Isolation of a New Ferulate Derivative from Niihime Fruit, a Sour Citrus Fruit, and Its Antioxidative Activity

Abstract

Niihime is a sour Citrus fruit produced along the coast of the Sea of Kumano in the Mie prefecture of Japan. A novel ferulate derivative was isolated from niihime fruit. The structure of the compound was elucidated by spectroscopic analyses and identified as saccharic acid 1,4-lactone 3,5-di-O-ferulate by ¹H-NMR, ¹³C-NMR, and MS analyses. The new compound exhibited significantly higher antioxidative activity than ferulic acid, a parent molecule of the compound, and trolox, a standard antioxidant, as measured by an ORAC assay (P < 0.05). Further, it was abundantly contained in the flavedo, albedo, and segment epidermis of the niihime peel. It was also found in the juice vesicles but not in the seeds.

Introduction

Plants contain phenolic compounds that have significant antioxidative and antiradical activities (Shahidi et al., 2007). The compounds provide protection against the harmful effects of free radicals and are known to reduce the risk of diseases associated with oxidative stress (Hertog et al., 1993). Citrus fruits have also been reported to contain numerous bioactive phenolic compounds (González-Molina et al., 2010). Niihime (Citrus unshiu x Citrus tachibana), a sour Citrus fruit, is produced in the Mie prefecture along the coast of the Sea of Kumano in Japan. It is thought to be an autogenous Citrus of a mandarin (C. unshiu) and a tachibana (C. tachibana). The phenolic compounds in niihime fruit have attracted attention and the fruit's flavonoid characteristics have been studied (Miyake, 2006). ORAC (oxygen radical absorbance capacity) assays have been widely used for the evaluation of the antioxidant capacity of foods and their compounds (Wu et al., 2004). In this study, we attempted to explore the active compound in niihime fruit and to examine the compound for antioxidative activity using an ORAC assay.

Materials and Methods

Materials and reagents Niihime fruit was obtained from the Kumano City Home Promotion Public Corporation (Kumano, Mie, Japan). The reagents used in the present study were of analytical or HPLC grade (Wako Pure Chemical Industries, Osaka, Japan).

Isolation of ferulate derivative from niihime fruit

The niihime peel (657 g) was soaked in methanol (2 L) for 2 days at room temperature. The extract was concentrated and applied to a reversed-phase column (ϕ 37 × 500 mm, Amberlite XAD-2 resin; Rohm and Haas Co., Philadelphia, PA, USA). The column was washed with 1.5 L of water and eluted with 1.5 L of methanol; the eluate was then concentrated under reduced pressure. The compounds in the methanol fraction were isolated by preparative HPLC (900 series; JASCO, Tokyo, Japan), using a YMC-ODS column (YMC-Pack ODS-A, ϕ20 × 250 mm, S-5 µmL; YMC Co., Kyoto, Japan), UV detection at 280 nm, mobile solvents of 30% methanol and 70% water containing 5% acetic acid, and a flow rate of 8 mL/min at room temperature. The peak of compound (1) was detected at 21.5 min (retention time). Compound (1) was isolated as a white amorphous powder (79.5 mg). Hesperidin, eriocitrin, narirutin, 1-feruloyl-β-glucoside, 1-sinapoyl-β-glucoside, 6,8-di-C-glucosylapigenin, and 6,8-di-C-glucosyldiosmetin, all of which have been previously reported as phenolic glucosides in niihime, were isolated from the methanol fraction (Miyake, 2006; Miyake et al., 2007).

Determination of compound (1) Optical rotations were recorded on a DIP-370 (JASCO) in methanol at 25°C. Elemental analysis was determined by JM-10 (JSL). UV spectra were recorded on a V-650 spectrophotometer (JASCO) in methanol. IR spectra were recorded on an FT/IR-4100 (JASCO). ¹H and ¹³C NMR, COSY, HMQC, and HMBC were measured on a JNMECP500 (JEOL) NMR spectrometer. Chemical shifts are shown in δ (ppm) with tetramethylsilane (TMS) as an internal reference. FabMS spectra were recorded on a JMS-HX110 (JEOL) spectrometer. The analytical data of compound (1) are shown below. [α]²⁵_D −5.3 (c = 0.113, methanol). Anal. Calcd for C26H24O13/3H2O: C, 52.18; H, 5.05. Found: C, 51.91; H, 5.24.; FabMS m/z 585 [M+H2O+Na]⁺, 567 [M+Na]⁺; UV (log ɛ, methanol) λ_max 218 (4.56), 234 (4.49), 296 (4.55), 322 (4.64) nm; IR (CHCl3)ν_max 3400, 1705, 1628, 1599 cm⁻¹; For ¹H, ¹³C NMR and HMBC see Table 1. Differential NOE: irradiation of 3′-OMe (δ 3.84) - 11% enhancement of H-2’ (δ 7.22), irradiation of 3″-OMe (δ 3.77) - 11% enhancement of H-2′’ (δ 7.17), irradiation of H-2 (δ 4.46) - 3% enhancement of H-5 (δ 5.35), irradiation of H-3 (δ 5.45) - 7% enhancement of H-4 (δ 4.72), irradiation of H-4 (δ 4.72) - 8% enhancement of H-3 (δ 5.45) and 10% enhancement of H-5 (δ 5.35), irradiation of H-5 (δ 5.35) - 4% enhancement of H-2 (δ 4.46) and 8% enhancement of H-4 (δ 4.72).

Table 1. NMR Spectral Data (500 MHz, acetone-d6) for Compound (1) Isolated from Niihime Fruit.

Compound (1)
Position	δC, type	δH (J in Hz)	HMBCa
1	173.2 C
2	70.7 CH	4. 46 (d, 6.4)	C-1, C-3, C-4
3	74.9 CH	5.45 (dd, 6.4, 3.8)	C-1, C-9′
4	69.9 CH	4.72 (dd, 3.8, 2.7)
5	74.6 CH	5.35 (d, 2.7)	C-4, C-6, C-9″
6	169.4 C
1′	127.4 C
2′	111.2 CH	7.22 (d, 1.9)	C-4′, C-6′, C-7′
3′	148.6 C
4′	150.0 C
5′	115.8 CH	6.81 (d, 7.9)	C-1′, C-3′
6′	124.0 CH	7.05 (dd, 7.9, 1.9)	C-2′, C-4′, C-7′
7′	146.6 CH	7.64 (d, 15.9)	C-2′, C-6′, C-9′
8′	115.0 CH	6.37 (d, 15.9)	C-1′
9′	166.6 C
3′-OMe	56.1 CH3	3.84 (3H, s)	C-3′
1″	127.2 C
2″	111.2 CH	7.17 (d, 1.9)	C-4″, C-6″, C-7″
3″	148.6 C
4″	150.0 C
5″	115.9 CH	6.73 (d, 7.9)	C-1″, C-3″
6″	124.0 CH	6.99 (dd, 7.9, 1.9)	C-2″, C-4″, C-7″
7″	146.4 CH	7.60 (d, 15.9)	C-2″, C-6″, C-9″
8″	115.0 CH	6.33 (d, 15.9)	C-1″
9″	166.5 C
3″-OMe	56.1 CH3	3.77 (3H, s)	C-3″

a  HMBC correlations are from proton(s) stated to the indicated carbon.

Antioxidative activity of compound (1) The antioxidative activity was measured by an ORAC assay, which reports the antioxidant capacity of the peroxyl radical relative to the trolox standard (Wu et al., 2004; Miyake et al., 2012). The compounds were dissolved in solvent (acetone: water: acetic acid = 70: 29.5: 0.5, v/v/v). The ORAC value of each sample was expressed as µmol of trolox equivalents (TE) per µmol of sample (µmol TE/µmol), and data were expressed as the mean ± SD (N = 3). The statistical significance (t test: two-sample equal variance, using two-tailed distribution) was determined.

Quantification of compound (1) Niihime fruit was separated to flavedo, albedo, segment epidermis, juice vesicles, and seeds of the fruit tissues using a knife. Each type of tissue was lyophilized and pulverized to a powder. Each powder (0.1 g) was extracted with a solution (5 mL, methanol and dimethyl sulfoxide, 1:1, v/v) by ultrasonic treatment for 60 min and left at room temperature overnight. The ferulate derivative in the extract was determined by HPLC, using a YMC-ODS column (YMC-Pack φ4.6 × 250 mm, S-5 µm; YMC Co.), UV detection at 280 nm, mobile solvents of 30% methanol and 70% water containing 5 % acetic acid, at a flow rate of 1 mL/min and a column temperature of 40°C.

Results and Discussion

Structure elucidation of compound (1) The molecular formula of compound (1) was determined as C₂₆H₂₄O₁₃ on the basis of the ¹³C-NMR spectral data, the positive-ion FabMS (m/z 585 [M+H₂O+Na]⁺, 567 [M+Na]⁺) and elemental analysis. The IR spectrum showed absorption bands due to a hydroxyl group (ν_max 3400 cm⁻¹) and carbonyl groups (ν_max 1705 and 1628 cm⁻¹). Its ¹H-, ¹³C-NMR, COSY, and HMQC spectra revealed the presence of two carboxyl groups (δc 173.2 and 169.4), four oxygen-bearing methines [δC 70.7, 74.9, 69.9, and 74.6, corresponding to d_H 4.46 (1H, d, J= 6.4 Hz), 5.45 (1H, dd, J= 6.4, 3.8 Hz), 4.72 (1H, d, J= 3.8, 2.7 Hz), and 5.35 (1H, d, J= 2.7 Hz), respectively]. The presence of two trans-4-hydroxy-3-methoxycinnamate (ferulate) in this molecule is suggested by the appearance of two pairs of doublets [δ 7.64 and 6.37 (each 1H, d, J= 15.9 Hz), δ 7.60 and 6.33 (each 1H, d, J= 15.9 Hz)], two ABC-type signals [d 6.81 (1H, d, J= 7.9 Hz), 7.05 (1H, dd, J= 7.9, 1.9 Hz), 7.22 (1H, d, J = 1.9 Hz) and d 6.73 (1H, d, J= 7.9 Hz), 6.99 (1H, dd, J= 7.9, 1.9 Hz), 7.17 (1H, d, J= 1.9 Hz)], and two OMe signals [d 3.84 and 3.77 (each 3H, s)] in the ¹H NMR (acetone-d₆) spectrum (Table 1). In the HMBC experiment (Table 1), the double-doublet signals at δ_H 5.45 (H-3) were correlated with two carboxyl signals at δ_C 173.2 (C-1) and 166.6 (C-9′), the latter being assignable to the carboxyl carbon of the trans-4-hydroxy-3-methoxycinnamate moiety, indicating that the trans-4-hydroxy-3-methoxycinnamate moiety was located at C-3. On the other hand, the doublet signals at δ_H 5.35 (H5) were correlated with two carboxyl signals at δ_C 169.4 (C-6) and 166.5 (C-9”), the latter being assignable to the carboxyl carbon of another trans-4-hydroxy-3-methoxycinnamate moiety, indicating that another trans-4-hydroxy-3-methoxycinnamate moiety was located at C-5. The proton chemical shift of the other signals of four oxygen-bearing methines indicated that these positions were not acylated. Furthermore, the positive-ion FabMS of 1 showed the [M + Na]⁺ ion peak at m/z 567, which was 18 mass units less than that of the ring opened compound, indicating the presence of a 1,4-lactone ring in the molecule. The relative configurations of the C-2 ∼ C-4 in compound (1) were confirmed by the analysis of the NOE experiment. The saccharic acid core was deduced by observation of NOEs between H-2 and H-5, between H-3 and H-4, and between H-4 and H-5 on the 1,4-lactone ring (Fig. 1). Based on the above evidence, the structure of compound (1) was concluded to be saccharic acid 1,4-lactone 3,5-di-O-ferulate. This is the first example of saccharic acid 1,4-lactone having two trans-4-hydroxy-3-methoxycinnamate moieties to be found in nature. Ferulate derivatives from Citrus fruits have been reported previously; 1-feruloyl-glucoside was isolated from lemon (Citrus limon) (Miyake et al., 2007) and 3-O-feruloyl saccharic acid 1,4-lactone methyl ester was isolated from sudachi (Citrus sudachi) (Nakagawa et al., 2006; Sengoku et al., 2012). In this study, a new ferulate derivative was isolated from niihime fruit.

Fig. 1.

Chemical Structure of Compound (1) Isolated from Niihime Fruit. The compound was identified as saccharic acid 1,4-lactone 3,5-di-O- ferulate.

Antioxidative activity of compound (1) The ferulate derivative, compound (1), isolated from the niihime fruit in this study and ferulic acid, a parent molecule of the compound, were examined for antioxidative activity by a hydrophilic ORAC_FL assay according to the reported method (Wu et al., 2004). The ferulate derivative and ferulic acid were shown to possess higher antioxidant capacities than trolox (Table 2). The ferulate derivative having two molecules of ferulic acid exhibited a significantly higher ORAC value than ferulic acid (P < 0.05), which has been studied for its antioxidative activity (Miyake et al., 2007; Kumazawa et al., 2010).

Table 2. Antioxidative Activity of Ferulate Derivative and Ferulic Acid.

	ORAC value (µmol TE/µmol)a
Saccharic acid 1,4-lactone 3,5-di-O-ferulateb	4.73 ± 0.03c
Ferulic acid	2.81 ± 0.01

a  ORAC value is expressed as µmol of trolox equivalent per µmol of compound. Each data value is represented as the mean ± SD (n= 3).

b  Compound (1), ferulic derivative, isolated from niihime fruit. The molecular weight used is 598, C₂₆H₂₄O₁₃/3H₂O.

4  Value is significantly different from that of ferulic acid at P < 0.05.

Determination of compound (1) The quantities of the ferulate derivative in the various fruit tissues (mg/g of dry fruit tissue) are shown as the mean ± SD (N = 3). It was contained abundantly in the flavedo (9.1 ± 0.6 mg/g), albedo (7.1 ± 0.7 mg/g), and segment epidermis (4.1 ± 0.4 mg/g) of the niihime peel. It was also found in the juice vesicles (1.6 ± 0.2 mg/g) but not in the seeds.

In this study, a novel ferulate derivative was isolated from niihime fruit and identified as saccharic acid 1,4-lactone 3,5-di-Oferulate. The compound possessed a high antioxidative property.

Acknowledgements We wish to thank Miss Tatsuko Sakai for elemental analysis. A part of this study was supported by a Grantin Aid for Scientific Research C (No. 23500975 to Y. M and No. 23590026 to C. I.) from the Japan Society for the Promotion of Science.

References

•  González-Molina,  E.,  Domínguez-Perles,  R.,  Moreno,  D.A., and  García-Viguera,  C. (2010). Natural bioactive compounds of Citrus limon for food and health. J. Pharm. Biomed. Anal., 51, 327-345.
•  Hertog,  M.G.,  Feskens,  E.J.,  Hollman,  P.C.,  Katan,  M.B., and  Kromhout,  D. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet, 342, 1007-1011.
•  Kumazawa,  S.,  Ahn,  M.R.,  Fujimoto,  T., and  Kato,  M. (2010). Radical-scavenging activity and phenolic constituents of propolis from different regions of Argentina. Nat. Prod. Res., 24, 804-812.
•  Miyake,  Y.,  Mochizuki,  M.,  Okada,  M.,  Hiramitsu,  M.,  Morimitsu,  Y., and  Osawa,  T. (2007). Isolation of antioxidative phenolic glucosides from lemon juice and their suppressive effect on the expression of blood adhesion molecules. Biosci. Biotechnol. Biochem., 71, 1911-1919.
•  Miyake,  Y. (2006). Characteristics of flavonoids in niihime fruit—a new sour citrus fruit. Food Sci. Technol. Res., 12, 186-193.
•  Miyake,  Y.,  Ito,  C., and  Itoigawa,  M. (2012). A novel trans-4hydroxycinnamic acid derivative from Meyer lemon (Citrus meyeri). Food Chem., 15, 135, 2235-2237.
•  Nakagawa,  H.,  Takaishi,  Y.,  Tanaka,  N.,  Tsuchiya,  K.,  Shibata,  H., and  Higuti,  T. (2006). Chemical constituents from the peels of Citrus sudachi. J. Nat. Prod., 69, 1177-1179.
•  Sengoku,  T,  Murata,  Y.,  Mitamura,  H.,  Takahashi,  M., and  Yoda,  H. (2012). Synthesis of novel mucic acid 1,4-lactone methyl ester 3-O-ferulate related to an extractive component isolated from the peels of Citrus sudachi. Tetrahedron Lett. 53, 435-437.
•  Shahidi,  F.,  Alasalvar,  C., and  Liyana-Pathirana,  C.M. (2007). Antioxidant phytochemicals in hazelnut kernel (Corylus avellana L.) and hazelnut byproducts. J. Agric. Food Chem., 55, 1212-1220.
•  Wu,  X.,  Beecher,  G.R.,  Holden,  J.M.,  Haytowitz,  D.B.,  Gebhardt,  S.E., and  Prior,  R.L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agric. Food Chem., 52,4026-4037.