sphingomyelin synthase (SMS)(IC50 = 1.5~3 μM)

In this study, we studied the mechanism of the cytotoxicity of malabaricone C (mal C) against human breast cancer MCF-7 cell line. Mal C dose-dependently increased the sub G1 cell population, associated with cytoplasmic oligonucleosome formation and chromatin condensation. The mal C-induced apoptosis led to mitochondrial damage as revealed by fluorescence microscopy and flow cytometry of the JC-1-stained cells as well as from the release of mitochondrion-specific nuclease proteins AIF and endo G. Mal C also released intracellular Ca(2+) from the MCF-7 cells, but the Ca(2+)-modulators BAPTA-AM and Ru360 only partially abrogated the apoptosis. The calpain activation by mal C did not have any effect on its cytotoxicity. On the other hand, after mal C treatment significant lysosomal membrane permeabilization (LMP), along with release of cathepsin B, as well as Bid-cleavage and its translocation to mitochondria were observed much earlier than the mitochondrial damage. This suggested that cytotoxicity of mal C against human MCF-7 human breast cancer cell line may proceed through LMP as the initial event that triggered a caspase-independent, but cathepsin B and t-Bid-dependent intrinsic mitochondrial apoptotic pathway. A significant accumulation of cells in the S or G2-M phases along with upregulation of the cyclins E and A due to mal C exposure promises it to be a potential anti-cancer agent.[3]

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malabaricone C (mal C) inhibited T-cell activation, proliferation, and cytokine production. malabaricone C (mal C) suppressed mitogen-induced activation of cell surface markers, MAPK and NF-κB. malabaricone C (mal C) modulated cellular redox status in lymphocytes[2].

The interaction between natural occurring inhibitors and targeted membrane proteins could be an alternative medicinal strategy for the treatment of metabolic syndrome, notably, obesity. In this study, we identified malabaricones A-C and E (1-4) isolated from the fruits of Myristica cinnamomea King as natural inhibitors for sphingomyelin synthase (SMS), a membrane protein responsible for sphingolipid biosynthesis. Having the most promising inhibition, oral administration of compound 3/malabaricone C (mal C) exhibited multiple efficacies in reducing weight gain, improving glucose tolerance, and reducing hepatic steatosis in high fat diet-induced obesity mice models. Liver lipid analysis revealed a crucial link between the SMS activities of malabaricone C (mal C)compound 3 and its lipid metabolism in vitro and in vivo. The nontoxic nature of compound 3/malabaricone C (mal C) makes it a suitable candidate in search of drugs which can be employed in the treatment and prevention of obesity. [1]

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malabaricone C (mal C) administration inhibited T-cell activation ex vivo [2]
In order to ascertain the ability of malabaricone C (mal C) as immunosuppressant in vivo, mice were given intra-peritoneal injection of vehicle (DMSO) or Mal C, and lymphocytes were isolated after 24 h. Then, the cells were stained with CFSE and stimulated with Con A. Mal C administration significantly decreased the percentage of proliferating cells as compared with vehicle control (figure 4A and B). Mal C administration resulted in significant decrease in mitogen-induced cytokine (IL-2, IFN-γ, and IL-6) secretion by T-cells ex vivo.
To investigate the utility of malabaricone C (mal C) for prophylaxis of acute GvHD, the lymphocytes from C57BL/6 mice were treated with Mal C (10 μM) for 4 h and used as allogenic donor cells. The recipient BALB/c mice were rendered lymphopenic by exposure to 6 Gy dose of WBI. The donor cells were adoptively transferred by a lateral tail vein injection (10 million cells per mouse) and GvHD was monitored for morbidity and mortality of the hosts. The mice reconstituted with vehicle-treated allogenic cells (GvHD group) died within 30 days. However, Mal C treatment of donor cells completely suppressed GvHD-associated mortality and morbidity (figure 4D and E). GvHD-associated weight loss was evident on day 10 as compared with the unirradiated control, which was significantly prevented in mice reconstituted with Mal C-treated cells (figure 4E). Mice receiving WBI (6 Gy) exhibited significantly elevated IL6 levels as compared with the un-irradiated control (figure 4F). GvHD-associated IFN-γ levels were significantly higher than WBI 6Gy group. Further, serum levels of IL6 and IFN-γ were decreased in hosts reconstituted with Mal C-treated donor cells as compared with the hosts that received vehicle-treated donor cells (figure 4F). Figure 4G shows the effect of Mal C treatment on homeostatic proliferation of syngeneic CD4+ T-cells in lymphopenic hosts. Mal C treatment did not affect the homeostatic proliferation of CD4+ T-cells [2].

Surface staining and intracellular staining [2]
Cells were stained with fluorochrome (PE or FITC)-conjugated mAbs as previously described (Sharma et al. 2012). Briefly, lymphocytes (2.5×106) were incubated with malabaricone C (mal C) (10 μM, 2 h) followed by stimulation with Con A (2.5 μg/mL, 24 h for surface staining, and 2.5 μg/mL, 2 h for intracellular staining) and subsequent staining with fluorochrome-conjugated CD69 or CD25 monoclonal antibodies. Unstained and isotype-stained cells were used as controls. Then, cells were acquired on a BD FACS Melody flow cytometer and analyzed using FlowJo software.

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Estimation of ROS and GSH [2]
For ROS estimation based on DCF fluorescence (excitation/emission at 485/535 nm), malabaricone C (mal C)- or vehicle-treated cells were stained with H2DCF-DA and monitored using a spectrofluorimeter (Synergy H1 Hybrid Microplate Reader, BioTek). For quantitation of GSH (reduced/oxidized) levels, cells were treated with Mal C or vehicle control, harvested, and lysed, and the cell extract was probed for GSH quantitation as previously described (Patwardhan et al. 2015). For estimation of cellular thiols, cells were stained with MCB (excitation/emission at 390/478 nm), followed by flow cytometric acquisition.

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HPLC analysis of malabaricone C (mal C) and NAC interaction [2]
We employed a multi-step gradient mobile phase with acetonitrile as solvent A and aqueous formic acid (0.1%) as solvent B to carry out chromatographic separation using a C-18 column with an optimized method. The gradient was set as: 40% solvent A for 2 min; 80% solvent A for 22 min; 100% solvent A for 2 min; 40% solvent A for 4 min, and the flow rate was maintained at 1 mL/min. The sample volume for injection was 20 μL and the total run time was 30 min and Mal C was detected at a retention time (Rt) of 15.773 min. Detection was carried out using a diode array detector at 274 nm. A stock solution of NAC of 100 mM concentration was prepared and dilution was done in 1X PBS. Mal C was added to NAC (Mal C 100 μM + NAC 100 µM) and was incubated at 37°C for 1 h and run on HPLC (Dionex Ultima 3000 series HPLC system with Chromeleon software (version 6.8)).

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Spectroscopic analysis of malabaricone C (mal C) and NAC interaction [2]
Mal C (100 μM) was incubated with NAC (100 μm) in water at 37°C for 1 h, and absorbance spectra were recorded from 250 to 700 nm using a Jasco Spectrophotometer. A graph showing absorbance versus wavelength (nm) was plotted using Jasco Spectra Manager Ver. 2 software.

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Western blotting [2]
Cells (10×106/mL) treated with malabaricone C (mal C) or Con A (2.5 μg/mL) or both were harvested, and lysed using 1X RIPA lysis buffer consisting of 50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1.0 mM EDTA, 0.1% (w/v) SDS and 0.01% (w/v) sodium azide, protease inhibitor cocktail, and phosphatase inhibitor at a pH of 7.4. Protein estimation was carried out using the Bradford assay followed by electrophoretic separation on SDS-PAGE (10%). Proteins were transferred onto a PVDF membrane and probed for pERK, pJNK, ERK, and JNK using monoclonal antibodies followed by the HRP-conjugated secondary antibody. The bands were visualized in SynGene: GBox Gel documentation system using an enhanced chemiluminescence kit (POD).

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Gel shift assay for transcription factor [2]
Cells (10×106/mL) were treated with vehicle or indicated concentrations of Mal C or Con A (2.5 μg/mL) or both. Nuclear extracts were prepared from the cells and incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotides from the human immunodeficiency virus long terminal repeat (5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′) as previously described (Patwardhan et al. 2015, 2016). The sample was run on a native polyacrylamide gel followed by drying of the gel and its subsequent exposure to a phosphor image plate for visualizing radioactive bands using a PhosphorImage plate scanner.

Ex vivo stimulation of lymphocytes [2]
The high lipophilicity and low water solubility of malabaricone C (mal C) leads to poor oral bioavailability, and hence mice were administered with malabaricone C (mal C) (10 mg/kg body weight) or vehicle intra-peritoneally (3 mice per group) and were sacrificed 24 h after the injection. Lymphocytes were stimulated with Con A (2.5 μg/mL) for 24 h for estimation of secreted cytokines using ELISA. Another set of lymphocytes was stained with CFSE and stimulated with Con A (2.5 μg/mL) for 72 h for assessment of cell proliferation by flow cytometry.

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Graft-versus-host disease [2]
Recipient BALB/c mice were exposed to whole-body irradiation (WBI) of 6 Gy at a dose rate of 1 Gy/min in a blood irradiator for induction of lymphopenia (6 mice per group). The lymphocytes from allogenic C57BL/6 donors were treated with vehicle control or malabaricone C (mal C) in vitro, and 10 million cells were administered intravenously into the lateral tail vein of lymphopenic recipients 48 h after WBI. After engraftment, lymphopenic recipient mice were monitored for assessment of changes in the body weight and survival up to 30 days. On day 5 post engraftment, blood was collected from lymphopenic recipient mice and the serum was separated to monitor the cytokine levels using ELISA.

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Monitoring homeostatic proliferation in vivo [2]
CD4+ T-cells were sorted from lymphocytes of BALB/c mice using magnetic beads and were stained with CFSE. The cells were incubated with malabaricone C (mal C) (10 μM) or vehicle for 4 h at 37°C in 5% CO2. The cells were washed and 1 million CFSE+ CD4+ T-cells were intravenously engrafted into lymphopenic syngeneic BALB/c mice (3 mice per group). After 72 h, lymphocytes from lymphopenic recipient mice were isolated and the frequency and proliferation of donor CFSE+ cells were monitored by flow cytometry.

[1]. Malabaricone C as Natural Sphingomyelin Synthase Inhibitor against Diet-Induced Obesity and Its Lipid Metabolism in Mice. ACS Med Chem Lett. 2019;10(8):1154-1158.

[2]. Malabaricone C, a constituent of spice Myristica malabarica, exhibits anti-inflammatory effects via modulation of cellular redox. J Biosci. 2023;48(2):9.

[3]. Mechanism of the malabaricone C-induced toxicity to the MCF-7 cell line. Free Radic Res. 2014 Apr;48(4):466-77.

malabarine C（mal C） is a butanone. It has a role as a metabolite.
Malabaricone C has been reported in Myristicaceae, Myristica fragrans, and other organisms with data available.

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In summary, malabarine C（mal C）, an acylphenol isolated from the fruits of M. cinnamomea, has been identified as a lead natural sphingomyelin synthase inhibitor. Having the same mechanisms of action as the previously reported SMS knockout studies, malabaricone C was highly efficacious in preventing oleic acid uptake across the membrane, which in turn reduced lipid droplet formation in vitro.15 Malabaricone C was also found to be able to reduce body weight gain, improve glucose tolerance, and decrease lipid accumulation in the liver in vivo, thus making this the first report involving a plant derived SMS inhibitor against high fat diet-induced obesity. Its nontoxic nature makes malabaricone C a suitable candidate for its further development as a new drug or medicinal supplement to treat and prevent obesity. [1]

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T-cells are important mediators of adaptive immune responses. However, under specific pathological conditions like autoimmunity, allergy, chronic inflammation, COVID-19, and acute GvHD, there is a need to suppress T-cell activation (Maurice et al. 1997; Griffiths et al. 2011; Weyand et al. 2018). The past few decades have seen unprecedented research on the development of prophylactic as well as therapeutic agents to suppress undesirable T-cell activation in the clinic. Many researchers, including our group, have earlier highlighted the influence of cellular redox status and surrounding redox environment in the control of T-cell activation, proliferation, and differentiation (Checker et al. 2009; Kesarwani et al. 2013; Gambhir et al. 2014). Many redox active agents including antioxidants as well as pro-oxidants have been shown to modulate T-cell activation. Previously we demonstrated that pro-oxidants like plumbagin, 1,4-naphthoquinone, and menadione could inhibit T-cell activation at sub-micromolar concentrations through the modulation of GSH levels in cells (Checker et al. 2010, 2011). Antioxidants like GSH, NAC, chlorophyllin, baicalein, and vitamin C have been shown to modulate the activation and differentiation of T-cells (Sharma et al. 2007; Patwardhan et al. 2016). GSH dysregulation is also implicated in early experimental GvHD severity (Suh et al. 2014). Activation of T-cells is also dependent on the intrinsic generation of ROS in the mitochondria and metabolic reprogramming (Sena et al. 2013). Bombay mace, or false nutmeg, is a spice derived from the fruit aril of an Indian medicinal plant Myristica malabarica (Myristicaceae; Ayurvedic name, Rampatri) (Patro et al. 2005). Based on its chemical structure and the known biological properties of its active ingredient, we hypothesized that malabarine C（mal C） may affect T-cell activation. It was indeed found that malabarine C（mal C） treatment significantly suppressed the activation, proliferation, and cytokine secretion in murine T-cells. Further, Mal C treatment suppressed Con A-induced ROS generation. Here we observed that inhibition of T-cell activation was accompanied by a decrease in total ROS levels. Incubation of lymphocytes with Mal C resulted in significant depletion of reduced GSH levels. Hence, a question arises as to whether the efficacy of Mal C as an immune-modulatory agent is due to its antioxidant nature or due to its thiol-seeking behaviour. Interestingly, the anti-proliferative effects of Mal C were abrogated by thiol antioxidants, suggesting possible interaction of Mal C with cellular thiols. Indeed, biophysical studies revealed that Mal C could physically interact with NAC in an aqueous environment. Interestingly, NAC supplementation restored cellular thiol levels in Mal C-treated cells, which could be responsible for the abrogation of anti-proliferative effects of Mal C on T-cells in the presence of thiol antioxidants. These studies revealed that Mal C could possibly interact with cellular protein and non-protein thiols, thereby disrupting cellular redox. Cellular redox changes affect biological responses such as proliferation, differentiation, survival, and apoptosis through redox sensory signaling molecules (Chiu and Dawes 2012; Hancock and Whiteman 2018). MAP kinases can respond to cellular redox changes in terms of their phosphorylation status through the action of redox-sensitive phosphatases (Kamata and Hirata 1999; Seth and Rudolph 2006). T-cell receptor ligation induces distinct MAPK signaling that regulates T-cell responses (Adachi and Davis 2011). Mitogenic stimulation of T-cells leads to phosphorylation of MAP kinases, in turn leading to the activation of immune-regulatory transcription factor NF-κB. Mal C treatment suppressed phosphorylation of ERK and JNK and the subsequent DNA binding of NF-κB following mitogenic stimulation. Upon activation through signals delivered by the T-cell receptor and costimulatory molecules, T-cells upregulate the expression of CD25 (IL2Rα) and CD69 (c-type lectin). While CD25 expression determines IL2 responsiveness, CD69 activation stimulates an influx of calcium ions and the activation of extracellular kinases ERK1/2, thereby facilitating T-cell proliferation (Chen et al. 2020). Mal C-mediated inhibition of both early (CD69) as well as late activation marker (CD25) in T-cells indicates that Mal C interferes with the early activation of T-cells and renders them unresponsive to mitogenic stimulation. Based on these results, we hypothesized that transient exposure of T-cells to Mal C may be beneficial for prophylaxis of acute GvHD. We used a murine model of complete allogenic lymphocyte transplantation and found that Mal C completely abrogated acute GvHD-associated morbidity and mortality of the lymphopenic hosts. Mal C treatment of donor T-cells also resulted in significant reduction in GvHD-associated serum cytokines in the host. In vivo suppression of T-cell proliferation can also result in the disruption of immune homeostasis. In order to study the effect of Mal C on the behaviour of syngeneic T-cells, we evaluated the homeostatic proliferation of purified CD4+ T-cells in syngeneic lymphopenic hosts. Mal C did not inhibit the homeostatic proliferation of T-cells. Mal C treatment led to non-classical cellular redox perturbation by simultaneous scavenging of ROS and thiols. However, glutathione is essential for antigen-induced T-cell proliferation but not for homeostatic proliferation (Sena and Chandel 2012; Sena et al. 2013). Hence, it is anticipated that Mal C treatment did not affect the homeostatic proliferation of T-cells. [2]
The signaling requirements for antigen-specific proliferation of T-cells during acute GvHD are different from those involved in homeostatic proliferation in response to lymphopenia. Our results establish a differential and specific inhibitory action of malabarine C（mal C） on antigen/mitogen-induced proliferation but not on the homeostatic proliferation of T-cells. [2]
In summary, this is the first report showing inhibition of T-cell activation, proliferation, and cytokine production by malabarine C（mal C） through modulation of cellular redox balance (figure 5). Since Mal C suppressed only mitogen/alloantigen-induced proliferation but not homeostatic proliferation, it may be useful for prophylaxis of acute GvHD without disruption of the host immune reconstitution. The present results warrant further mechanistic studies to elucidate the molecular mechanism and biochemical targets of Mal C in T-cells.[2]

C21H26O5

358.42814

358.178

C, 70.37; H, 7.31; O, 22.32

63335-25-1

100313

White to off-white solid powder

119 - 121 °C

4.665

4

5

10

26

403

0

C1=CC(=C(C(=C1)O)C(=O)CCCCCCCCC2=CC(=C(C=C2)O)O)O

HCOZRFYGIFMIEX-UHFFFAOYSA-N

InChI=1S/C21H26O5/c22-16-13-12-15(14-20(16)26)8-5-3-1-2-4-6-9-17(23)21-18(24)10-7-11-19(21)25/h7,10-14,22,24-26H,1-6,8-9H2

1-(2,6-dihydroxyphenyl)-9-(3,4-dihydroxyphenyl)nonan-1-one

Malabaricone C; 63335-25-1; 1-(2,6-dihydroxyphenyl)-9-(3,4-dihydroxyphenyl)nonan-1-one; CHEBI:69015; C9K53R3PRN; DTXSID40212721; NSC 287968; NSC-287968;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~278.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.97 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)