Isolation, Chemical Identification and Biological Characterization of Phytoestrogens from Wild Bitter Gourd (Momordica Charantia)
Date Issued
2011
Date
2011
Author(s)
Hsu, Chin
Abstract
Postmenopausal women suffer not only from the menopausal syndromes but also higher risks of osteoporosis, cardiovascular diseases and metabolic syndrome. Some phytoestrogens have been characterized as selected estrogen receptor modulator (SERM) and regarded as one of the substitute for hormone-replacement therapy which was shown to have adverse events. The Cucurbitaceae plant Momordica charantia (MC, bitter gourd) is a common tropical vegetable and has been used in traditional medicine. We have found the the non-saponifiable fraction (NS) of a wild bitter gourd (BG) extract activated transcription via estrogen receptors (ER). Studies in this thesis thus aimed at identifying and characterizing phytoestrogenic compounds in BG.
To assess the in vivo estrogen-related response of BG, 3 groups (n=4/group) of weanling C57BL/6J female mice were respectively fed a basal diet (B), basal diet supplemented with E2 (2 mg 17β-estradiol/kg diet) or BGP (5% bitter gourd powder). The vaginal opening of mice treated with E2 and BGP were respectively, 4.5 days (p<0.01) and 1.8 days (p=0.12) earlier than those of the B group. Results of vaginal smear examination indicated that the E2 group persisted on the estrus stage while BGP group showed a prolonged estrus stage (61%) that was longer than B group (41%)(p<0.01). To assess more in vivo estrogen-related response, ovariectomized mice fed a high fat diet (HF group) were supplemented with E2 (E2 group), 5% bitter gourd powder (BGP group) or 0.2% non-saponifiable fraction from ethyl acetate extract of BG (NS group) for 15 or 30 weeks. The uterus weight was significantly higher in the E2 group (p<0.05) but not in the BGP and the NS group. In the calcium balance analysis after 3 weeks feeding, the fecal Ca excretion was less (p<0.01), and the Ca balance (p=0.12) and Ca retention (p=0.07) were higher in the E2 group. The fecal and urinary Ca excretion of the BGP group were also less (p<0.01) than the control group. The Ca balance and Ca retention of BGP and NS groups were also improved and comparable to those of the E2 group (p>0.05). These results implied that BG may be beneficial for estrogen deficiency. To elucidate the phytoestrogenic compounds in BG, a cell-based ER transactivation assay were used to track the active compounds. Lyophilized powders of BG fruits were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The non-saponifiable fraction (NS) of the EA extract as well as the acid-hydrolyzed EtOH extract (AH) was fractionated by repeated column chromatography and isolates were further purified by preparative HPLC or RP-HPLC. Lutein, loliolide, phytol, and one known cucurbitane-type triterpenoid 5β,19-epoxycucurbita-6,24-dien-3β,23-diol (6) were identified from NS. Five new cucurbitane-type triterpenoids, cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1), 4β,19-epoxycucurbita-6,22(E),24-trien-3β,19-diol (2), 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4), together with 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5) were isolated from AH. Except for compound 3 and 4, the remaining compounds showed weak agonistic activity via ERα and β in the non-cytotoxic concentration range. Compounds 1, 2, 3, 5 and 6 showed significant antagonist activity on ERs in the transactivation assay. These ER active extract/compounds were then assessed for their effect on the proliferation of estrogen-dependent human breast cancer cell MCF-7 and endometrial-derived Ishikawa cells by MTT assay in the absence or presence of 1 nM E2. Lutein, compound 2, 3 and 6 significantly reduced the MTT values of MCF-7. Except for compound 4, lutein and the remaining 5 cucurbitane-type triterpenoids significantly reduced the MTT values of Ishikawa cells. All the 6 cucurbitane-type triterpenoids decreased the ER target gene ALP activity of Ishikawa cells. Besides, lutein and all the 6 cucurbitane-type triterpenoids antagonized the proliferation effect of 1 nM E2 in both of these estrogen-dependent cancer cell lines. These inhibition effects were not observed in the estrogen-independent MDA-MB-231 cells within a similar concentration range. In conclusion, five new cucurbitane-type triterpenoids were isolated and identified from the AH of Momordica charantia, and four of them showed estrogenic/antiestrogenic activities. Besides, another four known compounds were isolated and identified from the NS as phytoestrogens of MC. These compounds showed significant antagonistic activity toward E2. These results provide basic evidence that Momordica charantia might be a source of beneficial phytoestrogens isolated and identified.
To assess the in vivo estrogen-related response of BG, 3 groups (n=4/group) of weanling C57BL/6J female mice were respectively fed a basal diet (B), basal diet supplemented with E2 (2 mg 17β-estradiol/kg diet) or BGP (5% bitter gourd powder). The vaginal opening of mice treated with E2 and BGP were respectively, 4.5 days (p<0.01) and 1.8 days (p=0.12) earlier than those of the B group. Results of vaginal smear examination indicated that the E2 group persisted on the estrus stage while BGP group showed a prolonged estrus stage (61%) that was longer than B group (41%)(p<0.01). To assess more in vivo estrogen-related response, ovariectomized mice fed a high fat diet (HF group) were supplemented with E2 (E2 group), 5% bitter gourd powder (BGP group) or 0.2% non-saponifiable fraction from ethyl acetate extract of BG (NS group) for 15 or 30 weeks. The uterus weight was significantly higher in the E2 group (p<0.05) but not in the BGP and the NS group. In the calcium balance analysis after 3 weeks feeding, the fecal Ca excretion was less (p<0.01), and the Ca balance (p=0.12) and Ca retention (p=0.07) were higher in the E2 group. The fecal and urinary Ca excretion of the BGP group were also less (p<0.01) than the control group. The Ca balance and Ca retention of BGP and NS groups were also improved and comparable to those of the E2 group (p>0.05). These results implied that BG may be beneficial for estrogen deficiency. To elucidate the phytoestrogenic compounds in BG, a cell-based ER transactivation assay were used to track the active compounds. Lyophilized powders of BG fruits were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The non-saponifiable fraction (NS) of the EA extract as well as the acid-hydrolyzed EtOH extract (AH) was fractionated by repeated column chromatography and isolates were further purified by preparative HPLC or RP-HPLC. Lutein, loliolide, phytol, and one known cucurbitane-type triterpenoid 5β,19-epoxycucurbita-6,24-dien-3β,23-diol (6) were identified from NS. Five new cucurbitane-type triterpenoids, cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1), 4β,19-epoxycucurbita-6,22(E),24-trien-3β,19-diol (2), 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4), together with 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5) were isolated from AH. Except for compound 3 and 4, the remaining compounds showed weak agonistic activity via ERα and β in the non-cytotoxic concentration range. Compounds 1, 2, 3, 5 and 6 showed significant antagonist activity on ERs in the transactivation assay. These ER active extract/compounds were then assessed for their effect on the proliferation of estrogen-dependent human breast cancer cell MCF-7 and endometrial-derived Ishikawa cells by MTT assay in the absence or presence of 1 nM E2. Lutein, compound 2, 3 and 6 significantly reduced the MTT values of MCF-7. Except for compound 4, lutein and the remaining 5 cucurbitane-type triterpenoids significantly reduced the MTT values of Ishikawa cells. All the 6 cucurbitane-type triterpenoids decreased the ER target gene ALP activity of Ishikawa cells. Besides, lutein and all the 6 cucurbitane-type triterpenoids antagonized the proliferation effect of 1 nM E2 in both of these estrogen-dependent cancer cell lines. These inhibition effects were not observed in the estrogen-independent MDA-MB-231 cells within a similar concentration range. In conclusion, five new cucurbitane-type triterpenoids were isolated and identified from the AH of Momordica charantia, and four of them showed estrogenic/antiestrogenic activities. Besides, another four known compounds were isolated and identified from the NS as phytoestrogens of MC. These compounds showed significant antagonistic activity toward E2. These results provide basic evidence that Momordica charantia might be a source of beneficial phytoestrogens isolated and identified.
Subjects
Momordica charantia
estrogen receptor
phytoestrogen
transactivation assay
ovariectomized mice
estrogen-dependent carcinoma
SDGs
Type
thesis
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