Article

Discovering anti-osteoporosis constituents of maca (Lepidium meyenii) by combined virtual screening and activity verification

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Abstract

Maca (Lepidium meyenii Walp.) is a traditional medicine and nutritional supplement from South America with many pharmacological effects, such as enhancing female and male fertility, improving sexual dysfunction, preventing osteoporosis and relieving menopausal syndrome. Until now, the active principles behind most of these effects have not been clarified, severely hindering the exploitation and application of maca products. In this study, the effective anti-osteoporotic components of maca are uncovered. Through virtual screening against estrogen receptor and verification of pharmacological activity on osteoblasts, we found that N-benzyl-palmitamide is an active constituent of maca in preventing osteoporosis. As well, N-benzyl-palmitamide promotes osteoblast proliferation, differentiation and mineralization, plus it benefits bone formation through enhanced expression of osteogenesis-related genes, such as bone morphogenetic protein-2, core binding factor alpha 1, type 1 collagen and alkaline phosphatase. The effects of N-benzyl-palmitamide on enhanced bone formation are very likely through the estrogen receptor pathway, as N-benzyl-palmitamide also increases the expression of ERα and ERβ genes. Clarifying single-component biological activity will greatly enable exploitation and application of maca as a medication and health supplement to prevent osteoporosis.

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... The positive effect of maca on osteoporosis was also demonstrated in vitro using MC3-T3 E1 osteoblast-like cell. Macamide N-benzylpalmitamide, promoted cell-proliferation and a dose-response effect in increasing extracelluar matrix mineralisation at a concentration of 10 -8 M. Importantly, ethanolic extract with same content of Nbenzylpalmitamide, had better effect than the compound alone (Liu et al., 2015), suggesting a synergistic effect of the metabolites in maca in the protection against bone-loss. ...
... In-silico estrogen receptor type ER-alpha and ER-beta docking assay revealed no activity from the phytochemicals of maca (Powers and Setzer, 2015). However, another study found that the macamide N-benzylpalmitamide, virtually docked to estrogen receptor and increased expression of ER-alpha and ER-beta genes at 10 -7 M in MC3T3-E1 osteoblast-like cell (Liu et al., 2015). ...
... Maca extract showed chondro-protective effect in human cartilage samples (Miller et al., 2006). One in vitro assay demonstrated that the macamide N-benzylpalmitamide, promoted proliferation of osteoblast cell model MC3T3-E1 (Liu et al., 2015). Again, this highlights most likely, a protective effect of maca. ...
Article
Ethnopharmacological relevance: Maca - Lepidium meyenii Walp has been cultivated and used by Andean people for over 1,300 to 2000 years in Peru as food and medicine. Starting in the late 1990's it has developed into an important herbal medicine in China and is now cultivated there widely, too. Aim of study: This study aims to provide an insight into the emergence of maca on the global market as an alternative remedy to treat reproductive health related problems in both men and women and to critically assess these health claims. Methodology: A search of electronic databases such as EMBASE and a hand-search was done to acquire peer-reviewed articles and reports about maca. Results and discussion: Lepidium meyenii is used traditionally as a tonic, fertility enhancer for both humans and cattle, and to treat a variety of ailments such as rheumatism, respiratory disorders and anaemia among others. Maca root is cooked, baked, fermented as a drink and made into porridge. In the last twenty years, maca was introduced onto the global market and demand has dramatically grown over this time with its promotion on the internet, as the 'Peruvian Ginseng' for libido and fertility enhancement. It has also been said to treat menopausal symptoms, erectile dysfunction and benign prostatic hyperplasia. The sky-rocketing demand for the plant has seen a shift from traditional cultivation methods to mass production practices with the use of fertilisers and also pesticides; as maca is now grown in areas other than the Andes such as in the Yunnan province in China. This can potentially affect the phytochemistry and composition of the plant and thus, the quality, safety and efficacy of maca products. Meanwhile, research into maca's medicinal properties has followed the spike in popularity of maca and has been focused mainly on maca's aphrodisiac and fertility enhancing properties. So far, the in vivo studies and clinical trials conducted have yielded inconclusive results. Some of the key limitations reside in methodology and sample size. Chemical profiling, led to the discovery of new compounds unique to maca, such as, 'macamides' and also other active metabolites like the glucosinolates; to which the medicinal effects of maca have been ascribed but cannot be confirmed due to lack of data. Conclusions: To date, the health claims of maca cannot be fully supported from a scientific standpoint and more research is needed. It appears that the indigenous local knowledge about the health benefits of maca has been dragged out of context to fit the demands of a growing market for herbal remedies. This globalisation (or hype esp. in China) also has had serious consequences for the local producers in Peru. The lack of protocols to regulate the production and marketing of maca during this rapid expansion, poses a threat to both the safety of consumers and the sustainability of supply.
... The hypocotyls of L. meyenii, commonly referred to as BMaca,^are a wellknown functional food for improving immunity system, female fertility, sexual function, etc. (Gonzales 2011;León 1964). Currently, several bioactivities of Maca including aphrodisiac activity (Cicero et al. 2001), anti-osteoporosis (Liu et al. 2015), anti-cancer, and anti-inflammation (Bai et al. 2015) and its major chemical compositions (Chain et al. 2014;Dini et al. 1994;Muhammad et al. 2002;Zhao et al. 2005) have been increasingly reported. ...
... Because of its edible value and potential medicinal efficacy, Maca and its products are available as a popular nutraceutical in the market around the world. The functional properties of Maca have also raised worldwide attention in recent years (Bai et al. 2015;Choi et al. 2012;Chen et al. 2015;Liu et al. 2015;Zha et al. 2014). Among them, macamides, a group of benzylalkylamide compounds, were identified as the characteristic constituents while contributing to the major efficacies in Maca such as anti-fatigue (Choi et al. 2012), enhancing sexual behavior (Zheng et al. 2000), and improving fertility (Uchiyama et al. 2014). ...
Article
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Macamides with benzylalkylamides structure are a group of characteristic constituents isolated from functional food Maca (Lepidium meyenii Walp.). Previous study demonstrated that macamides are secondary amides, the accumulation of which is associated with drying process. In this study, a rapid method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is developed for the simultaneous detection and determination of macamides in Maca. Validation parameters including linearity, limit of detection, limit of quantification, accuracy, precision, repeatability, and stability were all within the required limits. Seven major macamides are determined by the UPLC method with detection wavelength at 205 nm in 12 min. Based on the fragmentation pattern of standard compounds, four fragment ions (m/z 91, 108, 121, and 138) are selected as diagnostic ions for detection of macamides through precursor ion acquisition mode. Additionally, comparative analysis is performed to examine the effects of different drying processes (air-drying, oven-drying, freeze-drying, and steaming) on Maca (whole hypocotyls and pieces) for macamides. Whole hypocotyls with oven-drying showed the highest content of macamides (2.528 mg/g), while freeze-drying and steaming significantly decrease the amount of macamides. The results provide a reliable method for analysis of macamides and extend previous work insights into the drying process of Maca.
... [11] Besides, Liu et al. reported that macamides showed a positive effect on preventing osteoporosis. [12] Therefore, the investigation of macamides in commercial maca products and the major influence factors have great significance for both edible and medicinal usage. ...
... Macamides have been suggested as a quality indicator for maca products due to their high bioactivities. [7][8][9][10][11][12] In the present study, we found the significant variations of macamides in commercial maca products and analyzed the influence factors for macamide biosynthesis. As shown in Fig. 1, (a) glucosinolate catabolism, (b) lipid hydrolysis, and (c) amide formation are considered to be the key steps for macamide biosynthesis. ...
Article
Macamides are bioactive and marker compounds of maca (Lepidium meyenii). Thirty-five commercial maca products were surveyed for macamide composition and content by HPLC-UV/MS. Significant variations of macamide content were found in these products (69-2738 μg/g). Analysis of the macamide biosynthetic pathway suggests that: a) glucosinolate catabolism, b) lipid hydrolysis and c) amide formation are key steps controlling macamide accumulation in the tissues during the postharvest drying process. Therefore, we further investigated the effects of sample forms, drying temperatures and storage times on macamide accumulation. Our results show that 1) powdered maca provided the largest macamide accumulation followed by sliced hypocotyls, while whole roots displayed a significantly reduced amide-generating potential; (2) the ideal temperature for macamide formation is about 30 °C; (3) macamide content increases continuously along with storage time; (4) exposure to air results in the percentage of unsaturated macamides decreasing. These findings provide useful insights which can be applied in the industrial manufacture of maca products with higher content of bioactive amides.
... For example, rapid fatigue, insomnia, amnesia, low fertility and sexual dysfunction have become common threats to human health. When screening for natural medicines that may cure or alleviate those symptoms, maca (Lepidium meyenii Walpers) has been found to be a potential candidate (Liu et al., 2015;Lee et al., 2016;Li et al., 2017;Tang et al., 2017;Wang & Zhu, 2019). Maca is an annual herbaceous plant of the Brassicaceae family that grows best in high altitude regions . ...
... Many nutritional properties and the traditional therapeutic effectiveness of maca have been confirmed, including improving fertility (Gonzales et al., 2004;Bustos-Obreg on et al., 2005;Rubio et al., 2006), antiproliferative function (Fahey et al., 2001;Lee et al., 2005), enhancing bone formation and preventing osteoporosis (Liu et al., 2015) and immunomodulatory effects (Zhang et al., 2016). Particularly of interest is the bioactivities of maca that improve sexual and reproductive performance (Zheng et al., 2000;Gonzales et al., 2002;Gonzales et al., 2003;Bogani et al., 2006;Lentz et al., 2007). ...
Article
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Macamides, functional compounds found only in Lepidium meyenii Walpers, have gained interest in many fields of research due to its multiple bioactivities. Nevertheless, the mechanism by which macamides are synthesized is not thoroughly understood. In this study, the synthesis of six macamides and the conversion of three glucosinolates were studied systematically during a six‐month natural air drying process. An investigation of synthesis profiles showed that the largest category of macamides, the major constituents of total macamides, have the same benzyl group as benzyl glucosinolate, which has higher content than other glucosinolates. The second largest group of macamides contains the same m‐methoxybenzyl group as m‐methoxybenzyl glucosinolate which is the second major constituent of total glucosinolates. The results from this study suggest that glucosinolates are one of the most important precursors of macamides. Compared with all other drying methods, natural air drying is the most suitable condition for the formation of macamides.
... 8S ). Subsequently, knowing the molar masses of these fatty acids, and that the macamides are formed through the reaction between the hydroxyl group of the fatty acid and the amino group of the benzylamine [33], we managed to determine the molar masses of the different macamides (▶Table 2S). ▶Table 5 shows the retention times, the m/z values of the peaks with greater abundance (▶ Fig. 2), as well as the ion corresponding to each of the macamides present in the sub-fraction IIID (▶ Figs. ...
Article
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Although Tropaeolum tuberosum tubers have been consumed cooked as a folk remedy for the treatment of skin, lungs, liver and kidneys diseases, these uses have very limited scientific basis. Therefore, this article develops a phytochemical analysis of the yellow tubers of T. tuberosum with the objective to assess whether the isolated compounds have anti-inflammatory potential in the CCD-1109Sk, MRC-5 and RWPE-1 cell lines. We performed an extraction of T. tuberosum tubers using different organic solvents, followed by a bioguided chromatographic separation. Four macamides were identified by LC/MS techniques, but only N-benzyllinoleamide (1) and N-benzyloleamide (2) were isolated and elucidated by NMR/MS techniques, given that they were present in a larger proportion in the tubers. The anti-inflammatory potential of macamides was evaluated by the inhibition of NF-κB and STAT3 activation. Both compounds displayed inhibition of NF-κB activation with IC50 values of 2.28±0.54 µM; 3.66±0.34 µM and 4.48±0.29 µM for compound (1) and 6.50±0.75 µM; 7.74±0.19 µM and 8.37 ±0.09 µM for compound (2) in CCD-1109Sk, MRC-5 and RWPE-1 cell lines, respectively. Moreover, both compounds inhibited the STAT3 activation with IC50 of 0.61±0.76 µM; 1.24±0.05 µM and 2.10±0.12 µM for compound (1) and 5.49±0.31 µM; 7.73 ±0.94 µM and 7.79±0.30 µM for compound (2). Therefore, isolated macamides of T. tuberosum tubers showed promising anti-inflammatory effects, suggesting a possible beneficial use to combat inflammatory processes of skin, lung and prostate.
... Additionally, improvements of bone mineralisation against osteoporosis and antioxidant activity against stress have been reported in several studies of rats consuming Maca and its extracts (ZHANG et al., 2006;VEČEŘA et al., 2007). The bioactive compounds in Maca, such as N-benzyl-palmitamide, benzylisothiocyanate, glucosinolates and phenolics, have effects on lipid, mineral and antioxidant metabolisms (VEČEŘA et al., 2007, CAMPOS et al., 2013LIU et al., 2015). However, there are a limited number of studies in which Maca was used as a plant feed additive for farm animals. ...
Article
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The present study investigated the effects of dietary supplementation with Maca powder on the performance, egg quality, serum parameters, hormones and antioxidant enzyme levels of laying hens in their post-peak period. A total of 150 hens, 56 week of age, were separated into three treatment groups (50 hens per group). Over 16 weeks, diets were supplemented with Maca powder at concentrations of 0 (control), 5 and 10 g/kg respectively. Performance and egg quality were not significantly influenced by the supplementation of Maca powder. Cholesterol contents of the yolk were not influenced by the experimental diets. At the end of the study, no significant difference in the levels of serum glucose, total triglyceride and total cholesterol, calcium, phosphorus, progesterone and oestradiol was detected. However, serum magnesium levels decreased as the rate of Maca powder increased ( P< 0.05). A significant increase in serum glutathione peroxidase (GPx) was measured in the hens fed with 10 g/kg Maca powder. In conclusion, Maca powder had neither positive nor adverse impacts on performance, egg quality, yolk cholesterol content, serum parameters (except magnesium) and hormones. Despite that serum magnesium levels were adversely affected, Maca powder may enhance the antioxidant status, specifically GPx, of laying hens in the post-peak period.
... (maca) has been used as both a dietary staple and a traditional medicine in the Andes for centuries (Gonzales, 2012). Macamides are the major active ingredients of maca, which play a stimulatory role in the central nervous system, showing positive effects for neuroprotection and prevention of osteoporosis ( Almukadi et al., 2013;Alquraini, Wag- gas, Bӧhlke, Maher, & Pino-Figueroa, 2014;Hajdu et al., 2014;Lewis & Pino-Figueroa, 2013;Liu et al., 2015;Wu, Kelley, Pino- Figueroa, Vu, & Maher, 2013). ...
Article
A novel technique of ultrasound-assisted freeze-thaw pretreatment (UFP) was developed to improve the drying efficiency of maca and bioactive amide synthesis in maca. The optimal UFP conditions are ultrasonic processing 90 min at 30 °C with 6 freeze-thaw cycles. Samples with freeze-thaw pretreatment (FP), ultrasound pretreatment (UP), and UFP were prepared for further comparative study. A no pretreatment (NP) sample was included as a control. The results showed that UFP improved the drying efficiency of maca slices, showing the highest effective moisture diffusivity (1.75 × 10−9 m2/s). This result was further supported by low-field nuclear magnetic resonance (LF-NMR) analysis and scanning electron microscopy (SEM). The rehydration capacity and protein content of maca slices were improved by UFP. More importantly, contents of bioactive macamides and their biosynthetic precursors were increased in 2.5- and 10-fold, respectively. In conclusion, UFP is an efficient technique to improve drying efficiency, physicochemical properties, and bioactive macamides of maca, which can be applied in the industrial manufacture of maca products.
... These chemical compounds in plants show antiviral and antibacterial effects in other living things that consume plants and cause many effects that strengthen the immune system. Bioactive compounds such as N-benzyl-palmitamide, benzylisothiocyanate, glucosinolates and phenolics in maca have important effects on lipid, mineral and antioxidant metabolisms [2][3][4][5]. Many plants and their extracts as feed additives in animal nutrition have been used for many years for growth, reproduction, improvement of product quality and health protection. ...
Article
Full-text available
Maca plant contains rich nutrients and in addition, it has various bioactive substances (N-benzyl-palmitamide, benzyl isothiocyanate, glucosinolates and phenolics). It is used to improve reproductive properties and has antioxidant effects for both humans and animals. The aim of this study was to determine the effects of adding maca plant powder to the ration at different levels on growth, slaughter carcass, partial egg production and some reproductive characteristics in Japanese quail (Coturnix coturnix japonica). The experimental groups were formed by adding 0% (control), 0.05% and 0.1% maca powder to the diet, and a total of 300 birds were used. Growth (weekly body weights, parameters of Gompertz growth function), feed efficiency and carcass characteristics of quails in the 42-day fattening trial were determined. Reproductive characteristics were measured up to 22 weeks of age. Addition of maca powder to the diet increased the feed consumption (p < 0.05) but did not affect body weights at 35 and 42 days of age, β0 and β1 parameters or point of inflection weight of the Gompertz model, carcass traits, partial egg production or fertility. It may be advisable to add 0.1% maca powder to the diets of breeders. Besides, maca powder reduced embryonic deaths and improved chick quality (both p < 0.05). It is thought that different results for reproductive traits can be obtained if maca powder is used for a longer period in the diets of breeder quail flocks.
... As a medicinal plant, maca has been therapeutically used for improving fertility and sexual function (Zheng et al. 2000;Flores et al. 2003). Other benefits of maca include anti-fatigue, anti-oxidant, anti-osteoporosis, inflammatory, immunomodulatory, and hepatoprotective effects against alcoholic liver damage (ALD) (Sandoval et al. 2002;Bai et al. 2015;Liu et al. 2015;Wang et al. 2016;Li et al. 2017a;Zhang et al. 2017). As a novel resource food, maca powder was approved by the Ministry of Health of the People's Republic of China in 2011, and maca products are now legalized in China . ...
Article
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Maca (Lepidium meyenii Walp.), an edible medicinal plant and popular dietary supplement, is native to South America in the Andes mountains of Peru. However, maca is frequently adulterated due to its high nutritional and medicinal value, which compromises its safety and effectiveness. In the present study, maca and its two adulterants, Brassica rapa (turnip) and Raphanus sativus (radish), were identified by one nuclear barcode (ITS) and four chloroplast barcodes (matK, psbA-trnH, rbcL, and trnL-trnF). All five DNA barcodes were perfectly identified in maca, turnip, and radish. Out of the five tested barcodes, trnL-trnF could be regarded as the ideal barcode for detecting turnip/radish adulteration in maca as it generated different sequence length among these three species, it was further validated by mixing turnip/radish into maca at various proportions, and trnL-trnF efficiently and sensitively detected adulteration even at a very low level (0.5% of adulteration).
... Macamides, product of Maca after drying process, are considered to be its unique iconic ingredient [6]. Based on recent pharmacological studies, macamides exhibit a variety of biological activities on neuroprotection, antioxidation, anti-osteoporosis, fatty acid hydrolase inhibition, anticancer, promoting leydig cells proliferation, and testosterone secretion [7][8][9][10][11]. Additionally, it is reported that macamides can scavenge free radicals, protect cells from OS, and play a key role in anti-fatigue [12]. ...
Article
In this paper, a UPLC-ESI-MS was established to analyze macamide and macaene fractions from Lepidium meyenii (Maca). The antioxidant activity of Maca crude extract (MCE), total macamides (TMM) and total macaenes (TME) was evaluated by DPPH radical scavenging, ABTS radical scavenging and reducing power. The ability of MCE, TMM and TME against multiple cancer cell lines (leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7, and colon cancer SW480) was used to evaluate their anticancer activity. The results demonstrate that TMM has the best free radical scavenging ability and reducing power compared to MCE, TME, and TMM, TME has the weakest antioxidant capacity compared to them. The difference in antioxidant properties between TMM and TME may be caused by benzylated alkamide in the chemical structure. TMM also has a good inhibitory effect on the five cancer cells. N-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide (NBot) is the most cytotoxicity inhibitory of the macamides tested. The inhibitory effect of N-benzyl-9Z, 12Z-octadecadienamide (NBod) on colon cancer SW480 is near that of control group, while the inhibitory rates of NBot on leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, and breast cancer MCF-7 are near those of the control group. Hence, NBot can be considered to have great potential as a natural drug with high efficiency and low side effects for cancer therapy.
... It is rich in minerals such as calcium, potassium, iron, manganese, copper, zinc, sodium, as well as fatty acids and secondary metabolites of pharmacological and nutritional importance such as macaene, macamides, macaridin and alkaloids that are only found in this plant [35][36][37][38]. In addition, maca has nutritional, energizing and male and female fertility enhancing properties, has effect on sexual dysfunctions, prostatic hyperplasia, alleviates menopausal syndrome, improves memory and learning, improves skin health [30,36,37,[39][40][41], prevents osteoporosis [42], has antioxidant and antitumor properties [43]. Therefore, maca is a great food that improves the health of people who consume it. ...
Article
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Heavy metal contamination of soil and agricultural products is an environmental problem, has an adverse effect on the quality of food crops, and is a danger to food security and public health. The concentration of arsenic (As), cadmium (Cd), lead (Pb), iron (Fe) and zinc (Zn) in surface soils and edible hypocotyls tissues of two ecotypes of Lepidium meyenii Walpers (maca) was evaluated in three districts of the Junín province, Peru. In addition, the risk to human health due to exposure to heavy metals from maca consumption was evaluated. Soil samples and maca hypocotyls were collected in areas influenced by mining and metallurgical activity. The mean concentration of Cd (0.32 ± 0.23 mg/kg) and Pb (0.20 ± 0.12 mg/kg) in maca samples exceeded the values established by the Food and Agriculture Organization and the World Health Organization. The bioconcentration factor was less than 1. The estimated daily intake of each metal was below the oral reference dose. The hazard quotient and hazard index were less than 1, it is unlikely to cause non-cancer adverse health outcome. The cancer risk for As and Cd was higher than the tolerable limit (1 × 10⁻⁶) in children and adults. In the district of Ondores, the cancer risk for As in children was higher than the acceptable limit (1 × 10⁻⁴). Residents of the Ondores district would be more exposed to As and Cd from consumption of maca hypocotyls. It is very important to carry out continuous monitoring of other toxic metals in different ecotypes of maca (red, black, yellow, purple, creamy white, pink) in order to evaluate the variation in the accumulation of heavy metals and the level of toxicity of each metal between ecotypes.
... [25][26][27] Maca lipophilic extract containing macamides has shown neuroprotective effects against middle cerebral artery occlusion in vivo and H 2 O 2 injury in vitro. 28,29 Macamides, a series of nonpolar and long-chain fatty acid N-benzylamides, are considered as unique constituents in maca, 30,31 exhibiting various biological activities, including anti-oxidative, antifatigue, and anti-osteoporotic activities, [32][33][34] and activities related to improving the nervous system. Macamides are acetylcholinesterase (AChE) inhibitors. ...
Article
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Maca has attracted considerable attention owing to its neuroprotective effects in vitro and vivo. Macamides, a series of nonpolar and long-chain fatty acid N-benzylamides, are considered unique constituents in maca. This study investigated the protective effects of ethanol extracts of maca (EEM) and macamides on corticosterone-induced (CORT) neurotoxicity in rat pheochromocytoma (PC12) cells. CORT reduced cell viability and increased LDH release, intracellular ROS levels, and MMP decline rate, and induced mitochondrial apoptosis. However, pretreatment with EEM and macamides ameliorated CORT-induced neurotoxicity. EEM increased the cell viability and reduced the LDH release. M 18:1, M 18:2, and M 18:3 increased cell viability and reduced LDH release and intracellular ROS generation. M 18:2 and M 18:3 inhibited MMP reduction and reduced the Bax/Bcl-2 ratios. M 18:1 reduced the intracellular ROS without affecting other factors. Moreover, M 18:3 prevented CORT-induced mitochondrial apoptosis, restrained the expression levels of pro-apoptotic proteins, namely, Bax, cytochrome C, cleaved-caspase-3, and cleaved-PARP, and increased the expression levels of Bcl-2. In addition, M 18:3 increased Akt phosphorylation and the ability of M 18:3 to protect against CORT-induced cytotoxicity was remarkably reduced by LY294002, a PI3K phosphorylation inhibitor. M 18:3 also elevated the phosphorylation of CREB and activated the BDNF protein levels in CORT-induced PC12 cells. In conclusion, macamides, especially M 18:3, exert protective effects on CORT-induced PC12 cells. The cellular mechanism of M 18:3 against CORT-induced cytotoxicity may involve inhibition of mitochondrial apoptosis, and activation of Akt and CREB phosphorylation. Overall, macamides may potentially treat neuronal damage induced by CORT.
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En este trabajo se estudiaron propiedades termofísicas y termoquímicas de la harina de maca comercial seca, utilizando calorimetría de combustión y calorimetría diferencial de barrido (DSC). Se determinó el valor calorífico neto o energía másica de combustión (419 ± 3 Kcal/100g), calores específicos (entre -4 y 70 oC) en el que se registra para 25 °C el c.e = 1.46 ± 0.03 J/g.oC; y parámetros térmicos, como la temperatura y entalpía, tanto del proceso de descomposición (Tmax = 210.1 ± 0.4 oC y ΔHdec = 82 ± 10 J/g) como de la gelatinización (Tp = 76.9 ± 0.4 oC; ΔHgel = 5.6 ± 0.5 J/g).
Article
Macamides, the major bioactive compounds of Lepidium meyenii (Walp.) or Maca, are a unique class of non-polar, long chain fatty acid N-benzylamides with fertility-enhancing, neuroprotective, neuro-modulatory, anti-fatigue and anti-osteoporosis effects. However, the relationship between the structures and pharmacological effects of macamides have not been established so far. In addition, little is known regarding their biosynthetic pathways and the mechanisms underlying the biological activities. In this review, we have summarized the methods currently used for the extraction, purification and synthesis of macamides. Their pharmacological effects, clinical prospects and biomedical applications have also been discussed. Current data strongly suggest that macamides are a promising bio-active agent, and further studies are warranted to elucidate their mechanisms of action.
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Macamides are the major and unique bioactive compounds of Lepidium meyenii (Walp.) or Maca. N-benzyl-(9Z, 12Z)-octadecadienamide (N-benzyl-linoleamide) is one of the most biologically active macamides with various pharmacological activities - anti-fatigue, neuroprotective, antioxidant, anti-tumoral activities, anti-inflammatory, and analgesic. In this study, the anti-fatigue properties of N-benzyl-(9Z, 12Z)-octadecadienamide were further evaluated by a weight-loaded forced swimming test. Results indicated N-benzyl-(9Z, 12Z)-octadecadienamide supplementation increased the forelimb grip strength of mice and exercising time remaining on the Rota-rod test. Furthermore, significant decreases in pro-inflammatory factors and reactive oxygen species (ROS) contents were observed in mice receiving N-benzyl-(9Z, 12Z)-octadecadienamide treatment after a 30 min swimming test, which was equivalent to that of caffeine. Histological analysis also indicated that N-benzyl-(9Z, 12Z)-octadecadienamide attenuated damage to the liver in mice by up-regulating the expression of heme oxygenase-1 (HO-1) and inhibiting the expression of Interleukin (IL)-1β during exercise. Pearson correlation analysis suggested peripheral fatigue indexes, including energy sources, metabolites were significantly correlated with inflammatory factors and ROS levels. Likewise, central fatigue parameters are also associated, including hippocampal inflammatory response and hypothalamic neurotransmitters. Hence, macamides can be considered to have great potential as a natural drug with high efficiency and low side effects for fatigue management.
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Lepidium meyenii Walp. (maca) has been utilized in the Andean region because of its edibleness and medicinal value. The aerial parts of maca (APM) were analyzed for protein, total sugar, vitamins, amino acids, and minerals and its characteristic active ingredients at five different growth stages. Results showed the high protein, total sugar, vitamin C, niacin, potassium, and calcium contents of APM. All 17 amino acids and the characteristic active ingredients, namely, macamide, glucosinolates, adenosine, and total saponins, were detected. We examined the effects of APM powders on gastric emptying and intestinal propulsion and the levels of serum motilin and gastrin in atropine-treated mice. Benzyl isothiocyanate (BITC) was investigated to identify the potential active material foundation of APM. Results revealed that both maca plant powders and BITC can promote gastrointestinal prokinetic efficacy. Thus, APM feature potential as new functional vegetable sources.
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Three new pyrrole alkaloids macapyrrolins A–C (1–3) were isolated from the roots of Lepidium meyenii collected from Lijiang, Yunnan province in China. Their structures were elucidated by extensive NMR and MS spectroscopic analyses. Interestingly, macapyrrolins A–C represent the first examples of pyrrole alkaloids found in Lepidium meyenii with a benzyl substituent attached to the pyrrole nucleus. All the isolated macapyrrolins were evaluated for their cytotoxicity against five human cancer cell lines. However, no significant activities were detected at concentrations up to 40 μM.
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Most of studies have focused on the maca (Lepidium Meyenii) hypocotyls, including the extraction, purification, characterization of bioactive components and their structures, but few on maca leaves. In this paper, we used response surface methodology to optimize the extraction conditions of maca leaves polysaccharides (MLPs). The optimal conditions were as follows: ratio of water to raw material (38 mL/g); extraction time, 150 min; extraction temperature, 100 °C and extraction times, 3. Under the condition, the experimental yield was 14.2 ± 0.25% (mL/g) (n = 3), which was close to the predicted value. The primary characterization of the crude MLPs was explored, and we carried on the FT-IR experiment. The FT-IR analysis revealed the general characteristic absorption peaks of the crude MLPs.
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Nine new pyrrole alkaloids, including two undescribed dimeric pyrrole 2‑carbaldehyde alkaloids, lepipyrrolins A-B (1-2), seven pyrrole-alkaloid derivatives, macapyrrolins D–J (3–9), along with three known ones (10-12) were isolated from the rhizomes of Lepidium meyenii. Their structures and absolute configurations were demonstrated by extensive spectroscopic data (1D, 2D NMR, HRESIMS), and calculated electronic circular dichroism (ECD) experiment. Compounds 1, 3-12 were tested for their nitric oxide inhibitory effects. Furthermore, compound 1 was evaluated for its cytotoxic activity against five human tumor cell lines (HL-60, SMMC-7221, A549, MCF-7, and SW480) in vitro, and displayed selective cytotoxicity against SMMC-7721 with IC50 value of 16.78 ± 0.49 μM.
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Maca (Lepidium meyenii) is a biennial plant of the crucifer family (Brassicaceae), cultivated on the Andean plateaus of Peru. According to traditional beliefs, maca is considered to be an aphro-disiac, also known as ‘Peruvian ginseng’. The edible parts of the plant are the bulbous root and hypocotyl, which have health-promoting properties. The analysis of the chemical composition showed that L. meyenii had a high nutritional value and contained biologically active compounds such as: polyphenols (flavonoids, anthocyanins), tannins, saponins, prostaglandins and alkaloids. Furthermore, the plant is a source of polyunsaturated fatty acids, sterols (β-sitosterol, campesterol, stigmasterol) and glucosinolates, which are valuable dietary components. According to the avail-able literature, maca is currently the only known plant that contains macamides – components improving sexual functions. The results of studies have confirmed its impact on fertility, physical performance and health improvement in animals and humans. Moreover, maca also shows anti- -cancer, anti-osteoporosis and neuroprotective potential.
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Lepidium meyenii (Maca) contains several active components, including alkaloids, glucosinolates, isothiocyanates, polysaccharides, polyphenols, and sterols, which make it have the traditional therapeutic uses. In this paper, we summarized the analytical progress of the active components associated with alkaloids, polysaccharides, glucosinolates, sterols, free fatty acids, flavonoids, and natural phenols in Maca by mass spectrometry (MS). Due to the effect of color and type on active components in Maca, we summarized the study of quality evaluation about Maca according to the type and the content of active components such as glucosinolates, essential oils, macamides, and macaenes by MS. Additionally, the research on the change of active components in Maca at different growth stages by MS will be beneficial to full utilization of active components in Maca and other natural resources. We reviewed the study in the visible distribution of amino acids, amide alkaloids, imidazolium alkaloids, and saccharides in Maca by imaging mass spectrometry (IMS). We also reviewed the pharmacology value associated with improvement of reproductive function, anti‐stress response, anti‐osteoporosis, antitumor activity, clinical research and toxicity of Maca, and so forth. Nevertheless, due to individual differences and limitations of the subjects, further high‐quality studies are needed to confirm the clinical efficacy of the plant.
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A novel polysaccharide named as MC-1 was isolated from the roots of Lepidium meyenii using a water extraction method. Structural characterization revealed that MC-1 had an average molecular weight of 11.3 kDa and consisted of arabinose (26.21%), mannose (11.81%), glucose (53.66%), and galactose (8.32%). The main linkage types of MC-1 were proven to be (1→5)-α-L-Ara, (1→3)-α-L-Man, (1→2,6)-α-L-Man, (1→)-α-D-Glc, (1→4)-α-D-Glc, (1→6)-α-D-Glc and (1→6)-β-D-Gal by methylation analysis, periodate oxidation-Smith degradation and NMR analysis. The immunostimulating assay indicated that MC-1 could significantly enhance the pinocytic and phagocytic capacity and promote the NO, TNF-α and IL-6 secretion of RAW 264.7 cells, involving toll-like receptor 2, complement receptor 3 and mannose receptor mainly. These results suggested the potential utilization of MC-1 as an attractive functional food supplement candidate for hypoimmunity population.
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This study was designed to determine the effect of different varieties of maca (Lepidium meyenii) on bone structure in ovariectomized (OVX) rats. 36 female rats were randomly divided into 6 groups: sham and OVX rats treated with vehicle, estradiol (40 microg/kg), black, yellow or red maca (63 mg/ml) for 4 weeks. At the end of the treatment, uterine weight, femoral bone and lumbar vertebra histomorphology were assessed. Ovariectomy reduced weight, diameter and width of the femoral bone. Estradiol, black and red maca treatment reduced the effect of ovariectomy on these variables. Histological analyses revealed that estradiol, black and red maca treatments reversed the effect of ovariectomy by increasing the trabecular bone area in the second lumbar vertebra. Uterine weight was reduced in OVX rats, and estradiol but neither black nor red maca increased uterine weight. Red and black maca have protective effects on bone architecture in OVX rats without showing estrogenic effects on uterine weight.
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Lepidium meyenii (Maca) is a Peruvian hypocotyl that grows exclusively between 4000 and 4500 m in the central Andes. Maca is traditionally employed in the Andean region for its supposed aphrodisiac and/or fertility-enhancing properties. This study was a 12-week double-blind, placebo-controlled, randomized, parallel trial in which active treatment with different doses of Maca Gelatinizada was compared with a placebo. The study aimed to test the hypothesis that Maca has no effect on serum reproductive hormone levels in apparently healthy men when administered in doses used for aphrodisiac and/or fertility-enhancing properties. Men aged between 21 and 56 Years received 1500 mg or 3000 mg Maca. Serum levels of luteinizing hormone, follicle-stimulating hormone, prolactin, 17-alpha hydroxyprogesterone, testosterone and 17-beta estradiol were measured before and at 2, 4, 8 and 12 weeks of treatment with placebo or Maca (1.5 g or 3.0 g per day). Data showed that compared with placebo Maca had no effect on any of the hormones studied nor did the hormones show any changes over time. Multiple regression analysis showed that serum testosterone levels were not affected by treatment with Maca at any of the times studied (P, not significant). In conclusion, treatment with Maca does not affect serum reproductive hormone levels.
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Computational approaches that 'dock' small molecules into the structures of macromolecular targets and 'score' their potential complementarity to binding sites are widely used in hit identification and lead optimization. Indeed, there are now a number of drugs whose development was heavily influenced by or based on structure-based design and screening strategies, such as HIV protease inhibitors. Nevertheless, there remain significant challenges in the application of these approaches, in particular in relation to current scoring schemes. Here, we review key concepts and specific features of small-molecule-protein docking methods, highlight selected applications and discuss recent advances that aim to address the acknowledged limitations of established approaches.
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TGF-beta1 is a ubiquitous growth factor that is implicated in the control of proliferation, migration, differentiation, and survival of many different cell types. It influences such diverse processes as embryogenesis, angiogenesis, inflammation, and wound healing. In skeletal tissue, TGF-beta1 plays a major role in development and maintenance, affecting both cartilage and bone metabolism, the latter being the subject of this review. Because it affects both cells of the osteoblast and osteoclast lineage, TGF-beta1 is one of the most important factors in the bone environment, helping to retain the balance between the dynamic processes of bone resorption and bone formation. Many seemingly contradictory reports have been published on the exact functioning of TGF-beta1 in the bone milieu. This review provides an overall picture of the bone-specific actions of TGF-beta1 and reconciles experimental discrepancies that have been reported for this multifunctional cytokine.
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The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name "maca"), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC(50) values of 3.46 +/- 0.16 and 0.71 +/- 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 mug of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.
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Objective: To evaluate the effect of long-term ethanol extract of Lepidium meyenii (Maca) on serum hormone levels in ovariectomized (OVX) rats and compare them with the effect of diethylstilbestrol. Materials and Methods: Fifty female Sprague-Dawley rats were ovariectomized or sham operated. Both sham and OVX control groups (n = 10, respectively) received the vehicle. The remaining OVX rats were oral administrated with ethanol extract of Maca (0.096, or 0.24g/kg; n = 10, respectively) and diethylstilbestrol (0.05 mg/kg; n = 10). The treatment continued for 28 weeks. At week 12 and week 28, the blood of rats was collected and serum hormone levels, including estradiol (E2), testosterone (T) and follicle-stimulating hormone (FSH) were measured by radioimmunoassay. Results: At week 12, the levels of serum E2 were slightly higher in Maca groups than that in OVX group; T levels were significantly decreased; and FSH levels were advanced slightly in Maca groups than that in sham group. After 28 weeks administration, serum E2 levels in Maca-treated animals did not differ significantly from sham control, the low dose of Maca increased serum E2 levels, and Maca prevented increase in serum FSH levels compared with OVX group. Conclusions: Long-term Maca supply modulates endocrine hormone balance in OVX rats, especially it decreases enhanced FSH levels. It is proposed that Maca may become a potential choice for postmenopausal women.
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The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.
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It is becoming increasingly apparent that the risk of developing osteoporosis is accrued throughout the entire lifecourse, even from as early as conception. Thus early growth is associated with bone mass at peak and in older age, and risk of hip fracture. Novel findings from mother-offspring cohorts have yielded greater understanding of relationships between patterns of intrauterine and postnatal growth in the context of later bone development. Study of biological samples from these populations has helped characterize potential mechanistic underpinnings, such as epigenetic processes. Global policy has recognized the importance of early growth and nutrition to the risk of developing adult chronic non-communicable diseases such as osteoporosis; testing of pregnancy interventions aimed at optimizing offspring bone health is now underway. It is hoped that through such programs, novel public health strategies may be established with the ultimate goal of reducing the burden of osteoporotic fracture in older age. © 2014 American Society for Bone and Mineral Research.
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Lepidium meyenii (Maca) is traditionally employed in the Andean region for its supposed fertility benefits. This study investigated the effect of Maca on the serum pituitary hormone levels during the pro-oestrus phase. Maca powder was made from the tubers of Lepidium meyenii Walp collected, dried, and reduced to powder at the plantation in Junín Plateau and was purchased from Yamano del Perú SAC. The Maca powder was identified by chemical profiling and taxonomic methods. Two groups of female Sprague-Dawley rats were provided feed with normal feed containing 5%, 25%, or 50% Maca powder ad libitum for 7 weeks. At 1800h of the proestrus stage, the rats were euthanised, and blood samples were collected for serum isolation. The serum pituitary hormone levels were measured using enzyme-linked immunosorbent assays (ELISAs). No significant differences in feed intake or growth rate were observed among the rats. During the pro-oestrus stage, a 4.5-fold increase (P<0.01) in luteinising hormone (LH) and a 19-fold increase (P<0.01) in follicle-stimulating hormone (FSH) were observed in the sera of rats fed with 50% Maca powder compared with the control rats. No significant differences were observed in the levels of the other pituitary hormones, including growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), and thyroid-stimulating hormone (TSH). A dose-dependent increase of LH serum levels was observed within the range of 3 to 30g Maca/kg. Furthermore, the enhancement of the LH serum levels was specific to the pro-oestrus LH surge. The present study demonstrates that Maca uniquely enhances the LH serum levels of pituitary hormones in female rats during the pro-oestrus LH surge and acts in a pharmacological, dose-dependent manner. These findings support the traditional use of Maca to enhance fertility and suggest a potential molecular mechanism responsible for its effects.
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Maca (Lepidium meyenii), a traditional food crop of the Peruvian Andes is now widely touted as a dietary supplement. Among the various chemical constituents isolated from the plant are a unique series of non-polar, long-chain fatty acid N-benzylamides known as macamides. We have synthesized 11 of the 19 reported macamides and have tested each as potential inhibitors of the human enzyme, fatty acid amide hydrolase (FAAH). The five most potent macamides were FAAH inhibitors (IC50=10-17μM). These amides were derivatives of oleic, linoleic and linolenic acids and benzylamine or 3-methoxybenzylamine. Of the three compounds evaluated in a pre-incubation time study, two macamides were not irreversible inhibitors of FAAH. The third, a carbamate structurally related to macamides, was shown to be an irreversible inhibitor of FAAH (IC50=0.153μM).
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Maca (Lepidium meyenii Walpers) hypocotyls are reported to have a favorable biological activity in man. The presumed bioactive constituents seem to vary among maca hypocotyl color types, but the residual variation is large. This study tested the hypothesis that, apart from color types, environmental factors have a distinct influence on the concentration of characteristic secondary metabolites in maca. In a field experiment at 4100 m altitude (Peru), maca of four hypocotyl color types was evaluated at two sites with different soil types. At each site, experimental areas were either never cultivated or cultivated with maca 2 to 3 yr ago followed by a fallow period. Applying four color replicates per site and area resulted in 64 plots (100 plants plot(-1)). Especially, color type largely influenced concentrations of macaene, macamides, beta-sitosterol, campesterol, and glucosinolates. Site (also clearly affecting growth yield) was weaker in effect on these metabolites but still significantly influenced concentrations of some constituents, while the effect of cultivation history was widely absent. Macaene, macamides, and beta-sitosterol were negatively correlated with glucosinolates. This shows that environmental conditions and color type have to be considered in producing maca with high concentrations of distinct bioactive metabolites.
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Osteoporosis results in approximately one-half of older white women and one-third of men sustaining fractures, which cause significant disability and increased mortality. Interventions are now available which reduce fracture risk by about one-half, and there is evidence that they also reduce mortality in frail elderly by about 10%. The mechanism of this reduced mortality is unclear but it has the potential to substantially impact on the cost-benefit of osteoporosis treatment. Available treatments are generally well-tolerated. Bisphosphonates cause gastrointestinal side-effects when administered orally, and acute phase responses when given intravenously. Osteonecrosis of the jaw is overwhelmingly a problem of cancer sufferers rather than those with osteoporosis, but atypical patterns of fracture in the upper femoral shaft sometimes occur in users of these drugs, though they are very rare in comparison with the other osteoporotic fractures which these drugs prevent. Thus, the cost-benefit of bisphosphonate use is clearly positive in those with osteoporosis. In contrast, calcium supplements probably increase the risk of myocardial infarction, admissions to hospital with acute gastrointestinal complaints and risk of renal calculi, whereas their impact on fracture is marginal (about a 10% reduction). Thus, they are not cost-effective, and a balanced diet is a safer way of obtaining one's calcium requirements.
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Several products derived from processed maca hypocotyls (Lepidium peruvianum Chacón, previously known as L. meyenii Walp.) were surveyed for glucosinolate content and quantified by HPLC analysis. These included pills, capsules, flour, liquor, tonic and mayonnaise. Different plant organs such as fresh hypocotyls and leaves, seeds, dry hypocotyls, and sprouts were also included in the survey. The most abundant glucosinolates detected in fresh and dry hypocotyls and leaves were the aromatic glucosinolates, benzylglucosinolate (glucotropaeolin) and p-methoxybenzylglucosinolate. Maca seeds and sprouts differed in profile from hypocotyls and leaves due to the modification of benzylglucosinolate. No glucosinolates were detected in liquor and tonic, while mayonnaise had only trace amounts of those glucosinolates. It had instead allylglucosinolate (sinigrin), which is an aliphatic glucosinolate. The pills, capsules and flour had the same glucosinolates as those observed in hypocotyls, but in variable amounts. The richest sources of glucosinolates were seeds, fresh hypocotyls and sprouts, in that order.
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Antimycin A treatment of cells blocks the mitochondrial electron transport chain and leads to elevated ROS generation. In the present study, we investigated the protective effects of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Osteoblastic MC3T3-E1 cells were pre-incubated with magnolol before treatment with antimycin A. Cell viability and mineralization of osteoblasts were assessed by MTT assay and Alizarin Red staining, respectively. Mitochondrial dysfunction in cells was measured by mitochondrial membrane potential (MMP), complex IV activity, and ATP level. The cellular antioxidant effect of magnolol in osteoblastic MC3T3-E1 cells was assessed by measuring cardiolipin oxidation, mitochondrial superoxide levels, and nitrotyrosine content. Phosphorylated cAMP-response element-binding protein (CREB ) was evaluated using ELISA assay. Pretreatment with magnolol prior to antimycin A exposure significantly reduced antimycin A-induced osteoblast dysfunction by preventing MMP dissipation, ATP loss, and CREB inactivation. Magnolol also reduced cardiolipin peroxidation, mitochondrial superoxide, and nitrotyrosine production induced by antimycin A. These results suggest that magnolol has a protective effect against antimycin A-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. All these data indicate that magnolol may reduce or prevent osteoblast degeneration in osteoporosis or other degenerative disorders.
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Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis.
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Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus. First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERβ1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.
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Maca (Lepidium meyenii), an Andean plant of the brassica (mustard) family has been used for centuries in the Andes as an adaptogenic plant to manage anemia, infertility and female hormone balance. The aim of this review was to assess the evidence for and against the effectiveness of the maca plant as a treatment for menopausal symptoms. We searched 17 databases from their inception up to June 2011 and included all randomized clinical trials (RCTs) that compared any type of maca-based intervention to a placebo for the treatment of menopausal symptoms. All studies were assessed for methodological quality using the Cochrane 'risk of bias' assessment tool. Four RCTs met all inclusion criteria. These RCTs tested the effects of maca on menopausal symptoms in healthy perimenopausal, early postmenopausal, and late postmenopausal women. Using the Kupperman Menopausal Index and the Greene Climacteric Score, all RCTs demonstrated favorable effects of maca. There have been very few rigorous trials of maca for menopausal symptoms. The results of our systematic review provide limited evidence for the effectiveness of maca as a treatment for menopausal symptoms. However, the total number of trials, the total sample size, and the average methodological quality of the primary studies, were too limited to draw firm conclusions. Furthermore, the safety has not been proved yet. Therefore, the efficacy and safety should be tested in larger studies.
Article
Polyunsaturated fatty acids and fish may influence bone health. We aimed to examine associations between dietary polyunsaturated fatty acid and fish intakes and hip bone mineral density (BMD) at baseline (1988-1989; n = 854) and changes 4 y later in adults (n = 623) with a mean age of 75 y in the Framingham Osteoporosis Study. BMD measures were regressed on energy-adjusted quartiles of fatty acid intakes [n-3 (omega-3): α-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and EPA+ DHA; n-6 (omega-6): linoleic acid (LA) and arachidonic acid (AA); and n-6:n-3 ratio] and on categorized fish intakes, with adjustment for covariates. Effect modification by EPA+DHA intake was tested for n-6 exposures. High intakes (≥3 servings/wk) of fish relative to lower intakes were associated with maintenance of femoral neck BMD (FN-BMD) in men (dark fish + tuna, dark fish, and tuna) and in women (dark fish) (P < 0.05). Significant interactions between AA and EPA+DHA intakes were observed cross-sectionally in women and longitudinally in men. In women with EPA+DHA intakes at or above the median, those with the highest AA intakes had a higher mean baseline FN-BMD than did those with the lowest intakes (quartile 4 compared with quartile 1: P = 0.03, P for trend = 0.02). In men with the lowest EPA+DHA intakes (quartile 1), those with the highest intakes of AA (quartile 4) lost more FN-BMD than did men with the lowest intakes of AA (quartile 1; P = 0.04). LA intake tended to be associated with FN-BMD loss in women (P for trend < 0.06). Fish consumption may protect against bone loss. The protective effects of a high AA intake may be dependent on the amount of EPA+DHA intake.
Article
Osteoporosis currently affects 10 million Americans and is responsible for more than 1.5 million fractures annually. The financial burden of osteoporosis is substantial, with annual direct medical costs estimated at 17 to 20 billion dollars. Most of these costs are related to the acute and rehabilitative care following osteoporotic fractures, particularly hip fractures. The societal burden of osteoporosis includes these direct medical costs and the monetary (eg, caregiver time) and nonmonetary costs of poor health. The aging of the US population is expected to increase the prevalence of osteoporosis and the number of osteoporotic fractures. Growth of the older adult population will pose significant challenges to Medicare and Medicaid, which bear most of the cost of osteoporosis. Efforts to address the looming financial burden must focus on reducing the prevalence of osteoporosis and the incidence of costly fragility fractures.
Article
We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.
Article
Glucocorticoids are important regulators of bone cell differentiation and mesenchymal lineage commitment. Using a cell-specific approach of osteoblast-targeted transgenic disruption of intracellular glucocorticoid signaling, we discovered a novel molecular pathway by which glucocorticoids, mainly through the mature osteoblast, regulate the cellular mechanisms that govern cranial skeleton development. Embryonic and neonatal transgenic mice revealed a distinct phenotype characterized by hypoplasia and osteopenia of the cranial skeleton; disorganized frontal, parietal and interparietal bones; increased suture patency; ectopic differentiation of cartilage in the sagittal suture; and disturbed postnatal removal of parietal cartilage. Concurrently, expression of Mmp14, an enzyme essential for calvarial cartilage removal, was markedly reduced in parietal bone and cartilage of transgenic animals. Expression of Wnt9a and Wnt10b was significantly reduced in osteoblasts with disrupted glucocorticoid signaling, and accumulation of beta-catenin, the upstream regulator of Mmp14 expression, was decreased in osteoblasts, chondrocytes and mesenchymal progenitors of transgenic mice. Supracalvarial injection of Wnt3a protein rescued the transgenic cranial phenotype. These results define novel roles for glucocorticoids in skeletal development and delineate how osteoblasts--under steroid hormone control--orchestrate the intricate process of intramembranous bone formation by directing mesenchymal cell commitment towards osteoblastic differentiation while simultaneously initiating and controlling cartilage dissolution in the postnatal mouse.
Article
Intermittent administration of parathyroid hormone (PTH) stimulates bone formation on the surface of cancellous and periosteal bone by increasing the number of osteoblasts. Previous studies of ours in mice demonstrated that intermittent PTH increases cancellous osteoblast number at least in part by attenuating osteoblast apoptosis, but the mechanism responsible for the anabolic effect of the hormone on periosteal bone is unknown. We report that daily injections of 100 ng/g of PTH(1-34) to 4-6 month old mice increased the number of osteoblasts on the periosteum of lumbar vertebrae by 2-3 fold as early as after 2 days. However, the prevalence of apoptotic periosteal osteoblasts was only 0.2% in vehicle treated animals, which is approximately 20-fold lower than is the case for cancellous osteoblasts. Moreover, PTH did not have a discernable effect on periosteal osteoblast apoptosis. Administration of BrdU for 4 days failed to label periosteal osteoblasts under either basal conditions or following administration of PTH. Cancellous osteoblasts, on the other hand, were labeled under basal conditions, but PTH did not increase the percentage of BrdU-positive cells. Thus, intermittent PTH does not increase cancellous or periosteal osteoblast number by stimulating the proliferation of osteoblast progenitors. Consistent with high turnover of cancellous osteoblasts as compared to that of periosteal osteoblasts, ganciclovir-induced ablation of replicating osteoblast progenitors in mice expressing thymidine kinase under the control of the 3.6 kb rat Col1A1 promoter resulted in disappearance of osteoblasts from cancellous bone over a 7-14 day period, whereas periosteal osteoblasts were unaffected. However, 14 days of pre-treatment with ganciclovir prevented PTH anabolism on periosteal bone. We conclude that in cancellous bone, attenuation of osteoblast apoptosis by PTH increases osteoblast number because their rate of apoptosis is high, making this effect of the hormone profound. However, in periosteal bone where the rate of osteoblast apoptosis is low, PTH must exert pro-differentiating and/or pro-survival effects on post-mitotic pre-osteoblasts. Targeting the latter cells is an effective mechanism for increasing osteoblast number in periosteal bone where the production of osteoblasts from replicating progenitors is slow.
Article
To examine the estrogenic and androgenic activity of Lepidium meyenii (Maca) and its effect on the hormonal profile and symptoms in postmenopausal women. Fourteen postmenopausal women completed a randomized, double-blind, placebo-controlled, crossover trial. They received 3.5 g/day of powered Maca for 6 weeks and matching placebo for 6 weeks, in either order, over a total of 12 weeks. At baseline and weeks 6 and 12 blood samples were collected for the measurement of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin, and the women completed the Greene Climacteric Scale to assess the severity of menopausal symptoms. In addition, aqueous and methanolic Maca extracts were tested for androgenic and estrogenic activity using a yeast-based hormone-dependent reporter assay. No differences were seen in serum concentrations of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin between baseline, Maca treatment, and placebo (P > 0.05). The Greene Climacteric Scale revealed a significant reduction in scores in the areas of psychological symptoms, including the subscales for anxiety and depression and sexual dysfunction after Maca consumption compared with both baseline and placebo (P < 0.05). These findings did not correlate with androgenic or alpha-estrogenic activity present in the Maca as no physiologically significant activity was observed in yeast-based assays employing up to 4 mg/mL Maca extract (equivalent to 200 mg/mL Maca). Preliminary findings show that Lepidium meyenii (Maca) (3.5 g/d) reduces psychological symptoms, including anxiety and depression, and lowers measures of sexual dysfunction in postmenopausal women independent of estrogenic and androgenic activity.
Article
We present an automatic method for docking organic ligands into protein binding sites. The method can be used in the design process of specific protein ligands. It combines an appropriate model of the physico-chemical properties of the docked molecules with efficient methods for sampling the conformational space of the ligand. If the ligand is flexible, it can adopt a large variety of different conformations. Each such minimum in conformational space presents a potential candidate for the conformation of the ligand in the complexed state. Our docking method samples the conformation space of the ligand on the basis of a discrete model and uses a tree-search technique for placing the ligand incrementally into the active site. For placing the first fragment of the ligand into the protein, we use hashing techniques adapted from computer vision. The incremental construction algorithm is based on a greedy strategy combined with efficient methods for overlap detection and for the search of new interactions. We present results on 19 complexes of which the binding geometry has been crystallographically determined. All considered ligands are docked in at most three minutes on a current workstation. The experimentally observed binding mode of the ligand is reproduced with 0.5 to 1.2 A rms deviation. It is almost always found among the highest-ranking conformations computed.
Article
Prediction of small molecule binding modes to macromolecules of known three-dimensional structure is a problem of paramount importance in rational drug design (the "docking" problem). We report the development and validation of the program GOLD (Genetic Optimisation for Ligand Docking). GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein, and satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Numerous enhancements and modifications have been applied to the original technique resulting in a substantial increase in the reliability and the applicability of the algorithm. The advanced algorithm has been tested on a dataset of 100 complexes extracted from the Brookhaven Protein DataBank. When used to dock the ligand back into the binding site, GOLD achieved a 71% success rate in identifying the experimental binding mode.
Article
In this paper we describe the search strategies developed for docking flexible molecules to macomolecular sites that are incorporated into the widely distributed DOCK software, version 4.0. The search strategies include incremental construction and random conformation search and utilize the existing Coulombic and Lennard-Jones grid-based scoring function. The incremental construction strategy is tested with a panel of 15 crystallographic testcases, created from 12 unique complexes whose ligands vary in size and flexibility. For all testcases, at least one docked position is generated within 2 A of the crystallographic position. For 7 of 15 testcases, the top scoring position is also within 2 A of the crystallographic position. The algorithm is fast enough to successfully dock a few testcases within seconds and most within 100 s. The incremental construction and the random search strategy are evaluated as database docking techniques with a database of 51 molecules docked to two of the crystallographic testcases. Incremental construction outperforms random search and is fast enough to reliably rank the database of compounds within 15 s per molecule on an SGI R10000 cpu.
Article
The tubers of Lepidium meyenii contain the benzylated derivative of 1,2-dihydro-N-hydroxypyridine, named macaridine, together with the benzylated alkamides (macamides), N-benzyl-5-oxo-6E,8E-octadecadienamide and N-benzylhexadecanamide, as well as the acyclic keto acid, 5-oxo-6E,8E-octadecadienoic acid. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR spectroscopic analyses, including 1H-1H COSY, 1H-13C HMQC, 1H-13C HMBC and 1H-1H NOESY experiments, as well as from 1H-15N NMR HMBC correlations for macaridine and N-benzylhexadecanamide.
Article
Despite decades of accumulated observational evidence, the balance of risks and benefits for hormone use in healthy postmenopausal women remains uncertain. To assess the major health benefits and risks of the most commonly used combined hormone preparation in the United States. Estrogen plus progestin component of the Women's Health Initiative, a randomized controlled primary prevention trial (planned duration, 8.5 years) in which 16608 postmenopausal women aged 50-79 years with an intact uterus at baseline were recruited by 40 US clinical centers in 1993-1998. Participants received conjugated equine estrogens, 0.625 mg/d, plus medroxyprogesterone acetate, 2.5 mg/d, in 1 tablet (n = 8506) or placebo (n = 8102). The primary outcome was coronary heart disease (CHD) (nonfatal myocardial infarction and CHD death), with invasive breast cancer as the primary adverse outcome. A global index summarizing the balance of risks and benefits included the 2 primary outcomes plus stroke, pulmonary embolism (PE), endometrial cancer, colorectal cancer, hip fracture, and death due to other causes. On May 31, 2002, after a mean of 5.2 years of follow-up, the data and safety monitoring board recommended stopping the trial of estrogen plus progestin vs placebo because the test statistic for invasive breast cancer exceeded the stopping boundary for this adverse effect and the global index statistic supported risks exceeding benefits. This report includes data on the major clinical outcomes through April 30, 2002. Estimated hazard ratios (HRs) (nominal 95% confidence intervals [CIs]) were as follows: CHD, 1.29 (1.02-1.63) with 286 cases; breast cancer, 1.26 (1.00-1.59) with 290 cases; stroke, 1.41 (1.07-1.85) with 212 cases; PE, 2.13 (1.39-3.25) with 101 cases; colorectal cancer, 0.63 (0.43-0.92) with 112 cases; endometrial cancer, 0.83 (0.47-1.47) with 47 cases; hip fracture, 0.66 (0.45-0.98) with 106 cases; and death due to other causes, 0.92 (0.74-1.14) with 331 cases. Corresponding HRs (nominal 95% CIs) for composite outcomes were 1.22 (1.09-1.36) for total cardiovascular disease (arterial and venous disease), 1.03 (0.90-1.17) for total cancer, 0.76 (0.69-0.85) for combined fractures, 0.98 (0.82-1.18) for total mortality, and 1.15 (1.03-1.28) for the global index. Absolute excess risks per 10 000 person-years attributable to estrogen plus progestin were 7 more CHD events, 8 more strokes, 8 more PEs, and 8 more invasive breast cancers, while absolute risk reductions per 10 000 person-years were 6 fewer colorectal cancers and 5 fewer hip fractures. The absolute excess risk of events included in the global index was 19 per 10 000 person-years. Overall health risks exceeded benefits from use of combined estrogen plus progestin for an average 5.2-year follow-up among healthy postmenopausal US women. All-cause mortality was not affected during the trial. The risk-benefit profile found in this trial is not consistent with the requirements for a viable intervention for primary prevention of chronic diseases, and the results indicate that this regimen should not be initiated or continued for primary prevention of CHD.
Article
Two new imidazole alkaloids (lepidiline A and lepidiline B) have been isolated from a root extract of Lepidium meyenii with the common name Maca and identified as 1,3-dibenzyl-4,5-dimethylimidazolium chloride (1) and 1,3-dibenzyl-2,4,5-trimethylimidazolium chloride (2), respectively. The structures of these two new compounds were determined by spectroscopic methods, as well as single-crystal X-ray diffraction performed on compound 1.
Article
Virtual screening uses computer-based methods to discover new ligands on the basis of biological structures. Although widely heralded in the 1970s and 1980s, the technique has since struggled to meet its initial promise, and drug discovery remains dominated by empirical screening. Recent successes in predicting new ligands and their receptor-bound structures, and better rates of ligand discovery compared to empirical screening, have re-ignited interest in virtual screening, which is now widely used in drug discovery, albeit on a more limited scale than empirical screening.
Article
Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappaB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10(-10) M) and high-dose (10(-7) M) 17beta-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT-PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10(-10) M (P < 0.05) and 10(-7) M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERalpha expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERbeta was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in osteoblasts.
Article
Bone remodeling, a coupled process involving bone resorption and formation, is initiated by mechanical signals and is controlled by local and systemic factors that regulate osteoblast and osteoclast differentiation and function. An excess of resorption over formation leads to the bone loss and increased propensity to fracture that is characteristic of osteoporosis. A newly described inhibitor of osteoblast differentiation, Ciz, interferes with bone morphogenic protein signaling. As a consequence, Ciz-deficient mice develop increased bone mass.
Article
Estrogen receptor (ER), which mediates the multiple effects of estrogen in brain, is regulated by several factors including its own ligand. In the present study, we have examined the effect of age, sex and gonadal steroids (estrogen and testosterone) on the level of ERalpha and ERbeta in the cerebral cortex of AKR mice. Adult and old mice of both sexes were divided into four groups: intact, gonadectomized, 17beta-estradiol treated and testosterone treated. Western blot analysis showed higher level of ERalpha and ERbeta in the cerebral cortex of adult female than male mice. ERbeta level decreased significantly with advancing age in both sexes, whereas 17beta-estradiol supplementation decreased ERalpha level in old male and increased in old female, it also increased ERbeta level in old male and adult female. On the other hand, testosterone treatment decreased ERalpha level significantly in old female and ERbeta level in adult female but increased ERbeta level in male mice of both ages. Thus, these findings showed that the expression of ERalpha and ERbeta protein is differentially influenced by age, sex and gonadal steroids in the mouse cerebral cortex, suggesting differences in ER-mediated brain functions.
Article
The aim of this study was to compare the efficiency of two well known approaches for the discovery of the bioactive principle/s in medicinal plants, namely the activity-guided isolation versus the computer-aided drug discovery by means of virtual screening (VS) techniques. Morus root bark of Morus sp. L. (Moraceae) was selected as application example for the discovery of compounds with anti-inflammatory activity. The two cyclooxygenase isoenzymes COX-1 and COX-2 were chosen as targets and the corresponding pharmacophore models were generated by our research. The activity-guided fractionation of the methanol extract of the root bark resulted in the isolation of nine compounds. Their structures were elucidated by mass spectrometry, 1- and 2-dimensional NMR experiments and identified as moracins B, M, the regioisomers O/P as a mixture, and sanggenons B, C, D, E and O. The COX-1 and COX-2 inhibiting activities of these compounds were established in an enzyme assay and compared with the predicted hits obtained from the VS. Sanggenons C, E, and O, that were tested the first time for an inhibitory effect on COX-1 and -2, showed IC50 values of 10 - 14 μM, and 40 - 50 μM, respectively. The results show that the COX activities obtained for the sanggenons are correctly predicted by the in silico filtering experiment. In the case of the isolated moracins, however, it failed because the COX inhibiting activities of moracins M and P/O were not retrieved by the VS. Structure-activity relationships of the isolated compounds are discussed as well as potential pitfalls and advantages of the applied strategies. Abbreviations COX:cyclooxygenase EIA:enzyme immunoassay HBA:hydrogen bond acceptor PGE:prostaglandin-E PGHS:prostaglandin-H synthase VS:virtual screening
Article
The macamides are a distinct class of secondary metabolites that have so far been found only in Lepidium meyenii Walp. (Maca). Using HPLC-UV-MS/MS, the main macamides have been identified as n-benzylhexadecanamide, n-benzyl-(9Z)-octadecenamide, n-benzyl-(9Z, 12Z)-octadecadienamide, n-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide and n-benzyloctadecanamide. The identities of n-benzyl-(9Z)-octadecenamide and n-benzyl-(9Z, 12Z)-octadecadienamide were confirmed by comparison of chromatographic and spectral properties with synthetic analogues. Total macamides have been quantified by HPLC-UV in plant material from different vendors using n-benzylhexadecanamide as an external standard. The amount of macamides in the dried plant material ranged from 0.0016 to 0.0123%.
Article
Maca (Lepidium meyenii Walp.) is a cruciferous plant from the Andes of Peru. The root of Maca is traditionally employed for its supposed properties in aphrodisiacs and improving fertility, it also has been widely used to help alleviate the symptoms of menopause. The purpose of this study was to evaluate the effect of ethanol extract of Maca on postmenopausal osteoporosis in ovariectomized rats. Female Sprague-Dawley rats were divided into four groups: Sham-operated and ovariectomized groups were fed with equivolume of distilled water, and the remaining ovariectomized groups were orally administrated with ethanol extract of Maca at 0.096 and 0.24 g/kg for 28 weeks. The findings derived from the basis of bone mineral density, biomechanical, biochemical and histopathological parameters indicated that higher dose of ethanol extract of Maca was effective in the prevention of estrogen deficient bone loss.
Article
In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption. Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology. We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage. Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen. Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.
Article
Estrogen actions are mainly mediated by estrogen receptor (ER)α and ERβ which in turn are regulated by several factors including age, sex and gonadal steroid hormones 17β-estradiol and testosterone. In the present study, we have used nuclear run-off assay to examine the effect of these factors on the rate of transcription of ERα and ERβ of mouse cerebral cortex. The run-off assay result was further corroborated with the measurement of steady state level of ERα and ERβ mRNA by semiquantitative RT-PCR method. Our results reveal that ERα transcription rate decreases in old mice of both sexes, whereas ERβ transcription rate decreases only in old females when compared to their adult counterparts. 17β-Estradiol supplementation reduces the transcription rate of ERα and ERβ in all groups except in adult male while testosterone treatment down regulates the transcription rate of ERα and ERβ in all groups. The semiquantitative RT-PCR analysis reveals that the level of ERα mRNA decreases in old male but shows no effect in old female as compared to adult counterpart. In contrast, ERβ transcript level decreases in old mice of both sexes. Furthermore, ERα mRNA level is higher in adult female than in adult male but no sex-dependent difference is seen in ERβ mRNA level. Supplementation of 17β-estradiol shows no significant alteration but testosterone reduces the ERα level in male mice, while 17β-estradiol and testosterone down regulate the ERα level in female mice of both ages. In case of ERβ, 17β-estradiol decreases the transcript level in all groups except adult male while testosterone treatment results in the down regulation of transcript level in all groups. Thus these findings suggest differential effects of age, sex, 17β-estradiol and testosterone supplementation on the transcription of mouse ER genes which may account for differences in the protein levels of ERα and ERβ and their functions in the brain.
Article
Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.