Estrogen like activity of Licorice Ext

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					Estrogen-Like Activity of Licorice Root Extract and its Constituents

                        Jacob Vaya1*, Snait Tamir1, Dalia Somjen2

    Laboratory of Natural Compounds for Medicinal Use, Migal – Galilee Technological

    Center, Kiryat Shmona 10200, Israel
    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


                                          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


      7.     Licorice Constituents Inhibit Serotonin Re-uptake

      8.     Whitening Effect of Licorice Extract and its Constituents

      9.     Summary

     10.     References

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

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.

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

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

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

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

(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

     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).

  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

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

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


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

     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.

   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


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.

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

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-


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


     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 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.

  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



Figure 1: Structures of several phytoestrogens, tamoxifen, raloxifene and estradiol

                                   N          O                                                          OH

                                                                        OH         HO
                                         HO              S

             Tamoxifen                       Raloxifene                                 17b-Estradiol

                                   HO                                                                   OH
       HO                                                    OH
                                                             OH             HO          O


            Enterolactone               Enterodiol                               Coumestrol

                                                                             HO             O
            HO           O
                                        HO           O

                                                                                    OH      O
                  OH     O                                                                               OH
                                   OH                O
                                                                                        Biochanin A
                       Genistein                  Daidzein

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

                                    OH                                       O
             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.

Figure 3: The binding of estradiol and licorice constituents to human estrogen

                         receptor α.


binding (%)

               50                                                                 glabrene

                10 -15     10 -11   10 -8 10 -7   10 -6   10 -5   10 -4   10 -3
                                          concentration (M)

Figure 4:            The effects of licorice constituents on the growth of estrogen-responsive

                     human breast cancer cells.

             300                                                                               estradiol
growth (%)



               10 -16 10 -13 10 -10   10 -9   10 -8        10 -7   10 -6   10 -5   10 -4   10 -3
                                         concentration (M)


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.


Table 1: The effect of licorice extract on the induction of CK activity in various

            female rat tissues.

                             Control          Estradiol     Licorice ext.   Glabridin

                                               0.5µg             µ
                                                               25µg             µ

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.

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.

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
  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
               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



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Description: Estrogen like activity of Licorice Ext