Effects of Compounds Isolated from Lindera erythrocarpa on Anti-Inflammatory and Anti-Neuroinflammatory Action in BV2 Microglia and RAW264.7 Macrophage

Lindera erythrocarpa contains various constituents such as cyclopentenedione-, flavonoid-, and chalcone-type components. In this study, a novel bi-linderone derivative and 17 known compounds were isolated from the leaves of L. erythrocarpa by using various chromatographic methods. The structures of the components were determined from nuclear magnetic resonance and mass spectrometry data. All isolated compounds were tested for anti-inflammatory and anti-neuroinflammatory activities in lipopolysaccharide (LPS)-induced BV2 and RAW264.7 cells. Some of these compounds showed anti-inflammatory effects by inhibiting the nitric oxide (NO) produced by LPS. In particular, linderaspirone A (16), bi-linderone (17) and novel compound demethoxy-bi-linderone (18) showed significant inhibitory effects on the production of prostaglandin E2 (PGE2), tumor necrosis factor-α, and interleukin-6. The three compounds also inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are pro-inflammatory proteins, and the activation of nuclear factor κB (NF-κB). Therefore, linderaspirone A (16), bi-linderone (17), and demethoxy-bi-linderone (18) isolated from the leaves of L. erythrocarpa have therapeutic potential in neuroinflammatory diseases.


Introduction
Neurodegenerative diseases are among the most common age-related diseases, affecting more than 50 million people worldwide [1]. Neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) [2]. Many studies have focused on developing treatments for neurodegenerative diseases, but these have not yet been developed. However, with the efforts of many researchers, neurodegenerative diseases have been found to begin with neuroinflammation [3][4][5]. Microglia and astrocytosis (gliosis) are involved in the regulation of neuroinflammation, and when they are activated during the induction of neurodegeneration, various inflammatory substances are secreted to induce neuroinflammation [6,7]. Thus, microglial activation and gliosis play a central role in neuroinflammation and the consequent neurodegeneration. Therefore, there is a need for more research on therapeutic agents for neurodegenerative diseases, with the signaling pathway of neuroinflammation as the target, and it is necessary to discover therapeutic agents that suppress neuroinflammation, the cause of the disease.
Microglia, which are the macrophages of the central nervous system, can activate and trigger innate immune responses by sensing exogenous neurotoxic substances and proinflammatory stimuli [8,9]. However, overactivation of microglia owing to various causes results in neuroinflammatory responses, nerve damage, and cognitive dysfunction [10]. Specifically, inflammation is mediated through the production of proinflammatory mediators including prostaglandin E2 (PGE 2 ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and nitric oxide (NO) [11]. Therefore, modulation of neuroinflammatory substances that can affect microglial activation represents a potential strategy for the alleviation of neurodegenerative diseases.
Lindera erythrocarpa is a plant belonging to the Lauraceae family and is widely distributed in Korea, Japan, and China [12]. The characteristic feature of L. erythrocarpa is that it blooms during April and May and bears fruits in September. Dried fruits have a distinctive aroma and bitter taste and are used in Japan as a digestive and pain reliever for neuralgia [13]. It has antifungal, digestive, and antibacterial properties and has been used as an herbal medicine for a long time. According to the findings of studies conducted thus far, various activities such as antioxidant, anti-inflammatory, anticancer, and antifungal activity of L. erythrocarpa have been reported. [14][15][16][17][18]. L. erythrocarpa contains various components such as linderone, lucidone, kanakuziol, camphene, and limonene [18][19][20]. However, among the compounds isolated from L. erythrocarpa, none of the studies have shown the inhibitory effects in neuroinflammation in microglia.
The purpose of this study was to identify bioactive and novel compounds in L. erythrocarpa leaves that inhibit neuroinflammation, a cause of neurodegenerative diseases. Therefore, using BV2 microglia and RAW264.7 macrophages, we attempted to isolate a novel component with an anti-inflammatory effect from the components present in the methanol extract of L. erythrocarpa leaves.

Structural Elucidation of Isolated Compounds
In this study, 18 compounds ( Figure 1) were isolated from the leaves of L. erythrocarpa using several chromatographic methods. The chemical structures of the isolated compounds were determined from nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. A novel bi-linderone derivative (18) and 17 known metabolites classified as chalcone, flavonoid, cyclopentenedione, and dimer of linderone were identified.

Inhibitory Effect of Nitrite Production of 18 Natural Compounds Isolated from L. erythrocarpa
To identify compounds that have an inhibitory effect on neuroinflammation and are present in L. erythrocarpa leaves, we investigated the anti-inflammatory effects of the 18 isolated compounds. First, toxicity was assessed to establish treatment concentrations for compounds isolated from BV2 microglia (Figure 2A-F). Compounds 3, 4, 7, 9-13, and 16-18 did not show toxicity even at a concentration of 40 µM, whereas compounds 1, 2, and 8 showed toxicity at a concentration of 40 µM. Compounds 5, 6, 14, and 15 showed toxicity at 10 µM; therefore, the cell treatment concentration was set at 5 µM, which did not show toxicity.
Next, toxicity was assessed to establish treatment concentrations for compounds isolated from RAW264.7 macrophages ( Figure 3A-F). Compounds 3, 4, 7-13, and 16-18 did not show toxicity even at a concentration of 40 µM, whereas compounds 1 and 2 showed toxicity at a concentration of 40 µM. In addition, compounds 14 and 15 were toxic at a concentration of 20 µM, whereas compounds 5 and 6 were toxic at 10 µM. Based on these results, non-toxic treatment concentrations were established for all compounds.
We investigated the inhibitory effect of all compounds on nitrite production in lipopoly saccharide (LPS)-induced BV2 microglia under varied treatment concentration settings based on toxicity assessment ( Figure 4A-F). The results confirmed that compounds 1-4, 7, 8, and 13-18 inhibited nitrite production. In particular, compounds 9-12 showed similar flavonoid glycoside structures, and it was confirmed that none of them exhibited an inhibitory effect on nitrite production. In addition, it was confirmed that the novel compound, compound 18, had a skeleton similar to those of compounds 16 and 17, which were dimer of the linderone types, and had a similar inhibitory effect on nitrite production.
Next, we investigated the inhibitory effect of the native compounds on nitrite production in LPS-induced RAW264.7 macrophages ( Figure 5A-F). It was confirmed that all compounds except compounds 6 and 9 had an inhibitory effect on nitrite production. In particular, it was confirmed that the novel compound 18 and the known compounds 16 and 17 had a nitrite production inhibitory effect that is, respectively, similar to or superior over the positive control at a concentration of 40 µM.

Inhibitory Effect of Compounds 16-18 on iNOS and COX-2 Expression
In this study, the inhibitory effect of the 18 natural compounds isolated from L. erythrocarpa leaves on nitrite production was confirmed using BV2 and RAW264.7 cells. Among them, additional activity evaluation was performed for compounds 16-18, which showed the best inhibitory effect on nitrite production. To investigate the additional antiinflammatory effect of the compound, the inhibitory effect of the pro-inflammatory proteins inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-induced BV2 and RAW264.7 cells was investigated ( Figure 6). The compounds 16-18 inhibited the expression of both iNOS and COX-2, which are pro-inflammatory proteins. In particular, compound 18 exhibited the strongest inhibitory effect on the expression of the pro-inflammatory proteins.

Inhibitory Effect of Compounds 16-18 on iNOS and COX-2 Expression
In this study, the inhibitory effect of the 18 natural compounds isolated from L. erythrocarpa leaves on nitrite production was confirmed using BV2 and RAW264.7 cells. Among them, additional activity evaluation was performed for compounds 16-18, which showed the best inhibitory effect on nitrite production. To investigate the additional antiinflammatory effect of the compound, the inhibitory effect of the pro-inflammatory proteins inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPSinduced BV2 and RAW264.7 cells was investigated ( Figure 6). The compounds 16-18 inhibited the expression of both iNOS and COX-2, which are pro-inflammatory proteins. In particular, compound 18 exhibited the strongest inhibitory effect on the expression of the pro-inflammatory proteins. shown. Data are expressed as the mean ± standard deviation value of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS.

Inhibitory Effect of Compounds 16-18 on PGE2, TNF-α, and IL-6 Production
First, we confirmed the effects on the production of inflammatory mediators and cytokine when each compound of 16, 17, and 18 was treated to the BV2 and RAW264.7 cells without stimulus. As a result, compounds 16-18 had no effect on nitrite, PGE2, TNF-α and IL-6 production when treated alone ( Figure S24). Through Western blotting, we confirmed that compounds 16-18 isolated from L. erythrocarpa leaves inhibited the expression of proinflammatory proteins. Therefore, we investigated whether these compounds modulated the release of PGE2, TNF-α, and IL-6, which are substances that modulate cellular inflammation associated with the expression of pro-inflammatory proteins. First, the inhibitory effect on the production of PGE2, an inflammatory mediator synthesized by the expression of the COX-2 protein in LPS-induced BV2 and RAW264.7 cells, was confirmed ( Figure  7A,B). All three compounds inhibited the production of PGE2, and compound 18 exhibited the best inhibitory effect on the production of PGE2. Next, we investigated whether it modulates the release of the inflammatory cytokines TNF-α and IL-6 from cells in response to inflammation ( Figure 7C-F). All three compounds inhibited the release of TNFα and IL-6 from cells, and compound 18 had the best inhibitory effect on the release of inflammatory cytokines in both BV2 and RAW264.7 cells.

Inhibitory Effect of Compounds 16-18 on PGE 2 , TNF-α, and IL-6 Production
First, we confirmed the effects on the production of inflammatory mediators and cytokine when each compound of 16, 17, and 18 was treated to the BV2 and RAW264.7 cells without stimulus. As a result, compounds 16-18 had no effect on nitrite, PGE 2 , TNF-α and IL-6 production when treated alone ( Figure S24). Through Western blotting, we confirmed that compounds 16-18 isolated from L. erythrocarpa leaves inhibited the expression of pro-inflammatory proteins. Therefore, we investigated whether these compounds modulated the release of PGE 2 , TNF-α, and IL-6, which are substances that modulate cellular inflammation associated with the expression of pro-inflammatory proteins. First, the inhibitory effect on the production of PGE 2 , an inflammatory mediator synthesized by the expression of the COX-2 protein in LPS-induced BV2 and RAW264.7 cells, was confirmed ( Figure 7A,B). All three compounds inhibited the production of PGE 2 , and compound 18 exhibited the best inhibitory effect on the production of PGE 2 . Next, we investigated whether it modulates the release of the inflammatory cytokines TNF-α and IL-6 from cells in response to inflammation ( Figure 7C-F). All three compounds inhibited the release of TNF-α and IL-6 from cells, and compound 18 had the best inhibitory effect on the release of inflammatory cytokines in both BV2 and RAW264.7 cells.

Inhibitory Effect of Compounds 16-18 on NF-κB Activation
We found that compounds 16-18 isolated from L. erythrocarpa leaves modulated inflammatory mediators. Therefore, it was necessary to investigate whether compounds 16-18 were involved in the activation of nuclear factor κB (NF-κB), which regulates iNOS and COX-2 expression. Therefore, it was investigated whether the compounds regulate the activation of NF-κB in LPS-induced BV2 and RAW264.7 cells (Figure 7). All three compounds inhibited the activation of NF-κB, and it was found that compound 18 had the strongest inhibitory effect on the activation of NF-κB.

Discussion
L. erythrocarpa has long been used in traditional medicine and has been extensively studied for its phytochemical components and biological activity. To date, several research papers have shown that L. erythrocarpa contains various components such as cyclopentedione-and flavonoid-type of compounds; their various activities have been studied as well. We attempted to isolate the bioactive and novel natural compound from L. erythrocarpa leaves, and as a result, one novel and 17 known compounds were isolated. Most compounds have already been reported as the components of L. erythrocarpa, but their bioactive for anti-neuroinflammatory effects have not yet been investigated. Therefore, we first attempted to compare the anti-inflammatory activities of the compounds isolated using LPS-induced BV2 microglia and RAW264.7 macrophages.
Macrophages and microglia are activated when harmful factors are present and play an important role in maintaining homeostasis by eliminating side effects in the body. However, when cells are overactivated, they secrete pro-inflammatory mediators and inflammatory cytokines [33]. The secretion of inflammatory mediators causes cell damage and, if left unattended, brain damage, leading to degenerative brain disease [34,35]. Therefore, a therapeutic agent to prevent degenerative brain disease should exhibit the effect of suppressing the secretion of inflammatory mediators. LPS is a major component of the

Inhibitory Effect of Compounds 16-18 on NF-κB Activation
We found that compounds 16-18 isolated from L. erythrocarpa leaves modulated inflammatory mediators. Therefore, it was necessary to investigate whether compounds 16-18 were involved in the activation of nuclear factor κB (NF-κB), which regulates iNOS and COX-2 expression. Therefore, it was investigated whether the compounds regulate the activation of NF-κB in LPS-induced BV2 and RAW264.7 cells (Figure 7). All three compounds inhibited the activation of NF-κB, and it was found that compound 18 had the strongest inhibitory effect on the activation of NF-κB.

Discussion
L. erythrocarpa has long been used in traditional medicine and has been extensively studied for its phytochemical components and biological activity. To date, several research papers have shown that L. erythrocarpa contains various components such as cyclopentedione-and flavonoid-type of compounds; their various activities have been studied as well. We attempted to isolate the bioactive and novel natural compound from L. erythrocarpa leaves, and as a result, one novel and 17 known compounds were isolated. Most compounds have already been reported as the components of L. erythrocarpa, but their bioactive for anti-neuroinflammatory effects have not yet been investigated. Therefore, we first attempted to compare the anti-inflammatory activities of the compounds isolated using LPS-induced BV2 microglia and RAW264.7 macrophages.
Macrophages and microglia are activated when harmful factors are present and play an important role in maintaining homeostasis by eliminating side effects in the body. However, when cells are overactivated, they secrete pro-inflammatory mediators and inflammatory cytokines [33]. The secretion of inflammatory mediators causes cell damage and, if left unattended, brain damage, leading to degenerative brain disease [34,35]. Therefore, a therapeutic agent to prevent degenerative brain disease should exhibit the effect of suppressing the secretion of inflammatory mediators. LPS is a major component of the outer cell membrane of Gram-negative bacteria. It is a substance in which lipids and polysaccharides are covalently bound to each other. As a pathogenic factor induced by an inflammatory response, it is widely used in anti-inflammatory studies, including microglia [36]. When macrophages and microglia are overactivated by LPS, inflammatory substances such as nitrite and PGE 2 are secreted. Therefore, confirming the nitrite production inhibitory effect is a measure for confirming the anti-inflammatory effect, and the inhibitory effect of the isolated compound on nitrite production was compared (Figures 4 and 5). Compounds  1-4, 7, 8, and 13-18 inhibited nitrite production in LPS-induced BV2 cells. In addition, compounds 1-5, 7, 8, and 10-18 inhibited nitrite production in LPS-induced RAW264.7 cells. Compounds 6 and 9 did not inhibit nitrite production in either BV2 or RAW264.7 cells. In particular, it was confirmed that the novel compound 18 and known compounds 16 and 17 had a nitrite production inhibitory effect that is similar to or superior over the positive control at a concentration of 40 µM in both macrophages and microglia.
The compounds isolated in this study were classified into five groups according to their structures. This is because similar activity was confirmed depending on the structure type while confirming the nitrite production inhibitory effect . Compounds 1-3, 7, and 8 were identified as chalcone-type compounds. Chalcone has an α,β-unsaturated ketone with various pharmacological activities, including antioxidant, anti-inflammatory, anticancer, antiviral, and immunosuppressive [37]. Kanakugiol (1) was isolated from L. erythrocarpa [17], and the activity of 1 has been reported as free radical scavenging, antifungal, and anticancer [17,18,38]. 2 -hydroxy-3 ,4 ,6 -trimethoxychalcone (2) was previously isolated from Polygonaceae, Annonaceae, Piperaceae, and Rosaceae, and this study is the first to report isolation from L. erythrocarpa [29,39]. Compound 2 is known to have cytotoxic effects, and most compounds with a chalcone structure are similar to that of various active ingredients, including antioxidant and anti-inflammatory effects [39]. Pashanone (3) has been reported to be isolated from Polygonaceae [39]. Its known activity was similar with compound 2 [39]. There are only a few reports on Dihydropashanone (7) and 2 -hydroxy-3 ,4 ,6 -trimethoxydihydrochalcone (8) and the activity of these compounds. However, it was expected to show general activity of the chalcone type, and it was confirmed that nitrite was significantly inhibited in BV2 microglia and RAW264.7 macrophages. In this study, we confirmed that compounds 1-3, 7, and 8 have similar inhibitory effects on nitrite production in BV2 microglia and RAW264.7 macrophages. However, the anti-inflammatory effects of chalcone-type compounds are well known; thus, no further studies have been conducted [37].
Compounds 4-6 were identified as flavanone-type compounds in this study. Flavanones have a stereogenic center (chiral center) at the C-2 position and various pharmacological activities, including antioxidant, cytotoxic, antibacterial, and quinone reductase activities [40]. Kanakugin (4) was isolated from L. erythrocarpa, and its various activities including skin protection have been reported [41]. 5-hydroxyl-7,8-dimethoxyflavanone (5) was discovered as a novel compound in Fistigma cupreonitens and has been reported to reverse drug resistance in cancer [42]. Onysilin (6) has been known as the component in the bark of L. oxyphylla and the same genus. In this study, flavanone-type compounds such as compounds 4-6 did not effectively inhibit nitrite production compared with other types of compounds. Therefore, we did not conduct follow-up studies.
Compounds 9-12 were considered as the flavonol glycosides group. Although flavonols are widely known for their antioxidant, antihyperlipidemic, and anticancer effects, especially, the antioxidant effect of flavonol glycosides is reported to be weaker than that of flavonol aglycones [43][44][45]. The flavonol glycosides isolated in this study had weaker effects than other types of flavonoid compounds. These results showed the same aspect as the above-mentioned antioxidant effect between flavonol glycosides and aglycones.
Compounds 13-15 are identified as cyclopentadione, and these are known as major components of L. erythrocarpa [16]. These compounds were previously reported to have anti-inflammatory effects on macrophages [16]. Therefore, we did not investigate the anti-inflammatory effects of these three compounds.
Compounds 16 and 17, as well as the epimer of 18, were previously isolated from Lindera aggregata [21,46,47]. Compounds 16 and 17 were reported as possible to synthesize from methyllinderone (14) reacted by UV. Furthermore, these two have also been reported to exhibit significant activity against glucosamine-induced insulin resistance in HepG2 cells [22]. However, studies on the anti-neuroinflammatory effects related to degenerative brain diseases have not yet been conducted. Both compounds showed a structure similar to that of the novel compound 18. In addition, among the various structural types isolated from L. erythrocarpa leaves, it was found that compounds 16, 17 and 18 of the dimer of linderone type showed the best anti-inflammatory effect. Therefore, we selected compounds 16-18 for further investigation of their anti-inflammatory activity.
In the process of macrophage inflammatory response, inflammatory mediators are generated by the expression of pro-inflammatory proteins. The most important proteins that generate inflammatory mediators are iNOS and COX-2. Expression of iNOS is induced by various cytokines released by inflammatory and immune responses, tissue damage, and oxidative stress at the gene transcription stage to generate large amounts of NO [48]. COX-2 plays a role in promoting cell proliferation by inducing the production of large amounts of prostaglandins in the inflammatory process and increasing the inflammatory response by suppressing immunity [49]. Therefore, we performed additional mechanistic studies of compounds 16-18 for understanding the regulation of iNOS and COX-2, which are important factors regulating the inflammatory response ( Figure 6). All compounds inhibited the expression of the pro-inflammatory proteins iNOS and COX-2 in LPS-induced BV2 and RAW264.7 cells. Next, the inflammatory cytokine and inflammatory mediator PGE 2 , which induces the expression of pro-inflammatory proteins, were investigated ( Figure 7). As expected, all compounds inhibited the inflammatory cytokines TNF-α and IL-6, including PGE 2 . By investigating the mechanisms of pro-inflammatory proteins and inflammatory cytokines, compound 18 was found to have the best anti-neuritis and anti-inflammatory effects.
We further investigated the anti-inflammatory mechanisms of the three compounds, including the novel ones, by measuring NF-κB binding activity (Figure 8). NF-κB belongs to the rel family and consists of the homo-and heterodimeric forms. The most widely studied form of NF-κB is a heterodimer of the p50 and p65 subunits, and it is a potent activator of gene transcription. In cells without activity, NF-κB is mainly present in the cytoplasm in an inactive state; however, when activated, the NF-κB heterodimer, which forms a complex of p65 and p50, enters the nucleus and is activated [50]. Activated NF-κB induces pro-inflammatory mediators and cytokines including NO, PGE 2 , TNF-α, IL-6, iNOS, and COX-2. In particular, it is significantly associated with NF-κB activation in chronic inflammation-related diseases such as asthma, arteriosclerosis, acquired immunodeficiency syndrome, AD, and PD [51,52]. This finding is consistent with the target disease of this study, and we need to identify compounds that inhibit NF-κB activation. Compounds 16-18 were confirmed to have inhibitory effects on NF-κB binding activity. In addition, the degree of inhibition of each compound showed a tendency similar to that of pro-inflammatory proteins and cytokines. Therefore, our results are the first to report the anti-neuroinflammatory effects of compounds 16-18, and one novel compound discovered in this study was confirmed to have potential as a therapeutic agent for neurodegenerative diseases. However, it is necessary to confirm the therapeutic effect of these compounds on neurodegenerative diseases using additional anti-neuroinflammatory mechanisms and in vivo models.

Plant Materials
One-and two-dimensional NMR spectra were recorded in chloroform-d, and dimethyl sulfoxide (DMSO)-d6 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). Electrospray ionization mass spectrometry (ESIMS) data were obtained using a quadrupole time-of-flight mass spectrometer (Q-TOF) micro-liquid chromatography-mass spectrometry (LC-MS/MS) instrument (Waters, Milford, MA, USA) at the Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ohchang, Korea. Solvents used for extraction and flash column chromatography (CC) were of reagent grade and were used without further purification. The solvents used for high-performance liquid chromatography (HPLC) were of analytical grade. Flash CC was performed using YMC octadecyl-functionalized silica gel (C18) and silica gel (Merck, Darmstadt, Germany). HPLC separations were performed on a prep-C18 column (21.2 × 250 mm; 5 μm particle size) with a flow rate of 10 mL/min, and a semiprep-C18 column (10 × 250 mm; 5 μm particle size) with a flow rate of 3 mL/min.

Plant Materials
One-and two-dimensional NMR spectra were recorded in chloroform-d, and dimethyl sulfoxide (DMSO)-d 6 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). Electrospray ionization mass spectrometry (ESIMS) data were obtained using a quadrupole time-of-flight mass spectrometer (Q-TOF) micro-liquid chromatographymass spectrometry (LC-MS/MS) instrument (Waters, Milford, MA, USA) at the Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ohchang, Korea. Solvents used for extraction and flash column chromatography (CC) were of reagent grade and were used without further purification. The solvents used for high-performance liquid chromatography (HPLC) were of analytical grade. Flash CC was performed using YMC octadecyl-functionalized silica gel (C18) and silica gel (Merck, Darmstadt, Germany). HPLC separations were performed on a prep-C18 column (21.2 × 250 mm; 5 µm particle size) with a flow rate of 10 mL/min, and a semiprep-C18 column (10 × 250 mm; 5 µm particle size) with a flow rate of 3 mL/min.