Estrogen-Like Activity of Licorice

28 Estrogen-Like Activity of Licorice

Root Extract and Its Constituents

Jacob Vaya Tel-Hai Academic College, Upper Galilee

and MIGAL-Galilee Technology Center Kiryat Shmona, Israel

Snait Tamir Tel-Hai Academic College

Upper Galilee, Israel Dalia Somjen

The Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel

I. INTRODUCTION

A. Estrogens Estrogens are steroid hormones that exhibit a broad range of physiological

activities. 17h-Hydroxyestradiol is the female sex hormone active in devel- oping the mammary gland and the uterus, maintaining pregnancy, relieving menopausal symptoms, and preventing cardiovascular and bone diseases (1). An apparent consequence of estrogen is the increase in short-term meno-

616 Vaya et al.

pausal symptoms including vasomotor hot flashes, urogenital 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 (2).

Estrogen is beneficial in reducing the risk of cardiovascular disease (3– 5). The incidence of heart disease among premenopausal women is low compared with men, whereas the incidence among postmenopausal women approaches that of men. The administration of estrogen to postmenopausal women decreases the incidence of heart disease (6). This protective effect of estrogen may partly be attributed to its influence in decreasing the ratio between LDL and HDL (7), to reduction of thrombus formation, and to improvement in vascular compliance. Secondary prevention trials, conducted on women with established coronary heart disease, failed to confirm the benefits of hormone replacement therapy (HRT) (8). A randomized con- trolled trial by the Women’s Health Initiative begun in 1993 and involving 16,608 participants who were undergoing HRT of estrogen plus progestin, was terminated before it reached its end, owing to a 26% increase in invasive breast cancer and, surprisingly, to an increase in the risk of heart attack (strokes, blood clots) among those taking the hormone regimen, compared with women taking a placebo (9).

Estrogen is known to be involved in osteoporosis (10), 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 develop- ment of estrogen-dependent tumors such as breast and uterus cancer (11). 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, ERa and ERh, which have different structure and tissue distributions (12). The biological effect of an ER ligand in a specific tissue is determined by the expression of ERa and ERh in that tissue. It was demonstrated that oxidative stress induced by

H 2 O 2 , Fe 2+ , AAPH, and activated macrophages affect the expression of ERa and ERh differently, demonstrating cell-specific response, which can be blocked by antioxidants (13). The two key events controlling the tissue selectivity of an estrogen are the receptor’s shape and the interaction with adaptor proteins (14,15). Compounds known as selective estrogen receptor

Estrogen-Like Activity of Licorice Extract 617

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 (16), whereas raloxifene functions as an agonist in the bone, breast, and cardio- vascular system but not in the uterus (17,18). 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 nonreproductive tissues (such as bone and cardiovascular tissues) may be of therapeutic use.

B. Studies on Structure-Activity Relationships Several studies have been carried out on the relationship between the structure

of compounds and their estrogen-like activity (12,19). 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 al. (20) evaluated 42 analogs of estradiol for their ER-binding affinity and toxicity to breast cancer cell lines, correlating the structure of these compounds with the above activities by means of three-dimensional quantitative structure activity (QSAR), employ- ing 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 electropos- itive properties near position 3. Sadler et al. (21) 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 com- pounds sharing the transstilbene structure were examined and results were compared to information from the ER-binding affinities of substituted estradiol analogs. This study demonstrated the importance of hydroxy substituents in nonsteroidal ligands that mimic the 3-OH and 17-OH of estradiol to obtain a high binding affinity. Grese et al. (22) 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 choles- terol, uterine weight gain, and uterine eosinophil peroxidase activity in an ovariectomized (OVX) rat model. In this study, they showed the importance of highly electronegative 4V-substituents, such as hydroxy or fluoro attached to the raloxifene molecule, in their ability to bind to the receptor. They also showed that increasing steric bulk at position 4V led to increased uterine stimulation in vivo and that additional substitutions at the 4-, 5-, or 7- positions of the benzothiophene moiety resulted in reduced biological activ- ity, while an additional substitution of the 2-aryl moiety had little effect.

618 Vaya et al.

Shiau et al. (23) investigated the crystal structure of the human LBD complex with an agonist (diethylstilbestrol), together with a peptide derived from an ERa coactivator and the crystal structure of LBD with an antagonist (4-hydroxy tamoxifen). They showed that the peptide binds as a short a 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 stabi- lizes 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.

F IGURE 1 Structures of several phytoestrogens, tamoxifen, raloxifene, and estradiol.

Estrogen-Like Activity of Licorice Extract 619

C. 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 (24,25). Phytoestrogens include the subclasses of lignans, coume- stans, isoflavones, and isoflavans (Fig. 1) that are widely distributed in oilseeds (flax, cereals), vegetables, soybeans, and roots. The main mammalian lignans are enterolactone (II) and enterodiol (III) (26), and of coumestan, coumestrol. The major food-active isoflavonoids are genistein (IV) and daidzein (V) (27) while the major isoflavan is glabridin (28). Epidemiological evidence indicates that soy intake (rich in isoflavonoids) is associated with lower breast cancer risk in women (29,30). Genistein is reported to prevent cancellous bone loss and to maintain or increase bone density in postmenopausal women (31,32). 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 to 15,000 times less than estradiol (33,34).

II. 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 (35) while others reported that it showed a low binding affinity (36). 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

T ABLE 1 The Effect of Licorice Extract on the Induction of CK Activity in Various Female Rat Tissue

Licorice extract Glabridin Control

Estradiol

(25 Ag) (25 Ag) Epiphysis

(0.5 Ag)

1.18 + 0.13 1.46 F 0.09 Diaphysis

4.17 + 0.07 3.41 F 0.19 Uterus

1.36 + 0.10 2.30 F 0.19 Aorta

1.42 + 0.18 1.42 F 0.09 Left ventricle

1.38 + 0.07 2.26 F 0.24 Pituitary

3.68 + 0.09 2.15 F 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 Ag/day/rat of estradiol, 25 Ag/day/rat of licorice extract, or 25 Ag/day/rat of glabridin for 4 weeks. CK activity was tested in various selected tissues.

620 Vaya et al.

T ABLE 2 Histomorphometric Analysis of OVX Bone Tissues of Female Rats Fed with Licorice Extract, Glabridin, or Estradiol for 4 Weeks

Licorice Glabridin Total bone

Control

Estradiol

37.0 F 8.0 40.4 F 3.3 volume (%) Cartilage

35.6 F 6.7

40.5 F 4.8

21.3 F 7.0 33.2 F 7.3 (width, Am) Growth plate

24.2 F 6.7

28.6 F 9.3

22.3 F 2.8 20.8 F 1.4 (height, Am) Width of

18.1 F 0.7

20.0 F 2.3

4.8 F 0.8 5.4 F 1.2 trabecules (Am)

4.2 F 0.9

5.0 F 0.7

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 Ag/day/rat of estradiol, 25 Ag/day/rat of licorice extract, or 25 Ag/ day/rat of glabridin for 4 weeks. The histomorphometric changes in the tested tissues are summarized.

with prostate cancer (37), which was attributed to licochalcone A present in the extract. The estrogen-like effects of licorice extract in vivo were tested in our laboratory (38). Ovariectomized female rats fed with licorice extract (25

A g/day/rat) or estradiol (0.5 Ag/day/rat) for 4 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 (39,40), and can therefore be used as an ER-response marker. These results showed that at 0.5

A g/day/rat, estradiol stimulated CK activity at the same level as licorice extract at 25 Ag/day/rat only in diaphyseal bone and the pituitary gland. A histomor- phometric 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 1 month may better clarify the licorice extract potential.

III. ESTROGEN-LIKE ACTIVITY OF LICORICE ROOT CONSTITUENTS

Licorice root constituents were isolated from the aqueous extract, such as glycyrrhizin and its glycone, glycyrrhetinic acid, and were used in the treatment of hyperlipemia, atherosclerosis, viral diseases, and allergic inflam- mation (41). The organic extract of licorice root (acetone or ethanol) is known

Estrogen-Like Activity of Licorice Extract 621

F IGURE 2 The structure of licorice constituents and estradiol. 2V-O-Methyl glabridin (2V-OMeG), and 2V,4V-O-Dimethtyl glabridin (2V,4V-OMeG) were synthesized from glabridin. *A revised structure for glabrene was assigned by Kinoshita, Tamura, et al. (1997) (74).

glabrol, glabrene, 3-hydroxyglabrol, 4V-O-methylglabridin (4V-OMeG), his- paglabridin A (hisp A), hispaglabridin B (hisp B), isoprenylchalcone deriv- ative (IPC), isoliquitireginin chalcone (ILC), and formononetin (28,42,43). 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 com- pound of this class having diverse biologically activities (see Aviram et al., in this book) and which is present in the extract in more than 10% w/w, also exhibits estrogen-like activity (38,44). 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, or a carbonyl group attached to carbon 4. This structure

622 Vaya et al.

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.

A. Glabridin Among the licorice constituents isolated and tested, the most active phytoes-

trogen in vitro and in vivo is glabridin (38,44). 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 (17h in estradiol and 2V in glabridin).

1. Binding of Glabridin to the ERa Glabridin binds the ER with IC 50 of 5 AM (44) and with approximately the

same affinity as genistein, the best known phytoestrogen (33), 10 4 times lower than estradiol (45) (Fig. 3).

2. Effect of Glabridin on Breast Cancer Cells Glabridin stimulated growth over a range of 0.1–10 AM, reaching a maximum

level at about 10 AM; at a higher level (15 AM), it inhibited cell growth (44) (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 (45–48).

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.

3. Effect of Glabridin on Cardiovascular Cells Animal and human studies indicate that estrogens are protective against cor-

onary atherosclerosis (4). 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 in- terest. 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) (49).

Estrogen-Like Activity of Licorice Extract 623

F IGURE 3 The binding of estradiol and licorice constituents to human estrogen receptor a (ERa). Competition of isolated licorice constituents for estrogen receptor with [ 3 H] labeled. 17h-Estradiol was tested in human breast cancer cells (T-47D). The cells were incubated with [ 3 H] 17h-estradiol and increasing concentrations of the tested compounds. 17h-Estradiol and 0.1% ethanol were used as controls. Radioactivity in cells’ nuclei was counted and ploted as % of control. Values are means F SD of >3 experiments.

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.

4. In Vivo Effects of Glabridin on Female Rat Tissues Ovariectomized female rats fed with estradiol or glabridin for 4 weeks (Table

1) showed that 0.5 Ag/day/rat of estradiol stimulated CK activity at the same level as 25 Ag/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 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.

B. 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

624 Vaya et al.

F IGURE 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 17h-estradiol or the isolated licorice constituents for 7 days. Proliferation was tested using the XTT cell proliferation reagent. Results are presented as the % of controls (0.1% ethanol). Values are means F SD of >3 experiments.

T ABLE 3 The Effect of Estradiol and Glabridin on Human Endothelial Cells and on Vascular Smooth Muscle Cells

Cells

VSMC Estradiol

0.40 + 0.22 Human endothelial cells (ECV304) and human primary vascular smooth muscle cells

3 AM

(VSMC) were exposed to increasing concentrations of glabridin. DNA synthesis was tested using 3 H-thymidine incorporation. Results are presented as an increased fold of control.

Estrogen-Like Activity of Licorice Extract 625

genistein. The hisp A and B, two additional isoflavans in the licorice root, were barely active, whereas IPC, another chalcone, was totally inactive. Glabrene and ILC showed ER-regulated growth-promoting effects such as glabridin (Fig. 4) and genistein. Glabrene produced dose-dependent tran- scriptional activation with half-maximal induction at 1 AM, 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 Ag/day/rat glabrene resulted in a similar effect to that of 5

A g/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, and are not active as phytoestrogens, such as quercetin, catechin, apigenin, etc. (reviewed in Ref. 50). 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 demon- strated phytoestrogenic activity in vitro and in vivo, which may suggest that conjugated double bonds between ring A to ring B are not essential for this activity. This phenomenon could be explained by the effect of ring C on the isoflavans stracture, 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 a, h double bond, a hydroxyl at position 2V (with two additional hydroxyls at positions 4 and 4V). However, only ILC, which does not contain the isoprenyl group, binds to the ER, whereas IPC, containing two isoprenyl groups, was totally inactive.

IV. THE EFFECT OF GLABRIDIN DERIVATIVES ON THEIR ER BINDING

Glabridin, which contains two hydroxyl groups at positions 2V and 4V, has a higher affinity to ER and a stronger effect on cell growth stimulation than 2V- O -MeG and 4V-O-MeG. 2V,4V-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 17h are required for binding (20,51). In glabridin,

626 Vaya et al.

hydroxyl 4V 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 17h but has ether oxygen in a parallel position (the g-pyran ring), which could contribute to the interaction to histidin 524 in the ligand-binding domain.

V. EFFECTS OF LICORICE CONSTITUENTS ON CELL PROLIFERATION

In contrast to the ER-regulated growth-promoting phytoerogenic effects of glabridin and glabrene in concentrations ranging from 100 nM to 10 AM, 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 path- ways. Antiproliferative effects of genistein were also observed in other non- breast carcinoma cell lines (52). The inhibited growth of ER-negative cells by glabridin supports the hypothesis that the actions of phytoestrogen on cell growth inhibition occur via different molecular mechanisms (53–55).

VI. DIFFERENTIAL EFFECTS OF GLABRIDIN AND GLABRENE ON ER

A AND ERB EXPRESSIONS Estrogen is known to offer protection from coronary artery disease in

postmenopausal 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 exhibit- ing some estrogen-like activities, can also regulate the expression of ERs.

Results showed that the phytoestrogens glabridin and glabrene pro- moted ERa and ERh expressions differently and in a cell-specific manner. ERh was significantly increased in human breast cancer cells only after being exposed to estradiol and glabridin (two- to fourfold increase), while vitamin D and glabrene inhibited ERh expression in these cells. On the other hand, ERa was significantly increased in all treatments (estradiol, fourfold; vitamin D, threefold; and glabridin, sixfold). Estradiol treatment inhibited ERh in colon and melanoma cells, while glabrene significantly increased ERh (two- to threefold). Glabridin had no significant effect in these cell lines, which only

Estrogen-Like Activity of Licorice Extract 627

exhibited ERh. Vitamin D showed the same effect as estradiol on ERh inhibition in colon cells but had the same stimulating effect on ERh (twofold) as glabrene in melanoma cells (unpublished data).

These data suggest that phytoestrogens not only mimic the estradiol function as physiological regulators of ERa and ERh expressions but also present tissue selectivity. They may also suggest that treatment using both estradiol and specific phytoestrogens 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 postmenopausal diseases and contribute to the development of novel therapeutic treatment strategies.

VII. LICORICE CONSTITUENTS INHIBIT SEROTONIN REUPTAKE—A POTENTIAL NATURAL TREATMENT FOR POSTMENOPAUSAL DEPRESSION

An increase in the prevalence of depressive symptoms in women undergoing menopause can be related to fluctuating estrogen levels (56). Depression in women seems to increase with a change in hormone levels (57). The seroto- nergic system appears to play a major role in depression, although other neurotransmitters are also involved (2,58,59).

Serotonin is a neurotransmitter in the central and peripheral nervous systems (60). Serotonin inactivation following its release is controlled by a specific reuptake 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 (59). 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 (61). Glabridin and 4V-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 2V-OMeG was totally inactive, proving the importance of hydroxyl 2V for the serotonin reuptake inhibition (62).

VIII. 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

628 Vaya et al.

absorbing the ultraviolet sunlight and removing reactive oxygen species (ROS). Various dermatological disorders arise from the accumulation of an excessive amount of epidermal pigmentation (melasma, 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 dopaqui- none. Subsequently, dopaquinone is converted to melanin by nonenzymatic reactions. Compounds may inhibit melanin biosynthesis through different mechanisms such as the absorption of UV light, the inhibition and prolifer- ation of melanocyte metabolism (63,64), 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 mutage- nicity (hydroquinone) or poor skin penetration (arbutin). Therefore, new de- pigmentation agents are needed that have improved properties. Yokota et al. (65) investigated the inhibitory effect of glabridin on melanogenesis in vitro in cell culture and found that glabridin inhibits tyrosinase activity at concen- trations of 0.1–1.0 Ag/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 tyro- sinase inhibitory activity using L -DOPA and L -tyrosine as substrates, and

melanin biosynthesis using human melanocytes (66). Glabrene (IC 50 = 16 Ag/ mL) proved to be active 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 owing to the steric effect. The inactivity of hisp B may be due to the absence of two free hydroxyl groups at positions 2V and 4V, as was found in glabridin. The importance of both hydroxyl groups is supported by the inactivation of the 2V-O-MeG and 4V-O-MeG.

IX. SUMMARY Although licorice has been known to be a useful medicinal plant for the past

3000 years, it is still luring investigators to explore new medicinal properties of this plant. In a separate chapter, Aviram et al. review the therapeutic effects of licorice extract and its major antioxidant constituents of glabridin on atherosclerosis via inhibition of the LDL oxidation molecular mechanism. The second part of the chapter reviews the potential of licorice extract and its constituents as HRT for postmenopausal 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 a and h, and inhibit serotonin reuptake, which may be beneficial for reducing postmeno- pausal hot flashes and depression. In the last part of the chapter, the depig-

Estrogen-Like Activity of Licorice Extract 629

mentation 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 inhibition of serotonin reuptake may all be related to the antioxidant properties of the extract and its constituents (see references in Aviram chapter). Antioxidants are known to increase LDL susceptibility and prevent atherosclerosis, and are potential therapeutic agents for ROS/ RNS-related diseases (67). All of the phytoestrogens known in the literature (lignans, coumestans, isoflavones, and isoflavans) have antioxidant activity, including the female hormone, the estradiol itself (68). The molecular mech- anism 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 (13,69). The molecular relationship between serotonin reuptake and antioxidant activity is unclear and has been only slightly investigated (70). The natural serotonin reuptake 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) (71). The other group of tyrosinase inhibitors are able to form complexes with transition metal ions such as copper ion (oxalic acid, kojic acid), an additional 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. Antioxidants or compounds with redox properties can prevent or delay pigmentation by different mechanisms: by scavenging reactive oxygen and nitrogen species (ROS and RNS), known to induce melanin synthesis (72), or by reducing o-quinones or other intermediates in the melanin biosynthesis, and thus delaying oxidative polymerization (73).

The chemical structure of isoflavans found to be important in all of the biological activities tested—inhibition of LDL oxidation, binding to ERs, effect on human breast cancer cell proliferation, inhibition of serotonin reuptake and tyrosinase inhibition—is the presence of free hydroxyl at the 2V 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

630 Vaya et al.

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.

REFERENCES 1. Korach KS. Insights from the study of animals lacking functional estrogen

receptor. Science 1994; 266:1524–1527. 2. Barton D, Loprinzi C, Wahner-Roedler D. Hot flashes: aetiology and manage- ment. Drugs Aging 2001; 18:597–606. 3. Seed M. Sex hormones, lipoproteins, and cardiovascular risk. Atherosclerosis 1991; 90:1–7. 4. Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR Jr, Lubahn DB, O’Donnell TF Jr, Korach KS, Mendelsohn ME. Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat Med 1997; 3:545– 548. 5. Sourander L, Rajala T, Raiha I, Makinen J, Erkkola R, Helenius H. Cardio- vascular and cancer morbidity and mortality and sudden cardiac death in post- menopausal women on oestrogen replacement therapy (ERT). Lancet 1998; 352: 1965–1969. 6. Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the nurses’ health study. N Engl J Med 1991; 325:756– 762. 7. Shewmon DA, Stock JL, Rosen CJ, Heiniluoma KM, Hogue MM, Morrison A, Doyle EM, Ukena T, Weale V, Baker S. Tamoxifen and estrogen lower circulating lipoprotein(a) concentrations in healthy postmenopausal women. Arterioscler Thromb 1994; 14:1586–1593. 8. Herrington DM, Reboussin DM, Brosnihan KB, Sharp PC, Shumaker SA, Snyder TE, Furberg CD, Kowalchuk GJ, Stuckey TD, Rogers WJ, Givens DH, Waters D. Effects of estrogen replacement on the progression of coronary-artery atherosclerosis. N Engl J Med 2000; 343:522–529. 9. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J. Risks and benefits of estrogen plus progestin in healthy postmeno- pausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 2002; 288:321–333. 10. Ettinger B, Genant HK, Cann CE. Long-term estrogen replacement therapy prevents bone loss and fractures. Ann Intern Med 1985; 102:319–324. 11. Russo IH, Russo J. Role of hormones in mammary cancer initiation and progression. J Mammary Gland Biol Neoplasia 1998; 3:49–61. 12. Katzenellenbogen BS, Sun J, Harrington WR, Kraichely DM, Ganessunker D, Katzenellenbogen JA. Structure-function relationships in estrogen receptors and the characterization of novel selective estrogen receptor modulators with unique pharmacological profiles. Ann NY Acad Sci 2001; 949:6–15.

Estrogen-Like Activity of Licorice Extract 631 13. Tamir S, Izrael S, Vaya J. The effect of oxidative stress on ERalpha and ERbeta

expression. J Steroid Biochem Mol Biol 2002; 81:323–332. 14. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L. Nuclear receptor coactivators and corepressors. Mol Endocrinol 1996; 10:1167–1177. 15. Smith CL, Nawaz Z, O’Malley BW. Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 1997; 11:657–666.

16. Pritchard KI. Breast cancer prevention with selective estrogen receptor modulators: a perspective. Ann NY Acad Sci 2001; 949:89–98. 17. Somjen D, Waisman A, Kaye AM. Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo. J Steroid Biochem Mol Biol 1996; 59:389–396. 18. Yang NN, Bryant HU, Hardikar S, Sato M, Galvin RJ, Glasebrook AL, Termine JD. 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 1996; 137:2075–2084. 19. Egner U, Heinrich N, Ruff M, Gangloff M, Mueller-Fahrnow A, Wurtz JM. Different ligands-different receptor conformations: modeling of the hER alpha LBD in complex with agonists and antagonists. Med Res Rev 2001; 21:523–539. 20. Wiese TE, Polin LA, Palomino E, Brooks SC. 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 1997; 40:3659–3669. 21. Sadler BR, Cho SJ, IshaqKS, Chae K, Korach KS. 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 1998; 41:2261–2267. 22. Grese TA, Cho S, Finley DR, Godfrey AG, Jones CD, Lugar CW 3rd, Martin MJ, Matsumoto K, Pennington LD, Winter MA, Adrian MD, Cole HW, Magee DE, Phillips DL, Rowley ER, Short LL, Glasebrook AL, Bryant HU. Structure- activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene. J Med Chem 1997; 40:146–167. 23. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. The structural basis of estrogen receptor/coactivator recognition and the antag- onism of this interaction by tamoxifen. Cell 1998; 95:927–937. 24. Cassidy A, Bingham S, Carson J, Setchell KDR. Biological effects of plant estrogens in premenopausal women. FASEB J 1993; 7(3 Pt II):5000. 25. Tham DM, Gardner CD, Haskell WL. Clinical review 97: Potential health benefits of dietary phytoestrogens: a review of the clinical, epidemiological, and mechanistic evidence. J Clin Endocrinol Metab 1998; 83:2223–2235. 26. Liggins J, Grimwood R, Bingham SA. Extraction and quantification of lignan phytoestrogens in food and human samples. Anal Biochem 2000; 287:102–109. 27. Wiseman H. The therapeutic potential of phytoestrogens. Expert Opin Invest Drugs 2000; 9:1829–1840. 28. Vaya J, Belinky PA, Aviram M. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxida- tion. Free Rad Biol Med 1997; 23:302–313.

632 Vaya et al. 29. Lee HP, Gourley L, Duffy SW, Esteve J, Lee J, Day NE. Dietary effects on

breast-cancer risk in Singapore [see comments]. Lancet 1991; 337:1197–1200. 30. Fournier DB, Erdman JW Jr, Gordon GB. Soy, its components, and cancer prevention: a review of the in vitro, animal, and human data. Cancer Epidemiol Biomarkers Prev 1998; 7:1055–1065. 31. Valente M, Bufalino L, Castiglione GN, D’Angelo R, Mancuso A, Galoppi P, Zichella L. Effects of 1-year treatment with ipriflavone on bone in postmeno- pausal women with low bone mass. Calcif Tissue Int 1994; 54:377–380. 32. Arena S, Rappa C, Del Frate E, Cenci S, Villani C. A natural alternative to menopausal hormone replacement therapy: phytoestrogens. Minerva Ginecol 2002; 54:53–57. 33. Zava DT, Blen N, Duwe G. Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture. Environ Health Perspect 1997; 105(suppl 3):637–645. 34. Bingham SA, Atkinson C, Liggins J, Bluck L, Coward A. Phyto-oestrogens: where are we now? Br J Nutr 1998; 79:393–406. 35. Zava DT, Dollbaum CM, Blen M. Estrogen and progestin bioactivity of foods, herbs, and spices. Proc Soc Exp Biol Med 1998; 217:369–378. 36. Liu J, Burdete JE, Xu H, Gu C, van Breemen RB, Bhat KP, Booth N, Constantinou AI, Pezzuto JM, Fong HH, Farnsworth NR, Bolton JL. Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms. J Agric Food Chem 2001; 49:2472–2479. 37. Rafi MM, Rosen RT, Vassil A, Ho CT, Zhang H, Ghai G, Lambert G, DiPaola RS. Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid. Anticancer Res 2000; 20:2653–2658. 38. Tamir S, Eizenberg M, Somjen D, Izrael S, Vaya J. Estrogen-like activity of glabrene and other constituents isolated from licorice root. J Steroid Biochem Mol Biol 2001; 78:291–298. 39. Malnick SD, Shaer A, SoreqH, Kaye AM. Estrogen-induced creatine kinase in the reproductive system of the immature female rat. Endocrinology 1983; 113: 1907–1909. 40. Somjen D, Waisman A, Weisman J, Kaye AM. 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 1998; 63:340–343. 41. Kimura Y, Okuda T, Okuda H. Effects of flavonoids from licorice roots (Glycyrrhiza inflata Bat.) on arachidonic acid metabolism and aggregation in human platelets. Phyt Res 1993; 7:341–347. 42. Saitoh T, Kinoshita T. New isoflavane and flavanone from licorice root. Chem Pharm Shibata, S Bull 1976; 24:752–755. 43. Mitscher LA, Park YH, Clark D, Beal JL. Antimicrobial agents from higher plants: antimicrobial isoflavonoids and related substances from Glycyrrhiza glabra L. var. typica. J Nat Prod 1980; 43:259–269. 44. Tamir S, Eizenberg M, Somjen D, Stern N, Shelach R, Kaye A, Vaya J. Estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells. Cancer Res 2000; 60:5704–5709.

Estrogen-Like Activity of Licorice Extract 633 45. Wang C, Kurzer MS. Phytoestrogen concentration determines effects on DNA

synthesis in human breast cancer cells. Nutr Cancer 1997; 28:236–247. 46. Gehm BD, McAndrews JM, Chien PY, Jameson JL. Reveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci USA 1997; 94:14138–14143. 47. Breinholt V, Larsen JC. Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay. Chem Res Toxicol 1998; 11:622–629. 48. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. 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 1998; 58:3833–3838. 49. Somjen D, Kohen F, Jaffe A, Amir-Zaltsman Y, Knoll E, Stern N. Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells. Hypertension 1998; 32:39–45. 50. Vaya J, Tamir S. The relation between the chemical structure of flavonoids and their estrogen-like activities. Curr Med Chem. In press. 51. Brzozowsk AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 1997; 389:753–758. 52. Zhou JR, Mukherjee P, Gugger ET, Tanaka T, Blackburn GL, Clinton SK. Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 1998; 58:5231–5238. 53. Peterson G, Barnes S. Genistein inhibits both estrogen and growth factor- stimulated proliferation of human breast cancer cells. Cell Growth Differ 1996; 7:1345–1351.

54. Shao ZM, Alpaugh ML, Fontana JA, Barsky SH. 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 1998; 69:44–54.

55. Shao ZM, Wu J, Shen ZZ, Barsky SH. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998; 58:4851–4857. 56. Archer JS. NAMS/Solvay Resident Essay Award. Relationship between estrogen, serotonin, and depression. Menopause 1999; 6:71–78. 57. Avis NE, Crawford S, Stellato R, Longcope C. Longitudinal study of hor- mone levels and depression among women transitioning through menopause. Climacteric 2001; 4:243–249. 58. Fuller RW. Uptake inhibitors increase extracellular serotonin concentration measured by brain microdialysis. Life Sci 1994; 55:163–167. 59. Barker EL, Blakely RD. Norepinephrine and serotonin transporter: molecular targets of antidepressant drugs. In: Bloom FE, Kupfer DJ, eds. Psychophar- macology: The Fourth Generation of Progress. New York: Raven, 1995:321– 333. 60. Fozzard J. Peripheral actions of 5-hydroxytryptamine. In: Fozzard J. ed. Periph- eral Actions of 5-Hydroxytryptamine. New York: Oxford University Press, 1989.

634 Vaya et al. 61. Chang AS, Chang SM. Nongenomic steroidal modulation of high-affinity

serotonin transport. Biochim Biophys Acta 1999; 1417:157–166. 62. Ofir R, Tamir S, Khatib S, Vaya J. Inhibition of serotonin reuptake by licorice constituents. J Mol Neurosci 2003; 20:135–140. 63. Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, Shapiro SS. Inhibition of melanosome transfer results in skin lightening. J Invest Dermatol 2000; 115:162–167. 64. Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, Shapiro SS. The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions. Exp Cell Res 2000; 254:25–32. 65. Yokota T, Nishio H, Kubota Y, Mizoguchi M. The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation. Pigment Cell Res 1998; 11:355–361. 66. Nerya O, Vaya J, Musa R, Izrael S, Ben-Arie R, Tamir S. Glabrene and iso- liquiritigenin as tyrosinase inhibitors from licorice roots. J Agric Food Chem 2003; 51:1201–1207. 67. Castro L, Freeman BA. Reactive oxygen species in human health and disease. Nutrition 2001; 17:161, 163–165. 68. Yen CH, Hsieh CC, Chou SY, Lau YT. 17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells. Life Sci 2001; 70:403–413. 69. Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA. Reactive oxygen species, cell signaling, and cell injury. Free Rad Biol Med 2000; 28:1456–1462. 70. Jiang XR, Wrona MZ, Dryhurst G. 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 1999; 12:429–436. 71. Kubo I, Kinst-Hori I, Kubo Y, Yamagiwa Y, Kamikawa T, Haraguchi H. Molecular design of antibrowning agents. J Agric Food Chem 2000; 48:1393– 1399. 72. Seo SY, Sharma VK, Sharma N. Mushroom tyrosinase: recent prospects. J Agric Food Chem 2003; 51:2837–2853. 73. Karg E, Odh G, Wittbjer A, Rosengren E, Rorsman H. Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells. J Invest Dermatol 1993; 100:209S–213S. 74. Kinoshita T, Tamura Y. Chemical studies on Glycyrrhiza glabra (licorice): isolation of two new 3-arylcoumarins and revised structure of an isoflav-3-ene glabrene. Nat Prod Lett 1997; 9:289–296.