The Efficient Synthesis and Anti-Fatigue Activity Evaluation of Macamides: The Unique Bioactive Compounds in Maca.

Macamides are a class of amide alkaloids that are only found in maca and are widely considered to be its bioactive marker compounds. More than thirty macamide monomers have been identified in recent years; however, it is difficult to obtain a single macamide monomer from the maca plant because of their similar structures and characteristics. We used the carbodiimide condensation method (CCM) to efficiently synthesize five typical macamides, including N-benzyl-hexadecanamide (NBH), N-benzyl-9Z,12Z,15Z-octadecenamide, N-(3-methoxybenzyl)-9Z,12Z-octadecenamide, N-benzyl-9Z,12Z-octadecenamide, and N-(3-methoxybenzyl)-9Z,12Z,15Z-octadecadienamide. All the synthesized macamides were purified by a one-step HPLC with a purity of more than 95%. NBH is the most abundant macamide monomer in natural maca, and it was selected to evaluate the anti-fatigue effects of macamides. The results indicated that NBH could enhance the endurance capacity of mice by increasing liver glycogen levels and decreasing blood urea nitrogen, lactate dehydrogenase, blood ammonia, and blood lactic acid levels. Macamides might be the active substances that give maca its anti-fatigue active function.

Safety evaluation and protective effects of ethanolic extract from maca (Lepidium meyenii Walp.) against corticosterone and H2O2 induced neurotoxicity.

Maca has been traditionally used to enhance sexual behavior and fertility. Recently, maca's neuroprotective effects have been reported. The purpose of this study was to investigate whether the ethanol extract of maca (EEM) (100 mg/kg/bw, 200 mg/kg/bw, 400 mg/kg/bw, p.o.) exerted neuroprotective effects in corticosterone (CORT)-induced (40 mg/kg/bw, s.c.) rats, to determine the neuroprotective effects of EEM (12.5, 25, 50 µg/ml) and macamides in H2O2-induced (50 µM) PC12 cells. The acute toxicity (2000 mg/kg/bw, p.o.) and subacute toxicity (200 mg/kg/bw, 500 mg/kg/bw, 1000 mg/kg/bw, p.o.) of EEM were evaluated by mouse models. EEM reversed CORT-induced abnormal behaviors, reduced the contents of TNF-α, IL-6 in hippocampi, and increased the positive cells of doublecortin (DCX), bromodeoxyuridine (BrdU) and DCX + BrdU in the hippocampus of rats. Moreover, EEM and 4 macamides remarkably increased the cell viability in H2O2-induced PC12 cells. EEM promoted the phosphorylation of IκBα and p65, suppressed the NF-κB activation, and inhibited the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and their mRNA levels in H2O2-induced PC12 cells. In conclusion, EEM could exert neuroprotective effects in CORT-induced rats and in H2O2-induced PC12 cells. Moreover, EEM did not present relevant toxicity after exposure to single and repeated doses.

[Quality evaluation method of Lepidium meyenii using UPLC-UV-Q-TOF-MS].

In order to evaluate the quality of different varieties of Maca(Lepidium meyenii), the main chemical components in Maca were investigated and a method for simultaneous determination of the main chemical components in Maca was established. UPLC-UV-Q-TOF-MS technology and reference materials were used to identify the structures of 19 main components in Maca. Seven compounds with UV absorption and high contents were selected to establish a simultaneous concentration determination method. The method was employed with a Waters Acquity I-Class~(TM) liquid chromatographic system coupled with a PDA detector and a Waters Acquity Cortecs C_(18)~+ column(2.1 mm×100 mm, 1.6 µm), and acetonitrile-0.2% phosphoric acid water was used as mobile phase(0.45 mL·min~(-1)). The detection wavelength was 195 nm and the column temperature was maintained at 40 ℃. There was efficient separation of seven compounds, p-hydroxybenzylglucosinolate, benzylglucosinolate, N-benzyl-9Z,12Z,15Z-octadecatrienamid, N-benzyl-9Z,12Z-octadecadienamide, N-(3-methoxybenzyl)-hexadecanamide, N-benzyl-hexadecanamide, and N-benzyl-9Z-octadecenamide. The stan-dard calibration curves were good(R~2>0.999). The precision, stability and repeatability were also good. The linearity ranges were 0.197-4.980 µg·mL~(-1) to 193.67-796.8 µg·mL~(-1), and the average recovery rate was 96.71%-103.9%. The average concentration of glucosinolates and macamides in Maca were 1.20% and 0.20%, respectively. Among four kinds of Maca grown in China, the concentration of glucosinolates in yellow Maca and black Maca were relatively high(1.55%), followed by white Maca(0.93%), and purple Maca(0.76%). The concentration of macamides in yellow, purple and white Maca was similar(0.23%-0.29%), however black Maca had significantly lower concentration(0.15%). Peru Maca tested in this study had the lowest concentration of these compounds. This qua-lity evaluation method was fast, accurate, and comprehensively reflects the concentration of the main chemical components in Maca, which provides a useful reference for the quality control and evaluation of Maca.

Neuroprotective activity of macamides on manganese-induced mitochondrial disruption in U-87 MG glioblastoma cells.

Macamides are a distinct class of secondary metabolites, benzylamides of long chain fatty acids, which were isolated from the Peruvian plant Lepidium meyenii (Maca). As structural analogues of the endocannabinoid anandamide (AEA), they have demonstrated neuroprotective effects in vitro and in vivo. The purpose of this study was to demonstrate the neuroprotective activity of the macamides: N-(3-methoxybenzyl)oleamide (MAC 18:1), N-(3-methoxybenzyl)linoleamide (MAC 18:2) and N-(3-methoxybenzyl)linolenamide (MAC 18:3) in a neurotoxic environment caused by exposure of U-87 MG glioblastoma cells to manganese chloride (MnCl2). The neuroprotective effects of these macamides were reversed by the CB1 antagonist AM251. The mechanism by which manganese (Mn) induces cell damage was investigated by studying its effects on mitochondria. Reactive oxygen species (ROS) increase intracellular calcium and enhance the opening of mitochondrial permeability transition pores (MPTP), which leads to decreased mitochondrial membrane potential (MMP), to disruption of mitochondria and to neuron death in neurodegenerative disorders. In this study, MnCl2 at 50µM was responsible for mitochondrial disruption, which was attenuated by all three of the macamides tested. Human peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to be a cannabinoid target, and PPARγ has also been demonstrated to mediate some of the longer-term vascular effects of the plant cannabinoid, ∆9-tetrahydrocannabinol. PPARγ activation was observed in response to exposures of cells to MAC 18:2 and MAC 18:3. These findings suggest that macamides achieve their neuroprotective effects by binding to CB1 receptors to protect against Mn-induced toxicity in U-87 MG glioblastoma cells. Additionally these macamides, in a manner similar to the analogous endocannabinoid AEA, interact with other targets such as PPARγ to regulate metabolism and energy homeostasis, cell differentiation and inflammation.

Optimization of Ultrasound-Assisted Extraction, HPLC and UHPLC-ESI-Q-TOF-MS/MS Analysis of Main Macamides and Macaenes from Maca (Cultivars of Lepidium meyenii Walp).

Ultrasound-assisted extraction (UAE), using petroleum ether as the solvent, was systematically applied to extract main macamides and macaenes from Maca hypocotyls. Extraction yield was related with four variables, including ratio of solution to solid, extraction temperature, extraction time, and extraction power. On the basis of response surface methodology (RSM), the optimal conditions were determined to be the ratio of solution to solid as 10:1 (mL/g), the extraction temperature of 40 °C, the extraction time of 30 min, and the extraction power of 200 W. Based on the optimal extraction method of UAE, the total contents of ten main macamides and two main macaenes of Maca cultivated in twenty different areas of Tibet were analyzed by HPLC and UHPLC-ESI-Q-TOF-MS/MS. This study indicated that UAE was able to effectively extract macamides alkaloids from Maca hypocotyls. Quantitative analysis showed that geographical origins, not ecotypes, played a more important role on the accumulation of active macamides in Maca.

Characteristic fingerprinting based on macamides for discrimination of maca (Lepidium meyenii) by LC/MS/MS and multivariate statistical analysis.

BACKGROUND: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS). RESULTS: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification. CONCLUSION: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry.

Exploring the chemical and pharmacological variability of Lepidium meyenii: a comprehensive review of the effects of maca.

Maca (Lepidium meyenii), a biennial herbaceous plant indigenous to the Andes Mountains, has a rich history of traditional use for its purported health benefits. Maca's chemical composition varies due to ecotypes, growth conditions, and post-harvest processing, contributing to its intricate phytochemical profile, including, macamides, macaenes, and glucosinolates, among other components. This review provides an in-depth revision and analysis of Maca's diverse bioactive metabolites, focusing on the pharmacological properties registered in pre-clinical and clinical studies. Maca is generally safe, with rare adverse effects, supported by preclinical studies revealing low toxicity and good human tolerance. Preclinical investigations highlight the benefits attributed to Maca compounds, including neuroprotection, anti-inflammatory properties, immunoregulation, and antioxidant effects. Maca has also shown potential for enhancing fertility, combating fatigue, and exhibiting potential antitumor properties. Maca's versatility extends to metabolic regulation, gastrointestinal health, cardio protection, antihypertensive activity, photoprotection, muscle growth, hepatoprotection, proangiogenic effects, antithrombotic properties, and antiallergic activity. Clinical studies, primarily focused on sexual health, indicate improved sexual desire, erectile function, and subjective wellbeing in men. Maca also shows promise in alleviating menopausal symptoms in women and enhancing physical performance. Further research is essential to uncover the mechanisms and clinical applications of Maca's unique bioactive metabolites, solidifying its place as a subject of growing scientific interest.

Deep Eutectic Solvent-Based Ultrasound-Assisted Strategy for Simultaneous Extraction of Five Macamides from Lepidium meyenii Walp and In Vitro Bioactivities.

This study aimed to develop an integrated approach of deep eutectic solvent-based ultrasound-assisted extraction (DES-UAE) to simultaneously extract five major bioactive macamides from the roots of Lepidium meyenii Walp. Ten different DESs containing choline chloride and selected hydrogen-bond donors were prepared and evaluated based on the extracted macamide content determination using high-performance liquid chromatography (HPLC). Choline chloride/1,6-hexanediol in a 1:2 molar ratio with 20% water exhibited the most promising extraction efficiencies under the optimized parameters verified using single-factor optimization as well as Box-Behnken design. Using the optimized DES-UAE method, the extraction efficiencies of the five macamides were up to 40.3% higher compared to those using the most favorable organic solvent petroleum ether and were also superior to those of the other extraction methods, such as heating and combination of heating and stirring. Furthermore, using the macroporous resin HPD-100, the recoveries of the five target macamides from the DES extraction reached 85.62-92.25%. The 20 µg/mL group of the five macamide extracts showed superior neuroprotective activity against PC12 cell injury than that of the positive drug nimodipine. The macamide extracts also showed higher NO inhibition in LPS-stimulated RAW264.7 cells. Thus, the developed approach was a green and potential alternative that can be used to extract bioactive macamide constituents from L. meyenii in the pharmaceutical and food industries.

Macamide B suppresses lung cancer progression potentially via the ATM signaling pathway.

Macamides are a class of bioactive natural products obtained from Lepidium meyenii (maca), which have been reported to exert inhibitory activity in cancer. However, their role in lung cancer is currently unknown. In the present study, macamide B was shown to inhibit the proliferation and invasion of lung cancer cells, as determined by Cell Counting Kit-8 and Transwell assays, respectively. By contrast, macamide B induced cell apoptosis, as determined by Annexin V-FITC assay. Moreover, combined treatment with macamide B and olaparib, an inhibitor of poly (ADP-ribose) polymerase, further suppressed the proliferation of lung cancer cells. At the molecular level, the expression of ataxia-telangiectasia mutated (ATM), RAD51, p53 and cleaved caspase-3 were significantly increased by macamide B, as determined by western blotting, whereas the expression levels of Bcl-2 were decreased. By contrast, when ATM expression was knocked down by small interfering RNA technology in A549 cells treated with macamide B, the expression levels of ATM, RAD51, p53 and cleaved caspase-3 were reduced, whereas those of Bcl-2 were increased. Consistently, cell proliferation and invasive ability were partially rescued by ATM knockdown. In conclusion, macamide B inhibits lung cancer progression by inhibiting cell proliferation and invasion, and inducing apoptosis. Furthermore, macamide B may participate in regulating the ATM signaling pathway. The present study provides a potential new natural drug for treating patients with lung cancer.

Anti-glycative and anti-inflammatory effects of macamides isolated from Tropaeolum tuberosum in skin cells.

Tropaeolum tuberosum, commonly known as Mashua, is an herbal remedy used on the skin in order to treat local pain and to heal wounds. This study aimed to evaluate the extracts and isolated compounds from T. tuberosum with anti-glycative and anti-inflammatory activities. Guided isolation by bioassay led to the isolation and characterisation by NMR and MS of (S)-(-)-N-(α-methylbenzyl)-oleamide (1) and (S)-(-)-N-(α-methylbenzyl)-linoleamide (2). Both compounds inhibited the production of TNF-α with IC50 values of 9.38 µM (NIH/3T3 cells) and 10.06 µM (PA317 cells) for compound 1, and 5.3 µM (NIH/3T3 cells) and 6.48 µM (PA317 cells) for compound 2. Compounds 1 and 2 showed the inhibitory effect on the BSA-MGO formation at concentrations of 9.38 µM (3.39%) and 5.30 µM (8.53%), respectively. Moreover, both compounds showed significant breaking properties on the MGO-AGE-protein crosslink with percent modification of 6.58% (9.38 µM) and 18.08% (5.30 µM), respectively.

Combination of a multiplatform metabolite profiling approach and chemometrics as a powerful strategy to identify bioactive metabolites in Lepidium meyenii (Peruvian maca).

Lepidium meyenii is an edible plant that has been used as a nutritional supplement worldwide due to its medicinal properties. However, most of the studies have focused on the pharmacological activities of the extracts rather than their chemical composition. Herein, we used a combination of a multiplatform metabolite profiling approach and chemometrics to identify bioactive metabolites in L. meyenii. Extracts obtained with ethyl acetate and ethanol showed the promising antioxidant, anti-glioma and antibacterial activities. Sixty metabolites were identified by HPLC-MS, whereas fifteen were identified by GC-MS. Partial least squares discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA), and Variable Importance in Projection (VIP) successfully discriminated extracts obtained in different organic solvents from in natura dry roots and commercial product samples of L. meyenii. Additionally, correlation analysis allowed us to pinpoint potential candidates responsible for each biological activity tested for the extracts, which could be extrapolate for other food-related species.

Antioxidant and antitumoral activities of isolated macamide and macaene fractions from Lepidium meyenii (Maca).

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.

Postharvest Ultrasound-Assisted Freeze-Thaw Pretreatment Improves the Drying Efficiency, Physicochemical Properties, and Macamide Biosynthesis of Maca (Lepidium meyenii).

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.

Is the hype around the reproductive health claims of maca (Lepidium meyenii Walp.) justified?

ETHNOPHARMACOLOGICAL RELEVANCE: Maca - Lepidium meyenii Walp. has been cultivated and used by Andean people for over 1300-2000 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.

Bioactive maca (Lepidium meyenii) alkamides are a result of traditional Andean postharvest drying practices.

Maca, Lepidium meyenii Walpers (Brassicaceae), is an annual herbaceous plant native to the high plateaus of the Peruvian central Andes. Its underground storage hypocotyls have been a traditional medicinal agent and dietary staple since pre-Columbian times. Reported properties include energizing and fertility-enhancing effects. Published reports have focused on the benzylalkamides (macamides) present in dry hypocotyls as one of the main bioactive components. Macamides are secondary amides formed by benzylamine and a fatty acid moiety, with varying hydrocarbon chain lengths and degree of unsaturation. Although it has been assumed that they are usually present in fresh undamaged tissues, analyses show them to be essentially absent from them. However, hypocotyls dried by traditional Andean postharvest practices or industrial oven drying contain up to 800µgg(-1) dry wt (2.3µmolg(-1) dry wt) of macamides. In this study, the generation of macamides and their putative precursors were studied during nine-week traditional drying trials at 4200m altitude and in ovens under laboratory conditions. Freeze-thaw cycles in the open field during drying result in tissue maceration and release of free fatty acids from storage and membrane lipids up to levels of 1200µgg(-1) dry wt (4.3µmolg(-1) dry wt). Endogenous metabolism of the isothiocyanates generated from glucosinolate hydrolysis during drying results in maximal benzylamine values of 4300µgg(-1) dry wt (40.2µmolg(-1) dry wt). Pearson correlation coefficients of the accumulation profiles of benzylamine and free fatty acid to that of macamides showed good values of 0.898 and 0.934, respectively, suggesting that both provide sufficient substrate for amide synthesis during the drying process.