Two Pairs of 7,7′-Cyclolignan Enantiomers with Anti-Inflammatory Activities from Perilla frutescens

Perilla frutescens (L.) Britt. (Labiatae), a medicinal plant, has been widely used for the therapy of multiple diseases since about 1800 years ago. It has been demonstrated that the extracts of P. frutescens exert significant anti-inflammatory effects. In this research, two pairs of 7,7′-cyclolignan enantiomers, possessing a cyclobutane moiety, (+)/(−)-perfrancin [(+)/(−)-1] and (+)/(−)-magnosalin [(+)/(−)-2], were separated from P. frutescens leaves. The present study achieved the chiral separation and determined the absolute configuration of (±)-1 and (±)-2. Compounds (+)-1 and (−)-1 have notable anti-inflammatory effects by reducing the secretion of pro-inflammatory factors (NO, TNF-α and IL-6) and the expression of pro-inflammatory mediators (iNOS and COX-2). These findings indicate that cyclolignans are effective substances of P. frutescens with anti-inflammatory activity. The present study partially elucidates the mechanisms underlying the effects of P. frutescens.


Introduction
Chirality is very important in pharmacological research and drug development [1]. Drugs are mainly derived from natural products or by chemical synthesis, and, hence, often mixtures of several isomers are produced. Racemates are two enantiomers that exhibit identical physical and chemical properties, except for optical activities. As chiral separation techniques advance rapidly, people come to realize that some enantiomers may possess different properties with respect to pharmacological effects, toxicity and pharmacokinetics [2][3][4]. Some chiral medicines even exert opposite effects. For instance, (+)-picenadol is a potent opiate agonist, while (−)-picenadol acts as an opioid antagonist [5]. Therefore, it is important to study the enantiomers of chiral substances.
Perilla frutescens (L.) Britton (Labiatae) is a perennial herb that is extensively dispersed and cultivated not only in China, but also in other Asian countries [6]. As a well-known traditional edible and medicinal herb, P. frutescens has been used to treat common colds with cough and nausea and food poisoning such as fish or crab to humans [7]. Recent studies have demonstrated that about 90 compounds are derived from P. frutescens, including terpenoids, lignans and flavonoids [8]. Most of them have various pharmacological effects, including antimicrobial [9], anti-allergic [10], anti-inflammatory [11], antioxidant [12] and anti-depressive activities [13]. As part of our systematic study of bioactive products from traditional Chinese medicines, the isolation, structural elucidation and anti-inflammatory activity evaluation of the isolated compounds from P. frutescens were explored in the current study.  Further analysis of the HSQC, 1 H, 1 H-COSY, HMBC and NOESY data confirm the planar structure and relative configuration of compound 1. Interestingly, no cotton effects are observed in its ECD spectrum, which indicates that 1 might be isolated as a racemic mixture. Subsequently, compound 1 was further enantioseparated on a chiral column to afford ( [11]. Moreover, magnosalin's absolute configuration has not been ascertained. Interestingly, X-ray diffraction data analysis of compound 2 ( Figure 3) suggests that it contains two racemates. The (+)-2 and (−)-2 enantiomers successfully obtained by chiral Further analysis of the HSQC, 1 H, 1 H-COSY, HMBC and NOESY data confirm the planar structure and relative configuration of compound 1. Interestingly, no cotton effects are observed in its ECD spectrum, which indicates that 1 might be isolated as a racemic mixture. Subsequently, compound 1 was further enantioseparated on a chiral column to afford ( Further analysis of the HSQC, 1 H, 1 H-COSY, HMBC and NOESY data confirm the planar structure and relative configuration of compound 1. Interestingly, no cotton effects are observed in its ECD spectrum, which indicates that 1 might be isolated as a racemic mixture. Subsequently, compound 1 was further enantioseparated on a chiral column to afford ( [11]. Moreover, magnosalin's absolute configuration has not been ascertained. Interestingly, X-ray diffraction data analysis of compound 2 (  Figure 2, the absolute configuration of (+)-2 is identified as 7R,8S,7 R,8 S based on the good agreement with the calculated ECD spectrum of (7R,8S,7 R,8 S)-2, naturally elucidating the absolute configuration of (−)-2 as (7S,8R,7 S,8 R)-2. Consequently, the latter configuration (7S,8R,7 S,8 R) is clarified as the reported compound (−)-magnosalin [11], whereas the former one (7R,8S,7 R,8 S) corresponds to an unreported compound labeled (+)-magnosalin.

Physicochemical Properties and Spectroscopic Data of Compounds 1 and 2
Perfrancin (1) Table 1 shows 1 H and 13 C NMR data. The

Effects of Compounds (+)-1 and (−)-1 on NO Production in RAW 264.7 Macrophages
NO has been considered an important mediator whose production increases in the case of inflammation [15]. Therefore, inhibition of excessive NO production is commonly used to assess anti-inflammatory effects of compounds [16]. Levels of NO production in the supernatant of RAW 264.7 macrophages activated by LPS were investigated using the Griess reagent Kits. Figure 5 shows that, in comparison with the control group, NO production was higher in the LPS-treated group. Administration of (+)-1 or (−)-1 inhibits NO synthesis in RAW 264.7 macrophages was activated by LPS in a dose-dependent manner. The IC50 values of (+)-1 and (−)-1 are 21.61 ± 2.35 and 28.02 ± 1.93 μM, respectively. In addition, we used curcumin as a positive control (IC50 = 20.37 ± 0.77 μM).

Effects of Compound (+)-1 on TNF-α and IL-6 Production in RAW 264.7 Macrophages
Macrophages were cultured in the presence of compound (+)-1 and LPS to further characterize the anti-inflammatory effects of compound (+)-1. The concentrations of TNFα (p < 0.01, Figure 6A) and IL-6 (p < 0.01, Figure 6B) rose to high levels following treatment

Effects of Compounds (+)-1 and (−)-1 on NO Production in RAW 264.7 Macrophages
NO has been considered an important mediator whose production increases in the case of inflammation [15]. Therefore, inhibition of excessive NO production is commonly used to assess anti-inflammatory effects of compounds [16]. Levels of NO production in the supernatant of RAW 264.7 macrophages activated by LPS were investigated using the Griess reagent Kits. Figure 5 shows that, in comparison with the control group, NO production was higher in the LPS-treated group. Administration of (+)-1 or (−)-1 inhibits NO synthesis in RAW 264.7 macrophages was activated by LPS in a dose-dependent manner. The IC 50 values of (+)-1 and (−)-1 are 21.61 ± 2.35 and 28.02 ± 1.93 µM, respectively. In addition, we used curcumin as a positive control (IC 50 = 20.37 ± 0.77 µM). macrophages at 3.13, 6.25, 12.5, 25 and 50 μM. Therefore, compounds (+)-1 and (−)-1 at 3.13-50 μM were used in subsequent experiments.

Effects of Compounds (+)-1 and (−)-1 on NO Production in RAW 264.7 Macrophages
NO has been considered an important mediator whose production increases in the case of inflammation [15]. Therefore, inhibition of excessive NO production is commonly used to assess anti-inflammatory effects of compounds [16]. Levels of NO production in the supernatant of RAW 264.7 macrophages activated by LPS were investigated using the Griess reagent Kits. Figure 5 shows that, in comparison with the control group, NO production was higher in the LPS-treated group. Administration of (+)-1 or (−)-1 inhibits NO synthesis in RAW 264.7 macrophages was activated by LPS in a dose-dependent manner. The IC50 values of (+)-1 and (−)-1 are 21.61 ± 2.35 and 28.02 ± 1.93 μM, respectively. In addition, we used curcumin as a positive control (IC50 = 20.37 ± 0.77 μM).

Effects of Compound (+)-1 on TNF-α and IL-6 Production in RAW 264.7 Macrophages
Macrophages were cultured in the presence of compound (+)-1 and LPS to further characterize the anti-inflammatory effects of compound (+)-1. The concentrations of TNFα (p < 0.01, Figure 6A) and IL-6 (p < 0.01, Figure 6B) rose to high levels following treatment

Effects of Compound (+)-1 on iNOS and COX-2 Protein Expression in RAW 264.7 Macrophages
Many critical enzymes are related to the establishment and progression of inflammation, including iNOS and COX-2 [17,18]. We carried out Western blot analysis on LPStreated RAW 264.7 macrophages with or without compound (+)-1 to determine protein expression levels. LPS treatment at 1 μg/mL for 24 h markedly increased iNOS (p < 0.01, Figure 7A and Table S3) and COX-2 (p < 0.01, Figure 7B and Table S3) protein expression, in comparison with the control group. Compound (+)-1 treatment significantly reversed the increased expression of iNOS (p < 0.01) and COX-2 (p < 0.01) at 50 μM in RAW 264.7 macrophages activated by LPS.

Effects of Compound (+)-1 on iNOS and COX-2 Protein Expression in RAW 264.7 Macrophages
Many critical enzymes are related to the establishment and progression of inflammation, including iNOS and COX-2 [17,18]. We carried out Western blot analysis on LPS-treated RAW 264.7 macrophages with or without compound (+)-1 to determine protein expression levels. LPS treatment at 1 µg/mL for 24 h markedly increased iNOS (p < 0.01, Figure 7A and Table S3) and COX-2 (p < 0.01, Figure 7B and Table S3) protein expression, in comparison with the control group. Compound (+)-1 treatment significantly reversed the increased expression of iNOS (p < 0.01) and COX-2 (p < 0.01) at 50 µM in RAW 264.7 macrophages activated by LPS.

Effects of Compound (+)-1 on iNOS and COX-2 Protein Expression in RAW 264.7 Macrophages
Many critical enzymes are related to the establishment and progression of inflammation, including iNOS and COX-2 [17,18]. We carried out Western blot analysis on LPStreated RAW 264.7 macrophages with or without compound (+)-1 to determine protein expression levels. LPS treatment at 1 μg/mL for 24 h markedly increased iNOS (p < 0.01, Figure 7A and Table S3) and COX-2 (p < 0.01, Figure 7B and Table S3) protein expression, in comparison with the control group. Compound (+)-1 treatment significantly reversed the increased expression of iNOS (p < 0.01) and COX-2 (p < 0.01) at 50 μM in RAW 264.7 macrophages activated by LPS.

Discussion
As lignans, no more than 200 7,7 -cyclolignans have been identified in nature, which are mainly reported from the Labiatae [19][20][21], Asteraceae [22,23], Piperaceae [24,25] and Ginkgoaceae families [26]. In total, Labiatae accounts for less than 30 compounds. Two pairs of 7,7 -cyclolignan enantiomers, (±)-perfrancin (1) and (±)-magnosalin (2), were separated from the leaves of P. frutescens. Perfrancin has been reported only once before in organic synthesis [14], and this is the first report of perfrancin from natural plants. Magnosalin has been isolated from Magnolia salicifolia [27], Piper sumatranum [24] and P. frutescens [11]. However, absolute configurations of these compounds have not been determined. In the current study, we found that two pairs of lignan enantiomers were further separated through chiral HPLC separation, and compounds' absolute configurations were successfully determined by X-ray diffraction analyses and ECD calculations.
As an essential inflammatory mediator, NO is overproduced in macrophages stimulated by LPS [28]. As reported before, compound 2 is regarded as an inhibitor of NO synthase [11]. In our present study, 7,7 -cyclolignan enantiomers (+)-1 and (−)-1 were assessed for their anti-inflammatory effect against the release of NO in RAW 264.7 macrophages stimulated by LPS. They have similar inhibitory effects on NO production. In acute and chronic diseases, TNF-α is an essential factor in the inflammatory reaction [29]. Another cytokine, IL-6, also plays a critical role in inflammation [30,31]. Thus, a therapeutic strategy to reduce inflammation could be suppressing the production of inflammatory mediators and inhibiting the release of pro-inflammatory cytokines. In the present study, LPS treatment significantly increased the secretion of TNF-α and IL-6, which was markedly suppressed by compound (+)-1.
Inflammatory processes are initiated and sustained by iNOS and COX-2, which play key roles in the production of NO [32,33]. Many studies have confirmed that the expression of iNOS and COX-2 is highly upregulated during infection [34][35][36]. Our Western blot results revealed that the excessive expression of iNOS and COX-2 induced by LPS in RAW 264.7 macrophages was significantly inhibited by compound (+)-1. Thus, our findings provide further evidence that compound (+)-1 has anti-inflammatory activity.

Extraction and Isolation
All chemical materials are shown in Supplementary Materials. After air-drying the P. frutescens leaves (10 kg), we extracted them with 95% EtOH (3 × 50 L) for 3 × 2 h under reflux. A yellow residue (2.1 kg) was obtained after evaporating the EtOH extract under reduced pressure, which was suspended in H 2

Statistical Analysis
Each experiment was conducted three times. Data are presented as the mean with SEM. Multiple groups were analyzed by ANOVA combined with Tukey's post hoc test. Statistical significance was defined as p < 0.05.