RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

Effect of nutmeg essential oil (Myristica fragrans Houtt.) on methane production, rumen fermentation, and nutrient digestibility in vitro.

The study evaluated the effect of adding of nutmeg (Myristica fragrans Houtt.) essential oil (NEO) as a feed additive on methane production, rumen fermentation parameters, rumen enzyme activity, and nutrient digestibility in vitro. This study was divided into three treatments based on the level of NEO addition, which included 0 µL/L (T0), 100 µL/L (T1), and 200 µL/L (T2). The feed substrate composition consisted of king grass as forage and concentrate in a 60:40 ratio. Feed fermentation was conducted using the Menke and Steingass gas production and two-step Tilley and Terry in-vitro digestibility technique. The data obtained from the study were analyzed using one-way ANOVA and if there were differences between means, they were further assessed using DMRT. The results showed that T2 treatment significantly decreased (P < 0.05) ammonia (NH3) levels, total VFA, acetate, propionate, butyrate, and microbial protein (P < 0.05). Methane production and the activity of rumen protease enzyme significantly decreased (P < 0.05) at T1 and T2 treatment. The T2 treatment significantly reduced (P < 0.05) protein digestibility (IVCPD) at 48 h, while IVCPD at 96 h significantly increased (P < 0.05). On the other hand, the addition of nutmeg essential oil did not effect the activity of the amylase, carboxymethyl cellulase, and ß-glucosidase enzymes, as well as the in-vitro digestibility of dry matter (IVDMD), crude fiber (IVCFD), and organic matter (IVOMD). The conclusion drawn from this study is that the optimum level for NEO is 200 µL/L, which can reduce methane production and increase crude protein digestibility at 96 h without any negative effect on rumen fermentation and nutrient digestibility.

Malabaricone C, a constituent of spice Myristica malabarica, exhibits anti-inflammatory effects via modulation of cellular redox.

The present study primarily focuses on the efficacy of Malabaricone C (Mal C) as an anti-inflammatory agent. Mal C inhibited mitogen-induced T-cell proliferation and cytokine secretion. Mal C significantly reduced cellular thiols in lymphocytes. N-acetyl cysteine (NAC) restored cellular thiol levels and abrogated Mal C-mediated inhibition of T-cell proliferation and cytokine secretion. Physical interaction between Mal C and NAC was evinced from HPLC and spectral analysis. Mal C treatment significantly inhibited concanavalin A-induced phosphorylation of ERK/JNK and DNA binding of NF-κB. Administration of Mal C to mice suppressed T-cell proliferation and effector functions ex vivo. Mal C treatment did not alter the homeostatic proliferation of T-cells in vivo but completely abrogated acute graft-versus-host disease (GvHD)-associated morbidity and mortality. Our studies indicate probable use of Mal C for prophylaxis and treatment of immunological disorders caused due to hyper-activation of T-cells.

Inhibitory and antioxidative capacity of nutmeg extracts on reduction of lipid oxidation and heterocyclic amines in pan-roasted beef patties.

Identification and inhibition of mutagenic and carcinogenic heterocyclic amines (HCAs) from pan-roasted beef patties were performed by adding (0.02%) tertiary butyl hydroquinone (TBHQ) and (0.05%) ethanol-extracted nutmeg (ENE) using HPLC and principal component analysis. Ten HCAs, including six polar and four non-polar, were assessed. The addition of (0.05%) ENE significantly (P < 0.05) reduced the cooking loss and shrinkage of patties during cooking and reduced the total formation HCAs by 73.97%, which proved the significant (P < 0.05) inhibitory effect as a natural antioxidant against lipid oxidation and HCA formation compared to TBHQ. The DPPH radical-scavenging activity, total phenolic content, and available active metabolites of ENE were estimated. Furthermore, a positive correlation was observed between pH, level of thiobarbituric acid reactive substances, and HCA formation in both the groups. TBHQ and ENE were significant HCAs inhibitors (P < 0.001), but ENE showed resilient oxidative stability during refrigeration storage. Therefore, ENE can be used to reduce HCAs formation in pan-roasted beef patties.

Hepatoprotective effect of nanoniosome loaded Myristica fragrans phenolic compounds in mice-induced hepatotoxicity.

In this study, nanoniosome-loaded Myristica fragrans' (MF) phenolic compounds (NLMP) were synthesized and characterized for their physical properties, and hepatoprotective effects on mice with liver toxicity induced by L-asparaginase (LA) injection. According to the results, NLMP has a spherical shape with a 263 nm diameter, a zeta potential of -26.55 mV and a polydispersity index (PDI) of 0.192. The weight and feed intake of mice induced with hepatotoxicity were significantly (p ≤ 0.05) increased after they were treated with NLMP (2.5 mg/kg body weight of mice). In addition, the blood levels of triglyceride (TG), cholesterol (Chol), liver enzymes (aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP)) and total bilirubin were significantly (p ≤ 0.05) decreased. A significant increase (p ≤ 0.05) in the blood levels of the antioxidant defence system (glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT)) were also reported after NLMP treatment. NLMP was also led to a significant decrease (p ≤ 0.05) in inflammatory-related gene expression of inducible nitric oxide synthase (iNOS) and Interferon-gamma (IFN-γ) in the liver, as well as a meaningful (p ≤ 0.05) increase in the expression of SOD as an antioxidant status biomarker. Consequently, the NLMP is recommended as a potential dietary supplement to alleviate the symptoms of LA-induced hepatotoxicity.

Malabaricone C derived from nutmeg inhibits arachidonate 5-lipoxygenase activity and ameliorates psoriasis-like skin inflammation in mice.

As pro-inflammatory lipid mediators, leukotrienes have pathophysiological activities in several inflammatory diseases, including psoriasis. In the biosynthesis of leukotrienes from arachidonic acid, 5-lipoxygenase catalyzes the first two steps. In the present study, we showed that nutmeg (Myristica fragrans) strongly inhibited the catalytic activity of 5-lipoxygenase. To characterize the bioactive component(s) of nutmeg, we performed 5-lipoxygenase inhibitory activity-guided fractionation of aqueous ethanol extract of nutmeg, resulting in the isolation of malabaricone C having antioxidant activity. Malabaricone C exhibited potent competitive inhibition of 5-lipoxygenase with an IC50 value of 0.2 µM. In mice with imiquimod-induced psoriasis-like skin lesions, topical application of 2 mM malabaricone C significantly ameliorated hyperplasia and inflammatory cell infiltration, and suppressed the expression of the psoriasis-associated genes S100a9, Krt1, Il17a, and Il22. Lipid metabolome analysis of these psoriasis-like skin lesions showed that malabaricone C markedly decreased the level of leukotriene B4 but did not significantly increase the other pro-inflammatory lipid mediators. These findings suggest that malabaricone C decreases LTB4 by the 5-lipoxygenase inhibition and ameliorates the symptoms of psoriasis-like skin inflammation.

Mechanisms and Safety of Antidepressant-Like Effect of Nutmeg in Mice.

Nutmeg, a dried seed kernel of a tall evergreen Myristicaceae tree, is widely used as a spice and herbal medicine and is known to have antidepressant-like effects. This study evaluates the mechanisms underlying this antidepressant-like effect and safety of nutmeg n-hexane extract (NNE) in mice. Tail suspension and open field tests showed that NNE (10 mg/kg, per OS (p.o.)) significantly decreased the immobility time of mice without effecting their spontaneous locomotor activity. The reduction of immobility time of mice elicited by NNE was significantly inhibited by ketanserin (5-hydroxytryptamine (5-HT)2A/2C receptor antagonist), ondansetron (5-HT3 receptor antagonist), and yohimbine (α2 receptor antagonist). WAY100635 (5-HT1A receptor antagonist) tended to inhibit the effect of NNE but without significance. Testing according to the Organisation for Economic Co-operation and Development Guidelines, no mice died due to administrated NNE (2000 mg/kg, p.o.), and behavioral and weight changes were not seen in the acute toxicity test. In the Ames test, no increase in the number of revertant colonies for each bacterial strain test strains TA98 and TA100 by nutmeg powder was observed either with or without metabolic activity by S9 mix. These results suggest that NNE shows an antidepressant-like effect involving various serotonergic and noradrenergic nervous systems and maybe a highly safe natural preparation.

Myristica fragrans bio-active ester functionalized ZnO nanoparticles exhibit antibacterial and antibiofilm activities in clinical isolates.

We provide a novel one-step/one-pot bio-inspired method of synthesis for Myristica fragrans leaf ester (MFLE) capped­zinc oxide nanoparticles (MFLE-ZnONPs). Antibacterial and antbiofilm efficacies of MFLE-ZnONPs were tested against the multi-drug resistant (MDR) Escherichia coli (E. coli-336), methicillin-resistant Staphylococcus aureus (MRSA-1) and methicillin-sensitive (MSSA-2) clinical isolates. Antibacterial screening using well diffusion assay revealed the cytotoxicity of MFLE-ZnONPs in the range of 500-2000 µg/ml. MFLE-ZnONPs significantly increased the zone of growth inhibition of E. coli-336 (17.0 ±â€¯0.5 to 19.25 ±â€¯1.0 mm), MSSA-2 (16.75 ±â€¯0.8 to 19.0 ±â€¯0.7 mm) and MRSA-1 (16.25 ±â€¯1.0 to 18.25 ±â€¯0.5 mm), respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) against E. coli-336, MRSA-1 and MSSA-2 were found to be 1500, 1000 and 500 µg/ml, and 2500, 2000 and 1500 µg/ml, respectively. A time and dose dependent reduction in the cell proliferation were also found at the respective MICs of tested strains. Scanning electron microscopy (SEM) of MFLE-ZnONPs-treated strains exhibited cellular damage via loss of native rod and coccoid shapes because of the formation of pits and cavities. E. coli-336 and MRSA-1 strains at their MICs (1500 and 1000 µg/ml) sharply reduced the biofilm production to 51% and 24%. The physico-chemical characterization via x-ray diffraction (XRD) ascertained the crystallinity and an average size of MFLE-ZnONPs as 48.32 ±â€¯2.5 nm. Gas chromatography-mass spectroscopy (GC-MS) analysis of MFLE-ZnONPs unravelled the involvement of two bio-active esters (1) butyl 3-oxobut-2-yl ester and (2) α-monoolein) as surface capping/stabilizing agents. Fourier transform infrared (FTIR) analysis of MFLE and MFLE-ZnONPs showed the association of amines, alkanes, aldehydes, amides, carbonyl and amines functional groups in the corona formation. Overall, our data provide novel insights on the rapid development of eco-friendly, cost-effective bio-synthesis of MFLE-ZnONPs, showing their putative application as nano-antibiotics against MDR clinical isolates.

Degranulation inhibitors from the arils of Myristica fragrans in antigen-stimulated rat basophilic leukemia cells.

A methanol extract of mace, the aril of Myristica fragrans (Myristicaceae), was found to inhibit the release of ß-hexosaminidase, a marker of antigen-IgE-stimulated degranulation in rat basophilic leukemia cells (RBL-2H3, IC50 = 45.7 µg/ml). From the extract, three new 8-O-4' type neolignans, maceneolignans I-K (1-3), were isolated, and the stereostructures of 1-3 were elucidated based on spectroscopic and chemical evidence. Among the isolates, maceneolignans A (5), D (6), and H (8), (-)-(8R)-∆8'-4-hydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan (13), (-)-(8R)-∆8'-3,4,5,3',5'-pentamethoxy-8-O-4'-neolignan (14), (-)-erythro-(7R,8S)-∆8'-7-acetoxy-3,4-methylenedioxy-3',5'-dimethoxy-8-O-4'-neolignan (17), (+)-licarin A (20), nectandrin B (24), verrucosin (25), and malabaricone C (29) were investigated as possible degranulation inhibitors (IC50 = 20.7-63.7 µM). These inhibitory activities were more potent than those of the antiallergic agents tranilast (282 µM) and ketotifen fumalate (158 µM). Compounds 5, 25, and 29 also inhibited antigen-stimulated tumor necrosis factor-α production (IC50 = 39.5-51.2 µM), an important process in the late phase of type I allergic reactions.

Sialidase inhibitory activity of diarylnonanoid and neolignan compounds extracted from the seeds of Myristica fragrans.

Sialidases are key virulence factors that remove sialic acid from host cell surface glycans, thus unmasking receptors to facilitate bacterial adherence and colonization. In this study, we report the isolation and characterization of novel inhibitors of the Streptococcus pneumoniae sialidases NanA, NanB, and NanC from Myristica fragrans seeds. Of the isolated compounds (1-12), malabaricone C showed the most pneumococcal sialidases inhibition (IC50 of 0.3µM for NanA, 3.6µM for NanB, and 2.9µM for NanC). These results suggested that malabaricone C and neolignans could be potential agents for combating S. pneumoniae infection agents.

Indirect modulation of the endocannabinoid system by specific fractions of nutmeg total extract.

CONTEXT: Nutmeg [Myristica fragrans Houtt. (Myristicaceae)] has a long-standing reputation of psychoactivity. Anecdotal reports of nutmeg use as a cheap marijuana substitute, coupled to previous studies reporting a cannabimimetic-like action, suggest that nutmeg may interact with the endocannabinoid system. OBJECTIVE: The study evaluates nutmeg fractions for binding capacity with various CNS receptors and their potential interaction with the endocannabinoid system. MATERIALS AND METHODS: Dichloromethane (DF) and ethyl acetate (EF) fractions were prepared from the methanol extract of powdered whole nutmeg. The HPLC-profiled fractions were assayed by the NIMH Psychoactive Drug Screening Program (PDSP) in a panel of CNS targets at a 10 µg/mL concentration. The fractions were also screened for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition, initially at a concentration of 500 µg/mL, then by concentration-dependent inhibition studies. RESULTS: None of the tested fractions showed significant binding to CNS receptors included in the PDSP panel. However, both fractions exerted significant inhibition of the FAAH and MAGL enzymes. The DF fraction inhibited FAAH and MAGL enzymes at IC50 values of 21.06 ± 3.16 and 15.34 ± 1.61 µg/mL, respectively. Similarly, the EF fraction demonstrated FAAH and MAGL inhibition with IC50 values of 15.42 ± 3.09 and 11.37 ± 6.15 µg/mL, respectively. DISCUSSION AND CONCLUSION: The study provides the first piece of evidence that nutmeg interacts with the endocannabinoid system via inhibition of the endocannabinoid catabolizing enzymes. This mechanism provides insight into reported cannabis-like action as well as expands the potential therapeutic utility of nutmeg.

Radioprotection of Swiss albino mice by Myristica fragrans houtt.

Nutmeg, the dried seed kernel of Myristica fragrans, MF (Family: Myristicaceae) possesses antifungal, hepatoprotective and antioxidant properties. Its radioprotective effect against 6, 8 and 10 Gy gamma radiation was evaluated by 30 day survival assay. Regression analysis yielded LD(50/30 )as 6.83 Gy and 8.89 Gy for irradiated only and (MF + radiation) groups, respectively. The dose reduction factor was computed as 1.3. Administration of MF significantly enhanced hepatic glutathione (GSH) and decreased testicular lipid peroxidation (LPO) level whereas acid phosphatase (ACP) and alkaline phosphatase (ALP) activity did not show any significant alteration. Irradiation resulted in significant elevation in LPO level and ACP activity, and decreased the GSH content and ALP activity. MF pretreatment effectively protected against radiation induced biochemical alteration as reflected by a decrease in LPO level and ACP activity, and an increase in GSH and ALP activity. The present study has implications for the potential use of MF as a radioprotector.