Determination of flavonoids from Perilla frutescens var. japonica seeds and their inhibitory effect on aldose reductase

Perilla frutescens var. japonica (PF) is an annual aromatic herb has been consumed as a food ingredient and medicinal crop in Asian countries. To evaluate the therapeutic efficacy of aldose reductase (AR) inhibition, we tested the PF seeds. The stepwise polarities of PF were tested for AR inhibition, and we determined the CH2Cl2 and EtOAc fractions to be good inhibitors (5.81 and 3.99 μg/mL, respectively). Compounds 1–3 were isolated from the CH2Cl2 and EtOAc fractions and identified as luteolin (1), apigenin (2), and diosmetin (3) by physicochemical and spectroscopic data. Among them, luteolin (1) and apigenin (2) had high AR inhibitory activity (1.89 and 4.18 μM). Deulsaem, a variety of PF, was determined to have the highest flavonoid content among ten PF seeds tested (2.10 mg/g). This study suggests that PF could be utilized as a natural source to treat diabetic complications.


Introduction
Aldose reductase (AR), part of aldo-keto superfamily, is the key enzyme in the polyol pathway, which catalyzes the reduction of glucose to fructose [1,2]. AR can be found in almost all mammalian cells; however, its accumulation in the lens, sciatic nerves, and retina can cause diabetic complications [3]. An influx in the polyol pathway can lead to the increased sorbitol levels, which results in the generation of osmotic stress and blindness [4,5]. Thus, preventing AR accumulation is vital.
Perilla frutescens var. japonica (PF) is an annual aromatic herbaceous plant that belongs to the Lamiaceae family. It has been consumed as a food ingredient and medicinal crop in Asian countries for many years [6]. It is grown primarily in East Asia, India, Japan, and Korea. In Korea, oil extracted from the seeds of PF by pressing is used. Presently, PF is used to extract oil and as an ornamental plant in Europe [7]. Previous research revealed that PF has antioxidant [8], antiallergic [9], and anti-inflammatory properties; it also promotes anti-tumor effects [10], and effects on gastrointestinal motility [11]. Therefore, its oil could be a good supplement for improving blood flow [12].
PF oil contains a high amount of omega-3 fatty acids such as a-linolenic acid (ALA), which constitutes approximately 60% of the total. Other researchers have reported that dietary intake of ALA can protect against bladder cancer [13] and limit the risk of coronary artery disease [14,15] and prevent diseases such as cardiovascular disorders, cancer, inflammatory, and rheumatoid arthritis [16]. Because of this, it is widely used as a functional food with pharmaceutical and nutritional value. Previous studies have reported on additional biological compounds of PF. Therefore, we investigated the chemical composition of PF.
The objective of this work was to investigate the chemical profile of PF. We isolated compounds from an EtOH extract, and their structures were elucidated by EI-MS, as well as 1 H-and 13 C-NMR. Additionally, fractions and compounds of PF were tested for AR inhibition.

Plant materials
PF was obtained in 2014 from Department of Southern Area Crop Science, National Institute of Crop Science, Rural Development Administration, Miryang, Korea. Voucher specimens of PF were deposited at our Department.

Extraction and fractionation of PF
Dried seeds of PF (6 kg) were finely powdered and extracted with EtOH for 3 h (21 L 9 3) under reflux at 65-75°C. After filtration and evaporation in vacuo, the EtOH extract residue (285.3 g) was collected. Collected extracts were suspended in distilled water and partitioned successively with n-hexane (222.8 g), CH 2 Cl 2 (9.3 g), EtOAc (10.4 g), and n-BuOH (11.4 g).

Isolation of compounds 1-3 from PF
EtOH extract from PF seeds was subjected to chromatographic separation on a silica gel and Sephadex LH-20. A portion of the EtOAc fraction (3.4 g) was chromatographed by silica gel column chromatography (6 9 80 cm, No. 7734) using a CH 2 Cl 2 -MeOH elution system to yield eight fractions (E 1 -E 8 ). Subfraction E 4 afforded compounds 1 and 2, which were isolated by semi-prep HPLC. A portion of the CH 2 Cl 2 (3.1 g) fraction from PF was separated by silica gel column chromatography (6 9 80 cm, No. 7734) eluted with a stepwise gradient of the n-hexane-EtOAc system and then repeatedly by the EtOAc-MeOH system. The CH 2 Cl 2 fraction yielded 18 additional fractions (M 1 -M 18 ). Fraction M 12 yielded compound 3 by recrystallization using MeOH.  Table 1.

Measurement of AR activity
Rat lenses were removed from Sprague-Dawley rats and preserved by freezing until use. Each sample of the EtOH extract, n-hexane, CH 2 Cl 2 , EtOAc, n-BuOH fractions and compounds 1-3 were dissolved in DMSO for the AR activity assay [17].

Sample preparation for HPLC
For analysis of compounds 1-3 using HPLC, the PF seeds were extracted with EtOH. Extracts were then filtered with a syringe filter (0.45 lm), and the solution was used for HPLC analysis.
Quantitative analysis of PF compounds 1-3 HPLC analysis of PF compounds 1-3 was conducted. A Waters Spherisorb Ò INNO C18 (4.6 9 250 mm, 5 lm) column was used for their simultaneous determination. The mobile phase included 0.5% acetic acid (reagent A) and MeOH (reagent B). The gradient system was initially set at 70 (A):30 (B), increased in linear gradient to 45:55 for 20 min, and then increased to 0:100 for 30 min and kept for 5 min at 0.5 mL/min. Finally, the gradient was increased to 70:30 for 10 min. The total analysis was conducted over 65 min. Flavonoids were detected at 340 nm. The limits of detection and quantification (LOD and LOQ, respectively) for flavonoids as standard compounds were used to validate the HPLC method.

Calibration curves
Flavonoid stock solutions (0.1-10 lg/mL) were prepared in MeOH. The flavonoid content of the samples was determined using the corresponding calibration curves. The calibration curve for flavonoids was calculated using the peak area (Y), concentration (X, mg/mL), and mean values (n = 3) ± standard deviation (SD), which is shown in Fig. 2.

Results and discussion
The PF EtOH extracts were tested for AR inhibition. The results are summarized in Table 2. The EtOAc and CH 2 Cl 2 fractions significantly inhibited AR in rat lens (IC 50 = 3.99 and 5.81 lg/mL, respectively). Previous researchers also studied the various biological activities of PF. Extracts from PF seeds have been shown to have antioxidant [18] and antimicrobial activity [19]. However, there are still limited reports regarding the biological activity of PF. Our results demonstrate that PF seed extracts inhibit AR.
The EtOAc and CH 2 Cl 2 fractions were chosen to be isolated and identified because they showed superior AR inhibitory properties. Specifically, they were chromatographed on a silica gel and Sephadex LH-20 column. Thus, three flavonoids (compounds 1-3) were isolated. The chemical structures were identified using 1 H-and 13 C-NMR and EI-MS. Compounds 1-3 were observed to have a typical flavonoid pattern. Spectrums of a singlet signal at d 12.96 and 12. 97 were observed in the presence of a 5-OH in an A-ring structure; 1 H-NMR spectra data are shown in Table 1. The four compounds were verified based on previous literature [20,21,22]. Additionally, spectroscopic NMR data were obtained. As shown in Fig. 1, the chemical structures of compounds 1-3 were identified as luteolin (1), apigenin (2), and diosmetin (3).
Compounds 1-3 were tested for inhibition of AR in rat lens. Data are shown in Table 3. Luteolin (1) exhibited a greater inhibitory effect than TMG, the positive control. The percent inhibition for luteolin (1) and TMG was 1.89 and 2.52 lM, respectively. Compounds 1 and 2 were isolated from the EtOAc fraction, whereas compound 3 was isolated from the CH 2 Cl 2 . The structures of compounds 1 and 2 are similar, except for the presence of additional hydroxyl group in B-ring. Compounds 1 and 2 have a diand monohydroxy group, respectively. The effects of compound 1 were greater than those of compound 2. The structures of compounds 1 and 3 were found to be except for B-ring; the structure of compound 3 contains a methoxy group. The AR inhibitory activity of compound 1 is greater than that of compound 3. These data demonstrate that a dihydroxyl in B-ring exhibited greater effect than a monomoiety, methoxy group at the B-ring, and positions at C-3 of skeleton do not affect the AR inhibitory activity. Generally, flavonoids are potent AR inhibitors. Previous studies demonstrated that the AR inhibitory activity of flavonoids is related to their structure [23,24,25]. They also have antioxidant and anti-inflammatory properties [26,27]. Consequently, PF seed extracts could be used to inhibit AR. Further studies are required to understand the mechanisms of inhibition. Four flavonoids showed significant AR inhibitory effects. HPLC/UV analysis was conducted to determine the concentration of compounds 1-3 in ten PF seeds (Dami, Danjo, Deulsaem, Daesil, Anyu, Yujin, Dayu, Yupseol, Hyangim, and Hwahong). The flavonoid contents are shown in Fig. 2 and Table 5; HPLC chromatograms are shown in Fig. 2. In all samples, luteolin (1) and apigenin (2) were found in higher amounts among the four compounds tested (1.45 and 5.17 mg/g extract, respectively). Among the ten PF seeds, Deulsaem contained the highest total concentration of flavonoids (2.1 mg/g extract). In contrast, Danjo contained no flavonoids. Almost PF seed has much amount of apigenin (2) and demonstrates that   [29] also studied the content of phenolic compounds including flavonoids in PF using HPLC/PDA and HPLC-ESI/QTOF/MS/MS. Quantitative analyses were conducted using a calibration curve. The linearity of standard curves and correlation (r 2 ) for compounds 1-3 are shown in Table 4. The LOD and LOQ of compounds 1-3 were 0.000-0.001 mg/mL and 0.001-0.005 mg/mL, respectively (Table 6). We evaluated the ability of PF seed extracts to inhibit AR; the EtOAc fraction and compound 1 had significant inhibitory activity. Deulsaem was determined to have the  a Y = peak area, X = concentration of standards (mg/mL) b r 2 = correlation coefficient for three data points in the calibration curves (n = 5) Table 6 Linearity of standard curves for compounds 1-3 Compound Calibration equation a r 2 b Linear range (mg/mL) LOD (mg/mL) LOQ (mg/mL)