Estrogenlike 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 inflammation (41). The organic extract of licorice root (acetone or ethanol) is known to contain isoflavans, isoflavene, and chalcones (Fig. 2) such as glabridin,

Hydroxyglabrol
Z-OMeG R1=CH3,R2=H 4'-OMeG RpH, R2=Me

Isoliquitireginin chalcone (ILC) Isoprenyl chalcone ( IPC)

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

Isoliquitireginin chalcone (ILC) Isoprenyl chalcone ( IPC)

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

glabrol, glabrene, 3-hydroxyglabrol, ^-O-methylglabridin (4'-OMeG), his-paglabridin A (hisp A), hispaglabridin B (hisp B), isoprenylchalcone derivative (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 compound 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 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.

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 (17p in estradiol and 2' in glabridin).

1. Binding of Glabridin to the ERa

Glabridin binds the ER with IC50 of 5 ||M (44) and with approximately the same affinity as genistein, the best known phytoestrogen (33), 104 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 |M, reaching a maximum level at about 10 |M; at a higher level (15 ||M), 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 coronary 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 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) (49).

10-15

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

10-15

Concentration (M)

hispB -o-ILC

- estradiol glabridin 4-O'MG 2-O'MG

glabrene

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

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 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 + SD of >3 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 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 + SD of >3 experiments.

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

Cells ECV304 VSMC

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

Cells ECV304 VSMC

Estradiol

0.3

nM

1.77

+

0.16

3.28

+

0.09

30

nM

2.44

+

0.11

0.53

+

0.19

Glabridin

30

nM

1.52

+

0.20

2.37

+

0.10

300

nM

3.34

+

0.30

0.89

+

0.22

3

am

8.72

+

0.28

0.40

+

0.22

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 increased fold of control.

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 increased fold of control.

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 transcriptional activation with half-maximal induction at 1 |M, 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 |ig/day/rat glabrene resulted in a similar effect to that of 5 Ag/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 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 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 2' (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.

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