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Medical applications of phytoestrogens from the Thai herb Pueraria mirifica
Abstract
Pueraria mirifica Airy Shaw et Suvatabandhu is a medicinal plant endemic to Thailand. It has been used in Thai folklore medicine for its rejuvenating qualities in aged women and men for nearly one hundred years. Indeed, it has been claimed that P. mirifica contains active phytoestrogens (plant substances with estrogen-like activity). Using high performance liquid chromatography, at least 17 phytoestrogens, mainly isoflavones, have been isolated. Thus, fairly considerable scientific researches, both in vitro in cell lines and in vivo in various species of animals including humans, have been conducted to date to address its estrogenic activity on the reproductive organs, bones, cardiovascular diseases and other climacteric related symptoms. The antioxidative capacity and antiproliferative effect on tumor cell lines have also been assessed. In general, P. mirifica could be applicable for preventing, or as a therapeutic for, the symptoms related to estrogen deficiency in menopausal women as well as in andropausal men. However, the optimal doses for each desirable effect and the balance to avoid undesired side effects need to be calculated before use.
... [10] It also reduces the signs of estrogen deficiency such as hair loss, wrinkles, and sagging breasts and may help in breast enhancement, and overall esthetics. [12][13][14] PM is available in herbal stores, pharmacies, and on various internet websites in formulations such as pills, capsules, crackers, lotions, and gels. ...
... The plant's dried roots are used as a dietary supplement, sometimes in conjunction with other medicinal plants. [13] It has also been used for centuries as an anti-aging and rejuvenation medicine. ...
... Deoxymiroestrol possesses 10 times higher estrogenic activity than miroestrol and isomiroestrol, and can be easily converted to miroestrol and isomiroestrol by oxidation. [11,13] Chansakaow et al. reported extracting 2-3 mg of miroestrol and deoxymiroestrol in 100 g powdered tuber of PM. [11] Recently, a novel chromene, methylisomiroestrol, was isolated from the roots of PM, with comparatively stronger estrogenic potency than isomiroestrol, but lower than those of miroestrol and deoxymiroestrol. [40] Isoflavonoids (derivatives of flavonone) are another major group of compounds found in PM, which include, daidzein, daidzin, genistin, genistein, kwakhurin, kwakhurin hydrate, tuberosin, puerarin, mirificin, and puemiricarpene. ...
... mirifica is a plant can be found in northern Thailand and belongs to the Leguminosae family. The tubers of PM have been consumed by native Thai for generations to relieve postmenopausal symptoms including skin wrinkle, hair and memory losses [1,2] . Many scientific studies have proven the pharmacological activities such as estrogenic [3,4] , [5] , antioesteroporosis [6,7] , anticancer [8] , antimutagenic [9] , neuroprotective [10] , antidiabetic [11] of PM extracts. ...
... The administration of PM extract at 20-100 mg/day for 6 months, or 100-200 mg/day for 12 months was found to relieve menopause symptoms without significant changes on hepatic, hematologic, and renal functions [1] . A recommended safe dose of PM supplement was 1-2 mg/kg body weight/day or about 50-100 mg/day [1] . ...
... The administration of PM extract at 20-100 mg/day for 6 months, or 100-200 mg/day for 12 months was found to relieve menopause symptoms without significant changes on hepatic, hematologic, and renal functions [1] . A recommended safe dose of PM supplement was 1-2 mg/kg body weight/day or about 50-100 mg/day [1] . However, the longterm effect of administration has not yet been clarified in detail. ...
Pueraria mirifica (PM) has traditionally been used to relief postmenopausal symptoms. Recently, its extract has been developed into various cosmetic products to promote skin rejuvenation and youthfulness. This study investigated the phytochemicals of PM tuber and compared between the tuber flesh and its outer peel. Puerarin which is one of the major isoflavones and being considered as the marker compound was used to determine the presence of PM extract in local cosmetic products. Puerarin could be ionized by a mass spectrometer at both negative and positive modes. The peak ionized at the negative mode showed to have a narrower peak width (0.2 min) and higher signal-to-noise ratio (30) for pueararin (1 mg/L). The results also found PM extract contained many C- and O-glycosylated isoflavones, especially from its peel extract. This explains the peel extract showed to have four times higher antiradical activity than those of flesh extract. Puerarin from the cosmetic products was recovered via successive methanolic sonication and followed by liquid-liquid extraction using ethyl acetate. Puerarin was successfully partitioned from the highly complex chemical mixture of cosmetic products with the recovery ranged from 89.1 % to 115 %. Hence, isoflavones was found to be higher at the outer peels than its tuber flesh. A simple and reliable method has been developed to analyse the presence of PM extract in cosmetic products based on the detection of puerarin after successive extraction via methanolic sonication and ethyl acetate partition.
... At present, white Kwao Krua is used in estrogen replacement therapy and as rejuvenation medicine. It is sold in herbal markets and drugstores and over the Internet as tablets, capsules, cookies, creams, and gels, and is used to alleviate symptoms of estrogen deficiency (hair loss, wrinkles, and sagging breasts), enlarge breasts, and enhance esthetics [4,[7][8][9][10]. The main components in white Kwao Krua are isoflavones (daidzin, daidzein, genistin, genistein, and puerarin), together with other phytoestrogens, such as chromenes (miroestrol and deoxymiroestrol), and others (β-sitosterol, stigmasterol, kwakhurin, mirificin, coumestrol, and mirificoumestrol) [7,[11][12][13][14][15][16][17][18]. ...
... It is sold in herbal markets and drugstores and over the Internet as tablets, capsules, cookies, creams, and gels, and is used to alleviate symptoms of estrogen deficiency (hair loss, wrinkles, and sagging breasts), enlarge breasts, and enhance esthetics [4,[7][8][9][10]. The main components in white Kwao Krua are isoflavones (daidzin, daidzein, genistin, genistein, and puerarin), together with other phytoestrogens, such as chromenes (miroestrol and deoxymiroestrol), and others (β-sitosterol, stigmasterol, kwakhurin, mirificin, coumestrol, and mirificoumestrol) [7,[11][12][13][14][15][16][17][18]. Especially isoflavones, it was reported that they showed strong estrogenic activity [14]. ...
... Pueraria candollei is distributed in the deciduous forests of northern, northeastern, and western Thailand [7,23]. To prevent wild P. candollei from extinction due to the high demand, limited resources, and soil erosion, the Thai government included P. candollei, M. macrocarpa, and B. superba in the conserved plant list of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) under Thai Plants Act B.E. 2518 (1975) [23][24][25][26]. ...
The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH–psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic “white Kwao Krua” as claimed on their labels.
... The tuberous root of Pueraria candollei and P. candollei var. mirifica (family Leguminosae), known as White Kwao Krua in Thai, has long been used for hormone replacement therapy (Malaivijitnond, 2012;Suntara, 1931). The estrogenic activity of the tuberous root has been attributed to the active compounds, which have similar structures to estrogen, isoflavonoids and chromenes (Malaivijitnond, 2012). ...
... mirifica (family Leguminosae), known as White Kwao Krua in Thai, has long been used for hormone replacement therapy (Malaivijitnond, 2012;Suntara, 1931). The estrogenic activity of the tuberous root has been attributed to the active compounds, which have similar structures to estrogen, isoflavonoids and chromenes (Malaivijitnond, 2012). The data from clinical trials indicate a recommended dose of 50-100 mg dry weight per day (Chandeying & Lamlertkittikul, 2007;Chandeying & Sangthawan, 2007;Lamlertkittikul & Chandeying, 2004). ...
... Although chromenes are present in small amounts, they possess a powerful phytoestrogenic effect (Chansakaow et al., 2000;Okamura et al., 2008). In previous reported studies, Malaivijitnond (2012) and Udomsuk, Chatuphonprasert, Monthakantirat, Churikhit, and Jarukamjorn (2012) claimed that the main estrogenic effect of White Kwao Krua was generated from miroestrol ( Figure 1b). Therefore, the existence of puerarin indicates the identification of Pueraria sp., while miroestrol precisely identifies P. candollei. ...
Pueraria candollei or White Kwao Krua (Leguminosae) is indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation particularly in elder women. Among bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold‐based detection system for the simultaneous detection of miroestrol and puerarin in a one‐step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limit of miroestrol and puerarin were 0.15 and 4.5 μg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme‐linked immunosorbent assay (ELISA) and presented a good correlation between these two methods. This is the first report for the development of an ICS that can detect miroestrol and puerarin in one‐step. The developed ICS indicates a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.
... In contemporary times, P. mirifica is available in various forms, such as tablets, extracts, creams, sprays, and powdered formulations. This versatility allows it to be incorporated into different medicinal preparations or combined with other herbs, as individual conditions may require varying applications and dosages [17][18][19][20]30]. Notably, with its 17 isoflavonoid compounds, P. mirifica is known to contain a high level of phytoestrogen [18,30], which earlier research has suggested may have beneficial effects in preventing cancer and benign prostate hyperplasia (BPH) [31][32][33]. ...
... This versatility allows it to be incorporated into different medicinal preparations or combined with other herbs, as individual conditions may require varying applications and dosages [17][18][19][20]30]. Notably, with its 17 isoflavonoid compounds, P. mirifica is known to contain a high level of phytoestrogen [18,30], which earlier research has suggested may have beneficial effects in preventing cancer and benign prostate hyperplasia (BPH) [31][32][33]. The goal of the current study is to use in vivo models to examine the histomorphology preservation effect of P. mirifica from the tuberous root on BPH. ...
Introduction: Benign prostatic hyperplasia (BPH) is the most prevalent prostatic disease in ageing men, characterised by an excessive proliferation of the prostatic epithelial and stromal cells. Despite the extensive choices of pharmaceutical therapies, the current treatments possess side effects, necessitating the search for new alternative options, including herbal substances such as Pueraria mirifica. This tuberous root of P. mirifica is a medicinal plant that contains numerous phytoestrogens, traditionally used for health rejuvenation in aged men and women. This study was carried out to access the inhibitory effect of 5α-reductase of P. mirifica and its histoprotective effect in a rat model of testosterone-induced BPH. Methods: Adult Sprague Dawley (12 weeks) were subcutaneously injected with testosterone propionate (3 mg/kg) daily to induce BPH. Rats (n=6) in all groups (aqueous extract of P. mirifica (APM): 10, 100, and 1000 mg/kg, p.o.; finasteride: 2mg/kg, p.o., BPH model, and sham groups) were treated for 30 days. The determination of serum dihydrotestosterone (DHT) level, prostatic index and prostate structural changes were investigated. Results: APM and finasteride-treated groups showed significantly lesser prostatic weight and prostatic index, serum DHT levels compared to the model group (p<0.05). Furthermore, there was a significantly lower prostate score with improved prostate histomorphology, demonstrating fewer epithelial involutions of glandular tissues and improved stromal and epithelial cells. Conclusion: In conclusion, the aqueous extract of P. mirifica tuberous root mitigates the development of BPH and it can be inferred that aqueous extract of P. mirifica tuberous root may possess the active agents for anti-BPH treatment.
... mirifica (Airy Shaw & Suvat.) Niyomdham, an endemic Thai medicinal plant of the family Fabaceae, is considered a phytoestrogen-rich plant [1]. P. mirifica root (PMR) has been widely used for over 100 years in Thai folk medicine as a rejuvenating drug for aged people [1]. ...
... Niyomdham, an endemic Thai medicinal plant of the family Fabaceae, is considered a phytoestrogen-rich plant [1]. P. mirifica root (PMR) has been widely used for over 100 years in Thai folk medicine as a rejuvenating drug for aged people [1]. High-performance liquid chromatography (HPLC) analysis revealed that the major phytoestrogens found in PMR are puerarin, daidzin, daidzein, genistin, genistein, and miroestrol [2,3]. ...
Pueraria candollei var. mirifica (Fabaceae) root (PMR) has recently been developed as a potential selective estrogen receptor modulator (SERM) in menopausal women. Nowadays, many premenopausal women also take dietary PMR supplements, however, the exact biological effects of PMR have not been evaluated. This study included the application of the OECD guideline 407 for the assessment of 28-day oral exposure to PMR on pituitary-ovarian (PO) axis function and metabolic parameters in the premenopausal rat model. Ovary-intact adult rats were orally administrated with 10, 100, 750, 1,000, and 1,500 mg/kg body weight (BW)/day of PMR powder. The positive estrogenic group was given 2 mg 17β-estradiol (E2)/kg BW/day. Serum levels of reproductive hormones, lipid and thyroid parameters, estrous cycle determination, and histomorphometric and histopathological evaluations of the anterior pituitary, ovary, uterus, vagina, mammary gland, and liver were investigated. PMR displayed neutral effects on uterine, vaginal, and body weights, and circulating E2 and prolactin levels. PMR exerted E2-like effects by i) reducing ovarian and increasing hepatic weights, ii) decreasing serum gonadotropins, iii) lowering serum lipids without altering thyroid parameters, iv) increasing the prevalence of abnormal estrous cycles with prolonged estrus, v) increasing nuclear diameter of anterior pituitary cells, vi) decreasing ovarian size and follicular numbers and increasing follicular degeneration, vii) thickening of uterine myometrium and luminal epithelium, and vaginal epithelium, and viii) induction of mammary alveolar hyperplasia and ductal secretion. Unlike E2, the appearance of very small numbers of focal microvesicular steatosis in hepatocytes demonstrated mild toxicity at high PMR doses. This is the first report that high-dose PMR exerted actions exactly like E2 on gonadotrope-ovarian axis function and histology, lipid, and thyroid parameters without affecting uterine and vaginal growth in ovary-intact rats according to OECD guidelines.
... Recently, Anukulthanakorn et al. (2016) reported that oral administration of the crude extract of Pueritia mirifica herb (PME) could exhibit neurotherapeutic effects on the early-and late-stages of spatial cognitive impairment in ovariectomy-induced estrogen deficient rats. Pueritia mirifica is known to contain many phytoestrogenic substances, such as puerarin, miroestrol, deoxymiroestrol, genistein, genistin, daidzin, and daidzein, and its estrogenic activity has been widely tested in cells, laboratory animals, and humans on the reproductive organs, bones, and cancers (Malaivijitnond, 2012). The beneficial effects of these phytoestrogens on regulating the synaptic plasticity and preventing the formation of neurofibrillary tangles and amyloid plaques in the hippocampus have also been examined in vitro, and in female animals (Anukulthanakorn et al., 2016;Hong et al., 2016;Li et al., 2019;Liu et al., 2015;Monthakantirat et al., 2014). ...
... The neuroprotective effects of PME on the spatial learning and memory in ovariectomized rats has recently been reported (Anukulthanakorn et al., 2016). Since PME is renowned for its phytoestrogenic constituents and its high estrogenic activity, which has a high similarity in chemical structure to E 2 , especially the phenolic ring that is a prerequisite for binding to ERs (Malaivijitnond, 2012), its effects on cognitive function was, therefore, proposed to resemble those of E 2 . Comparing the effects of PME on the spatial learning and memory, and expression of genes (at mRNA and protein levels) associated with synaptic plasticity, neurofibrillary tangles, and amyloid plaques to those of DHT and E 2 , revealed that PME exerted the lowest efficacy on spatial learning and memory, but was in-between DHT and E 2 for the other effects. ...
This study investigated the neuroprotective effects of dihydrotestosterone (DHT), 17β-estradiol (E2), and Pueraria mirifica herb extract (PME; an alternative source of natural estrogens) on the (i) learning and memory in androgen-deficient male rats, and on the hippocampus expression levels of (ii) mRNA of genes associated with synaptic transmission and structure, neurofibrillary tangles, and amyloid plaques, and (iii) total and phosphorylated tau proteins. The four-month-old male rats were sham-operated or orchidectomized (ODX). The ODX rats were divided into four groups, and orally treated for 2 months with either 1 mL/d of distilled water or 100 mg/kg/d of PME; or subcutaneously injected with 1 mg/kg/d of DHT or 80 μg/kg/d of E2. The impairment of spatial learning behavior and memory capacity in the ODX rats was prevented by DHT, E2, and PME. Recovery of the orchidectomy-induced deterioration of the synaptic plasticity in the hippocampus of rats was ranked as E2 ≥ PME > DHT. Both DHT and PME mitigated the increased Tau3 and Tau4 mRNA levels, and Tau-5 and P-Tau Ser³⁹⁶ protein levels more than E2 (DHT ≥ PME > E2). Only DHT tended to decrease App mRNA expression level. In conclusion, DHT showed a stronger efficacy for mitigation of the impaired spatial learning behavior and memory capacity in androgen-deficient male rats compared to E2 and PME, and their mechanisms of action are slightly different.
... Thai people also used it to recover black hair, promote an appetite, and increase their longevity [17]. The ordinary dosage of PM for women is a peppercorn-sized piece, which is equivalent to approximately 250 mg/kg body weight, taken once daily at night [18]. The ethnobotanical application of PM in folk medicine implies the recognition of its multiple effects in the elderly without knowledge of hormones in that era. ...
... They include puerarin, puerarin-6'-monoacetate, daidzin, genistin, daidzein, genistein, mirificin, kwakhurin, kwakhurin hydrate, coumestrol, miroestrol, isomiroestrol, and deoxymiroestrol [20]. The estrogenic activities of PM extracts have been tested widely in cell cultures, animals, and humans [10,16,18,21]. We previously reported that miroestroltreated OVX mice exhibited improved cognitive function and neuroprotective effects in the hippocampal region [10]. ...
The effects of the phytoestrogen-enriched plant Pueraria mirifica (PM) extract on ovari-ectomy (OVX)-induced cognitive impairment and hippocampal oxidative stress in mice were investigated. Daily treatment with PM and 17β-estradiol (E2) significantly elevated cognitive behavior as evaluated by using the Y maze test, the novel object recognition test (NORT), and the Morris water maze test (MWM), attenuated atrophic changes in the uterus and decreased serum 17β-estradiol levels. The treatments significantly ameliorated ovariectomy-induced oxidative stress in the hippocampus and serum by a decrease in malondialdehyde (MDA), an enhancement of superoxide dismutase, and catalase activity, including significantly down-regulated expression of IL-1β, IL-6 and TNF-α proinflammatory cytokines, while up-regulating expression of PI3K. The present results suggest that PM extract suppresses oxidative brain damage and dysfunctions in the hippocampal antioxidant system, including the neuroinflammatory system in OVX animals, thereby preventing OVX-induced cognitive impairment. The present results indicate that PM exerts beneficial effects on cognitive deficits for which menopause/ovariectomy have been implicated as risk factors.
... Previously, PM application for treatment of a range of conditions, including those related to the aging process, has been reported [1]. Dried and powdered tuberous roots of PM contain at least 17 chemical compounds with estrogenic biological activities, usually divided into three groups: the first group includes teniso flavonoids such as genistin, genistein, daidzein, daidzin, kwakhurin, kwakhurin hydrate, tuberosin, puerarin, mirificin, and puemiricarpene [2,3]; the second group of coumestans comprises coumestrol, mirificoumestan, mirificoumestan glycol, and mirificoumestan hydrate; and the third features chromenes, such as miroestrol, deoxymiroestrol, and isomiroestrol [2]. All these substances are phytoestrogens with Vaginal smears were obtained daily before and after starting the treatment, dried and stained with an aqueous solution of methylene blue. ...
... Trophic effects of estrogenic compounds on the mammary gland and uterus were previously suggested to be due to activation of signaling through estrogen receptors (ERs) ERα and ERβ [17]. It was reported that PM phytoestrogens at high doses could effectively outcompete 17 β-estradiol binding to ERα in MCF-7 cells [3]. We have previously shown that IA at an estrogenic dose of 150 mg/kg b.w./day activated ERα or ERβ and downstream AP1 and NF-κB transcriptional factors, also potentiating F-actin signaling in mammary and uterine adenocarcinomas [15]. ...
Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3% and, more pronouncedly, of 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA.
... White kwao krua is a phytoestrogens enriched plant (10)(11)(12)(13)(14)(15)(16)(17). Previous studies revealed that at least 17 chemical components containing estrogenic activity were found in its tuberous roots, leave and stem, but predominantly found in tuberous roots (56). Among these components, they could be categorized into 3 major groups including isoflavonoids, coumestrans, and chromenes. ...
... Among these components, they could be categorized into 3 major groups including isoflavonoids, coumestrans, and chromenes. Ten isoflavonoids included daidzin, daidzein genistin, genistein, kwakhurin, kwakhurin hydrate, tuberosin, puerarin, mirificin and puemiricarpene; four coumestrans included coumestrol, mirificoumestan, mirificoumestan glycol and mirificoumestan hydrate; and three chromenes included deoxymiroestrol, miroestrol, and isomiroestrol (56)(57)(58)(59). All of these are structurally similar to 17β-estradiol. ...
Background and aim
White kwao krua is an edible plant that grows in Southeast Asia. It is very rich in natural phytoestrogens. Previous clinical studies revealed that the use of White kwao krua as a hormone replacement therapy has beneficial effects on the lipid profile of menopause women. In this present study, we utilized the hypercholesterolemia rabbit model to demonstrate the effect of White kwao krua on the daily intake of high-fat diet.
Experimental procedure
We induced hypercholesterolemia in rabbits by feeding with high-fat diet (1% cholesterol-containing diet). The animals were maintained 12 weeks for the experimentation. The White kwao krua supplement was administered 100 mg/kg/day, and the effects were monitored comparing with Statins and turmeric. Blood was collected periodically to monitor the plasma cholesterol level and the oxidative susceptibility of isolated LDL-cholesterol. At the end of the experiment, the aorta was collected from the animal and performed endothelial-dependent relaxation and endothelial-independent relaxation assays. The relative ratio of intima to media layer was microscopically evaluated from hematoxylin/eosin-stained tissues.
Results and conclusion
We showed that the White kwao krua supplement reduced LDL-cholesterol about 40% compared with high-fat diet consumption alone. Administration of White kwao krua had significantly prolonged the susceptibility of LDL-cholesterol to oxidation. Besides, it led to the improvement of vascular function by recovering endothelium-dependent relaxation and alleviating vascular structure impairment induced by high-fat dietary intake. Together, we suggest that White kwao krua should be used as a dietary supplement to reduce the atherogenesis in high-fat dietary consumption.
Section
Dietary therapy/nutrients supplements.
Taxonomy
Inflammation, Disease.
... P. candollei var. mirifica is also used to pacify cataracts in the eyes, help with memory loss, increase blood circulation and appetite, and alleviate sleep disorders (Malaivijitnond 2012). Currently, P. candollei var. ...
... Currently, P. candollei var. mirifica has been included in cosmetics, dietary supplements, and pharmaceutical products (Cain 1960;Malaivijitnond 2012). ...
The Acknowledgements section in the original article was incomplete. The complete section is shown below. Acknowledgements This research was supported by Rachadapisek Sompote Fund for Postdoctoral Fellowship and for New Faculty Member Chulalongkorn University, Bangkok and Thailand Research Fund Project No. MRG5680090. TM was a recipient of the Research assistant scholarship from the Graduate School, Chulalongkorn University.
... Its extracts include isoflavonoids, coumestrans, and chromenes. 17,18 The effects for combination of extracts on the stemness properties of DP cells via stabilize β-catenin are not well understood. The insights gained from studying stem cell-stimulating properties of these extracts via stabilized β-catenin could lead to new therapeutic options for hair rejuvenation. ...
Objective: Recent advancements in stem cell biology have revealed the potential of natural active compounds to boost hair growth and prevent hair loss by enhancing the stemness of dermal papilla (DP) cells. Methods: The level of upstream stabilized β-catenin was assessed using western blot analysis and immunofluorescence. The stemness was determined by real-time RT-qPCR to examine the mRNA level of stem cell factors and confirm the protein level by immunofluorescence. Results: Our cytotoxicity assay on DP cells treated with various extracts, including AV, CA, GT, PM, and a combination of these extracts (ACGP), indicated no impact on cell viability even at high concentrations (4000 μg/ml). Moreover, the DP cells treated with ACGP showed 1.5-fold high cells proliferation rate compared to single treatment DP cells. Western blot analysis revealed a 1.5-fold increase in the protein expression levels of both pAkt and β-catenin in ACGP-treated DP cells. The results demonstrated that cells treated with the combined extracts (ACGP) exhibited significantly increased mRNA levels of OCT4, NANOG, and SOX2 by 40-, 110-, and 30-fold, respectively, in the downstream stabilize β-catenin pathway for stem cell expression. Corresponding protein levels also increased by 2-, 3-, and 2-fold, respectively, compared to cells treated with each extract individually. Conclusion: These findings revealed the synergistic effect of natural products in stimulating DP stem cells via stabilize β-catenin and enhancing their cellular functions.
... It primarily grows in the deciduous forests of northern, western, and northeastern Thailand. The plant is characterized by its bluish-purple, legume-shaped flowers and white tubers [2,3]. ...
Pueraria candollei var. mirifica produces and accumulates various phytoestrogen compounds in its tuberous roots, including daidzein and genistein. Plant cell culture methods have been established to alleviate the problems associated with producing valuable phytochemicals from natural or field-cultivated plants, and hairy root culture is one of the most promising methods for the in vitro production of plant secondary metabolites. Thus, this study aimed to produce daidzein and genistein from hairy root cultures of P. candollei var. mirifica. The influences of cultivation parameters, including the culture medium, light conditions, sugar content in the culture medium, incubation temperature, and agitation speed, on biomass and daidzein and genistein production in hairy root cultures of this medicinal plant were investigated. The results revealed that the optimal cultivation conditions for biomass and bioactive compound production were Murashige & Skoog (MS) medium, a sucrose concentration of 30 g/L, a 16/8 h light/dark photoperiod, an incubation temperature of 26 °C, and an agitation speed of 90 rpm. The highest biomass and daidzein and genistein contents achieved in this study were 17.76 g/L, 6.85 mg/g DW, and 0.96 mg/g DW, respectively. Interestingly, the daidzein and genistein contents obtained from hairy roots were approximately 45.7- and 12.0-fold greater than those obtained from normal roots, respectively, suggesting that hairy root culture is a suitable method for the sustainable production of phytoestrogen, daidzein, and genistein from this medicinal plant.
... Puerarin is a natural phytonutrient rich in isoflavones, which can improve blood circulation and appetite, relieve sleep disorders, and enhance human vitality [6]. Mood disorders are common mental health issues that affect sleep. ...
More and more evidence suggests that puerarin, a potential remedy for gut inflammation, may have an ameliorative effect on sleep disturbances. However, the relationship between puerarin and sleep disruption has not been extensively researched. This study aims to explore the role and mechanisms of puerarin in improving sleep disorders. We established a light-induced sleep disorder model in mice and assessed the effects of puerarin on cognitive behavior using open field and water maze tests. Pathological detection demonstrated that sleep disturbances resulted in observable damage to the liver, lung, and kidney. Puerarin reversed multi-organ damage and inflammation. Further, puerarin activated paneth cells, resulting in increased lysozyme and TGF-β production, and stimulating intestinal stem cell proliferation. Puerarin also effectively inhibited the expression of F4/80, iNOS, TNF-α, and IL-1β in the small intestine, while it increased Chil3, CD206, and Arg-1 levels. Moreover, puerarin treatment significantly decreased P-P65, TLR4, Bcl-xl, and cleaved caspase-3 protein levels while increasing barrier protein levels, including ZO-1, Occludin, Claudin 1 and E-cadherin suggesting a reduction in inflammation and apoptosis in the gut. Overall, puerarin diminished systemic inflammation, particularly intestinal inflammation, and enhanced intestinal barrier integrity in mice with sleep disorders. Our findings suggest a potential new therapeutic pathway for sleep disorders.
Graphical Abstract
... Pueraria candollei var. mirifica (shortened as Pueraria mirifica) belongs to the genus Pueraria of the Fabaceae family and has been widely known as a traditional medicinal herb (Malaivijitnond, 2012). Due to their high bioactive accumulation, P. mirifica is useful for multiple areas such as the food, cosmetic, and pharmaceutical industries (Wang et al., 2020). ...
... Pueraria candollei var. mirifica (shortened as Pueraria mirifica) belongs to the genus Pueraria of the Fabaceae family and has been widely known as a traditional medicinal herb (Malaivijitnond, 2012). Due to their high bioactive accumulation, P. mirifica is useful for multiple areas such as the food, cosmetic, and pharmaceutical industries (Wang et al., 2020). ...
Pueraria mirifica is a popular medicinal plant and is rich in phytoestrogens, especially isoflavonoids. The chalcone isomerase (CHI) gene is well-known as a key factor in biosynthetic pathways such as flavonoid, isoflavonoid, and anthocyanin biosynthesis. Among four CHI subfamilies, group IV remained largely uncharacterized in P. mirifica. By isolation and gene cloning, the CHI4A gene was amplified and sequenced. A dataset that included CHI4A genes from isolation, transcriptome, and Glycine max CHI4A was constructed for analysis. A total of 31 single nucleotide polymorphism (SNPs) was detected among sequences and 14 SNPs were found in P. mirifica subgroups. Thirteen SNPs changed the amino acid sequences of CHI4A genes; however, they do not affect core active residues in the protein structure. Two active sites typical for group 4 CHI in the Fabaceae family: Ile50 and Tyr106; and one conserved site Asn113, were found in this study, which is similar to the result of the previous study on CHI4. A three-dimensional protein model of isolated P. mirifica CHI4A enzyme was constructed with a high confidence level. The result provides more information about the variation and protein profile of the CHI4A gene for further experiments.
... However, it has been reported that the estrogenic activity of deoxymiroestrol and miroestrol is almost the same as that of 17beta-estradiol [5]; therefore, products containing P. mirifica might possess estrogen-like effects. Indeed, the effects of P. mirifica on the reproductive organs, bones, cardiovascular diseases, and other symptoms related to estrogen deficiency in menopausal women have been studied [6]. In contrast, there have been a series of reports of adverse events related to the use of products containing P. mirifica among young women in Japan, and the National Consumer Affairs Center of Japan cautions consumers on P. mirifica [7,8]. ...
Recently, adverse events, such as irregular vaginal bleeding and menstrual disorders, associated with the use of dietary supplements containing Pueraria mirifica, have been reported in Japan. P. mirifica contains phytoestrogens, such as deoxymiroestrol and miroestrol. Therefore, we investigated the use of supplements that claim to have estrogen-like effects (i.e., estrogen-like supplements) in Japanese women aged from 15 to 69 years old in an online survey. The prevalence of estrogen-like supplement use was 5%, accounting for approximately 15% of the sample, including ex-users. The majority of the users were in their 40s and 50s, mainly using these supplements for the treatment of menopausal symptoms. In contrast, the younger generation mainly used them for beauty purposes, such as weight loss, mastogenic effects, and skin care. Many of them visited a clinic or took medicines for menstrual-related troubles. In all age groups, soybeans/isoflavones were the most commonly used, followed by equol and placenta. Participants in their teens and 20s also used P. mirifica. Among them, 16.2% had experienced adverse events, including irregular vaginal bleeding, breast swelling and pain, and heavy menstruation. In conclusion, estrogen-like supplement use is associated with adverse events; thus, it is necessary to pay attention to the use of these supplement. Furthermore, because the purpose of use differs depending on generation, caution according to each generation is necessary.
... Pueraria candollei var. mirifica (hereafter shortened as P. mirifica) has been well known for a long time, especially for treating or preventing various diseases in Thai traditional medicine (Malaivijitnond 2012). There is considerable scientific evidence supporting this plant's biological activities, showing that it is highly beneficial to human health as a source of phytoestrogens (Wang et al. 2020). ...
White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens have been well-known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by LC-MS/MS. Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.
... mirifica (Airy Shaw & Suvat.) Niyomdham (PM) is an economically important medicinal crop, and its roots are used by the local people in Thailand because of their rejuvenating properties to treat menopause and andropause (Malaivijitnond, 2012). PM is currently used in cosmeceutical and nutraceutical products because of its antiaging properties (Chattuwatthana and Okello, 2015;Dorni et al., 2017). ...
Magnetic fields can alter plant growth and development. In this study, the effect of magnetized water on the growth and secondary metabolite production of Pueraria candollei var. mirifica (PM) suspension cells was evaluated. Magnetized water was generated by passing water through a magnetic field (400 mT) at a 6 L/min flow rate. The suspension cells exposed to magnetized water for 10 (M2, 1.37 g) and 5 min (M1, 1.23 g) showed 21% and 9% higher biomass accumulation, respectively, compared with the control cells after 5 days. Compared with that in the control cells, the biomass was increased by approximately 43% when the cells were exposed to M2 (3.01 g) after 28 days. In addition, the use of magnetized water altered the accumulation pattern of isoflavonoids after 5 days of treatment. Isoflavonoid accumulation was the highest after treatment with M2 for 5 days and yielded 4.20 mg/g dry weight (DW) daidzein, 0.58 mg/g DW genistein, and 3.98 mg/g DW kwakhurin. When the cells were maintained in the treatment for a relatively long duration (28 days), a short span of magnetization (M1) resulted in the highest accumulation of isoflavonoids with the amounts of 3.51, 0.55, and 2.58 mg/g DW for daidzein, genistein, and kwakhurin, respectively. After 28 days of exposure to magnetized water, the expression of 2-hydroxyisoflavanone synthase and 2-hydroxyisoflavanone dehydratase increased, and this phenomenon coincided with the increased amount of isoflavonoids in the suspension cells. The magnetized water-treated PM cells also exhibited higher antioxidant activities than the control cells. The lowest half-maximal inhibitory concentration (37.93 mg/mL) was observed in the cells treated with magnetized water after 15 min (M3). The enhanced total antioxidant activity of the cells treated with M3 may be attributed to the presence of other compounds in addition to isoflavonoids. This study represents an innovative attempt at a low-cost and environmentally friendly approach that can enhance the growth, secondary metabolite production, and antioxidant activity of PM suspension cells.
... Other alternatives for hormonal aging treatment are topical estrogen (Manela-Azulay and Bagatin, 2009) and phytoestrogen; the latter causes fewer side effects compared to oral or topical estrogen (Kapuscinska and Nowak, 2015). Phytoestrogens are natural selective estrogen receptor modulators (Kapuscinska and Nowak, 2015;Stevenson and Thornton, 2007) and nonsteroidal chemical compounds derived from plants (Malaivijitnond, 2012); one, in particular, is the seeds of Trigonella foenum graecum (fenugreek) (Agustini, 2012). Several studies on fenugreek have demonstrated its benefits toward skin elasticity, moisture, and melanin concentration, but subjects involved in the studies were young males and females (Gade et al., 2015;Naveed et al., 2010;Waqas et al., 2010). ...
... 1 PM phytochemicals potentially protect against bone loss, act as antioxidants, and provide neurodegeneration protection. 1 PM phytoestrogens have been divided into three groups: (i) isoavonoids [kwakhurin, puerarin (PUE), daidzin (DZ), genistin (GT), daidzein (DZe), and genistein (GTe)], (ii) chromenes [miroestrol (MI), isomiroestrol, deoxymiroestrol (DMI), and methoxyisomiroestrol], and (iii) coumestans (coumestrol). 1 DMI was identied as the most estrogenic constituent, and it is more estrogenic than MI and isomiroestrol. 2 In raw materials, the glycoside isoavones of PM phytoestrogens were found to be more abundant. ...
Pueraria candollei var. mirifica (PM) has a significant beneficial effect on postmenopausal symptoms associated with estrogen deficiency. However, the estrogenic activity and intestinal absorption of isoflavonoid glycosides derived from PM, such as daidzin and genistin, are significantly lower than those of their aglycones. To enhance the estrogenic activity of the PM extract, we developed β-glucosidase and its immobilized form to increase the PM aglycone content (daidzein and genistein). The enzyme immobilization was done by alginate beads, and the resulting β-glucosidase alginate beads have a diameter of about 0.20 cm. Response surface methodology (RSM) was used to optimize certain parameters, such as the pH, temperature, and ethanol concentration. The optimal conditions of β-glucosidase for daidzein and genistein production were pH of 4.8-4.9, a temperature in the range 46.3-49.1 °C, and ethanol concentration of 10.0-11.0%. The ANOVA results indicated that the design experiment involving free and immobilized β-glucosidase was the best fit by quadratic models, which had adjusted R 2 values between 0.8625 and 0.9318. Immobilized β-glucosidase can be reused up to nine times and maintained efficacy of greater than 90%. Treatment of the PM extract with β-glucosidase increased the estrogenic activity of the PM extract by 8.71- to 23.2-fold compared to that of the untreated extract. Thus, β-glucosidase has a high potential for enhancing the estrogenic activity of PM constituents, and it can be applied on an industrial scale to increase the utility of these natural products.
... We report a concealed type 1 LQTS case (KCNQ1-T587M) that was unmasked by the ingestion of Pueraria mirifica, a commercially available rejuvenating supplement, containing estrogen-like substances. 10,11 Estrogen was reported to prolong action potential duration and QT interval. [5][6][7] One of the proposed mechanisms is that 17β-estradiol (E2) can suppress I Kr in a receptor-independent manner. ...
After taking an estrogen‐containing supplement derived from a tropical plant Pueraria mirifica, a 24‐year‐old woman presented marked QT prolongation and repetitive torsade de pointes. The patient was found to carry a heterozygous KCNQ1‐T587M mutation. This is the first report on Pueraria mirifica‐related acquired long QT syndrome.
... The phytochemicals in the tuberous roots of PM are known for their remarkable phytoestrogens, which are structurally classified as either chromenes or isoflavonoids. Chromene compounds, including deoxymiroestrol, miroestrol, and isomiroestrol, are found exclusively in PM, and they comprise a unique estrogen-like structure (Cain, 1960;Chansakaow et al., 2000a;Malaivijitnond, 2012;Yusakul et al., 2011). Deoxymiroestrol and miroestrol are defined as estrogenic markers of PM roots due to their potent in vitro and in vivo estrogenic activities, which are comparable to those of estradiol (Chansakaow et al., 2000a;Jones et al., 1961;Matsumura et al., 2005;Monthakantirat et al., 2014;Udomsuk et al., 2012bUdomsuk et al., , 2012a. ...
As a source of phytoestrogens, Pueraria candollei var. mirifica (PM) is popular in phytopharmaceutical product development for estrogen replacement therapy. It is a challenging process to extract a large amount of phytoestrogens from a complex plant mixture and to quantify these compounds for standardization. In this study, the 95% (v/v) crude ethanol extract of the PM root cortex was reextracted with 0-20% (v/v) aqueous ethanol. The results from the quantitative analysis showed that 10% (v/v) ethanol is the lowest solvent ratio that provided a high phytoestrogen content. More than 90% of the potent phytoestrogens (i.e., deoxymiroestrol and miroestrol) and 80% of the isoflavonoids (i.e., puerarin, daidzin, daidzein, genistein, and kwakhurin) were distributed into the derived soluble extract. In contrast, the insoluble extract contained a low level of phytoestrogens and was concentrated with less polar unidentified compounds. The extraction efficiency was confirmed by the estrogenic activity of the soluble extract on the proliferation of MCF-7 cells. The results also indicated that deoxymiroestrol mainly contributed to the total proliferative effects of PM, while the antiproliferative or cytotoxic effects occurred from treatment with the insoluble compounds, which were removed by the extraction method. Therefore, this extraction method is helpful to enhance the known phytoestrogen content and simplify the extraction of chemical constituents from PM as an ingredient in healthcare products.
... Native to Thailand and in documented us for over 100 years, Pueraria Mirifica has shown a marked antioxidant effect and been used to treat menopause and cardiovascular disease both of which may have symptoms that spur vertigo (Malaivijitnond, 2012). ...
Though nearly 40% of Americans will be affected by vertigo at some point during their lifetime, the current treatments are costly, time consuming, and focus on correction versus prevention. A more cost effective, over the counter method is needed. Based on this brief case study, the herbal supplement Vertigone by HumanX may be that option. Further research into the role herbal supplements play in vertigo treatment and prevention is warranted.
... For ages, PM has been used individually or in combination with other herbs in traditional practices. The roots of this plant, because of its rejuvenating qualities, are used by local people in Thailand to treat menopausal women and andropausal men (Malaivijitnond 2012). Due to the antioxidant, anticollagenase and anti-elastase properties of PM, it is presently used as an active ingredient in numerous nutraceuticals and cosmeceuticals (Chattuwatthana and Okello 2015;Dorni et al. 2017). ...
Elicitors that trigger the defense mechanism of plants could be a promising approach for elevating the bioactive compounds in tissue-cultured plant suspension cells. The objective of this study was to determine the effects of salicylic acid (SA), chitosan (CHI) and methyl jasmonate (MeJA) elicitation applied at different concentrations to suspension cells of Pueraria candollei var. mirifica (PM) on the contents of isoflavonoid, antioxidant, and antiaging activities. Six standards of isoflavonoids, namely, daidzein, genistein, daidzin, genistin, khwakhurin and puerarin, were determined by reversed-phase high-performance liquid chromatography (HPLC). To our knowledge, SA elicitation was first reported in this study and showed the highest accumulation of daidzein (2.37 mg/g DW), khwakhurin (1.05 mg/g DW), genistin (0.14 mg/g DW) and puerarin (5.13 mg/g DW). CHI elicitation gave favorable results, and the amounts of daidzein, genistein, khwakhurin and puerarin quantified were 2.24, 0.29, 1.04, and 1.80 mg/g DW, respectively. MeJA elicitation was the least effective method of all three elicitors. The results from DPPH-based assays suggested that extract from SA elicitation had the greatest antioxidant activity, followed by nonelicited cells (control), CHI, and MeJA elicitation. We also further evaluated the biological activities of PM extracts with elicitors on human dermal fibroblasts. We found that salicylic acid is the most promising elicitor to enhance the biological activities of PM extracts. The PM extract from SA-elicited cells promoted the proliferation of fibroblasts by approximately 20%. Furthermore, pretreatment with SA-elicited extract can prevent fibroblasts from experiencing oxidative insults, such as H2O2. The pharmacological data on fibroblasts support the antioxidant activities, implying the antiaging activities of PM extract. Taken together, our study clearly supports the potential utility of cell suspension culture of PM in the pharmaceutical and cosmeceutical industries to enhance the production of isoflavonoids and other active components.
... mirifica root compounds, elucidating a distinct number of unique phytoesterols, isoflavonoids and coumestans present in the tubers (17). Key players in its efficacy are deoxymiroestrol and miroestrol, compounds from plants that mimic the biological activity of estrogen in humans and have been shown to be the most potent phytoestrogens in nature to date (18,19). ...
Plants in the genus Pueraria DC., including the invasive kudzu, produce large, deep tubers that can weigh upwards of 400 pounds. Within its native Asia, tubers from various Pueraria species have been used for millennia for food and medicine, with more contemporary uses including soil stabilization and enrichment. Three species in particular provide unique ethnobotanical and economic uses: Pueraria tuberosa has been used as a food source and contraceptive for centuries in India; Pueraria candollei var. mirifica is purported to have strong rejuvenating properties, with laboratory research confirming numerous potential medical uses to treat symptoms of menopause in women; Pueraria lobata (kudzu) is a mainstay in traditional and contemporary Chinese medicine and a source of highly-prized starch used to produce fine delicacies in Japan. Here, a synopsis of the ethnobotanical and economical uses of tubers from three Pueraria species are reviewed.
... ex Benth. (Fabaceae) is a leguminous plant that is native to Thailand, and it is known asẄhite Kwao KruaörKwao Krua Khao.The remarkable estrogenic activity of this plant, together with its antiproliferative and antioxidative activities, has attracted great attention for its use as an alternative treatment for hormone replacement therapy [1]. Plant materials, especially tuber extracts, are incorporated in many health products in Thailand and other Asian countries. ...
Pueraria candollei is a phytoestrogen-rich herb used to treat estrogen deficiency disorders; however, quality control of P. candollei-related health products is required for consistency of clinical outcomes. Estrogenically active (+)-7-O-methylisomiroestrol could be a potential chemical marker that facilitates the prediction of the overall estrogenic activity of P. candollei. The analytical performance of ELISA using newly produced monoclonal antibodies against methylisomiroestrol was compared with HPLC analysis. The developed indirect competitive ELISA (icELISA) was highly sensitive to methylisomiroestrol for detection, with an LOQ of 2.9 ng/mL, whereas the LOQ was 1.15 μg/mL by HPLC. The results from method validation indicated acceptable precision (1.71–6.37 % and 0.13–2.40 %) and accuracy (99.23–102.54 % and 96.84–101.88 %) of the methylisomiroestrol analysis using icELISA and HPLC. These methods were effectively applied for the determination of the methylisomiroestrol content in P. candollei samples. Apart from the plant tubers, the stem was observed as a source of methylisomiroestrol. The developed ELISA was more effective than HPLC in detecting a small quantity of methylisomiroestrol in the plant samples [0.23 × 10⁻³% (w/w) to 0.628 × 10⁻³% (w/w) dry weight]. Therefore, the ELISA could be a useful tool for the standardization of P. candollei, which is the crucial step to improve the quality of plant-derived products.
... In Thai traditional medicine, PM has been prepared as honey-based traditional pills. According to clinical trials, [6,7] PM has been indicated to treat estrogen deficiency symptoms because the extract of its tuberous root exhibits oestrogenic activity. Among the bioactive compounds in PM is the isoflavonoid glycoside daidzin. ...
Background: Honey has been widely used as a traditional vehicle of herbal medicines. Honey behaves as a natural deep eutectic solvent (NADES) containing β-glucosidase; therefore, it can be used for the extraction and bio-activation of the bioactive compounds of herbs. Objectives: This study aims to apply honey (H-NADES) and a sugar-based NADES (S-NADES) for the extraction, analysis, and bioconversion of daidzin from Pueraria candollei var. mirifica (PM) root. Materials and Methods: Various concentrations of H-NADES and S-NADES (water:sucrose:glucose: fructose, 18:3:18:22 by weight) were used as solvents for extraction. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated for monitoring the extraction efficacy. The catalytic reactivity against daidzin of β-glucosidase purified from honey was investigated. Results: Using NADESs as solvents, icELISA was suitable for the reliable determination of daidzin with high sensitivity (1.95-125 ng/mL), specificity (% cross-reactivity ≤ 2.60), and accuracy (98.3-106% daidzin recovery). Daidzin at a concentration of 75.8 ± 3.67 μg/mL was extracted using 50% (v/v) S-NADES, which was the most effective for the extraction compared to H-NADES, water and ethanol. In addition, daidzin was converted to daidzein by honey β-glucosidase. Conclusion: Both S-NADES and H-NADES were useful for the extraction, analysis, and bioconversion of daidzin, and β-glucosidase from honey might enhance the oestrogenic activity and bioavailability of PM phytochemicals.
... White Kwao Krua (Pueraria candollei var. mirifica, hereafter shortened to P. mirifica, shown in Additional file 1: Figure S1), has been extensively used in Thai traditional medicine as a rejuvenating herb because of its numerous phytoestrogenic constituents [1]. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogen, and they have been applied to treat different forms of cancer, heart disease, menopausal symptoms, and osteoporosis [2]. ...
Background: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.
... White Kwao Krua (Pueraria candollei var. mirifica, hereafter shortened to P. mirifica, shown in Additional file 1: Figure S1), has been extensively used in Thai traditional medicine as a rejuvenating herb because of its numerous phytoestrogenic constituents [1]. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogen, and they have been applied to treat different forms of cancer, heart disease, menopausal symptoms, and osteoporosis [2]. ...
Background: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown.
Results: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein.
Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.
... White Kwao Krua (Pueraria candollei var. mirifica, hereafter shortened to P. mirifica, shown in Additional file 1: Figure S1), has been extensively used in Thai traditional medicine as a rejuvenating herb because of its numerous phytoestrogenic constituents [1]. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogen, and they have been applied to treat different forms of cancer, heart disease, menopausal symptoms, and osteoporosis [2]. ...
Background: Pueraria candollei var. mirifica , a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results: A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers.A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidatesinvolved in these biosyntheticpathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica . To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor ( At MYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins ledto the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for unraveling biosynthetic pathways in plants.
... One of the major obstacles is the lack of effective standardization procedures for PM, which leads to clinical inconsistency outcomes (Kongkaew et al. 2018). The root of PM is composed of isoflavonoids, coumestans and potent oestrogenic chromenes as the active chemical groups (Malaivijitnond 2012). Using the in vitro MCF-7 cell line as an oestrogenic evaluation model, the potency of the PM constituents are in the order deoxymiroestrol, miroestrol, coumestrol, genistein and daidzein (Matsumura et al. 2005). ...
(+)-7-O-Methylisomiroestrol (MeI), a novel chromene, was discovered as a phytoestrogen in the Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM) root having been used as an active agent against oestrogen depletion disorders. The identification of PM phytochemicals is crucial for the development of standardised botanical drugs of PM. MeI was purified from the root cortex of PM, and its structure was elucidated using NMR and mass spectrometry. The content of MeI in the root bark of the PM root was 2.1–6.5 × 10⁻³% (w/w). The oestrogenic potency of MeI was stronger than that of isomiroestrol but less than that of deoxymiroestrol and miroestrol. Therefore, MeI is a new oestrogenic biomarker for the effective chemical standardisation of the PM extract for health product development.
... White Kwao Krua (Pueraria candollei var. mirifica, hereafter shortened to P. mirifica, shown in Additional file 1: Figure S1), has been extensively used in Thai traditional medicine as a rejuvenating herb because of its numerous phytoestrogenic constituents [1]. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogen, and they have been applied to treat different forms of cancer, heart disease, menopausal symptoms, and osteoporosis [2]. ...
Background:
Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown.
Results:
Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein.
Conclusions:
Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.
... Pueraria candollei var. mirifica is a medicinal plant used in Thai traditional medicine for its rejuvenating and estrogenic effects [7]. For this reason, this plant has been widely investigated for its constituents and pharmacological activities. ...
Daily treatment of ovariectomized (OVX) ICR mice with puerarin, a glycosyl isoflavone isolated from the root bark of Pueraria candollei var. mirifica, and 17β-estradiol attenuated ovariectomy-induced depression-like behavior, as indicated by a decrease in immobility times in the tail suspension test (TST) and the forced swimming test (FST), an increase in the uterine weight and volume, a decrease in serum corticosterone levels, and dose-dependently normalized the downregulated transcription of the brain-derived neurotrophic factor (BDNF) and estrogen receptor (Erβ and Erα) mRNAs. Like 17β-estradiol, puerarin also inhibited ovariectomy-induced suppression of neurogenesis in the dentate gyrus of the hippocampus (increased the number of doublecortin (DCX)-immunosuppressive cells). These results suggest that puerarin exerts antidepressant-like effects in OVX animals, possibly by attenuating the OVX-induced hyperactivation of the HPA axis and/or normalizing the downregulated transcription of BDNF and ER mRNA in the brain.
... 2,3 Miroestrol was the first phytoestrogen isolated from PM, which has the strong estrogenic potency. 1 In traditional medicine, PM has been used for a long time with the purpose of improving tonic, beauty for women. Pharmacological studies have shown that PM has anti-osteoporotic effects, anti-collagenase, anti-elastase and antioxidant and also improving estrogenic activity. ...
Background: Pueraria candollei variety mirifica (PM) has been widely used as ingredient in many rejuvenating products. In this study, we aimed to assess the estrogenic activity of PM extract grown in Vietnam.Methods: Estrogenic activity of PM extract was estimated on immature female rats by using uterotrophic method to measure the weight of the reproductive organs. Estrogenic activity of PM extract also was investigated in mature female ovariectomized rats by evaluating the vaginal cells growth, reproductive organs weight, serum estradiol concentration.Results: Our results showed that PM extract at doses of 100 mg/kg, 200 mg/kg had increased the reproductive organs weight in immature rats and female ovariectomized rats. In addition, PM extract had increased the serum estradiol concentration and the vaginal cells growth by increasing the percentage of keratinocytes in female ovariectomized rats.Conclusions: Our results showed that PM extract has strong estrogenic activity in rats.
... It contains many chemical constituents, especially bioactive phytoestrogens, which not less than 17 chemical compounds with estrogenic activities have been found. PM was reported to use for increasing hair growth in both men and women [4]. ...
Pueraria mirifica (PM) extract is locally used to promote hair growth. However, the effective transdermal delivery system should be prepared to deliver the extract through the skin barrier. The objective of this study was to develop solid lipid nanoparticles (SLN) containing PM ethanolic extract for hair growth promotion. The cell viability and proliferation of human follicle dermal papilla cells (HFDPCs) treated with PM extract were evaluated by MTT assay. SLN formulations were developed as a transdermal delivery system of the PM extract, compared with liposomes. The physicochemical properties of these nanoparticles were determined. The in vitro skin permeation study was also evaluated by Franz type diffusion cells. For the result, PM extract was a good safety herbal extract, which no cytotoxicity at the concentrations from 1 to 1,000 μg/ml. The cell proliferation of PM extract treated HFDPCs significantly increased in a dose-dependent manner, indicating the possibility to promote hair growth at the concentrations from 10 to 100 μg/ml. For formulation development, 5% (w/v) PM extract-loaded SLN exhibited small particle size (93.83 ± 0.32 nm) with narrow size distribution and negatively charged. This formulation had the highest percent entrapment efficiency (42.64 ± 0.47%), followed by SLN containing 1% (w/v) PM extract (8.84 ± 0.24%) and undetectable in liposomes. For the skin permeation result, SLN containing 5% (w/v) of PM extract could penetrate through the skin more than solution form. Therefore, the small particle size and high PM extract entrapped in SLN exhibited higher PM extract penetrated through the skin barrier and hair follicles than PM ethanolic extract solution. PM extract-loaded SLN might be an effective formulation for hair growth disorders treatment.
... mirifica, PM) is a woody climbing plant that belongs to the Leguminosae family. Its root has been recognized as a rejuvenating herb, and several studies have demonstrated its estrogenic potency, which allows it to influence the prevention of bone loss and the reduction of postmenopausal symptoms [1]. Its anti-oxidant, anti-collagenase, and anti-elastase properties have also been noted [2,3]. ...
To address the high demand for Pueraria candollei var. mirifica (PM) used as the active ingredient in health products and its difficulty to cultivate in the field, the growth and production of deoxymiroestrol (DME) and isoflavonoid (ISF) phytoestrogens in PM cell suspensions were studied. In a 125-mL shake flask, the cell suspension produced DME [78.7 ± 8.79–116 ± 18.2 μg/g dry weight (DW)] and ISF (140 ± 6.83–548 ± 18.5 μg/g DW), which are the predominant ISF glycosides. While ISF aglycones accumulated in the PM cell suspension cultured in the airlift bioreactor. The DME content was increased to 976 ± 79.6 μg/g DW when the PM cell suspension was cultured in the 5-L scale bioreactor. The production of DME and ISF was enhanced by elicitors including methyl jasmonate (MJ), yeast extract (YE), and chitosan (CHI). MJ produced the highest induction of DME accumulation, while ISF accumulation was the highest with YE treatment. Analysis of catalase activity implied that the elicitors enhanced ROS production, which resulted in the enhancement of DME and ISF production and accumulation in PM cell suspension cultures. PM cell suspension culture is a promising source of beneficial PM phytoestrogens that exhibit bioactivity that may useful for the treatment of menopausal symptoms.
... This interesting and promising effect is attributed to the high degree of similarity between the structures of naturally occurring compounds (isoflavonoids, chromenes, and coumestans) contained in the roots of P. candollei and estradiol. These natural compounds act as agonists of estrogen receptor [1][2][3], leading to various estrogenic effects, and are known [4][5][6]. For this reason, P. candollei-derived products, including dietary supplements and cosmetics in the form of capsules, tablets and cream, have attracted much attention, resulting in a drastic increase in their demand throughout Asia, especially in Japan and China. ...
Pueraria candollei (P. candollei) is a traditional Thai herb widely used for estrogen replacement therapy because it contains many unique chromenes that possess potent estrogenic activity, one of which is known as isomiroestrol. Since isomiroestrol is a promising compound that is solely present in P. candollei, it can be used as an identifying marker for standardization of P. candollei. Here, we developed a lateral-flow immunochromatographic strip (ICS) test using a colloidal gold nanoparticle-conjugated anti-isomiroestrol monoclonal antibody (12C1-mAb) for the detection of isomiroestrol in plant samples and products of P. candollei. The advantages of the developed ICS over an enzyme-linked immunosorbent assay are its simplicity and rapidity, as the ICS test can be completed 15 min after dipping the strip into the analyte solution. The detectable concentration of isomiroestrol was 7.0 µg/mL. Considering the demand for the standardization of P. candollei due to concerns regarding its quality, our ICS test using isomiroestrol as an identifying marker would be effective and useful to assess the presence of isomiroestrol.
The use of natural substance-based supplements and treatments for mental wellness is increasingly gaining attention. Southeast Asia, with its rich heritage of medicinal practices and cultural reliance on natural remedies, presents a unique opportunity to explore such interventions. Delightex is actively collaborating with research partners in Southeast Asia to investigate natural substances that may enhance mental well-being and create enriching experiences. Memory, defined as the capacity to record, retain and recall sensory stimuli, events and information, is a fundamental aspect of mental health. Memory loss and Alzheimer’s Disease (AD) are significant and growing concerns worldwide, particularly due to aging populations. Nootropics are generally well tolerated and typically mild. However, occasional complications can still occur. Hence, it is important to explore more natural alternatives for memory enhancement or treatment of memory loss. In this review, following an initial comprehensive literature search on mental well-being, we focused on memory improvement, identified and summarized 57 natural substances from 31 families with potential memory-enhancing effects. This review highlights their traditional use in Southeast Asia and examines the scientific evidence supporting their efficacy in enhancing memory and potential as nootropics alternatives.
Cognitive impairment is a neurological symptom caused by reduced estrogen levels in menopausal women. The Thai traditional medicine, Yakae-Prajamduen-Jamod (YPJ), is a formula consisting of 23 medicinal herbs and has long been used to treat menopausal symptoms in Thailand. In the present study, we investigated the effects of YPJ on cognitive deficits and its underlying mechanisms of action in ovariectomized (OVX) mice, an animal model of menopause. OVX mice showed cognitive deficits in the Y-maze, the novel object recognition test, and the Morris water maze. The serum corticosterone (CORT) level was significantly increased in OVX mice. Superoxide dismutase and catalase activities were reduced, while the mRNA expression of IL-1β, IL-6, and TNF-α inflammatory cytokines were up-regulated in the frontal cortex and hippocampus of OVX mice. These alterations were attenuated by daily treatment with either YPJ or 17β-estradiol. HPLC analysis revealed that YPJ contained antioxidant and phytoestrogen constituents including gallic acid, myricetin, quercetin, luteolin, genistein, and coumestrol. These results suggest that YPJ exerts its ameliorative effects on OVX-induced cognitive deficits in part by mitigating HPA axis overactivation, neuroinflammation, and oxidative brain damage. Therefore, YPJ may be a novel alternative therapeutic medicine suitable for the treatment of cognitive deficits during the menopausal transition.
Objective
To compare the effects of 6% Pueraria mirifica vaginal gel with those of placebo gel on vaginal blood flow, vaginal maturation index (VMI), vaginal health index (VHI), endometrial thickness and genitourinary symptoms in postmenopausal women.
Study design
In a randomized, double-blinded, placebo-controlled study (TCTR20200624007), 72 postmenopausal women were randomized into the P. mirifica or the placebo gel group. Both groups were followed up at week 4 and week 12.
Main outcome measure
Doppler ultrasonography pulsatility index (PI) and resistance index (RI), VMI, VHI, endometrial thickness and genitourinary symptoms were evaluated at baseline, at week 4 and week 12 of treatment.
Results
Sixty-three participants completed the study. After 4 and 12 weeks of treatment, PI and RI had significantly decreased in the P. mirifica group compared with the placebo group. At week 12, PI in the P. mirifica group and in the placebo group were 3.03 + 1.09 and 6.88 + 2.16, respectively (p = 0.002). Similar changes were also demonstrated in the resistance indices. The P. mirifica group demonstrated a markedly higher mean VMI at week 12 compared with the placebo group, 55.19 ± 18.53 and 20.29 ± 28.46 (p = 0.012). In addition, all parameters of VHI based on the vaginal physical findings at week 12 in the P. mirifica group were significantly higher than in the placebo group (p < 0.001).
Conclusion
In this study, 6% P. mirifica vaginal gel for 12 weeks in postmenopausal women with GSM appeared to increase vaginal artery circulation and restore atrophic vaginal tissue.
Clinical Trial Registration Number: TCTR20200624007.
Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250 ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.Graphic abstract
Introduction
Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei ), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei ‐derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei ‐derived products has gained a sense of urgency in recent years.
Objective
This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products.
Material and Method
A rapid and sensitive immunoassay was developed with a combination of Kwa‐specific monoclonal antibody (MAb 11F) and Kwa–magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time.
Result
This novel MPs‐based enzyme immunoassay (MPs‐EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs‐EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.
Objective
To compare the effects of a 12-week course of 5%Pueraria mirifica gel and placebo gel on the prevalence of bacterial vaginosis, vaginal fungi, vaginal pH, vaginal health index (VHI), and genitourinary symptoms in postmenopausal women.
Study design
In a randomized, double-blinded, placebo-controlled study (TCTR20160517002), 60 postmenopausal women were randomly assigned to a 12-week course of eitherP. mirifica gel or identical placebo gel.
Main outcome measure
Vaginal Nugent score, fungal culture, pH, VHI, and genitourinary symptoms were evaluated at baseline and after 12 weeks of treatment.
Results
After 12 weeks of treatment, the proportion of participants with an abnormal Nugent score in the P. mirifica and the placebo groups were 6.7% (2/30) and 23.3% (7/30), respectively (p = 0.006). The mean changes in Nugent scores and VHI were significantly higher in the P. mirifica group (p < 0.05). There were no significant decreases in the prevalence of symptoms between the two groups after treatment (p > 0.05).
Conclusion
A 12-week course of treatment with 5%P. mirifica vaginal gel in postmenopausal women with GSM has been proved to be effective in reducing indicators of bacterial vaginosis compared with placebo gel. Nevertheless, the effect on alleviating genital symptoms was not demonstrated.
Background Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. The assembly of 166,923 contigs was used for a functional annotation against protein databases, and as a library for the identification of genes possibly involved in the biosynthesis of isoflavonoid and miroestrol. Twenty-one differential expressed genes (DEGs) from four separate libraries were identified as candidates involved in the biosynthesis pathways and had their expressions validated by qRT-PCR. Notably, isoflavonoid profiling generated by LC-MS/MS was positively correlated to the expression levels of isoflavonoid biosynthetic genes across the four types of tissues. In addition, R2R3 MYB transcription factors were also identified that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of the key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase (PmIFS) identified in our library was transiently co-expressed with the Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of those proteins led to the production of isoflavone genistein. Conclusions Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for unraveling biosynthetic pathways in plants.
Neurodegenerative diseases (NDD) are a range of debilitating conditions of the brain involving progressive loss of neurons, many of which are still currently incurable despite enormous efforts on drug discovery and development in the past decade. As NDD is closely linked to old age, the rapid worldwide growth in the aging population contributes to an increasing number of people with one of these incurable diseases and therefore it is considered a significant global health issue. There is an urgent need for novel effective treatments for NDDs, and many new research strategies are centered on traditional medicine as an alternative or complementary solution. Several previous findings have suggested that glutamate toxicity drives neurodegeneration in many NDDs, and the medicinal plants with anti-glutamate toxicity properties can be potentially used for their treatment. In order to obtain data relating to natural products against glutamate toxicity, six candidate plant species of Thailand were identified. Studies utilizing these herbs were searched for using the herb name (Latin and common names) along with the term “glutamate” in the following databases across all available years: PubMed, Scopus, and Google Scholar. This review emphasizes the importance of glutamate toxicity in NDDs and summarizes individual plants and their active constituents with the mechanism of action against glutamate toxicity-mediated neuronal cell death that could be a promising resource for future NDD therapy.
Taxonomy (classification by evise)
Alzheimer's disease, Neurodegenerative diseases, Cell culture, Molecular Biology, Traditional herbal medicine, Oxidative stress, Glutamate neurotransmitter.
Introduction
Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products.
Objectives
To prepare a single‐chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv‐based enzyme‐linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products.
Methods
A gene encoding anti‐miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti‐miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv‐based ELISA method was validated for its sensitivity, specificity, accuracy and precision.
Results
Anti‐miroestrol scFv antibody was highly specific to miroestrol. The scFv‐based ELISA was applied to determine miroestrol in the range 0.06–7.81 μg/mL, with the limit of quantification of 0.06 μg/mL miroestrol. The accuracy of the assay was validated by its 95.08–103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody‐based ELISA. The relative standard deviation of the intra‐ and inter‐assay were less than 6.0%.
Conclusion
The developed scFv‐based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.
Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.
Ten isoflavonoids including daidzein, daidzin (daidzein-7-O-glucoside), puerarin (daidzein-8-C-glucoside), genistein and coumestrol have been isolated from a methanolic extract of Pueraria mirifica roots. Apart from these known compounds and mirificin, a novel apioside derivative of puerarin, the roots have also been found to contain three minor coumestans and one 5-deoxyisoflavone for which only limited spectroscopic data are currently available. © 1986, Verlag der Zeitschrift für Naturforschung. All rights reserved.
The isoflavone aglycone kwakhurin hydrate, and the glycosides genistin (genistein-7-O-glucoside) and puerarin-6“-monoacetate have been isolated from a methanolic extract of Pueraria mirifica roots.
Three isoflavonoids obtained from a methanolic extract of Pueraria mirifica roots have been identified as 3,9-dihydroxy-8-methoxy-7-(3,3-dimethylallyl)-coumestan (mirificoumestan),3,9-dihydroxy-8-methoxy-7-(3-hydroxy-3-methylbutyl)-coumestan (mirificoumestan hydrate), and 3,9-dihydroxy-8-methoxy-7-(2,3-dihydroxy-3-methylbutyl)-coumestan (mirificoumestan glycol).These new coumestans co-occur with coumestrol (3,9-dihydroxycoumestan), a compound already found in P.mirifica roots.
The clinical trial to evaluate the estrogenic effects of the crude drug derived from dry powder of a phytoestrogen-rich Thai herb Pueraria mirifica (White Kwao Krua) in five female volunteers with menopausal symptoms showed that the crude drug clearly improved the signs and symptoms related to menopause such as, hot flushes, frustration, sleep disorder, skin dryness, high blood cholesterol, oligomenorrhoea and amenorrhoea, with no change in the blood cells, liver and kidney functions, as well as other physiological status after four months of treatment. In four volunteers, treatments were continued to complete the one-year test period with half the dose and was found to maintain their satisfied menopausal relief status. The crude drug dosage was administered at 200 mg daily for three weeks a month during the first four months of treatment and 200 mg every other day for 20 days per month for the remaining of eight months. These doses were effective and safe as phytoestrogen treatment of menopausal symptoms.
We measured bone mineral density (BMD) of the proximal femur, lumbar spine, or both by dual photon absorptiometry in 205 normal volunteers (123 women and 82 men; age range 20 to 92 yr) and in 31 patients with hip fractures (26 women and 5 men; mean age, 78 yr). For normal women, the regression of BMD on age was negative and linear at each site; overall decrease during life was 58% in the femoral neck, 53% in the intertrochanteric region of the femur, and 42% in the lumbar spine. For normal men, the age regression was linear also; the rate of decrease in BMD was two-thirds of that in women for femoral neck and intertrochanteric femur but was only one-fourth of that in women for lumbar spine. This difference may explain why the female/male ratio is 2:1 for hip fractures but 8:1 for vertebral fractures. The standard deviation (Z-score) from the sex-specific age-adjusted normal mean in 26 women with hip fracture averaged −0.31 (P < 0.05) for the femoral neck, −0.53 (P < 0.01) for the intertrochanteric femur, and +0.24 (NS) for the lumbar spine; results were similar for 5 men with hip fractures. By contrast, for 27 additional women, ages 51-65 yr, with only nontraumatic vertebral fractures, the Z-score was −1.92 (P < 0.001) for the lumbar spine. Thus, contrary to the view that osteoporosis is a single age-related entity, our data suggest the existence of two distinct syndromes. One form, “postmenopausal osteoporosis,” is characterized by excessive and disproportionate trabecular bone loss, involves a small subset of women in the early postmenopausal period, and is associated mainly with vertebral fractures. The other form, “senile osteoporosis,” is characterized by proportionate loss of both cortical and trabecular bone, involves essentially the entire population of aging women and, to a lesser extent, aging men, and is associated with hip fractures or vertebral fractures or both.
Pueraria mirifica Airy Shaw & Suvatabandhu has been used by Thai women for centuries as a rejuvenating herb. The scientific claim for its phytoestrogenic property leads to the popular use of this plant in several herbal formulations. This research project was carried out to examine estrogenic activity of P. mirifica from Lampang Province, Thailand, one of the areas that it is harvested for using as raw material in herbal industries. Estrogen bioassay of aqueous extract from P. mirifica at concentrations of 0.1 and 0.2 g/ml was performed in immature ovariectomized mice in comparison to both the negative and positive control groups. Vaginal orifice of mice treated with the extract in all groups and with the positive control group which injected with estradiol benzoate (EB) opened earlier than the negative control mice and vaginal cornification was also found in these 3 groups only. Moreover, the extract significantly increased uterine weight and uterine epithelium height as compared to negative control group with the comparable degree of increment to those of EB treated group. The data of this present study indicated that dried powder of P. mirifica which is used as raw material for herbal products of Lampang Province, exhibited the potent estrogenic activity.
Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.
Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.
We measured bone mineral density (BMD) of the proximal femur, lumbar spine, or both by dual photon absorptiometry in 205 normal volunteers (123 women and 82 men; age range 20 to 92 yr) and in 31 patients with hip fractures (26 women and 5 men; mean age, 78 yr). For normal women, the regression of BMD on age was negative and linear at each site; overall decrease during life was 58% in the femoral neck, 53% in the intertrochanteric region of the femur, and 42% in the lumbar spine. For normal men, the age regression was linear also; the rate of decrease in BMD was two-thirds of that in women for femoral neck and intertrochanteric femur but was only one-fourth of that in women for lumbar spine. This difference may explain why the female/male ratio is 2:1 for hip fractures but 8:1 for vertebral fractures. The standard deviation (Z-score) from the sex-specific age-adjusted normal mean in 26 women with hip fracture averaged -0.31 (P < 0.05) for the femoral neck, -0.53 (P < 0.01) for the intertrochanteric femur, and +0.24 (NS) for the lumbar spine; results were similar for 5 men with hip fractures. By contrast, for 27 additional women, ages 51-65 yr, with only nontraumatic vertebral fractures, the Z-score was -1.92 (P < 0.001) for the lumbar spine. Thus, contrary to the view that osteoporosis is a single age-related entity, our data suggest the existence of two distinct syndromes. One form, "postmenopausal osteoporosis," is characterized by excessive and disproportionate trabecular bone loss, involves a small subset of women in the early postmenopausal period, and is associated mainly with vertebral fractures. The other form, "senile osteoporosis," is characterized by proportionate loss of both cortical and trabecular bone, involves essentially the entire population of aging women and, to a lesser extent, aging men, and is associated with hip fractures or vertebral fractures or both.
This work reported on the antioxidant capacities of five different medicinal plants generally found throughout the country. The medicinal plants including Pueraria mirifica, Stevia rebaudiana Bertoni, Curcuma longa Linn., Andrographis paniculata (Burm.f.) Nees. and Cassia alata Linn. were subjected to extraction using a variety of solvents including ethanol, methanol, acetone, acetic acid, and distilled water. The method was based on inhibition in absorption of ABTS [2,2′-azino-bis (3-ethylbenthiazoline-6-sulfonic acid)] technique and the antioxidant capacity was recorded as TEAC (Trolox equivalent antioxidant capacity). The result showed that ethanol was the best solvent for extraction of all five medicinal plants to give the highest antioxidant capacities, followed by acetone, methanol, distilled water, and acetic acid, respectively. The highest antioxidant capacity was found in Stevia rebaudiana Bertoni., followed by Cassia alata Linn., Curcuma longa Linn., Andrographis paniculata (Burm.f.) Nees., and Pueraria mirifica, respectively. Moreover, the interaction effect between solvents and medicinal plants on antioxidant capacity showed that the highest antioxidant capacity was found in Stevia rebaudiana Bertoni extracted with acetone (2.96) and methanol (2.85), followed by Stevia rebaudiana Bertoni extracted with ethanol (2.64), and Cassia alata Linn. extracted with ethanol (2.57), while, the lowest antioxidant capacity was found in Andrographis paniculata (Burm.f.) Nees. extracted with acetone (0.04).
Objective: Phytoestrogens have been reported to exhibit antiproliferation to human breast cancer cells in vitro. We tested the phytoestrogen-rich, Pueraria mirifica against rat breast cancer induction in vivo. Methods: The weanling female Spargue-Dawley rats were pretreated with P. mirifica tuberous powder at a dosage of 0, 10, 100 and 1000 mg/kg BW/day for four consecutive weeks. Mammary tumor development was then induced with a single dose of 7,12-DMBA, 80 mg/kg BW, followed by a weekly examination for size and multiplicity of mammary tumors for 20 weeks and finally a necropsy. Mammary tissues were investigated for the virulence of tumor and also monoclonal antibody stained against ER alpha and ER beta. Results: Pretreatment of 1000 mg/(kg BW day) of P. mirifica tuberous powder resulted in decreasing of the virulence of rat tumor development. The mammary tumor tissues exhibited lower profile of ER alpha and ER beta as well as ER alpha/ER beta. Conclusion: P mirifica exhibited prevention of 7,12-DMBA-induced rat mammary tumors, with a proposed mechanism of strong competitive binding of its phytoestrogens to ER alpha and/or synthesis suppressor of ER alpha. (c) 2007 Elsevier Ireland Ltd. All rights reserved.
We have reviewed the literature regarding the food sources, potency, population intakes, and known biological effects of phytoestrogens in humans using MEDLINE data base from the years 1975-1996. Over 600 articles pertinent to the metabolism of phytoestrogens, including female reproduction (in particular menstruation and menopause), cardiovascular disease, osteoporosis, and cancer were assessed including relevant case control or cohort studies, as well as randomized trials and review articles. Epidemiological studies regarding human data were included, as well as human cell line and animal studies when there were no relevant human data available. We conclude that phytoestrogens exhibit physiological effects in humans. Mild estrogenic changes occur in postmenopausal women. Benefits are seen regarding hypercholesterolaemia. Epidemiological, animal, and in vitro data encourage further assessment of the role of phytoestrogens in cancer prevention.
Pueraria mirifica or White Kwao Krua has been extensively studied for its estrogenic effects on reproductive organs and bones using rodents as experimental animals. Commercial rodent diets are usually formulated with soybean products and therefore deliver a high dose of isoflavone phytoestrogens. Using high performance liquid chromatography, we determined the quantities of five major isoflavones (puerarin, daidzin, genistin, daidzein, and genistein) in five lots of standard rodent diets, a soybean-free diet, and two lots of P. mirifica 'Wichai-III'. The concentrations of total isoflavones were 38.6-72.4 mg/100 g in the standard rodent diets, 6.1 mg/100 g in the soybean-free diet, and 123.2-157.3 mg/100 g in the P. mirifica. While absent in the rodent diets, puerarin accounted for about half of the isoflavone content in P. mirifica. The levels of genistein and genistin in P. mirifica were very low compared to the level found in the standard rodent diets. Given the same dose of 50 mg/kg BW/day of P. mirifica for 14 days, rats fed with standard rodent diet showed a significantly higher percentage of cornified cells than those fed with soybean-free diet. These findings suggest the potential presence of phytoestrogens in standard rodent diets and its liability to be a confounding factor in estrogenic or phytoestrogenic research.
Mirœstrol, isolated from Pueraria mirifica by a new method, has been converted into a monobromo-derivative, indicated by its molecular formula and light absorption to be a simple substitution product of the parent compound. X-Ray crystallographic analysis of this derivative by other authors 1 has given its complete structure (I), from which that of mirestrol (II) follows. The absorption spectra and some chemical properties of mircestrol are described in the light of this structure. Various other derivatives of miraestrol have been prepared, including products of methylation, acetylation, and reduction by potassium borohydride. With bromine in methanol mircestrol yields a dibromo-methoxy-derivative, and with mineral acid gives an isomeric compound (isomircestrol); the structures (V) and (VIII; R=H) respectively are proposed for these products.
A novel estrogen receptor, estrogen receptor β (ERβ), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ERα has been reported to be present preferentially in osteoblasts, but the mechanism of action of estrogen in bone is still not known. In the present study, we examined expression of ERβ mRNA in bone. Expression of ERβ mRNA was evident in primary osteoblastic cells isolated from 1-day-old rat calvaria and rat osteosarcoma cells (ROS 17/2.8), and its level was higher than that of ERα mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERβ mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ERα mRNA was very low at the beginning of culture and it gradually increased during the differentiation of osteoblastic cells. Various tissues including bone were isolated from 8-week-old rats of both sexes, and total RNA was extracted to compare the tissue distribution of expression levels of ERβ mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERβ mRNA was obvious, and its level was equivalent to those in the uterus and testis, but lower than those in the ovary and prostate. The level of ERβ mRNA in femoral cortical bone was lower than that in cancellous bone. There was no appreciable differences between female and male rats in the distribution and expression levels of ERβ mRNA in bone. These results indicate that ERβ mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERβ in bone.
The rat estrogen receptor (ER) exists as two subtypes, ERα and ERβ, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes.
Saturation ligand binding analysis of in vitro synthesized human ERα and rat ERβ protein revealed a single binding component for 16α-iodo-17β-estradiol with high affinity[ dissociation constant (Kd) = 0.1 nm for ERα protein and 0.4 nm for ERβ protein]. Most estrogenic substances or estrogenic antagonists compete with 16α-[125I]iodo-17β-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17β-estradiol > coumestrol, ICI-164384 > estrone, 17α-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3β,17β-diol, genistein for the ERα protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17β-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3β,17β-diol > 17α-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ERβ protein. The rat tissue distribution and/or the relative level of ERα and ERβ expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ERα and prostate, ovary, lung, bladder, brain, uterus, and testis for ERβ. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.
Pueraria mirifica is a leguminous herbal plant whose tuberous roots are used in estrogen replacement. Leaves were collected for 39 locations across Thailand with seed pods and flowers also collected when available from a subset of 14 and 11 of these locations respectively. Morphometric analysis revealed a low level of variation between cultivars. Linear regression analysis suggested that leaves trended to decrease in size from the West to the East whilst pods trended to increase in size from the South to the North and also the West to the East. Genetic analysis was conducted by direct sequencing of one nuclear (rDNA ITS region) and one chloroplast (trnLF) region, and also by random genome analysis by RAPD-PCR using five primers. All chloroplast sequences obtained revealed a low level of variation between isolates although the rDNA ITS sequences displayed a divergence of up to 25.2 %. All of 93 bands generated by the five RAPD primers were polymorphic. The average genetic distance varied from 0 to 42. NJ based phylogenies derived from ITS and RAPD data revealed poor resolution. In summary, both analyses indicate low variation amongst cultivars.
We investigated a non-invasive method of specimen collection for determining the changes of reproductive hormones in aged menopausal monkeys after a long-term feeding of the Thai herb Pueraria mirifica (PM) containing phytoestrogens. Three groups of aged menopausal monkeys (n = three in each group) were fed daily with 10, 100, or 1000 mg of PM for a 90 day treatment period, and fed with distilled water for 30 and 60 days of the pre- and post-treatment periods, respectively. Urine samples were collected for 14 h daily every 5 days and assayed for follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol levels. The result showed that monkeys fed with PM 10, PM 100, and PM 1000 had a decrease in urinary FSH levels during the treatment period, followed by a rebound increase during the post-treatment period. Urinary estradiol levels tended to decrease and fluctuated between 4.28 and 266.71, 2.85–42.27, and 6.24–203.50% of the pre-treatment levels in those three groups, respectively. Decreases in urinary LH levels could not be observed in all the three groups. These results suggest that FSH could be a candidate marker to detect the estrogenic effects of phytoestrogens in aged menopausal monkeys when changes of urinary hormones need to be used as an indicator.
Miroestrol and deoxymiroestrol are phytoestrogens isolated from tuberous root of Pueraria candollei var. mirifica. Modulatory effects of miroestrol and deoxymiroestrol on enzymes involved in sex-hormone synthesis pathway in male C57BL/6 mice were investigated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Miroestrol and deoxymiroestrol suppressed the expressions of 3β-HSD, 17β-HSD1, and CYP17 while CYP19 mRNA expression was slightly decreased. In addition, the expression of 17β-HSD2 was induced in correlation with those did by estradiol. These observations supported that miroestrol and deoxymiroestrol could exhibit the same effect as estradiol regarding regulation of testicular gene related sex hormone synthesis pathway.
Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation.
The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation.
The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of β-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, β-naphthoflavone (βNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by βNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2.
The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.
A high-performance liquid chromatography (HPLC) method was developed to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and P. candollei var. candollei. The linear detection ranges were 0.78-25.00 μg/mL for miroestrol and 1.56-25.00 μg/mL for deoxymiroestrol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 0.78 μg/mL, respectively, for miroestrol and 0.78 and 1.56 μg/mL, respectively, for deoxymiroestrol. Our results suggest that both varieties of P. candollei can produce miroestrol and deoxymiroestrol and that the developed HPLC method can be applied for quality control of plants and their products.
Neuronal degeneration is known to be due to oxidative stress acting through a pathway involving the excessive activation of glutamate receptors. We studied the neuroprotection potential of an ethyl acetate-ethanol extract of Pueraria mirifica (P. candollei var. mirifica) root (PM extract). PM extract was evaluated for its antioxidant and neuroprotective activities against glutamate toxicity in mouse hippocampal HT22 neuronal cells. The extract at concentrations of 10 and 50 μg/ml exhibited considerable antioxidant activity with significant neuroprotection, based on the microscopic observations of cell morphology and the determination of cell viability and cell number. Studies of the possible mechanisms of action indicated that the neuroprotection exerted by PM extract was related to its scavenging activity against H(2)O(2) and related reactive oxygen species. High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) analyses showed that the extract contained daidzein and genistein as identified constituents, as well as additional components with antioxidant activity. While daidzein and genistein individually and in combination were observed not to be neuroprotective, we propose that the antioxidant and neuroprotective activities of PM extract are derived from the combined properties of its constituents.
A weak estrogenicity of puerarin on reproductive organs was addressed in female rats. In short-term treatment, immature ovariectomized rats were injected with 0.7 mg/kg BW/day of puerarin, for 14 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the percent of cornified cells (%Co), but it increased the number of uterine glands. In long-term treatment, mature rats were injected with 7.0mg/kg BW/day of puerarin for 140 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the number of uterine glands, but a significant increase in the %Co was observed from day 98 onwards.
Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.
To evaluate the effect of Pueraria mirifica on vaginal and urethral cytology, bladder pressure and capacity, residual urine, and leak point pressure in ovariectomized rats.
Seventy-two adult, ovariectomized, female Sprague-Dawley rats were placed into one of four groups: control, estradiol, or 100 or 1,000 mg/kg of Pueraria mirifica (PM-100 and PM-1000, respectively). The vaginal and urethral smears were checked after 30 days of ovariectomy at pretreatment and at day 28 of treatment.A single cystometry, defined as the micturition interval, filling pressure, threshold pressure, micturition pressure, and voided volume, was performed. Peak bladder pressure was calculated for each leak point pressure measured at half bladder capacity by slowly and manually increasing abdominal pressure until a leak occurred, at which point external pressure was rapidly released. Leak point pressure was tested three times per rat.
After 28 days of treatment, the estradiol, PM-100, and PM-1000 groups had significantly higher numbers of vaginal and urethral superficial cells compared with the control group (P < 0.05). Regarding the urodynamic parameters, the threshold pressure, micturition pressure, and leak point pressure were higher in the estradiol, PM-100, and PM-1000 groups compared with the control group (P < 0.05). The control, PM-100, and PM-1000 groups had the same values for micturition interval, bladder capacity, voided volume, and residual volume (P > 0.05) but lower values compared with the estradiol group (P < 0.05).
Pueraria mirifica 100 and 1,000 mg/kg/day showed an estrogen-like effect on the vaginal and urethral epithelium of ovariectomized rats. They did not change bladder capacity and residual urine volume but increased leak point pressure according to urodynamic study.
Impaired lipid metabolism is an important health problem in postmenopausal women with insufficient estrogens, because dyslipidemia is a risk factor for development of atherosclerosis and the incidence of cardiovascular disease markedly increases after menopause. Pueraria mirifica (PM), a Thai herb, has been noticed as a source of phytoestrogens, estrogen-mimicking plant compounds. However, the clinical effects of PM on lipid metabolism and the underlying molecular mechanisms remain undetermined. Therefore, we examined the effects of PM on serum lipid parameters in a randomized, double-blind, placebo-controlled clinical trial. Nineteen postmenopausal women were randomly assigned to receive oral administration of PM powder or placebo. After 2 months of treatment, the PM group showed a significant increase in serum concentrations of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-1 (34% and 40%, respectively), and a significant decrease in low-density lipoprotein (LDL) cholesterol and apo B (17% and 9%, respectively), compared with baseline measurements. Moreover, significant decreases were observed in the ratios of LDL cholesterol to HDL cholesterol (37%) and apo B to apo A-1 (35%). Next, we determined the effects of PM phytoestrogens on the activation of estrogen receptor (ER)-mediated transactivation by transient expression assays of a reporter gene in cultured cells. Among PM phytoestrogens, miroestrol and coumestrol enhanced both ERalpha- and ERbeta-mediated transactivation, whereas other phytoestrogens, including daidzein and genistein, preferentially enhanced ERbeta-mediated transactivation. In conclusion, PM has a beneficial effect on lipid metabolism in postmenopausal women, which may result from the activation of gene transcription through selective binding of phytoestrogens to ERalpha and ERbeta.
To evaluate the influences of seasonal changes and plant cultivars on estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica.
Three cultivars of P. mirifica; PM-I, PM-II and PM-V, were grown in the same field trial for 3 years and random tubers collected during the summer, rainy season and winter seasons. Female Wistar rats were ovariectomized, kept for 14 days, randomly segregated into groups and treated with one of DW, 200microg/100g BW 17beta-estradiol (E2) or tuberous powder of PM-I, PM-II and PM-V at dosages of 100, or 1000mg/kg BW for the next 14 days. For the last 7 days of post-treatment period, rats received only DW. The vaginal cornification was recorded during the treatment and post-treatment period. The uterine tissues of the treated rats at the treatment and post-treatment periods were analyzed for uterine gland number and for the surface area of the myometrium, endometrium and lumen. In addition, ethanol tuberous extracts of PM-I, PM-II and PM-V was submitted to DPPH analysis.
Vaginal cornification exhibited a dose-dependent response with plant samples collected during the winter and summer being more active than those collected in the rainy season. All plant samples-induced uterotrophic effects in the analysis at the treatment and post-treatment periods in a dose-dependent manner. The P. mirifica treated rats exhibited increasing uterine gland numbers and thickness of the endometrium and myometrium but a decreasing size of lumen, in comparison to the negative control. The results were more prominent in PM-I than other plants and also in plant samples collected during the winter and summer seasons than in the rainy season. DPPH assay of the ethanol tuberous extracts revealed variance in antioxidant activity.
The results of uterotrophic and vaginal cornification assays reveal that P. mirifica exhibits a dose-dependent estrogenic activity under the influence of both seasonal changes and plant cultivars, which is confirmed by DPPH assay.
Puerarin and daidzein are the major naturally occurring isoflavones in leguminous plants. These two compounds are metabolized to equol by human intestinal flora. Here we isolated two intestinal bacteria capable of metabolizing puerarin and daidzein, respectively, from human feces. One of them, strain PUE, converted puerarin to daidzein by cleaving a C-glucosyl bond, whereas the other, strain DZE, converted daidzein to equol by reducing a double bond in ring C followed by elimination of an oxo group. Based on the 16S ribosomal RNA gene sequence, strain DZE showed 85% similarity with Eggerthella lenta. Equol produced by strain DZE was identified as (3S)-equol through several analytical methods. Moreover, we obtained (3S)-equol from puerarin by co-incubation with strain PUE and DZE. In addition, 5-hydroxyequol was obtained from genistein by incubation with strain DZE.
The effects of 17 beta-estradiol and tamoxifen (TAM) on the proliferation of responsive MCF-7 and unresponsive HBC-4 human breast cancer cells were studied in a defined culture medium containing insulin (2 micrograms/ml), transferrin (2 micrograms/ml), ethanolamine (2 microM), and selenite (25 nM). MCF-7 cells grew at a population-doubling rate of 2.0 days in serum-free medium and at a rate of 1.7 days in the medium containing 1 mg/ml of the 55-70% ammonium sulfate fraction of bovine serum or 1% dextrancoated charcoal-treated fetal bovine serum. Increasing concentrations of the ammonium sulfate fraction and/or dextran-coated charcoal-treated fetal bovine serum increasingly inhibited the growth of MCF-7 cells but did not inhibit HBC-4 cell growth, indicating that such serum preparations contain some growth inhibitor specific for estradiol-responsive MCF-7 cells. A sufficiently high concentration of exogenous estradiol (100 pM) had the dual action of neutralizing the growth inhibition by the 55-70% ammonium sulfate fraction of bovine serum and dextran-treated charcoal-treated fetal bovine: serum and enhancing directly the MCF-7 cell growth maximally 2-fold. Bovine serum albumin fraction V containing globulin remnants also inhibited growth, but globulin-free bovine serum albumin did not. Eliminating growth inhibition by the use of globulin-free bovine serum albumin enabled us to develop an ideal medium for assaying the direct effects of estradiol and TAM on MCF-7 cells. With this medium, we clearly identified (a) a direct mitogenic effect of exogenous estradiol on MCF-7 cells which was initiated at 3 pM and maximized at 0.2 to 10 nM, (b) an acute lethal effect of 1 microM TAM and its prevention by 100 pM estradiol, and (c) a nearly 50-fold increase in the concentration of exogenous estradiol (10 nM) required for maximum growth enhancement in the presence of 1 microM TAM than without TAM (0.2-0.3 nM).
Estrogen-binding macromolecules were identified in sexual skin cytosol and nuclear fractions from female Japanese monkeys (Macaca fuscata fuscata), by sucrose-glycerol gradient analysis and dextran-coated charcoal adsorption technique. Furthermore, using the highly potent synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione; promegestone), progestin-binding macromolecules were detected in sexual skin cytosols from ovariectomized estrogen-primed monkeys. Both cytosol components sedimented at the 8S region on sucrose-glycerol gradients and had a high affinity for the respective steroids, and competition studies showed a specificity for estrogenic, or progestational compounds. The concentration of cytosol progestin receptors was markedly increased with the elevated level of nuclear estrogen receptors following estrogen priming. The above results demonstrate the existence of cytosol and nuclear estrogen receptor sites, and estrogen-induced progestin receptors in the sexual skin of the female monkeys.
Examination was made of the urinary and biliary excretion of the metabolites of puerarin, the major component of the roots of Pueraria lobata OHWI (Leguminosae) in rats. The urine of rats administered puerarin orally contained puerarin and four major metabolites, daidzein 4',7-di-O-sulfate (M-I), daidzein 7-O-beta-D-glucuronide (M-II), daidzein 4'-O-sulfate (M-III), daidzein (M-IV), as determined from spectroscopic and chemical data. Total cumulative amounts of the puerarin and four metabolites excreted in the urine at 48 h following the oral administration of puerarin were approximately 3.6% the doses administered. The bile of rats administered puerarin orally contained puerarin and two major metabolites, which were identified as puerarin 4'-O-sulfate (PB1) and puerarin 7-O-beta-D-glucuronide (PB2) on the basis of chemical and spectroscopic data. These experimental data suggest that C-glycoside puerarin is partially hydrolyzed to aglycone in the body, but mainly excreted in the urine as unchanged puerarin.
The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.
Most instances of endometrial cancer are potentially preventable. Unopposed endogenous estrogen stimulation of the endometrium has been shown to be the predisposing risk factor in most cases. Risk factors have been well-delineated, and it is important to recognize and treat the progesterone-deficient patient. Low-dose oral contraceptive pills in healthy, nonsmoking, older reproductive-aged women are an underutilized treatment modality. The many noncontraceptive benefits of longterm oral contraceptive use until the menopause should be explained to the patient, including the prevention of ovarian and endometrial cancer, the maintenance of bone density, and a reduction in the many surgical procedures performed for menstrual disorders. Progestin therapy in older reproductive-aged women and postmenopausal women with unopposed estrogen production is mandatory to prevent endometrial cancer. Knowledge and skill in simple endometrial sampling techniques performed in patients with known risk factors for endometrial cancer will often detect premalignant lesions that are treatable with progestin therapy or surgery.
A novel estrogen receptor, estrogen receptor beta (ERbeta), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ER alpha has been reported to be present preferentially in osteoblasts, but the mechanism of action of estrogen in bone is still not known. In the present study, we examined expression of ERbeta mRNA in bone. Expression of ERbeta mRNA was evident in primary osteoblastic cells isolated from 1-day-old rat calvaria and rat osteosarcoma cells (ROS 17/2.8), and its level was higher than that of ER alpha mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERbeta mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ER alpha mRNA was very low at the beginning of culture and it gradually increased during the differentiation of osteoblastic cells. Various tissues including bone were isolated from 8-week-old rats of both sexes, and total RNA was extracted to compare the tissue distribution of expression levels of ERbeta mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERbeta mRNA was obvious, and its level was equivalent to those in the uterus and testis, but lower than those in the ovary and prostate. The level of ERbeta mRNA in femoral cortical bone was lower than that in cancellous bone. There was no appreciable differences between female and male rats in the distribution and expression levels of ERbeta mRNA in bone. These results indicate that ERbeta mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERbeta in bone.
The effect of genistein on bone resorption in vitro was investigated. Femoral-metaphyseal tissues obtained from elderly female rats were cultured for 48 hr in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-3) M genistein. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M), prostaglandin E2 (PGE2; 10(-5) M), and lipopolysaccharide ( 10 microg/mL) caused a significant decrease in bone calcium content. The decrease in bone calcium content induced by bone-resorbing factors was inhibited completely by genistein (10(-7) to 10(-5) M). In addition, this isoflavonoid (10(-5) M) completely inhibited the PTH (10(-7) M)- or PGE2 (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by bone tissues. Moreover, genistein (10(-5) M) blocked both PTH (10(-7) M)-increased acid phosphatase and -decreased alkaline phosphatase activities of bone tissues. The inhibitory effect of genistein (10(-5) M) on PTH (10(-7) M)-stimulated bone resorption was clearly prevented by the presence of 10(-6) M tamoxifen, an anti-estrogen reagent. Genistein (10(-5) M) did not further enhance the inhibitory effect of estrogen (10(-9) M) on PTH-stimulated bone resorption. These findings indicate that genistein has a direct inhibitory effect on bone resorption in tissue culture in vitro.