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 compound with the function of a metabolite. Malabarine C has been reported to exist in plants of the Myristaceae family, nutmeg, and other organisms with relevant data. In summary, the acylphenol compound Malabarine C (mal C), isolated from the fruit of cinnamon nutmeg (M. cinnamomea), has been identified as a natural sphingomyelin synthase inhibitor. The mechanism of action of Malabarine C is the same as that of previously reported SMS gene knockout studies, which can effectively prevent the transmembrane absorption of oleic acid, thereby reducing the formation of lipid droplets in vitro. ¹⁵ Malabarine C has also been found to reduce weight gain, improve glucose tolerance and reduce lipid accumulation in the liver, thus this is the first reported case of a plant-derived SMS inhibitor against high-fat diet-induced obesity. The non-toxic nature of Malabarine C makes it an ideal candidate for further development of new drugs or health products for the treatment and prevention of obesity. [1]

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T cells are important mediators of adaptive immune responses. However, under specific pathological conditions such as autoimmunity, allergy, chronic inflammation, COVID-19, and acute graft-versus-host disease (GvHD), suppression of T cell activation is necessary (Maurice et al., 1997; Griffiths et al., 2011; Weyand et al., 2018). Over the past few decades, unprecedented research has been achieved in developing prophylactic and therapeutic agents to suppress clinically unwanted T cell activation. Many researchers, including our group, have previously emphasized the regulatory role of cellular redox state and the surrounding redox environment on T cell activation, proliferation, and differentiation (Checker et al., 2009; Kesarwani et al., 2013; Gambhir et al., 2014). Many redox-active substances, including antioxidants and pro-oxidants, have been shown to modulate T cell activation. We have previously demonstrated that pro-oxidants such as dextrin, 1,4-naphthoquinone, and menadione can inhibit T cell activation at sub-micromolar concentrations by regulating intracellular glutathione (GSH) levels (Checker et al., 2010, 2011). Antioxidants, such as GSH, N-acetylcysteine (NAC), chlorophyll, baicalin, and vitamin C, have been shown to regulate T cell activation and differentiation (Sharma et al., 2007; Patwardhan et al., 2016). GSH dysregulation has also been associated with the severity of early experimental graft-versus-host disease (GvHD) (Suh et al., 2014). T cell activation also depends on the intrinsic generation and metabolic reprogramming of reactive oxygen species (ROS) in mitochondria (Sena et al., 2013). Bombay nutmeg, also known as false nutmeg, is a spice extracted from the aril of the fruit of the Indian medicinal plant Myristica malabarica (Myristaceae family; Ayurvedic name: Rampatri) (Patro et al. 2005). Based on its chemical structure and the known biological properties of its active ingredients, we hypothesize that Malabarine C (mal C) may affect T cell activation. Studies have found that treatment with Malabalin C (mal C) significantly inhibited the activation, proliferation, and cytokine secretion of mouse T cells. Furthermore, Mal C treatment suppressed Con A-induced reactive oxygen species (ROS) production. We observed that the inhibition of T cell activation was accompanied by a decrease in total ROS levels. Incubation of lymphocytes with Mal C resulted in a significant decrease in reduced glutathione (GSH) levels. Therefore, a question arises: does the efficacy of Mal C as an immunomodulator stem from its antioxidant properties or its thiol-binding capacity? Interestingly, thiol antioxidants can eliminate the antiproliferative effect of Mal C, suggesting that Mal C may interact with cellular thiols. In fact, biophysical studies have shown that Mal C can physically interact with NAC in an aqueous environment. Even more interestingly, NAC supplementation can restore thiol levels in Mal C-treated cells, which may explain why the antiproliferative effect of Mal C on T cells is eliminated in the presence of thiol antioxidants. These studies suggest that Mal C may interact with cellular protein and non-protein thiols, thereby disrupting cellular redox balance. Cellular redox changes influence biological responses such as proliferation, differentiation, survival, and apoptosis through redox-sensing signaling molecules (Chiu and Dawes 2012; Hancock and Whiteman 2018). MAP kinases can alter their phosphorylation status in response to changes in cellular redox state through the action of redox-sensitive phosphatases (Kamata and Hirata 1999; Seth and Rudolph 2006). T cell receptor ligand binding can induce different MAPK signaling pathways, thereby regulating T cell responses (Adachi and Davis 2011). T cell mitogen stimulation leads to MAP kinase phosphorylation, which in turn activates the immunomodulatory transcription factor NF-κB. Mal C treatment inhibits the phosphorylation of ERK and JNK and subsequent DNA binding of NF-κB after mitogen stimulation. Following activation by signals transmitted by T cell receptors and co-stimulatory molecules, T cells upregulate the expression of CD25 (IL2Rα) and CD69 (C-type lectin). CD25 expression determines IL-2 responsiveness, while CD69 activation stimulates calcium influx and activation of extracellular kinases ERK1/2, thereby promoting T cell proliferation (Chen et al. 2020). Inhibition of Mal C-mediated early T cell activation markers (CD69) and late activation markers (CD25) indicates that Mal C interferes with early T cell activation, rendering them unresponsive to mitogen stimulation. Based on these results, we hypothesized that transient T cell exposure to Mal C may be beneficial in preventing acute graft-versus-host disease (GvHD). Using a mouse model of complete allogeneic lymphocyte transplantation, we found that Mal C completely eliminated acute GvHD-related morbidity and mortality in lymphopened hosts. Mal C treatment of donor T cells also significantly reduced the levels of GvHD-related cytokines in host serum. In vivo T cell proliferation inhibition can also lead to immune homeostasis dysregulation. To investigate the effects of Mal C on syngeneic T cell behavior, we evaluated the homeostatic proliferation of purified CD4+ T cells from syngeneic lymphopened hosts. Mal C did not inhibit T cell homeostatic proliferation. Mal C treatment leads to non-classical cellular redox dysregulation by simultaneously scavenging reactive oxygen species (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). Therefore, Mal C treatment is not expected to affect T cell homeostatic proliferation. [2]
The signaling requirements for T cell antigen-specific proliferation during acute graft-versus-host disease (GvHD) are different from those for homeostatic proliferation in lymphopenia. Our results show that malbalin C (mal C) has a differential and specific inhibitory effect on T cell antigen/mitogen-induced proliferation, but no inhibitory effect on T cell homeostatic proliferation. [2] In summary, this is the first study to report that malbalin C (mal C) inhibits T cell activation, proliferation and cytokine production by regulating cellular redox balance (Fig. 5). Since Mal C only inhibits mitogen/allogeneic antigen-induced proliferation and not homeostatic proliferation, it may help prevent acute graft-versus-host disease (GvHD) without disrupting the host's immune reconstitution. Current findings suggest that further mechanistic studies are needed to elucidate the molecular mechanisms 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.)