Estimation of the Bioactivity of Seven Aromatic Compounds Isolated from Niihime Fruit by Simultaneously Monitoring Superoxide Generation and Intracellular Calcium Ion Levels in Neutrophils

Abstract

This study attempted to isolate bioactive compounds from niihime, a sour Citrus fruit, and to evaluate their bioactivity by simultaneously monitoring superoxide generation and intracellular calcium ion levels in neutrophils. Seven aromatic compounds were isolated from a methanol extract of niihime peel and identified as 6-demethoxynobiletin, 6-demethoxytangeretin, nobiletin, tangeretin, sinensetin, 5,7-dimethoxycoumarin, and saccharic acid 1,4-lactone 3,5-di-O-ferulate. 6-Demethoxynobiletin, 6-demethoxytangeretin, and 5,7-dimethoxycoumarin were newly shown to be contained in niihime fruit. Bioactivity assay of 10 µM saccharic acid 1,4-lactone 3,5-di-O-ferulate revealed a significant increase in both superoxide generation and intracellular calcium ion concentration (p<0.05). This result indicates that saccharic acid 1,4-lactone 3,5-di-O-ferulate has immunostimulatory activity in neutrophils.

Introduction

Niihime is a sour Citrus fruit first discovered in Atashika town, Kumano city, Mie Prefecture, Japan and registered for seeds and seedlings in 1997. It is produced in the area along the coast of the Sea of Kumano (Miyake, 2006; Ichinokiyama et al., 2012). The juice is widely used for the acidic seasoning of cold beverages, distilled alcoholic liquor, cooked fish, confectionery and so on. The dry powder of niihime peel is also used as an ingredient in processed foodstuffs.

Foods have biological regulatory functions in the body, including in the immune, endocrine, nervous, circulatory, and digestive systems, as the ‘tertiary’ function of foods. Functional foods, defined as those that have the potential to reduce the risk of lifestyle-related diseases and associated abnormal modalities, have garnered global interest (Arai et al., 2008). Various foods have been investigated for the bioactive properties of their compounds (Abuajah et al., 2015). Aromatic compounds such as flavonoids, phenylpropanoids, and coumarin derivatives in Citrus fruit have been reported to display antioxidative activity, antitumor activity, and so on (Yu et al., 2005; González-Molina, et al., 2010). For niihime, the characteristic flavonoids of the fruit have been studied for the existence of flavanone glycosides (eriocitrin, narirutin, hesperidin, didymin) and polymethoxyflavones (nobiletin, sinensetin, tangeretin) (Miyake, 2006; Ichinokiyama et al., 2012). The flavanone glycosides were reported to show antioxidative activity in vitro and in vivo (Miyake et al., 1998; González-Molina et al., 2010). Saccharic acid 1,4-lactone 3,5-di-O-ferulate, a phenylpropanoid, was isolated from niihime, and was shown to have antioxidative activity using an ORAC (oxygen radical absorbance capacity) assay in vitro (Miyake et al., 2014). However, it is suggested that the result of in vitro methods for evaluating antioxidative activity may not precisely reflect in vivo conditions in the human body. To address this issue, an in vivo-like method capable of easily and rapidly evaluating such activity has been investigated and devised. A conventional assay method was reported, which involved the simultaneous monitoring of superoxide and intracellular calcium ion levels in neutrophils by chemiluminescence measurement (Ishibashi et al., 2006). Bioactive compounds could be evaluated for antioxidative activity, anti-inflammatory activity, and immunostimulatory activity using the assay (Yang et al., 2006; Kazumura et al., 2013; Kazumura, 2015). In the present study, bioactive compounds in niihime fruit were assessed. Furthermore, the bioactivity of compounds was assayed by simultaneous monitoring of superoxide generation and intracellular calcium ion levels in neutrophils.

Materials and Methods

Materials and reagents    Niihime fruit was obtained from the Kumano City Home Promotion Public Corporation (Kumano, Mie, Japan). Standard compounds of nobiletin, tangeretin, sinensetin, and 5,7-dimethoxycoumarin were obtained from Funakoshi Co., Ltd., Tokyo, Japan. Saccharic acid 1,4-lactone 3,5-di-O-ferulate was isolated according to the reported method (Miyake et al., 2014). Fluo-3 AM was obtained from Dojindo Laboratories (Kumamoto, Japan). MCLA (2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo [1,2]-a pyrazin-3-one hydrochloride) was obtained from Tokyo Kasei (Tokyo, Japan). fMLP (N-formyl-methionyl-leucyl-phenylalanine) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical or HPLC grade (Wako Pure Chemical Industries, Osaka, Japan). HL-60 (human acute promyelocytic leukemia cell line) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Isolation of compounds from niihime peel    Niihime peel (500 g) was soaked in methanol (2 L) for 4 days at room temperature. The obtained extract was concentrated under reduced pressure and applied to a reversed-phase column (ø37 × 500 mm, Amberlite XAD-2 resin; Rohm and Haas Co., Philadelphia, PA, USA). The column was washed with 1.5 L water, eluted with 1.5 L of 50% methanol, and then eluted continuously with 1.5 L of 100% methanol. The two fractions eluted using 50% methanol and 100% methanol were concentrated under reduced pressure. Compound 1 (7.4 mg), compound 2 (3.8 mg), nobiletin (30.5 mg), tangeretin (12.4 mg), sinensetin (14.9 mg), and 5,7-dimethoxycoumarin (0.9 mg) were isolated from the 100% methanol fraction by preparative HPLC (900 series; JASCO, Tokyo, Japan), using a YMC-ODS column (YMC-Pack ODS-A, ø20 × 250 mm, S-5 µm; YMC Co., Kyoto, Japan), UV detection at 280 nm, mobile solvents of 70% methanol and 30% water, and a flow rate of 8 mL/min at room temperature. Saccharic acid 1,4-lactone 3,5-di-O-ferulate (61.1 mg) was isolated from the 50% methanol fraction by preparative HPLC according to the previously reported method (Miyake et al., 2014).

Identification of compounds    Nobiletin, tangeretin, sinensetin, 5,7-dimethoxycoumarin, and saccharic acid 1,4-lactone 3,5-di-O-ferulate were identified by their retention times from HPLC analysis in reference to standard substances. 6-Demethoxynobiletin (compound 1) and 6-demethoxytangeretin (compound 2) were identified by ¹H-NMR analysis. The ¹H-NMR spectrum was recorded on a JNM-ECP-500 (JEOL) instrument (500 MHz for ¹H, Jeol, Tokyo, Japan). 6-Demethoxynobiletin (1): ¹H-NMR (CDCl₃) δ: 7.59 (1H, dd, J=8.7, 2.3 Hz, H-6′), 7.42 (1H, d, J=2.3 Hz, H-2′), 6.99 (1H, d, J=8.7 Hz, H-5′), 6.62 (1H, s, H-3), 6.44 (1H, s, H-6), 4.01 (3H, s, OMe), 3.99 (3H, s, OMe), 3.98 (3H, s, OMe), 3.96 (3H×2, s, OMe). 6-Demethoxytangeretin (2): ¹H-NMR (CDCl₃) δ: 7.89 (2H, d, J=8.7 Hz, H-2′, 6′), 7.02 (2H, d, J=8.7 Hz, H-3′, 5′), 6.60 (1H, s, H-3), 6.44 (1H, s, H-6), 4.01 (3H, s, OMe), 3.99 (3H, s, OMe), 3.96 (3H, s, OMe), 3.89 (3H, s, OMe).

Quantification of compounds in the juice and peel of niihime    The niihime fruit was cut in half and hand-squeezed to obtain the peel and juice. The peel was lyophilized and pulverized to a powder. The powder (0.1 g) was extracted with a solution (5 mL, methanol and DMSO (dimethyl sulfoxide), 1:1, v/v) by ultrasonic treatment for 60 min and left at room temperature overnight. The pulp of the juice was removed by centrifugation at 20,627 g for 5 min. The seven aromatic compounds in the peel extract and juice were determined by HPLC, using a YMC-ODS column (YMC-Pack ø4.6 × 250 mm, S-5 µm; YMC Co.) at a flow rate of 1 mL/min and a column temperature of 40°C. The compounds (6-demethoxynobiletin, 6-demethoxytangeretin, nobiletin, tangeretin, sinensetin, 5,7-dimethoxycoumarin) were analyzed with UV detection at 320 nm, mobile solvents of methanol and water (0–15 min; 50%→100% MeOH, 15–20 min; 100% MeOH), a flow rate of 1 mL/min, and a column temperature of 40°C. Saccharic acid 1,4-lactone 3,5-di-O-ferulate was analyzed with UV detection at 280 nm, mobile solvents of 30% methanol and 70% water containing 5% acetic acid according to a previously reported method (Miyake et al., 2014). Quantification values were presented as the means of duplicate measurement levels.

Measurement of the bioactivity of compounds    The bioactivity of the seven aromatic compounds (6-demethoxynobiletin, 6-demethoxytangeretin, nobiletin, tangeretin, sinensetin, 5,7-dimethoxycoumarin, saccharic acid 1,4-lactone 3,5-di-O-ferulate) was assayed according to the reported method (Kazumura et al., 2013). HL-60 cells were maintained in GIT medium (Wako Pure Chemical Industries), and were cultured with 1.3% (v/v) DMSO for 96 h to differentiate neutrophil-like cells at 37°C with 5% CO₂. The cells were loaded with 3µM fluo-3 AM, and were incubated for 45 min at 37°C with 5% CO₂. The cells were then washed with 15 mL Ringer-Hepes buffer (154 mM NaCl, 5.6 mM KCl, and 10 mM Hepes, pH 7.4) twice and suspended in the same buffer of 3-5 mL. A cell suspension of 1.0 × 10⁵ cells/mL in a solution containing 1 mM CaCl₂ and 0.5 µM MCLA in the presence of the chemical substance to be tested was placed in a cuvette. The mixture was kept stirred and incubated at 37°C during the measurement. After 3 min, the mixture was stimulated by injection of 1 µM fMLP. Chemiluminescence and fluorescence of the stimulated cells were monitored and recorded as time courses of the cellular responses using the instrument (Hamamatsu Photonics K.K., Hamamatsu, Japan; Kazumura et al., 2013). The data was shown with the values (sample/control ratio) for the peak area of the test substance relative to the peak area of the control.

Cell viability was determined by the MTT assay using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), and HL-60 cells (1.0 × 10⁵ cells/mL) were treated with a series of concentrations of the test substance for 30 min at 37°C. The values of cell viability were presented as the means of measurement levels (n=7).

Statistical analysis    The experimental data of the bioactivity assay was expressed as mean ± SD (n=3). The statistical significance between the control and the test compounds at each concentration level was determined by a paired t-test. Differences at p< 0.05 were considered to be significant.

Results and Discussion

Identification and quantification of compounds from niihime fruit Citrus    fruits have been reported to contain bioactive compounds such as flavonoids, phenylpropanoids, and coumarin derivatives (Yu et al., 2005; González-Molina, et al., 2010). In the present study, compounds in the 50% and 100% methanol fractions from the niihime peel extract were analyzed by HPLC. Eriocitrin, narirutin, hesperidin, and didymin as flavanone glycosides in flavonoids were not isolated from the 50% methanol fraction, although they were detected in the fraction. As such, they were reported as components of niihime (Miyake, 2006; Ichinokiyama et al., 2012) and to have antioxidative activity in vitro and in vivo (Miyake et al., 1998; González-Molina et al., 2010). Saccharic acid 1,4-lactone 3,5-di-O-ferulate (Fig. 1), a phenylpropanoid, was isolated from the fraction. While the in vivo bioactivity of this compound has not been assessed, it was previously isolated from niihime and reported as an antioxidant in vitro (Miyake et al., 2014). 5,7-Dimethoxycoumarin (Fig. 1), a coumarin derivative, was isolated from the 100% methanol fraction, and it has been reported to have antiproliferative activity against a melanoma cell line (Wolny et al., 2014). 5,7-Dimethoxycoumarin was newly shown to be contained in niihime fruit in the present study, although it was reported as a component of Citrus meyeri, commonly called the Meyer lemon (Miyake et al., 2011). Compounds 1 and 2 were isolated from the 100% methanol fraction. They were analyzed by ¹H-NMR analysis because they could not be identified by HPLC analysis. The two compounds were identified respectively as 6-demethoxynobiletin and 6-demethoxytangeretin (Fig. 1) by the ¹H-NMR data in reference with the reported data of Nagase et al. (2005). The two compounds, polymethoxyflavones of flavonoids, were newly shown to be contained in niihime fruit in the present study, although they were reported to be contained in Citrus depressa, commonly known as Shiikuwasha (Nagase et al., 2005). 6-Demethoxynobiletin was reported to show bioactivity by enhancing phosphorylation of extracellular signal-regulated kinases, which is related to the amelioration of cognitive dysfunction (Kimura et al., 2013). 6-Demethoxytangeretin was reported to show bioactivity by suppressing the production of interleukin-6 in mast cells, which is related to anti-allergic activity (Kim et al., 2014). In the 100% methanol fraction, nobiletin, tangeretin, and sinensetin (Fig. 1) as polymethoxyflavones were also detected by HPLC. These compounds were previously reported to be contained in niihime (Ichinokiyama et al., 2012). In the present study, they were isolated from the fraction, and their bioactivity was assayed in comparison with 6-demethoxynobiletin and 6-demethoxytangeretin. From these results, niihime was newly shown to contain the bioactive compounds 5,7-dimethoxycoumarin, 6-demethoxynobiletin, and 6-demethoxytangeretin. The quantities of the seven aromatic compounds in the peel and juice of niihime are shown in Table 1. Flavonoids and coumarin derivatives were reported to be contained abundantly in Citrus peel (Miyake et al., 1998; Dugrand, et al., 2013). In the present study, higher levels of the seven aromatic compounds were found in the peel of niihime than the juice. Meanwhile, 5,7-dimethoxycoumarin was found at lower levels than the other compounds. Saccharic acid 1,4-lactone 3,5-di-O-ferulate was found at higher levels in the juice than the other compounds.

Fig. 1.

Chemical Structures of the Seven Aromatic Compounds Isolated from Niihime peel.

Compounds 1 and 2 were identified as 6-demethoxynobiletin and 6-demethoxytangeretin, respectively.

Table 1. Content of Seven Aromatic Compounds in the Peel and Juice of Niihime.

	Peel	Juice
	(µg/g of dry weight)	(µg/mL)
6-Demethoxynobiletin	800	0.62
6-Demethoxytangeretin	956	0.45
Nobiletin	7156	3.56
Tangeretin	1256	0.58
Sinensetin	2984	2.10
5,7-Dimethoxycoumarin	95	trace
Saccharic acid 1,4-lactone 3,5-di-O-ferulate	5554	9.46

Niihime is thought to be an autogenous Citrus of a mandarin (C. unshiu) and a tachibana (C. tachibana) (Miyake et al., 2006; Ichinokiyama etal., 2012). The composition of polymethoxyflavones (nobiletin, tangeretin, and sinensetin) in Citrus species has been reported; these compounds were abundantly contained in tachibana, and were found in small amounts in mandarin (Nogata et al., 2006). The characteristic composition of the compounds in niihime was suggested to be derived from tachibana, since the levels obtained in the present study were higher than those found in mandarin.

Determination of the bioactivity of compounds by simultaneously monitoring superoxide generation and intracellular calcium ion levels in neutrophils    The bioactivity of the seven aromatic compounds isolated from niihime in the present study was assayed by monitoring superoxide generation and calcium ion levels in neutrophils (Fig. 2). In regards to superoxide generation, 10 µM saccharic acid 1,4-lactone 3,5-di-O-ferulate exhibited a significantly high level compared to the control (p<0.01). The other compounds did not exhibit variation in superoxide generation in this assay. Saccharic acid 1,4-lactone 3,5-di-O-ferulate did not show antioxidative activity by scavenging superoxide but did show enhanced superoxide generation in this assay. Notably, its superoxide scavenging activity had been anticipated because of the reported radical scavenging activity by ORAC assay in vitro (Miyake et al., 2014). The compound showed superoxide generation activity in the in vivo-like method using neutrophils. Saccharic acid 1,4-lactone 3,5-di-O-ferulate is suggested to have greater superoxide generation activity than superoxide scavenging activity in neutrophils. In regard to calcium ion levels, 0.1 µM 6-demethoxytangeretin and 10 µM saccharic acid 1,4-lactone 3,5-di-O-ferulate exhibited significantly high levels compared to the control (p<0.01, p<0.05). The compounds showed greater than 90% cell viability, indicating that the compounds did not show cell cytotoxicity. From these results, saccharic acid 1,4-lactone 3,5-di-O-ferulate exhibited significantly high levels of both superoxide generation and intracellular calcium ion levels compared to control at 10 µM, a concentration showing no inhibition of cell viability. The increase in both superoxide generation and intracellular calcium ion levels of neutrophils in this assay was reported to be related to an increase in immunostimulatory activity (Kazumura et al., 2012; 2015). By simultaneously monitoring superoxide generation and intracellular calcium ion levels of neutrophils in the present study, saccharic acid 1,4-lactone 3,5-di-O-ferulate was newly suggested to have immunostimulatory activity. 6-Demethoxytangeretin at a concentration of 0.1 µM did not affect superoxide generation, although it significantly increased the calcium ion concentration. The increase in calcium ion concentration alone was not thought to indicate immunostimulatory activity. The aromatic compounds other than saccharic acid 1,4-lactone 3,5-di-O-ferulate did not exhibit bioactivities in this assay. The present study revealed the novel bioactivity of saccharic acid 1,4-lactone 3,5-di-O-ferulate.

Fig. 2.

Effects of the Seven Aromatic Compounds Isolated from Niihime on Superoxide Generation, Intracellular Calcium Ion Concentration, and Cell Viability in Neutrophils.

Data of the bioactivity assay is expressed as mean ± SD (n=3).

* Value shows a significant difference vs. control at P < 0.05.

** Value shows a significant difference vs. control at P < 0.01.

*** The values of cell viability are presented as means of measurement levels (n=7).

Acknowlegements    This study was supported in part by a Grant-in-Aid for the Cross-ministerial Strategic Innovation Promotion Program (Cabinet Office, Government of Japan).

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