Glutaminase inhibitory activity of umbelliferone isolated from kabosu (Citrus sphaerocarpa Hort. ex Tanaka)

Abstract Kabosu (Citrus sphaerocarpa Hort. ex Tanaka) fruits have pleasant and fresh odors and have been used as raw materials for vinegar, seasonings, jams, marmalades and juices in Japan. The n-butanol extracts from kabosu fruits were prepared and a component in the extract was purified by column chromatography and HPLC to afford compounds 1-3. Three compounds, 5-(hydroxymethyl)-2-furaldehyde (1), umbelliferone (2) and oxypeucedanin hydrate (3), have been isolated from kabosu, and the structures of compounds 1-3 were elucidated by 1 D and 2 D NMR as well as EI-MS. Compound 2 exhibited potent glutaminase inhibitory activity with an IC50 value of 1.33 mM. This is the first report on glutaminase inhibitory activity of 2 and the isolation of three compounds 1-3 from kabosu fruits.


Introduction
L-Glutamate is the major excitatory neurotransmission in the mammalian central nervous system (CNS) and is responsible for normal brain function such as cognition, memory, movement and sensation (Sugiyama et al. 2017). Since high glutamateexposure triggers neuronal death and is responsible for chronic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Huntington's disease and Alzheimer's disease, it is important to lower extracellular glutamate concentration to below neurotoxic levels (Tanaka 2005). Glutaminase plays a critical role in the generation of glutamate and is thought to play a central role in the generation of excitotoxic glutamate in neuroinflammatory CNS disorders (Thomas et al. 2014). Glutaminase inhibitors could be valuable for the prevention of neurodegenerative diseases, but there are few known potent and selective glutaminase inhibitors available. Although 6-diazo-5-oxo-L-norleucine (DON), a glutaminase inhibitor, was developed, it could not be widely applied due to their non-specific selectivity and poor solubility (Thomas et al. 2014). On the other hand, glutaminase is responsible for glutaminolysis, which is a process harnessed by cancer cells to feed their accelerated growth and proliferation in many malignant tumors. Thus, inhibition of glutaminase reduced proliferation of cancer cells, since glutaminase knockdown suppressed the Wnt/b-catenin signaling pathway which played a key role in the regulation of tumor progression (Zhang et al. 2019). It has been widely accepted that cancer cells favoured glutamine as a source of energy (Thangavelu et al. 2014). Consequently, glutaminase has also become an attractive target for anti-cancer therapy due to its crucial role of glutamine metabolism in cancer cell growth (Sun et al. 2018). In addition, the use of plant extracts and phytochemicals can contribute to reduce the intake of pharmaceutical drugs and side effects. Moreover, balanced diet could prevent lifestyle related diseases and neurodegenerative diseases. Therefore, we need to seek new glutaminase inhibitors from the daily intake of food.
Citrus fruits are rich sources of various health-promoting substances and have been used for the treatment of lifestyle related diseases such as diabetes mellitus, dyslipidemia and hypertension. (Kawai et al. 1999). Kabosu (Citrus sphaerocarpa Hort. ex Tanaka) fruits have pleasant and fresh odors and have been used as raw materials for vinegar, seasonings, jams, marmalades and juices in Japan. This fruit has been known as healthy food (Shimada 2016) ), but there is little information on the psychotropic and anti-cancer activities of kabosu fruit. Our investigation of the metabolites of this fruit has now led to the isolation of umbelliferone as a glutaminase inhibitor. Here, we report the isolation, structural identification of three compounds from kabosu fruits and glutaminase inhibitory activity of umbelliferone.

Results and discussion
The n-butanol extracts from kabosu fruits inhibited glutaminase activity by 45% at a concentration of 10 mg/mL. This activity prompted investigation of its chemical constituents in order to isolate the corresponding bioactive constituents. A component in the extract was purified by MCI gel CHP 20P and silica gel column chromatography and HPLC to afford compounds 1-3. Three compounds were identified on the basis of spectroscopic data, especially using 1 D and 2 D NMR, and comparison of the data with literature values as 5-(hydroxymethyl)-2-furaldehyde (1) (Li et al. 2009), umbelliferone (2) (Iqbal et al. 2009) and oxypeucedanin hydrate (3) (Harkar et al. 1984), respectively (Figure 1).
Compound 1 is previously identified from Asparagus officinalis L and exhibits antioxidant and antimyocardial ischemia effects (Ito et al. 2013). Compound 2 is a benzopyrone in nature and widely found in many plants such as grapefruit, lime, bergamot and bitter orange (Mercolini et al. 2013). Compound 2 exhibits antioxidant activity (Salem et al. 2013), anti-inflammatory effects (Kabeya et al. 2013) and cytotoxic activities against HCT 116 and HT-29 cancer cell lines (Salem et al. 2013). Compound 3 is previously identified from roots of Angelica dahurica and exhibits antioxidant activity (Bai et al. 2016). However, there is no information regarding glutaminase inhibitory activities of compounds 1-3.
Compounds 1-3, DON and quercetin used as controls were examined for their effects on glutaminase. Compound 2 exhibited potent glutaminase inhibitory activity with an IC 50 value of 1.33 mM (Table S1). Although the glutaminase inhibitory activity of 2 was weaker than that of DON, compounds 1, 3 and quercetin did not show any inhibitory activity against glutaminase. Those results suggested that the phenolic hydroxy group and a-pyrone ring in the molecule of 2 played an important role in the inhibitory activity and might bind to the hydroxy group of Ser74 at the active center of glutaminase but the hydrogen bond between 2 and Ser74 might be weaker than the formation of the covalent bond between DON and the Ser74 hydroxyl oxygen (Brown et al. 2008). Consequently, the daily intake of kabosu fruits might play an important role in the prevention of chronic neurodegenerative diseases and anti-cancer therapy because biological activities of 2 had already been reported to exhibit antidepressant-like effect (Tingting et al. 2017) and antitumor activity (Wang et al. 2019) in addition to showing glutaminase inhibitory activity. This is the first report on glutaminase inhibitory activity of 2 and the isolation of compounds 1-3 from kabosu fruits.

Conclusions
The n-butanol extracts from kabosu fruits were purified by column chromatography and HPLC to afford three compounds. Those structures were established on the basis of 1 D and 2 D NMR spectroscopic data as 5-(hydroxymethyl)-2-furaldehyde (1), umbelliferone (2) and oxypeucedanin hydrate (3). Compound 2 exhibited potent glutaminase inhibitory activity with an IC 50 value of 1.33 mM. In addition, the biological activities of polysaccharides from kabosu fruits had already been reported to inhibit angiogenesis and breast cancer cell migration (Park et al. 2016). This is the first report on glutaminase inhibitory activity of 2 and the isolation of three compounds 1-3 from kabosu fruits.