glabrene resulted in a similar effect to that of 5 µg/rat of estradiol in specific skeletal and cardiovascular tissues. Glabrene, glabridin and genistein all exhibited phytoestrogenic activity and are characterized by the connection of ring B to position 3 of the isoflavan and isoflavone, respectively. On the other hand, many compounds have a flavonol or flavonone structure whereby ring B is attached to carbon 2, are not active as phytoestrogen (such as quercetin, catechin, apigenin, etc.). This may emphasize the importance of the former structure for performing phytoestrogenic activity. Results also show that the glabrene structure, having a double bond between carbons 3 and 4, resembles that of trans-diphenyl stilbene, a structure critical for the antagonistic and agonistic activities of the two drugs, tamoxifen and raloxifene (Fig. 1). However, glabridin lacks this double bond in ring C but nonetheless demonstrated phytoestrogenic activity in vitro and in vivo, which may suggest that conjugated double bonds between ring A to ring B 12 are not essential for this activity. This phenomenon could be explained by the special structure containing ring C of the isoflavans, which fixed the position of rings A and B, similar to the effect of the double bond in trans-stilbene, thus enabling them to bind efficiently to the ER. Both chalcones of the licorice constituents tested, ILC and IPC, contain an α, β double bond, a hydroxyl at position 2’ (with two additional hydroxyls at positions 4 and 4’). However, only ILC, which does not contain the isoprenyl group, binds to the ER; IPC, containing two isoprenyl groups, was totally inactive on the other hand. 4. The effect of glabridin derivatives on their ERs biding Glabridin, which contains two hydroxyl groups at positions 2’ and 4’, has a higher affinity to ER and a stronger effect on cell growth stimulation than 2’-O-MeG and 4’-O- MeG. 2’,4’-O-MeG did not bind to the human estrogen receptor and showed no proliferative activity. This suggests that when both hydroxyl groups are free, binding and cell growth promotion are more pronounced. Previous reports on the involvement of the two hydroxyl groups of estradiol in binding to the human estrogen receptor showed that both hydroxyl groups 3 and 17β are required for binding (Brzozowski, Pike et al. 1997; Wiese, Polin et al. 1997). In glabridin, hydroxyl 4’ may play the same role as hydroxyl 3 of estradiol, forming hydrogen bonds with Arg 394 and Glu 353 in the binding site. Glabridin lacks the additional hydroxyl group of estradiol at position 17β but has ether oxygen in a parallel position (the γ-pyran ring), which could contribute to the interaction to histidin 524 in the ligand-binding domain. 5. Effects of Licorice Constituents on Cell Proliferation 13 In contrast to the ER-regulated growth-promoting phytoesrogenic effects of glabridin and glabrene in concentrations ranging from 100 nM – 10µM, higher concentrations abruptly inhibited the proliferation of ER positive and ER negative breast cancer cells. The most plausible explanation for this biphasic effect of glabridin and glabrene on human breast cancer cells is that it mediates its actions, not only via the ER as an estrogen agonist, but also by interacting at higher concentrations with other ER- independent cellular mechanisms to inhibit cell proliferation induced by glabridin via ER pathways. Antiproliferative effects of genistein were also observed in other non- breast carcinoma cell lines (Zhou, Mukherjee et al. 1998). The inhibited growth of ER negative cells by glabridin supports the hypothesis that the actions of phytoestrogens on cell growth inhibition occur via different molecular mechanisms (Peterson and Barnes 1996; Shao, Alpaugh et al. 1998; Shao, Wu et al. 1998). 6. Differential Effects of Glabridin and Glabrene on ERα and ERβ Expressions α β Estrogen is known to offer protection from coronary artery disease in post- menopausal women, to be involved in Alzheimer’s disease, and to inhibit oxidative stress-induced nerve cell death and apoptosis, which are implicated in a variety of pathologies including strokes and Parkinson’s disease. The existence of estrogen receptors in these cells and tissues, and the possibility that some of these estrogen effects are ER-dependent, led to the investigation of whether phytoestrogens, known to bind the estrogen receptor and exhibiting some estrogen-like activities, can also regulate the expression of ERs. 14 Results showed that the phytoestrogens glabridin and galbrene promoted ERα and ERβ expressions differently and in a cell-specific manner. ERβ was significantly increased in human breast cancer cells only after being exposed to estradiol and glabridin (two- to four-fold increase), while Vitamin D and glabrene inhibited ERβ expression in these cells. On the other hand, ERα was significantly increased in all treatments (estradiol – four-fold, Vitamin D – three-fold and glabridin – six-fold). Estradiol treatment inhibited ERβ in colon and melanoma cells, while glabrene significantly increased ERβ (two- to three-fold). Glabridin had no significant effect in these cell lines, which only exhibited ERβ. Vitamin D showed the same effect as estradiol on ERβ inhibition in colon cells but had the same stimulating effect on ERβ (two-fold) as glabrene in melanoma cells. These data suggest that phytoestrogens not only mimic the estradiol function as physiological regulators of ERα and ERβ expressions but also present tissue selectivity (by using studies on structure activity, we showed that they can also differentiate between receptors). They may also suggest that treatment using both estradiol and specific phytoestrogen may increase tissue sensitivity to estradiol, enabling fewer hormones to be used, thus leading to favorable effects of estradiol and a reduction in the deleterious effects. All of this may provide new insight into the ER- dependent protective action of estrogen and phytoestrogens in various post- menopausal diseases and contribute to the development of novel therapeutic treatment strategies. 15 7. Licorice Constituents Inhibit Serotonin Re-uptake – A Potential atural Treatment for Post-menopausal Depression An increase in the prevalence of depressive symptoms in women undergoing menopause can be related to fluctuating estrogen levels (Archer 1999). Depression in women seems to increase with a change in hormone levels (Avis, Crawford et al. 2001). The serotonergic system appears to play a major role in depression, although other neurotransmitters are also involved (Fuller 1994; Barker and Blakely 1995; Barton, Loprinzi et al. 2001). Serotonin is a neurotransmitter in the central and peripheral nervous systems (Fozzard 1989). Serotonin inactivation following its release is controlled by a specific re-uptake of the transmitter from the synaptic cleft into the presynaptic nerve terminal by the plasma membrane 5HT transporter (SERT3). Selective blockage of central nervous system SERTs in humans is the initial step in the pharmacological improvement of a wide variety of disorders, including major depression (Barker and Blakely 1995). The ability of steroids to modulate 5HT transport was investigated, and it has been shown that estradiol exhibits a nongenomic, possibly allosteric, inhibition of 5-HT serotonin transport (Chang and Chang 1999). Glabridin and 4’- OMeG were found to be the most effective inhibitors (60% inhibition) of licorice constituents of 5-HT uptake, expressing a slightly higher activity than that of glabrene (47% inhibition). The 2’-OMeG was totally inactive, proving the importance of hydroxyl 2’ for the serotonin re-uptake inhibition. 16 8. Whitening Effect of Licorice Extract and its Constituents The color of mammalian skin and hair is determined by a number of factors, the most important of which is the degree and distribution of melanin pigmentation. Melanin protects the skin from ultraviolet (UV) lesion by absorbing the ultraviolet sunlight and removing reactive oxygen species (ROS). Various dermatological disorders arise from the accumulation of an excessive amount of epidermal pigmentation (melasama, age spots, actinic damage sites). Melanin is formed through a series of oxidative reactions involving the conversion of the amino acid tyrosine in the presence of the enzyme tyrosinase to dihydroxyphenylalanine (DOPA) and then to dopaquinone. Subsequently, dopaquinone is converted to melanin by non-enzymatic reactions. Compounds may inhibit melanin biosynthesis through different mechanisms such as the absorption of UV light, the inhibition and proliferation of melanocyte metabolism (Seiberg, Paine et al. 2000; Seiberg, Paine et al. 2000), or the inhibition of tyrosinase, the major enzyme in melanin biosynthesis. Existing inhibitors suffer from several drawbacks such as low activity (kojic acid), high cytotoxicity and mutagenisity (hydroquinone) or poor skin penetration (arbutin). Therefore, new de- pigmentation agents are needed that have improved properties. Yokota et al., (Yokota, Nishio et al. 1998) investigated the inhibitory effect of glabridin on melanogenesis in vitro in cell culture and found that glabridin inhibits tyrosinase activity at concentrations of 0.1 to 1.0 µg/ml; in vivo it prevented UVB-induced pigmentation on guinea pig skins by topical applications of 0.5% glabridin. In our laboratory, the effects of other constituents of licorice extract were tested for their tyrosinase inhibitory activity using L-DOPA and L-Tyrosine as substrates, and melanin biosynthesis using human melanocytes. Glabrene (IC50=16µg/ml) proved to be active 17 while hisp A and hisp B were not. The inactivity of hisp A could be attributed to the presence of the isoprenyl groups, which may prevent interaction with the enzyme due to the steric effect. The inactivity of hisp B may be due to the absence of two free hydroxyl groups at positions 2’ and 4’, as was found in glabridin. The importance of both hydroxyl groups is supported by the inactivation of the 2’-O-MeG and 4’-O- MeG. 9. Summary Although licorice has been known to be a useful medicinal plant for the past 3,000 years, it is still luring investigators to explore new medicinal properties of this plant. In the first part of this section, Aviram et al. reviewed the therapeutic effects of licorice extract and its major antioxidant constituents of glabridin on atherosclerosis via the inhibition of the LDL oxidation molecular mechanism. The second part reviewed the potential of licorice extract and its constituents as HRT for post- menopausal women. The licorice extract and its constituents were found to bind to estrogen receptors, affect endothelial and smooth muscle cells known to have a role in cardiovascular diseases, inhibit a decrease in bone mass, affect the expression of estrogen receptors α and β, and inhibit serotonin re-uptake, which may be beneficial for reducing post-menopausal hot flashes and depression. In the last part of the review, the de-pigmentation effect of the licorice extract and its constituents via the inhibition of tyrosinase, the major enzyme in the biosynthesis of melanin, is discussed. Are the above activities just random phenomena or do they have something in common? The inhibition of LDL oxidation, the estrogen agonistic activities and the 18 inhibition of serotonin re-uptake may all be related to the antioxidant properties of the extract and its constituents (see References in the first part of this review). Antioxidants are known to increase LDL susceptibility and prevent atherosclerosis, and are potential therapeutic agents for ROS/RNS related diseases (Castro and Freeman 2001). All of the phytoestrogens known in the literature (lignans, coumestans, isoflavones and isoflavans) have antioxidant activity, including the female hormone, the estradiol itself (Yen, Hsieh et al. 2001). The molecular mechanism that relates the antioxidant activity of a compound to its estrogen-like activity is not yet clear. A possible mechanism that relates antioxidants to phytoestrogens may result from the known effects of antioxidants on the level and type of ROS/RNS associated with the induction of ERs (Hensley, Robinson et al. 2000). The molecular relationship between serotonin re-uptake and antioxidant activity is unclear and has been only slightly investigated (Jiang, Wrona et al. 1999). The natural serotonin re-uptake inhibitors that were found in our laboratory are isoflavans, which are also known to be antioxidants. The relationship between tyrosinase inhibitors and antioxidants may be explained by the fact that many of the tyrosinase inhibitors contain phenolic hydroxyl(s) (hydroquinone, resveratrol derivatives, galic acid), which is one of the main features of antioxidant activity (donation of an electron or hydrogen atom) (Kubo, Kinst-Hori et al. 2000). The other group of tyrosinase inhibitor compounds are able to form complexes with transition metal ions such as copper ion (oxalic acid, kojic acid), another mechanism by which antioxidants may exert their activity. Tyrosinase is an enzyme containing copper ions in its active site, and one of the suggested mechanisms for its inhibition is by chelating the ion. 19 The chemical structure of isoflavans found to be important in all of the biological activities tested below – inhibition of LDL oxidation, binding to ERs, effect on human breast cancer cell proliferation, inhibition of serotonin re-uptake and tyrosinase inhibition – is the presence of free hydroxyl at the 2’ position of ring B. Additional knowledge of structure activity relationships between natural compounds and their specific bioactivity could shed some light on the mechanisms by which these compounds manifest different activities in different target cells, and may contribute to the development of novel therapeutic treatment strategies. In the case of estrogen-like compounds, this knowledge will contribute to the design and development of new HRT agents that have beneficial effects on bone and cardiovascular tissue and block the deleterious effect of estrogen on breast and uterus cancer. 20 FIGURES Figure 1: Structures of several phytoestrogens, tamoxifen, raloxifene and estradiol N O OH O N O OH HO HO S Tamoxifen Raloxifene 17b-Estradiol O HO OH HO OH O OH HO O O O OH OH Enterolactone Enterodiol Coumestrol HO O HO O HO O OH O OH O OH OH O OH Biochanin A Genistein Daidzein 21 Figure 2: The structure of licorice constituents and estradiol 7 O OH 5 17 HO O A OH 4 7 A C 2' 3 C O 3 B B 3 2 O HO 4' HO 2' OH Glabridin 17b-Estradiol Glabrene O O O O OH OH O O OR1 OH O OR2 Hispaglabridin A Hispaglabridin B 2'-OMeG R1=CH3, R2=H 4'-OMeG R1=H, R2=Me 2',4'-OMeG R1=R2=Me OH OH HO OH HO OH O O Isoliquitireginin chalcone (ILC) Isoprenyl chalcone ( IPC) 2’-O-Methyl glabridin (2’-OMeG), and 2’,4’-O-Dimethtyl glabridin (2’,4’-OMeG) were synthesized from glabridin. 22 Figure 3: The binding of estradiol and licorice constituents to human estrogen receptor α. 100 glabridin 4-O'MG 2-O'MG binding (%) 2,4-diO'MG estradiol 50 glabrene hispA hispB ILC 0 10 -15 10 -11 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 concentration (M) 23 Figure 4: The effects of licorice constituents on the growth of estrogen-responsive human breast cancer cells. 400 glabridin 4-O'MG 2-O'MG 2,4-diO'MG 300 estradiol glabrene growth (%) ILC IPC 200 100 0 10 -16 10 -13 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 concentration (M) 24 FIGURE LEGE DS Figure 1: Structures of several phytoestrogens, tamoxifen, raloxifene and estradiol Figure 2: The structure of licorice constituents and Estradiol Figure 3: The binding of estradiol and licorice constituents to human estrogen receptor α. Competition of isolated licorice constituents for estrogen receptor with [3H] labeled 17β-estradiol was tested in human breast cancer cells (T-47D). The cells were incubated with [3H] 17β-estradiol and increasing concentrations of the tested compounds. 17β-estradiol and 0.1% ethanol were used as controls. Radioactivity in cells nuclei was counted and plotted as % of control. Values are means ± SD of > three experiments. Figure 4: The effects of licorice constituents on the growth of estrogen-responsive human breast cancer cells. T-47D cells were incubated with increasing concentrations of 17β - estradiol or the isolated licorice constituents for seven days. Porliferation was tested using the XTT cell proliferation reagent. Results are presented as the % of controls (0.1% ethanol). Values are means ± SD of > 3 experiments. 25 TABLES A D TABLE LEGE DS Table 1: The effect of licorice extract on the induction of CK activity in various female rat tissues. Control Estradiol Licorice ext. Glabridin µ 0.5µg µ 25µg µ 25µg Epiphysis 1 + 0.07 1.42 + 0.12 1.18 + 0.13 1.46 ± 0.09 Diaphysis 1 + 0.30 1.99 + 0.10 4.17 + 0.07 3.41 ± 0.19 Uterus 1 + 0.25 2.15 + 0.14 1.36 + 0.10 2.30 ± 0.19 Aorta 1 + 0.25 1.7 + 0.20 1.42 + 0.18 1.42 ± 0.09 Left ventricle 1 + 0.15 1.74 + 0.17 1.38 + 0.07 2.26 ± 0.24 Pituitary 1 + 0.16 2 + 0.20 3.68 + 0.09 2.15 ± 0.23 The effect of licorice extract, glabridin and estradiol feeding on the induction of creatine kinase activity in Ovariectomized female rat tissues. Rats were fed with 0.5 µg/day/rat of estradiol, 25 µg/day/rat of licorice extract or 25 µg/day/rat of glabridin for four weeks. CK activity was tested in various selected tissues. 26 Table 2: Histomorphometric analysis of OVX bone tissues of female rats fed with licorice extract, glabridin or estradiol for four weeks. Control Estradiol Licorice Glabridin Total Bone Volume (%) 35.6 ± 6.7 40.5 ± 4.8 37.0 ± 8.0 40.4 ± 3.3 Cartilage (width µm) 24.2 ± 6.7 28.6 ± 9.3 21.3 ±7.0 33.2 ± 7.3 Growth Plate (height µm) 18.1 ± 0.7 20.0 ± 2.3 22.3 ± 2.8 20.8 ± 1.4 Width of Trabecules (µm) µ 4.2 ± 0.9 5.0 ± 0.7 4.8 ± 0.8 5.4 ± 1.2 The effect of licorice extract, glabridin and estradiol feeding on bone volume, cartilage, epiphysal growth plate and the trabecules was tested in ovariectomized females. Rats were fed with 0.5 µg/day/rat of estradiol, 25 µg/day/rat of licorice extract or 25 µg/day/rat of glabridin for four weeks. The histomotphometric changes in the tissued tested are summarized. 27 Table 3: The effect of estradiol and glabridin on human endothelial cells and on vascular smooth muscle cells. Cells ECV304 VSMC Estradiol 0.3 1.77 + 0.16 3.28 + 0.09 nM 2.44 + 0.11 0.53 + 0.19 30 nM Glabridin 30 1.52 + 0.20 2.37 + 0.10 nM 3.34 + 0.30 0.89 + 0.22 300 8.72 + 0.28 0.40 + 0.22 nM 3 µM Human endothelial cells (ECV304) and human primary vascular smooth muscle cells (VSMC) were exposed to increasing concentrations of glabridin. DNA synthesis was tested using 3H-thymidine incorporation. Results are presented as an increase fold of control. 28 FEFERE CES Archer, J. S. (1999). "NAMS/Solvay Resident Essay Award. Relationship between estrogen, serotonin, and depression." Menopause 6(1): 71-8. Arena, S., C. Rappa, et al. (2002). "A natural alternative to menopausal hormone replacement therapy. Phytoestrogens." Minerva Ginecol 54(1): 53-7. Avis, N. E., S. Crawford, et al. (2001). "Longitudinal study of hormone levels and depression among women transitioning through menopause." Climacteric 4(3): 243-9. Barker, E. L. and R. D. Blakely (1995). Norepinephrine and serotonin transporter: molecular targets of antidepressant drugs. Psychopharmacology: the Fourth Generation of Progress. Raven, New York, Bloom, F. E Kupfer, D. J.: 321- 333. Barton, D., C. Loprinzi, et al. (2001). "Hot flashes: aetiology and management." Drugs Aging 18(8): 597-606. Bingham, S. A., C. Atkinson, et al. (1998). "Phyto-oestrogens: where are we now?" Br J Nutr 79(5): 393-406. Breinholt, V. and J. C. Larsen (1998). "Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay." Chem Res Toxicol 11(6): 622-9. Brzozowski, A. M., A. C. Pike, et al. (1997). "Molecular basis of agonism and antagonism in the oestrogen receptor." Nature 389(6652): 753-8. 29 Cassidy, A., S. Bingham, et al. (1993). "Biological effects of plant estrogens in premenopausal women." FASEB J. 7 (3 Pt II), 5000. Castro, L. and B. A. Freeman (2001). "Reactive oxygen species in human health and disease." Nutrition 17(2): 161, 163-5. Chang, A. S. and S. M. Chang (1999). "Nongenomic steroidal modulation of high- affinity serotonin transport." Biochim Biophys Acta 1417(1): 157-66. Egner, U., N. Heinrich, et al. (2001). "Different ligands-different receptor conformations: modeling of the hER alpha LBD in complex with agonists and antagonists." Med Res Rev 21(6): 523-39. Ettinger, B., H. K. Genant, et al. (1985). "Long-term estrogen replacement therapy prevents bone loss and fractures." Ann Intern Med 102(3): 319-24. Fournier, D. B., J. W. Erdman, Jr., et al. (1998). "Soy, its components, and cancer prevention: a review of the in vitro, animal, and human data." Cancer Epidemiol Biomarkers Prev 7(11): 1055-65. Fozzard, J. (1989). Peripheral Actions of 5-Hydroxytryptamine. Peripheral Actions of 5-Hydroxytryptamine. J. Fozzard. Oxford, New York, University Press. Fuller, R. W. (1994). "Uptake inhibitors increase extracellular serotonin concentration measured by brain microdialysis." Life Sci 55(3): 163-7. Gehm, B. D., J. M. McAndrews, et al. (1997). "Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor." Proc Natl Acad Sci U S A 94(25): 14138-43. 30 Grese, T. A., S. Cho, et al. (1997). "Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene." J Med Chem 40(2): 146-67. Hensley, K., K. A. Robinson, et al. (2000). "Reactive oxygen species, cell signaling, and cell injury." Free Radic Biol Med 28(10): 1456-62. Horwitz, K. B., T. A. Jackson, et al. (1996). "Nuclear receptor coactivators and corepressors." Mol Endocrinol 10(10): 1167-77. Hsieh, C. Y., R. C. Santell, et al. (1998). "Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo [published erratum appears in Cancer Res 1999 Mar 15;59(6):1388]." Cancer Res 58(17): 3833-8. Iafrati, M. D., R. H. Karas, et al. (1997). "Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice." Nat Med 3(5): 545-8. Jiang, X. R., M. Z. Wrona, et al. (1999). "Tryptamine-4,5-dione, a putative endotoxic metabolite of the superoxide-mediated oxidation of serotonin, is a mitochondrial toxin: possible implications in neurodegenerative brain disorders." Chem Res Toxicol 12(5): 429-36. Katzenellenbogen, B. S., J. Sun, et al. (2001). "Structure-function relationships in estrogen receptors and the characterization of novel selective estrogen receptor modulators with unique pharmacological profiles." Ann N Y Acad Sci 949: 6- 15. 31 Kimura, Y., T. Okuda, et al. (1993). "Effects of flavonoids from licorice roots (Glycyrrhiza inflata Bat.) on arachidonic acid metabolism and aggregation in human platelets." Phyt. Res 7: 341-347. Korach, K. S. (1994). "Insights from the study of animals lacking functional estrogen receptor." Science 266(5190): 1524-7. Kubo, I., I. Kinst-Hori, et al. (2000). "Molecular design of antibrowning agents." J Agric Food Chem 48(4): 1393-9. Lee, H. P., L. Gourley, et al. (1991). "Dietary effects on breast-cancer risk in Singapore [see comments]." Lancet 337(8751): 1197-200. Liggins, J., R. Grimwood, et al. (2000). "Extraction and quantification of lignan phytoestrogens in food and human samples." Anal Biochem 287(1): 102-9. Liu, J., J. E. Burdette, et al. (2001). "Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms." J Agric Food Chem 49(5): 2472-9. Malnick, S. D., A. Shaer, et al. (1983). "Estrogen-induced creatine kinase in the reproductive system of the immature female rat." Endocrinology 113(5): 1907- 9. Mitscher, L. A., Y. H. Park, et al. (1980). "Antimicrobial agents from higher plants. Antimicrobial isoflavanoids and related substances from Glycyrrhiza glabra L. var. typica." J Nat Prod 43(2): 259-69. 32 Peterson, G. and S. Barnes (1996). "Genistein inhibits both estrogen and growth factor-stimulated proliferation of human breast cancer cells." Cell Growth Differ 7(10): 1345-51. Pritchard, K. I. (2001). "Breast cancer prevention with selective estrogen receptor modulators: a perspective." Ann N Y Acad Sci 949: 89-98. Rafi, M. M., R. T. Rosen, et al. (2000). "Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid." Anticancer Res 20(4): 2653-8. Russo, I. H. and J. Russo (1998). "Role of hormones in mammary cancer initiation and progression." J Mammary Gland Biol Neoplasia 3(1): 49-61. Sadler, B. R., S. J. Cho, et al. (1998). "Three-dimensional quantitative structure- activity relationship study of nonsteroidal estrogen receptor ligands using the comparative molecular field analysis/cross-validated r2-guided region selection approach." J Med Chem 41(13): 2261-7. Saitoh, T. and T. Kinoshita (1976). "New isoflavane and flavanone from licorice root. Chem. Pharm." Shibata, S.. Bull 24: 752-755. Seed, M. (1991). "Sex hormones, lipoproteins, and cardiovascular risk." Atherosclerosis 90(1): 1-7. Seiberg, M., C. Paine, et al. (2000). "Inhibition of melanosome transfer results in skin lightening." J Invest Dermatol 115(2): 162-7. Seiberg, M., C. Paine, et al. (2000). "The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions." Exp Cell Res 254(1): 25-32. 33 Shao, Z. M., M. L. Alpaugh, et al. (1998). "Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis." J Cell Biochem 69(1): 44-54. Shao, Z. M., J. Wu, et al. (1998). "Genistein exerts multiple suppressive effects on human breast carcinoma cells." Cancer Res 58(21): 4851-7. Shewmon, D. A., J. L. Stock, et al. (1994). "Tamoxifen and estrogen lower circulating lipoprotein(a) concentrations in healthy postmenopausal women." Arterioscler Thromb 14(10): 1586-93. Shiau, A. K., D. Barstad, et al. (1998). "The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen." Cell 95(7): 927-37. Smith, C. L., Z. Nawaz, et al. (1997). "Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen." Mol Endocrinol 11(6): 657-66. Somjen, D., F. Kohen, et al. (1998). "Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells." Hypertension 32(1): 39-45. Somjen, D., A. Waisman, et al. (1996). "Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo." J Steroid Biochem Mol Biol 59(5-6): 389-96. 34 Somjen, D., A. Waisman, et al. (1998). "Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up- regulate their responsiveness to estrogens." Steroids 63(5-6): 340-3. Sourander, L., T. Rajala, et al. (1998). "Cardiovascular and cancer morbidity and mortality and sudden cardiac death in postmenopausal women on oestrogen replacement therapy (ERT)." Lancet 352(9145): 1965-9. Stampfer, M. J., G. A. Colditz, et al. (1991). "Postmenopausal estrogen therapy and cardiovascular disease. Ten-year follow-up from the nurses' health study." N Engl J Med 325(11): 756-62. Tamir, S., M. Eisenberg, et al. (2000). "Estrogenic and Antiproliferative properties of Glabridin, Isoflavan isolated from Licorice in Human Breast Cancer Cells (manuscript in preparation)." Tamir, S., M. Eizenberg, et al. (2001). "Estrogen-like activity of glabrene and other constituents isolated from licorice root." J Steroid Biochem Mol Biol 78(3): 291-8. Tham, D. M., C. D. Gardner, et al. (1998). "Clinical review 97: Potential health benefits of dietary phytoestrogens: a review of the clinical, epidemiological, and mechanistic evidence." J Clin Endocrinol Metab 83(7): 2223-35. Valente, M., L. Bufalino, et al. (1994). "Effects of 1-year treatment with ipriflavone on bone in postmenopausal women with low bone mass." Calcif Tissue Int 54(5): 377-80. 35 Vaya, J., P. A. Belinky, et al. (1997). "Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation." Free Radic Biol Med 23(2): 302-13. Wang, C. and M. S. Kurzer (1997). "Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells." Nutr Cancer 28(3): 236-47. Wiese, T. E., L. A. Polin, et al. (1997). "Induction of the estrogen specific mitogenic response of MCF-7 cells by selected analogues of estradiol-17 beta: a 3D QSAR study." J Med Chem 40(22): 3659-69. Wiseman, H. (2000). "The therapeutic potential of phytoestrogens." Expert Opin Investig Drugs 9(8): 1829-40. Yang, N. N., H. U. Bryant, et al. (1996). "Estrogen and raloxifene stimulate transforming growth factor-beta 3 gene expression in rat bone: a potential mechanism for estrogen- or raloxifene-mediated bone maintenance." Endocrinology 137(5): 2075-84. Yen, C. H., C. C. Hsieh, et al. (2001). "17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells." Life Sci 70(4): 403-13. Yokota, T., H. Nishio, et al. (1998). "The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation." Pigment Cell Res 11(6): 355- 61. 36 Zava, D. T., M. Blen, et al. (1997). "Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture." Environ Health Perspect 105 Suppl 3: 637-45. Zava, D. T., C. M. Dollbaum, et al. (1998). "Estrogen and progestin bioactivity of foods, herbs, and spices." Proc Soc Exp Biol Med 217(3): 369-78. Zhou, J. R., P. Mukherjee, et al. (1998). "Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis." Cancer Res 58(22): 5231-8.