Anti-Inflammatory, Neurotrophic, and Cytotoxic Oxylipins Isolated from Chaenomeles sinensis Twigs

Oxylipins are important biological molecules with diverse roles in human and plants such as pro-/anti-inflammatory, antimicrobial, and regulatory activity. Although there is an increasing number of plant-derived oxylipins, most of their physiological roles in humans remain unclear. Here, we describe the isolation, identification, and biological activities of four new oxylipins, chaenomesters A–D (1–4), along with a known compound (5), obtained from Chaenomeles sinensis twigs. Their chemical structures were determined by spectroscopic (i.e., NMR) and spectrometric (i.e., HRMS) data analysis including 1H NMR-based empirical rules and homonuclear-decoupled 1H NMR experiments. Chaenomester D (4), an omega-3 oxylipin, showed a potent inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells (NO production, 8.46 ± 0.68 μM), neurotrophic activity in C6 cells through the induction of the secretion of nerve growth factor (NGF, 157.7 ± 2.4%), and cytotoxicity in A549 human cancer cell lines (IC50 = 27.4 μM).


Introduction
Oxylipins are a family of oxygenated fatty acids found in diverse living organisms such as fungi, plants, and animals. In general, oxylipins are classified into two categories, omega-3 and omega-6. Interestingly, while the only difference in the chemical structure of omega-3 and 6 oxylipins is the existence of an additional double bond in omega-3 compared to omega-6 molecules, they have opposite actions in the inflammatory response [1]. Omega-3 oxylipins, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are anti-inflammatory, whereas omega-6 oxylipins are pro-inflammatory [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most well-known and widely used medicines which inhibit the biosynthesis of inflammation-related prostaglandins from omega-6 oxylipin arachidonic acid by blocking human cyclooxygenases (COX) [3][4][5][6]. In plants, jasmonic acid, synthesized from omega-3 α-linolenic acid, is a plant hormone regulating plant responses to abiotic and biotic stress as well as plant growth and development [7].
To find more potent anti-inflammatory oxylipins from C. sinensis, we further investigated its chloroform (CHCl3) and n-butanol (n-BuOH) fractions. Herein, we report the isolation, structure characterization, and biological activities of four new oxylipins, chaenomesters A-D (1)(2)(3)(4), along with a known compound (5) from C. sinensis twigs (Figure 1). Their chemical structures were elucidated by an intensive analysis of NMR and HRMS data. Among the isolated compounds, the new compound chaenomester D (4) showed potent anti-inflammatory, neurotrophic, and cytotoxic activities.

Plant Material
Twigs of two-year-old C. sinensis (7.0 kg) were purchased in January 2012 from Yangjae Flower Market in Seoul, Korea. A voucher specimen of the plants (SKKU-NPL 1206) was authenticated by Prof. Kang Ro Lee and stored in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Nitric Oxide (NO) Assay
BV-2 cells, developed by Dr. V. Bocchini, were employed to evaluate the antiinflammatory effect of the isolated compounds [13]. The BV-2 microglioma cell line was used for measuring the produced levels of nitrite (NO 2 -) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in 5% CO 2 . The cells were seeded in a 96-well plate (4 × 10 4 cells/well) and then treated with the tested compounds (1-5) at 20 µM for 30 min prior to treatment with 100 ng/mL of lipopolysaccharide (LPS) and incubation for 1 day. The produced NO 2 levels, a soluble oxidized product of NO, was evaluated with Griess reagent including 0.1% N-1-napthylethylenediamine dihydrochloride and 1% sulfanilamide. The supernatant (50 µL) was mixed with an equal volume of Griess reagent. The absorbance was measured at 570 nm in 10 min. A graded sodium nitrite solution was used as a standard to determine the nitrite concentrations. In addition, the cytotoxicity of each compound was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay at the same concentration, and we calculated the concentration of compound that caused a 50% inhibition of cell proliferation. All samples were dissolved in DMSO. Before treatment, the final concentration of 0.01% DMSO was used, which did not show cytotoxicity [14]. The reported nitric oxide synthase (NOS) inhibitor N G -monomethyl-L-arginine (L-NMMA) was used as a positive control.

Nerve Growth Factor (NGF) Assay
In order to measure the induction of nerve growth factor (NGF) release, C6 glioma cells were used and maintained in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in 5% CO 2 . The tested cells were seeded onto a 24-well plate (1 × 10 5 cells/well) and incubated for 1 day. The cells were treated with the isolated compounds at a 20 µM concentration (1)(2)(3)(4)(5) in DMSO, together with serum-free DMEM for 1 day. To measure the released NGF levels in the cell supernatants after treatment, an ELISA development kit (R&D System, Minneapolis, MN, USA) was used. In addition, an MTT assay was carried out to evaluate the cell viability in comparison with cells treated with 6-shogaol (positive control), and the results are presented as percentage viability with respect to the control group. The NGF secretion values for each treatment were divided by the respective cell viability values to calculate normalized NGF secretion levels which were further statistically analyzed with the Student's t-test using GraphPad Prism 8. Statistical significance was considered for * p < 0.05, ** p < 0.01, and *** p < 0.001.

Cytotoxicity Assignment
The cytotoxic activity of the compounds (1)(2)(3)(4)(5) in four cultured human cancer cell lines was evaluated utilizing the sulforhodamine B colorimetric (SRB) method [15]. Each cell line was inoculated into standard 96-well flat-bottom microplates and incubated for 24 h at 37 • C in a humidified atmosphere with 5% CO 2 . The attached cells were incubated with the compounds serially diluted in DMSO (30, 10, 3, 1, 0.3, and 0.1 µM). After continuous exposure to these compounds for 48 h, the culture medium was removed, and the cells were fixed with 10% cold trichloroacetic acid at 4 • C for 1 h. After washing with tap water, the cells were stained with 0.4% SRB dye and incubated for 30 min at room temperature. These cells were washed again and then solubilized with a 10 mM unbuffered Tris base solution (pH 10.5). The absorbance was measured spectrophotometrically at 520 nm using a microtiter plate reader. The cell lines used for this study were A549 (non-small cell lung adenocarcinoma), SK-OV-3 (from an ovary malignant ascite), SK-MEL-2 (skin melanoma), and MKN-1 (adenosquamous carcinoma) and were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained at the Korea Research Institute of Chemical Technology. Etoposide (≥ 98%; Sigma Chemical Co., St. Louis, MO, USA) was used as a positive control.

Structure Elucidation of Compounds 1-5
Chaenomester A (1) was obtained as a colorless gum. The HRFAMMS analysis indicated that its molecular formula is C 15  ] groups. These spectroscopic data were quite similar to those of pinellic acid methyl ester [16], except for the absence of four methylene signals in 1 compared to pinellic acid methyl ester, suggesting 1 to be a C14 oxylipin rather than a C18 oxylipin. We performed a 2D NMR analysis of 1, including 1 H-1 H COSY, HSQC, and HMBC (Supplementary Materials), and the data were analyzed to elucidate the planar structure of 1. The 1 H-1 H COSY correlations from H-2 to H-14 including two olefinic (H-6 and H-7) and three oxygenated methine protons (H-5, H-8, and H-9), and the HMBC cross-peaks of H-2/C-1 and C-4, H-3/C-1, H-4/C-6, H-5/C-6 and C-7, H-8/C-7, and C-9, and H-14/C-12 and C-13 confirmed a C14 oxylipin scaffold for 1 and the location of a double bond and three hydroxy groups ( Figure 2). Additional HMBC correlation of H-15/C-1 indicated the methoxy group to be connected at C-1 through an ester bond. Antioxidants 2023, 12, x 6 of 13 The geometry of the double bond at C-6/C-7 was determined to be E by observing a relatively large coupling constant of H-6/H-7 (15.5 Hz) [17]. The relative configuration of the three hydroxy groups in 1 was initially assumed to be the same as that of pinellic acid, a natural product isolated from Pinellia ternata [18], and further confirmed by intensive comparative 1 H NMR data analysis as follow. In 2006, Shirahata et al. synthesized eight possible stereoisomers of pinellic acid and established 1 H NMR (in methanol-d4)-based empirical rules for the assignment of the relative configuration of three hydroxy groups [19]. In brief, a more downfield-shifted 1 H NMR signal of H-10 (δH 5.70/5.72) than of H-11 (δH 5.64/5.67) indicated a (12,13)-syn configuration, and the chemical shift of H-13 in (12,13)-syn-form, δH 3.41, was smaller than that in (12,13)-anti-form, δH 3.48 ( Figure 3A). Discrimination of the (9,12)-syn/anti configuration was achieved by calculating the ΔδH-   The geometry of the double bond at C-6/C-7 was determined to be E by observing a relatively large coupling constant of H-6/H-7 (15.5 Hz) [17]. The relative configuration of the three hydroxy groups in 1 was initially assumed to be the same as that of pinellic acid, a natural product isolated from Pinellia ternata [18], and further confirmed by intensive comparative 1  The geometry of the double bond at C-6/C-7 was determined to be E by observing a relatively large coupling constant of H-6/H-7 (15.5 Hz) [17]. The relative configuration of the three hydroxy groups in 1 was initially assumed to be the same as that of pinellic acid, a natural product isolated from Pinellia ternata [18], and further confirmed by intensive comparative 1 H NMR data analysis as follow. In 2006, Shirahata et al. synthesized eight possible stereoisomers of pinellic acid and established 1 H NMR (in methanol-d4)-based empirical rules for the assignment of the relative configuration of three hydroxy groups [19]. In brief, a more downfield-shifted 1 H NMR signal of H-10 (δH 5.70/5.72) than of H-11 (δH 5.64/5.67) indicated a (12,13)-syn configuration, and the chemical shift of H-13 in (12,13)-syn-form, δH 3.41, was smaller than that in (12,13)-anti-form, δH 3.48 ( Figure 3A). Discrimination of the (9,12)-syn/anti configuration was achieved by calculating the ΔδH-10/H-11 value. A relatively smaller ΔδH-10/H-11 value indicated a (9,12)-anti configuration [(9,12)-syn: 0.06 ppm, (9,12)-anti: 0.05 ppm]. By observing a larger δH-6 value (5.72 ppm) than δH-7 value (5.69 ppm), a small δH-9 value (3.41 ppm), and a small Δ δH-6/H-7 value (0.03 ppm) for 1, we were able to determine the relative configuration of 1 as (5,8)-anti and (8,9)syn ( Figure 3). Therefore, the structure of 1 was elucidated as methyl (5R*,8R*,9R*)-5,8,9trihydroxy-6E-tetradecenoate.    1H, m, H-11), and δ C 134.4 (C-12) and 126.4 (C-11) for 2 instead of two methylene signals, as observed for 1. The location of the double bond was confirmed at C-11/C-12 by 1 H-1 H COSY correlations of H-10/H-11 and H-12/H-13 and HMBC correlations of H-9/C-11, H-11/C-10, H-12/C-10, H-13/C-11, H-14/C-12, and C-13 ( Figure 2). The geometry of the double bond at C-11/C-12 was suggested to be Z based on the identical 1 H and 13 C NMR signals of the olefinic functionality in 2 and corchorifatty acid F, which have Z configuration ( Figure 4A) [20]. Hence, the structure of 2 was determined as methyl (5R*,8R*,9R*)-5,8,9-trihydroxytetradeca-6E,11Z-dienoate. Chaenomester B (2) has the molecular formula C15H26O5, identified from the sodiated molecular ion at m/z 309.1672 [M + Na] + (calcd. for C15H26O5Na + , m/z 309.1672). The 1 H and 13 C NMR data of 2 resembled those of 1, except for the presence of double bond signals at δH 5.46 (1H, m, H-12) and 5.44 (1H, m, H-11), and δC 134.4 (C-12) and 126.4 (C-11) for 2 instead of two methylene signals, as observed for 1. The location of the double bond was confirmed at C-11/C-12 by 1 H-1 H COSY correlations of H-10/H-11 and H-12/H-13 and HMBC correlations of H-9/C-11, H-11/C-10, H-12/C-10, H-13/C-11, H-14/C-12, and C-13 ( Figure 2). The geometry of the double bond at C-11/C-12 was suggested to be Z based on the identical 1 H and 13 C NMR signals of the olefinic functionality in 2 and corchorifatty acid F, which have Z configuration ( Figure 4A) [20]. Hence, the structure of 2 was determined as methyl (5R*,8R*,9R*)-5,8,9-trihydroxytetradeca-6E,11Z-dienoate.   Figure 2). We tried to measure the coupling constant between H-15 and H-16 to assign the geometry of the double bond, but we were not successful because of the complex splitting patterns of H-15 and H-16. To overcome this issue, we employed a classical selective 1 H homonuclear decoupling technique in the 1 H NMR measurement [21,22]. Briefly, if a proton resonance A is irradiated during acquisition, its coupling partner B loses its coupling to the irradiated proton. As a consequence, we could observe a more simplified splitting of B resonance and then calculate the coupling constant of B with other proton signal(s). As shown in the middle of Figure 4B, when H-17 was irradiated in 4, the splitting pattern of H-16 became simplified from a multiplet to a doublet, with a coupling constant of 10.9 Hz, indicating that the geometry of the double bond at C-15/C-16 was the cis-form. The same result was obtained by analyzing the H-14-irradiated homonuclear decoupled 1 H NMR spectrum ( Figure 4B, bottom). Collectively, we propose the structure of 4 to be ethyl (9R*,12R*,13R*)-9,12,13-trihydroxy-10E-octadecenoate.
The known compound 5 was identified as 1'-hexadecanoic acid-2',3'-dihydroxypropylester by the comparison of its spectroscopic data with those of the reference [23].

Biosynthetic Proposal
With the characterized the structures of 1-4 in hand, we then were able to propose their biosynthetic pathway ( Figure 5). Pinellic acid has been previously suggested to be synthesized from linoleic acid by lipoxygenase, epoxy alcohol synthase, and epoxide hydrolase [24]. Further ethylation on pinellic acid would produce compound 3. Similarly, goshuyic acid, a C14 fatty acid, would be utilized to generate the acid i, and then compound 1 would be synthesized by methylation on i. We propose that compounds 2 and 4 would be produced from (5Z,8Z,11Z)-5,8,11-tetradecatrienoic acid and α-linolenic acid, respectively, by the same enzymatic reaction as those necessary for 1 and 3.
(C-15) observed in 4. The analysis of the 1 H-1 H COSY and HMBC data of 4 around the double bond led us to propose the location of the double bond at C-15/C-16 ( Figure 2). We tried to measure the coupling constant between H-15 and H-16 to assign the geometry of the double bond, but we were not successful because of the complex splitting patterns of H-15 and H-16. To overcome this issue, we employed a classical selective 1 H homonuclear decoupling technique in the 1 H NMR measurement [21,22]. Briefly, if a proton resonance A is irradiated during acquisition, its coupling partner B loses its coupling to the irradiated proton. As a consequence, we could observe a more simplified splitting of B resonance and then calculate the coupling constant of B with other proton signal(s). As shown in the middle of Figure 4B, when H-17 was irradiated in 4, the splitting pattern of H-16 became simplified from a multiplet to a doublet, with a coupling constant of 10.9 Hz, indicating that the geometry of the double bond at C-15/C-16 was the cis-form. The same result was obtained by analyzing the H-14-irradiated homonuclear decoupled 1 H NMR spectrum ( Figure 4B, bottom). Collectively, we propose the structure of 4 to be ethyl (9R*,12R*,13R*)-9,12,13-trihydroxy-10E-octadecenoate.
The known compound 5 was identified as 1'-hexadecanoic acid-2',3'-dihydroxy-propylester by the comparison of its spectroscopic data with those of the reference [23].

Biosynthetic Proposal
With the characterized the structures of 1-4 in hand, we then were able to propose their biosynthetic pathway ( Figure 5). Pinellic acid has been previously suggested to be synthesized from linoleic acid by lipoxygenase, epoxy alcohol synthase, and epoxide hydrolase [24]. Further ethylation on pinellic acid would produce compound 3. Similarly, goshuyic acid, a C14 fatty acid, would be utilized to generate the acid i, and then compound 1 would be synthesized by methylation on i. We propose that compounds 2 and 4 would be produced from (5Z,8Z,11Z)-5,8,11-tetradecatrienoic acid and α-linolenic acid, respectively, by the same enzymatic reaction as those necessary for 1 and 3.

Anti-Neuroinflammatory Activity of the Isolated Compounds (1-5)
Chemically, 2 and 4 are classified as omega-3 oxylipins since they possess a double bond at the omega-3 position, and 1 and 3 are omega-6 fatty acid-derived oxylipins. Given the important roles of the omega-3 and 6 oxylipins in the inflammatory response, as we mentioned earlier, we first tested the isolated compounds (1-5) for their anti-inflammatory effect by measuring the level of NO production after treatment with the compounds at a concentration of 20 µM in LPS-stimulated BV-2 microglia cells. Among the tested compounds including the positive control, L-NMMA (produced NO concentration = 22.37 ± 0.69 µM), the most potent inhibitor was 4, an omega-3 oxylipin, with an NO level of 8.46 ± 0.68 µM and without cell toxicity (Table 2). Compounds 3 and 5 also showed strong NO inhibitory activity, with reduced values of 30.70 ± 1.38 and 24.09 ± 1.11 µM, respectively, and compounds 1 (38.56 ± 1.61 µM) and 2 (38.69 ± 1.41 µM) were not active, as the NO levels were similar to that observed in the LPS-treated group (39.95 ± 1.17 µM). With these NO inhibition data for 1-5 and other four structurally similar compounds our group reported previously [8], we suggest a structure-activity relationship (SAR) for these oxylipin compounds as follow ( Figure 6). Collectively, we propose that the presence of a double bond at C-15/C-16 (omega-3 position) and of an ethyl ester functionality is important for a strong anti-inflammatory activity, which is consistent with the fact that some omega-3 oxylipins such as EPA and DHA are anti-inflammatory agents [2,25]. In addition, an ethyl ester is generally more than two-fold stable than a methyl ester during hydrolysis, indicating that an ethyl ester would lend not only anti-neuroinflammatory activity but also stability to the molecule [26].  Figure 6. Structure-activity relationship (SARs) for the isolated metabolites (1)(2)(3)(4) and other four structurally similar metabolites our group reported previously [8].

Neurotrophic Activity of the Isolated Compounds (1-5)
In addition to the anti-neuroinflammatory effect, several omega-3 oxylipins have also shown neuroprotective effect by decreasing inflammation and apoptosis [27][28][29], inhibiting astrogliosis [30], activating mitochondria [29], promoting angiogenesis, and regulating the blood flow [30,31]. Therefore, we also evaluated the compounds 1-5 for their neurotrophic effects and ability to induce NGF secretion from C6 glioma cells (Table 3). Compound 4 exhibited a significant NGF-stimulating effect, inducing NGF secretion that was 157.7 ± 2.4% ( *** p < 0.001) of that measured for the untreated control, without cell toxicity, and stronger than that induced by 6-shogaol (154.0 ± 5.6%, ** p < 0.01), the positive control. Compared with the untreated control group, compounds 3 and 5 were mild NGF inducers, with 126.7 ± 2.4% ( * p < 0.05) and 123.8 ± 11.0% ( * p < 0.05) of stimulating potency, respectively, and compounds 1 and 2 were inactive (NGF secretion ≈100%). 123.8 ± 11.0 * 101.6 ± 1.4 6-shogaol 3 154.0 ± 5.6 ** 97.1 ± 0.2 1 C6 cells were treated with the compounds at 20 µM concentration. After 24 h, the level of NGF secretion in C6-conditioned medium was measured by ELISA. The level of secreted NGF from the cells is expressed as a percentage of the level measured for the untreated control divided by the respective cell viability values. The data shown represent the means ± SD of three independent experiments performed in triplicate. Significant differences between the experimental groups were determined using one-way analysis of variance followed by the Newman-Keuls post hoc test, used to measure statistical significance using GraphPad Prism 8. Statistical significance was set to * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group 2 Cell viability after treatment with each compound at 20 µM concentration was determined by the MTT assay and is expressed in percentage (%).
The results are averages of three independent experiments, and the data are expressed as mean ± SD; 3 6-shogaol was the positive control.

Conclusions
Our current study revealed four new oxylipins, chaenomesters A-D (1-4), isolated from the twigs of C. sinensis along with the structurally similar known compound 5. The structural elucidation of the isolates was performed mainly by HRMS and NMR data analysis, and the stereochemical assignments were achieved by 1 H NMR-based empirical rules and homonuclear decoupled 1 H NMR experiments. Among the isolates, chaenomester D (4), an omega-3 oxylipin, showed potent neurotrophic activity in C6 glioma cells, mild anti-neuroinflammatory activity in LPS-stimulated BV-2 microglia cells, and mild cytotoxic activity in A549 cancer cells. The SAR study suggested the double bond at C-15/C-16 and the ethyl ester functionality in chaenomester D (4) would play an important role in the production of a potent anti-inflammatory effect. Collectively, we propose that chaenomester D (4) could contribute to the development of new drugs to treat neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.