GABase and glutaminase inhibitory activities of herbal extracts and acylated flavonol monoglycosides isolated from the leaves of Laurus nobilis L.

2.1 General experimental procedures

NMR spectra were recorded on a Bruker AVANCE™ III 600 (Karlsruhe, Germany) operating at 600 MHz for ¹H and 150 MHz for ¹³C NMR at room temperature in CD₃OD, or pyridine-d₅ using a 5 mm probe. Coupling constants were expressed in hertz, and chemical shifts were recorded in ppm (δ) with respect to the corresponding solvent as the internal standard. ESI-MS spectra were recorded using a Shimadzu LCMS-8040 mass spectrometer (Kyoto, Japan). ESI parameters were as follows: Nebulizer gas flow, 3.0 L/min (N₂); drying gas flow, 15.0 L/min (N₂); desolvation line temperature, 200 °C; and heat block temperature, 200 °C. GC-MS analysis was performed on a Shimadzu GCMS-QP2010 system. The capillary column was used an InertCap 5MS/Sil (0.25 mm i.d. × 30 m, film thickness: 0.5 μm, GL Sciences, Inc., Tokyo, Japan). The inlet temperature was set at 250 °C in the split mode (1:50). Helium was used as a carrier gas at a flow rate of 1.77 mL/min. The temperature program used for the analysis was as follows: the initial temperature at 40 °C, held for 3 min, and 16 °C/min up to 350 °C, held for 5 min. The temperature of the interface was set at 220 °C and the ion source was set at 250 °C. The mass spectrometer was operated in the electron ionization mode at 70 eV. Column chromatography was performed on 75–150 μm MCI-gel CHP20P (Mitsubishi Chemical Co. Ltd., Tokyo, Japan), 25–100 μm Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) and 40–63 μm silica gel 60 (Merck KGaA, Darmstadt, Germany).

Semipreparative HPLC was performed on a Jasco Gulliver series liquid chromatography system coupled with a PU-980 HPLC pump and UV-970 UV–visible detector (Jasco Co., Tokyo, Japan) using an InertSustain PFP column (10 mm i.d. × 250 mm, 5 μm, GL Sciences, Inc., Tokyo, Japan). The detection wavelength was 320 nm.

2.2 Chemicals

Dimethylsulfoxide (DMSO), dithiothreitol, DON, GABA, l-glutamine, HBA, α-ketoglutarate, Nessler’s reagent and tris (hydroxymethyl) aminomethane (Tris) were obtained from FUJIFILM Wako Pure Chemical Corporation, Ltd (Osaka, Japan). GABase (a mixture of GABA-aminotransferase and succinic semialdehyde dehydrogenase from Pseudomonas fluorescens) and glutaminase (l-glutamine amidohydrolase from Escherichia coli) were bought from Sigma-Aldrich Co. (St. Louis, USA). NADP⁺ was purchased from Oriental Yeast (Tokyo, Japan). HBA was used as a control of the assay for GABase inhibition since it had already been reported to show GABase inhibitory activity [2]. DON was used as a control of the assay for glutaminase inhibition since it had already been reported to show glutaminase inhibitory, neuroprotective, anticancer [5], anti-type 2 diabetes, and anti-obesity effects [9, 10].

2.3 Plant material and extraction

Twenty dried herbs were purchased through Jewel Colours Japan PT Lab. Co., Ltd (Tokyo, Japan). These plants are distributed throughout 14 families. The botanical names of the samples are listed in Table 1. The collected samples were identified by comparing the shape features of those with preserved samples by Prof. Masanori Inagaki. The voucher specimens of the tested herbs were deposited at the herbarium of Nakamura Gakuen University Junior College. 2 g of the air-dried herb was crushed and extracted with 70 % EtOH (20 mL × 2) for 3 weeks at room temperature to give each EtOH extract after evaporation under reduced pressure [13, 14]. The extracts were redissolved in 1 % DMSO at a concentration of 10 mg/mL prior to use in the individual assays.

2.4 Collection and identification of laurel

The laurel trees were planted in Fukuoka City (E130°36′ N33°57′; altitude 7 m), which has a temperate climate, generally warm with an average annual temperature of 17.1 °C and an average annual rainfall of 1774 mm. The trees were planted in well-drained soils under natural sunlight and were fed with humus and compost every summer and winter.

Fresh laurel leaves were harvested in October 2021. The collected samples were identified based on the leaf shape features and the GC-MS analysis of the main volatile constituents such as 1,8-cineole and α-terpinyl acetate of the essential oil [15] by Prof. Masanori Inagaki. A voucher specimen (NGAS202110) was deposited at the herbarium of Nakamura Gakuen University.

2.5 Extraction and isolation procedures

Fresh laurel leaves were air-dried at room temperature in the shade for 1 week. Air-dried leaves (196 g) were crushed and extracted with 70 % EtOH (1 L × 2 times) for 3 weeks at room temperature to give the EtOH extract (45.6 g) after evaporation under reduced pressure. The extract (67 % and 75 % inhibition of GABase and glutaminase at 10 mg/mL concentrations) was first fractionated on an MCI gel CHP 20P column chromatography (50 mm i.d. × 243 mm) with distilled water–MeOH (1: 0 v/v to 0: 1 v/v) to give four fractions. Fraction 3 (5.1 g; 23 % and 33 % inhibition of GABase and glutaminase at 1 mg/mL concentrations) eluted with MeOH was fractionated on a silica gel 60 column chromatography (32 mm i.d. × 302 mm) and eluted with the solvent system CHCl₃–MeOH (97: 3 v/v to 1: 4 v/v) to obtain 16 fractions. Sub-fraction 3–13 (306 mg; 43 % and 46 % inhibition of GABase and glutaminase at 1 mg/mL concentrations) eluted with 20 % MeOH was fractionated on a Sephadex LH-20 column chromatography (20 mm i.d. × 333 mm) and eluted with the solvent system CHCl₃–MeOH (95: 5 v/v to 0: 1 v/v) to obtain 10 fractions. Sub-fraction 3-13-8 (89 mg; 64 % and 54 % inhibition of GABase and glutaminase at 1 mg/mL concentrations) eluted with MeOH was further purified by semipreparative HPLC using 50 % AcCN to yield 1 (2.6 mg), 2 (6.4 mg) and 3 (12.2 mg).

2.6 Essential oil analysis

Fresh laurel leaves (20.5 g) were cut into small pieces and were steam distilled by a water steam distillation unit for 60 min, at 100 °C [14]. The essential oil (247 mg) was collected, and its volatile constituents were established by GC-MS analysis.

2.7 Spectral data of isolated compounds

Compound 1 [kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside]: ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) spectroscopic data see Supplementary Material Tables S1 and S2. ESI-MS (positive mode): m/z = 601.20 [M + Na]⁺, 579.20 [M + H]⁺.

Compound 2 [kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside]: ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) spectroscopic data see Supplementary Material Table S1 and S2. ESI-MS (positive mode): m/z = 747.45 [M + Na]⁺, 725.40 [M + H]⁺.

Compound 3 [kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside]: ¹H (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) spectroscopic data see Supplementary Material Tables S1 and S2. ESI-MS (positive mode): m/z = 747.45 [M + Na]⁺, 725.40 [M + H]⁺.

2.8 GABase inhibitory assay

GABase inhibitory assay was assayed according to the modified method of Tsukatani et al. [16]. The reaction mixture (1.0 mL) contained 0.1 mL of a sample solution in 5 % DMSO, 0.1 mL of enzyme solution [1.0 U/mL GABase in 80 mM Tris-HCl buffer (pH 9.0)], 0.2 mL of substrate solution (2 mM GABA in the same buffer) and 0.6 mL of the same buffer containing 750 mM sodium sulfate, 10 mM dithiothreitol, 1.4 mM NADP⁺ and 2.0 mM α-ketoglutarate, was incubated at 37 °C for 30 min. The formation of NADPH was measured as absorbance at 340 nm. The percentage inhibition of the GABase inhibitory activity was calculated according to the following equation: the inhibitory activity (%) = [(Control Abs—Control blank Abs)—(Sample Abs—Sample blank Abs)]/(Control Abs—Control blank Abs) × 100, where control is the activity of the enzyme with distilled water instead of sample solution and blank is the activity without the enzyme. HBA was used as a positive control [2].

2.9 Glutaminase inhibitory assay

Glutaminase inhibitory activity was assayed according to the modified method of Elshafei et al. [17]. The reaction mixture (0.5 mL) contained 0.1 mL of a sample solution in 5 % DMSO, 0.1 mL of 0.1 M acetate buffer (pH 4.9), 0.1 mL of enzyme solution (2.0 U/mL glutaminase in the same buffer) and 0.2 mL of substrate solution (0.04 mM l-glutamine in the same buffer), was incubated at 37 °C for 30 min. The reaction was stopped by adding 0.5 mL of 0.5 M H₂SO₄ solution. The precipitated protein was removed by centrifugation (3000 rpm, 10 min) and 0.2 mL of supernatant was added to 3.8 mL of distilled water. Thereafter, 0.5 mL of Nessler’s reagent was added, and the absorbance was measured at 420 nm within 10 min. The percentage inhibition of the glutaminase inhibitory activity was calculated according to the following equation: the inhibitory activity (%) = [(Control Abs—Control blank Abs)—(Sample Abs—Sample blank Abs)]/(Control Abs—Control blank Abs) × 100, where control is the activity of the enzyme with distilled water instead of sample solution and blank is the activity without the enzyme. DON was used a positive control [5].

2.10 Data analysis

All experiments were performed in triplicate and the data were expressed as means. The half-maximal inhibitory concentration (IC₅₀) values (mM) obtained from log dose inhibition curves are expressed as the mean ± SD (n = 3), since the IC₅₀ value (mM) is commonly used as a measure of the effect of a compound in inhibiting specific bioactivity. All statistical analyses were achieved using Microsoft 365 Excel (Microsoft Co. Redmond, WA, USA).

3.1 GABase and glutaminase inhibitory activities of herbal extracts

As shown in Table 1, laurel extract showed higher GABase inhibitory activity compared to the other tested herbal extracts and three extracts from laurel, Filipendula ulmaria and Cinnamomum verum had high glutaminase inhibitory activities.

3.2 Acylated flavonol monoglycosides from laurel and their biological activities

The extract from laurel leaves was purified by column chromatography and semipreparative HPLC to afford three compounds. Compounds 1–3 were considered to be kaempferol-3-O-rhamnopyranosides mono or di-substituted with p-coumaroyl groups according to the NMR spectroscopic data (see Supplementary Material Tables S1 and S2) with identical quasi-molecular ions at m/z: 579.20 [M + H]⁺ or 725.40 [M + H]⁺ in the ESI MS spectra. 2D NMR experiments showed clear coupling connections around the rhamnopyranoside ring and the p-coumaroyl groups. The E-configurations of the p-coumaroyl groups and α-linkage of the rhamnose were assigned from the ¹H NMR coupling constants. In the ¹H NMR spectra of 1–3, down-field shifts were observed at H-4″ in 1, H-3″ and H-4″ in 2 and H″-2 and H-4″ in 3, compared with those published data on kaempferol-3-O-α-l-rhamnopyranoside [17]. Hence, three compounds were identified as kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside (1), kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (2) and kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (3) [18, 19] on the basis of the spectroscopic data in agreement with the published data (Figure 1). Compounds 1–3, acylated flavonol monoglycosides, are secondary plant metabolites widespread in herbs and spices [20], but there is no information regarding GABase and glutaminase inhibitory activities of 1–3.

Figure 1:

Chemical structures of 1–3.

Compounds 1–3, obtained by bioassay-guided fractionation, were evaluated for their inhibitory effects against GABase and glutaminase (see Supplementary Material Table S3). The IC₅₀ values of GABase inhibitory activity of 1–3 and HBA as control were 0.24 mM, 0.14 mM, 0.12 mM, and 0.43 mM, respectively. The GABase inhibitory activities of 1–3 showed stronger than that of HBA. Additionally, the IC₅₀ values of glutaminase inhibitory activity of 1–3 and DON as control were 0.34 mM, 0.13 mM, 0.14 mM, and 0.33 mM, respectively. The glutaminase inhibitory activities of 2 and 3 exhibited stronger than that of DON. Consequently, 1–3 might be able to become lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases, since 1–3 showed potent GABase and glutaminase inhibitory activities. This is the first report on GABase and glutaminase inhibitory activities of 1–3.