\nKeywords:<\/strong> Pueraria mirifica, Ovariectomized rabbits, Endothelial function, Nitric oxide, Effects of Pueraria Mirifica, Effects of Pueraria Mirifica<\/p>\n
Effects of Pueraria Mirifica<\/h2>\nINTRODUCTION<\/strong><\/h2>\nEstrogen plays a significant protective role in the cardiovascular system. Postmenopausal women experience a markedly increased risk of cardiovascular disorders, and some studies suggest that hormone replacement therapy reduces the mortality rate due to cardiovascular disease. Accumulating evidence indicates that estrogen improves the vascular endothelial function via nitric oxide (NO)-dependent pathway, which may contribute to its cardiovascular protective effects . Moreover, low-dose hormone replacement treatment (HRT) improves lipid profiles and brachial artery endothelial function in women at risk for coronary heart disease . However, several studies suggest that estrogens may increase the risk of developing breast and endometrial cancer. Recently, selective estrogen receptor modulators (SERMs) have been introduced for postmenopausal new therapy. SERMs are compounds that interact with the estrogen receptors and have tissue-specific effects distinct from those of estradiol that they are estrogen agonists in certain tissues and antagonists in others. The main potential benefit of SERMs is that they selectively interact with specific receptors, coactivators and corepressors in different organ systems proving a better risk:benefit profile relative to standard hormone replacement therapy. While many attempts and several clinical trials have been conducted toward the development of “ideal” SERMs, benefits from the use of phytoestrogens in menopause has increasingly become evident. Several phytoestrogens has been identified such as, genistein, daidzein, formononetin, equol, biochanin A while a search for “phyto-SERMs” is on the way. For instance, a soy-derived product (DT56a (Tofupill\/ Femarelle) and an extract from Cimicifuga racemosa (BNO 1055) possess SERM characteristics and may be used as alternative HRT.
\nRecent studies show that phytoestrogens from dietary sources, such as soybeans, improve the impaired endothelial-dependent relaxation. In Thailand, the rhizome of Pueraria Mirifica<\/a> (“Kwao Kreu”) has long been used as a tonic for women in folk medicine. It contains several known compounds, including isoflavones, coumestans, and deoxymiroestrol. The phytoestrogenenic effects were demonstrated in recombinant yeast, MCF-7 cell proliferation and HepG2 cell transient transfection assay. However, its effect on endothelial function has not been investigated. Here, the authors use the experiment model of ovariectomized female rabbits treated with P. mirifica to determine its effect on vascular reactivity in vitro. In addition, its effects on blood lipid profile, lipid peroxidation and liver enzyme function were also determined.<\/p>\nMaterial and Method Animals<\/strong><\/h3>\nFemale mature New Zealand White rabbits (3.2-4.0 Kg) were subjected to bilateral ovariectomy and they were divided into two groups; control (OVX, n = 5), and Pueraria Mirifica<\/a> (PM) treated group (OVX + PM, n = 4). Four weeks after surgery, control rabbits (OVX) received saline and OVX + PM rabbits received ground powder of PM (100 mg\/kg\/day) orally for 90 days. The PM powder was kindly provided by Assoc. Prof. Chaiyo Chaichantipyuth, Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Plant material was standardized as a commercial roduct. Vascular reactivity studies At the end of the treatment period, rabbits were euthanized with an overdose of sodium pentobarbital. Vascular study was performed as previously described. Briefly, thoracic aortas were removed and placed in cold Krebs’ solution. Aortic rings (5 mm in length) were then placed under 1 g of tension in an organ bath containing 10 ml Krebs solutions at 37\u00acC and bubbled with 95% O2 and 5% CO2. The vascular reactivity studies included contractile response to norepinephrine (NE), relaxation response to acetylcholine (Ach) (Sigma), 17\u00ac-estradiol (Sigma) in the presence or absence of L-NG-Nitroarginine methyl ester, hydrochloride (L-NAME) (Sigma), and sodium nitroprusside (SNP) (Sigma).<\/p>\nSerum lipid profile and blood chemistry<\/strong><\/h3>\nBlood was drawn from the ear vein and serum\/plasma samples were corrected at week 0, 4, 8 and 12 of PM treatment. The analysis of cholesterol, LDL, triglyceride, HDL, ALT (SGOT), AST (SGPT) and alkaline phosphatase was determined by automatic analysis technique using commercially available reagents and performed at the Laboratory of Faculty of Allied Health Sciences, Chulalongkorn Universty.<\/p>\n
Serum lipid peroxidation<\/strong><\/h3>\nThe extent of cellular lipid peroxidation was determined by measuring the concentrations of thiobarbituric acid-reactive substances (TBARS) as escribed by Menendez et al with minor modification. Briefly, sample oxidation was terminated by adding butylated hydroxytoluene (Sigma) to the final concentration of 10 \u00b5M. Malondialdehyde (MDA) (Sigma) solutions (0 to 10 \u00b5M) were used to generate standard curve. Two hundred microliters of standards or samples were incubated with thiobarbituric acid solution containing 0.5% thiobarbituric acid (Sigma), 6% trichloroacetic acid (Merck), and 1 mM EDTA (Sigma), at 95 C for 90 min. The reaction mixtures were then centrifuged at 9,000 x g for 10 min. The super-natants were removed and measured the absorbance at 532 nm (Shimadzu UV-1601). Total protein content in serum was determined by Bradford assay using bovine serum albumin as protein standard (Biorad). TBARS values were expressed as MDA equivalences (nmol MDA\/mg protein).<\/p>\n
Data analysis<\/strong><\/h3>\nData are expressed as mean + SEM. Statistical analysis was performed using t test or one-way ANOVA with repeated measurement and with Bonferroni post hoc test. A value of p < 0.05 wasJ Med Assoc Thai Vol. 88 Suppl.1 2005 S23 onsidered significant.<\/p>\n
Effects of Pueraria Mirifica<\/h2>\nResults<\/strong><\/h2>\nChanges in vascular reactivity<\/strong><\/h3>\nIsolated vascular contractile responses are shown in Table 1 and Fig. 1. Significant alteration in acetylcholine-induced relaxation was observed (Fig. 1A). A statistically significant 3.5-fold reduction in EC50 (522 + 131 vs 150 + 44 nM, OVX vs OVX + PM) was observed, with significant change (24% increase) in maximal relaxant response (Emax 62.5 + 0.6% vs 82.4 + 0.8%). In contrast to diminished acetylcholine responses, no significant change in vasorelaxant response to the endothelium independent and ‘spontaneous’ NO donor sodium nitroprusside as observed between teatments (Table 1 and Fig. 1B). Significant increases in minimal and maximal relaxation responses to 17\u03b2-estradiol were observed (Emin 5.11 + 1.62% vs 29.80 + 9.04%; Emax 63.76 + 0.55% vs 76.45 + 0.49%, OVX vs OVX + PM, p < 0.05). In addition, the EC50 of 17\u03b2\u2212estradiol in PM treated rabbits increased by 3.8-fold (Table 1 and Fig. 1C). In the presence of a nitric oxide synthase inhibitor L- NAME, significant reduction in Emax was detected whereas no modification in EC50 was found in aortic rings from OVX + PM rabbits (Table 1 and Fig 1D). In addition to alteration in vasorelaxant effect, maximal vascular contractile response to a-receptor agonist phenylephrine was significantly decreased in the PM-treated group (OVX + PM vs OVX, p < 0.05) without change in EC50 (Table 1, Fig 1E).<\/p>\n
Serum lipid profile, liver enzyme, and alkaline phosphatase activities<\/strong><\/h3>\nShown in Table 2 are serum lipid profiles of ovariectomized rabbits at week 0, 4, 8, and 12 following saline (OVX) and 100 mg\/kg\/day PM (OVX + PM) treatments. There was no significant change in total cholesterol, LDL, and HDL both when
\nTable 1<\/strong>. Vascular reactivities of aorta from ovariectomized rabbits<\/p>\n
Estrogen plays a significant protective role in the cardiovascular system. Postmenopausal women experience a markedly increased risk of cardiovascular disorders, and some studies suggest that hormone replacement therapy reduces the mortality rate due to cardiovascular disease. Accumulating evidence indicates that estrogen improves the vascular endothelial function via nitric oxide (NO)-dependent pathway, which may contribute to its cardiovascular protective effects . Moreover, low-dose hormone replacement treatment (HRT) improves lipid profiles and brachial artery endothelial function in women at risk for coronary heart disease . However, several studies suggest that estrogens may increase the risk of developing breast and endometrial cancer. Recently, selective estrogen receptor modulators (SERMs) have been introduced for postmenopausal new therapy. SERMs are compounds that interact with the estrogen receptors and have tissue-specific effects distinct from those of estradiol that they are estrogen agonists in certain tissues and antagonists in others. The main potential benefit of SERMs is that they selectively interact with specific receptors, coactivators and corepressors in different organ systems proving a better risk:benefit profile relative to standard hormone replacement therapy. While many attempts and several clinical trials have been conducted toward the development of “ideal” SERMs, benefits from the use of phytoestrogens in menopause has increasingly become evident. Several phytoestrogens has been identified such as, genistein, daidzein, formononetin, equol, biochanin A while a search for “phyto-SERMs” is on the way. For instance, a soy-derived product (DT56a (Tofupill\/ Femarelle) and an extract from Cimicifuga racemosa (BNO 1055) possess SERM characteristics and may be used as alternative HRT. Female mature New Zealand White rabbits (3.2-4.0 Kg) were subjected to bilateral ovariectomy and they were divided into two groups; control (OVX, n = 5), and Pueraria Mirifica<\/a> (PM) treated group (OVX + PM, n = 4). Four weeks after surgery, control rabbits (OVX) received saline and OVX + PM rabbits received ground powder of PM (100 mg\/kg\/day) orally for 90 days. The PM powder was kindly provided by Assoc. Prof. Chaiyo Chaichantipyuth, Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Plant material was standardized as a commercial roduct. Vascular reactivity studies At the end of the treatment period, rabbits were euthanized with an overdose of sodium pentobarbital. Vascular study was performed as previously described. Briefly, thoracic aortas were removed and placed in cold Krebs’ solution. Aortic rings (5 mm in length) were then placed under 1 g of tension in an organ bath containing 10 ml Krebs solutions at 37\u00acC and bubbled with 95% O2 and 5% CO2. The vascular reactivity studies included contractile response to norepinephrine (NE), relaxation response to acetylcholine (Ach) (Sigma), 17\u00ac-estradiol (Sigma) in the presence or absence of L-NG-Nitroarginine methyl ester, hydrochloride (L-NAME) (Sigma), and sodium nitroprusside (SNP) (Sigma).<\/p>\n Blood was drawn from the ear vein and serum\/plasma samples were corrected at week 0, 4, 8 and 12 of PM treatment. The analysis of cholesterol, LDL, triglyceride, HDL, ALT (SGOT), AST (SGPT) and alkaline phosphatase was determined by automatic analysis technique using commercially available reagents and performed at the Laboratory of Faculty of Allied Health Sciences, Chulalongkorn Universty.<\/p>\n The extent of cellular lipid peroxidation was determined by measuring the concentrations of thiobarbituric acid-reactive substances (TBARS) as escribed by Menendez et al with minor modification. Briefly, sample oxidation was terminated by adding butylated hydroxytoluene (Sigma) to the final concentration of 10 \u00b5M. Malondialdehyde (MDA) (Sigma) solutions (0 to 10 \u00b5M) were used to generate standard curve. Two hundred microliters of standards or samples were incubated with thiobarbituric acid solution containing 0.5% thiobarbituric acid (Sigma), 6% trichloroacetic acid (Merck), and 1 mM EDTA (Sigma), at 95 C for 90 min. The reaction mixtures were then centrifuged at 9,000 x g for 10 min. The super-natants were removed and measured the absorbance at 532 nm (Shimadzu UV-1601). Total protein content in serum was determined by Bradford assay using bovine serum albumin as protein standard (Biorad). TBARS values were expressed as MDA equivalences (nmol MDA\/mg protein).<\/p>\n Data are expressed as mean + SEM. Statistical analysis was performed using t test or one-way ANOVA with repeated measurement and with Bonferroni post hoc test. A value of p < 0.05 wasJ Med Assoc Thai Vol. 88 Suppl.1 2005 S23 onsidered significant.<\/p>\n Isolated vascular contractile responses are shown in Table 1 and Fig. 1. Significant alteration in acetylcholine-induced relaxation was observed (Fig. 1A). A statistically significant 3.5-fold reduction in EC50 (522 + 131 vs 150 + 44 nM, OVX vs OVX + PM) was observed, with significant change (24% increase) in maximal relaxant response (Emax 62.5 + 0.6% vs 82.4 + 0.8%). In contrast to diminished acetylcholine responses, no significant change in vasorelaxant response to the endothelium independent and ‘spontaneous’ NO donor sodium nitroprusside as observed between teatments (Table 1 and Fig. 1B). Significant increases in minimal and maximal relaxation responses to 17\u03b2-estradiol were observed (Emin 5.11 + 1.62% vs 29.80 + 9.04%; Emax 63.76 + 0.55% vs 76.45 + 0.49%, OVX vs OVX + PM, p < 0.05). In addition, the EC50 of 17\u03b2\u2212estradiol in PM treated rabbits increased by 3.8-fold (Table 1 and Fig. 1C). In the presence of a nitric oxide synthase inhibitor L- NAME, significant reduction in Emax was detected whereas no modification in EC50 was found in aortic rings from OVX + PM rabbits (Table 1 and Fig 1D). In addition to alteration in vasorelaxant effect, maximal vascular contractile response to a-receptor agonist phenylephrine was significantly decreased in the PM-treated group (OVX + PM vs OVX, p < 0.05) without change in EC50 (Table 1, Fig 1E).<\/p>\n Shown in Table 2 are serum lipid profiles of ovariectomized rabbits at week 0, 4, 8, and 12 following saline (OVX) and 100 mg\/kg\/day PM (OVX + PM) treatments. There was no significant change in total cholesterol, LDL, and HDL both when
\nRecent studies show that phytoestrogens from dietary sources, such as soybeans, improve the impaired endothelial-dependent relaxation. In Thailand, the rhizome of Pueraria Mirifica<\/a> (“Kwao Kreu”) has long been used as a tonic for women in folk medicine. It contains several known compounds, including isoflavones, coumestans, and deoxymiroestrol. The phytoestrogenenic effects were demonstrated in recombinant yeast, MCF-7 cell proliferation and HepG2 cell transient transfection assay. However, its effect on endothelial function has not been investigated. Here, the authors use the experiment model of ovariectomized female rabbits treated with P. mirifica to determine its effect on vascular reactivity in vitro. In addition, its effects on blood lipid profile, lipid peroxidation and liver enzyme function were also determined.<\/p>\nMaterial and Method Animals<\/strong><\/h3>\n
Serum lipid profile and blood chemistry<\/strong><\/h3>\n
Serum lipid peroxidation<\/strong><\/h3>\n
Data analysis<\/strong><\/h3>\n
Effects of Pueraria Mirifica<\/h2>\n
Results<\/strong><\/h2>\n
Changes in vascular reactivity<\/strong><\/h3>\n
Serum lipid profile, liver enzyme, and alkaline phosphatase activities<\/strong><\/h3>\n
\nTable 1<\/strong>. Vascular reactivities of aorta from ovariectomized rabbits<\/p>\n
![\"Vascular](\"https:\/\/www.puerariathai.com\/wp-content\/uploads\/2015\/11\/Vascular-responses-in-isolated-aortic-rabbit-rings.png\")
<\/a><\/p>\n![\"Effect](\"https:\/\/www.puerariathai.com\/wp-content\/uploads\/2015\/11\/Effect-of-Pueraria-Mirifica-treatment-on-serum-lipid-peroxidation.png\")
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