We have previously shown that RJ has estrogenic activities and suggested it contains estrogenic components (10
). In the current study, we isolated four compounds from RJ (10H2DA, 10HDA, 2DEA and 24MET) that exhibit ERβ-binding activity. We used several assays to evaluate the estrogenic activity of these compounds. ERE-based reporter gene expression assays suggested that these compounds activated ERs, leading to transcriptional activation via an ERE (). We further observed that these compounds stimulated the proliferation of MCF-7 cells, which have been shown to respond to estrogens (). Concomitant treatment with tamoxifen blocked this effect. Thus, all in vitro
data indicate that 10H2DA, 10HDA, 2DEA and 24MET elicit the full sequence of estrogenic action.
RJ comprises 60–70% water, 12–15% proteins, 10–12% carbohydrates, 3–7% lipids (including sterols and fatty acids) and traces of mineral salts and vitamins (19
). 24MET constitutes 49–58% of RJ sterols (20
). 10H2DA and 10HDA are characteristic fatty acids of RJ, together representing 60–80% of the organic acid content of RJ, whereas 2DEA is present at a much lower level (21
). 10H2DA has been reported to have antibacterial (22
), antitumor (23
) and insulin-like (24
) activities. 10H2DA and 10HDA have also been shown to promote collagen production by skin fibroblasts (25
). However, little information is available concerning the pharmacological effects of either 24MET or 2DEA. To our knowledge, this is the first report demonstrating the estrogenic effects of 24MET, 2DEA, 10H2DA or 10HDA, although these compounds are not as potent as steroid estrogens.
10H2DA, 10HDA, 2DEA and 24MET have rather weak binding affinities for ERβ compared with diethylstilbestrol (10−2
-fold) (), circulating E2
-fold), genistein (10−3
-fold) or daidzein (10−1
). However, the concentrations of these compounds in RJ are much greater than those of the endogenous steroid estrogens found under physiological conditions. The high concentrations of these compounds in RJ could, partly at least, account for the pharmacological effects attributed to RJ, including improvement of menopausal symptoms (8
), bone formation (27
) and prevention of osteoporosis (28
), although further analysis is necessary.
The weight increase induced in the immature uterus of the rat by exposure to ethynylestradiol is characteristic of the response expected for a potent ER agonist (17
). The uterine wet weight increase induced by 17αEE2
results from hypertrophy and hyperplasia, as well as accumulation of fluid in the lumen (15
). Although we did not detect an increase in the uterine wet weight in response to exposure of the immature rat uterus to 10H2DA, 10HDA, 2DEA or 24MET, we did observe mild hypertrophy of the luminal epithelium ( and ; ). These observations may be explained by differences in the pattern of expression of ER subtypes in rat tissues. ERα rather than ERβ is predominantly expressed in the uterus (29
). Our data shows that 10H2DA, 10HDA, 2DEA and 24MET bind preferentially to ERβ rather than ERα (), which may explain why we only observe a slight increase in epithelial cell height, and no increase in uterus weight, in response to treatment of immature rats with these compounds.
Earlier studies have shown that unsaturated fatty acids, but not saturated fatty acids, modulate estrogen and/or ER(s) by alterations in estradiol binding to receptors (30
), and/or by cleaving native ER(s) (31
). Linoleic acid, an unsaturated fatty acid, was isolated from chaste-tree berries as an estrogenic compound utilizing ERβ binding as a monitor (32
). Thus, unsaturated fatty acids have been shown to modulate and/or induce some estrogenic effects via interaction with estrogen and/or ERs. Other studies suggest that non-esterified fatty acids may influence cell growth and proliferation by modifying membrane fluidity (33
). Our data indicate that 10H2DA, 10HDA, 2DEA and 24MET interact with ER, activate ERs resulting in enhanced transcription of reporter gene via the ERE and enhance MCF-7 cell proliferation. 10H2DA, 10HDA and 2DEA are similar in their molecular structure, although 10HDA is saturated whereas 10H2DA and 2DEA are both unsaturated fatty acids. The results suggest that saturated fatty acids can also exhibit estrogenic activity.
In summary, we isolated and identified four compounds associated with the estrogenic effects of RJ. Further understanding of these compounds should provide a scientific basis for the development of better therapeutic applications of dietary supplement for the improvement of quality of life in menopausal women.