Separation, characterization and anti-inflammatory activities of galactoglycerolipids from Perilla frutescens (L.) Britton

Abstract The study was to optimize the separation procedures, characterize the galactoglycerolipids and explore their anti-inflammatory activities. Two monogalactosyldiacylglycerols (MGDGs) and three digalactosyldiacylglycerols (DGDGs) from Perilla frutescens (L.) Britton were obtained through one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The presence of additional MGDG (1-O-9Z,12Z,15Z-octadecatrienoyl-2-O-7Z,10Z,13Z-hexadecatrienoyl-3-O-(β-D-galactopyranosyl)-sn-glycerol) and DGDG (1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Z-octadecatrienoyl-3-O-(β-D-galactopyranosyl-(1'→6'')-α-D-galactopyranosyl)-sn-glycerol) was concluded for the first time in perilla, by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In lipopolysaccharide (LPS)-induced RAW264.7 cells, five galactoglycerolipids exhibited good inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner, suggesting that fatty acid chain length and unsaturation degree affected their anti-inflammatory activities. Graphical Abstract


Introduction
Monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols (DGDGs) account for 75% of cell membrane lipids in higher plants (D€ ormann and Benning 2002), and are attracting much attention due to their anti-inflammatory activities (Banskota et al. 2013;de Los Reyes et al. 2016).Although many studies indicated that fatty acids or galactoses might affect the anti-inflammatory activities of galactoglycerolipids (Lopes et al. 2014;Tena Perez et al. 2020), the structure-anti-inflammatory activity relationship was unclear.Therefore, more glycoglycerolipids and related researches are needed for a comprehensive understanding of the structure-anti-inflammatory activity relationship.
Perilla frutescens (L.) Britton is widely distributed and cultivated in East Asia, and is famous for its medicinal properties (Bachheti et al. 2014).Galactoglycerolipids are more abundant in leaves than that in stems and roots of higher plants (Christensen 2009).They were also reported to be abundant in perilla (Sugawara and Miyazawa 1999).Therefore, perilla leaves would be good resources for the extraction of galactoglycerolipids.
Previous studies mostly used repeated silica gel column chromatography to obtain galactoglycerolipids (Kiem et al. 2012;Sashidhara et al. 2012), which is time-consuming with low recovery.Therefore, we carried out the study on a simple separation method of galactoglycerolipids from Perilla frutescens (L.) Britton by one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light detector (HPLC-ELSD).Their structures were identified by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR).In addition, all isolated galactoglycerolipids were evaluated for their anti-inflammatory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 cells.

Results and discussion
The separation and characterization protocol for galactoglycerolipids from Perilla frutescens (L.) Britton is shown in Figure S1.One-step silica gel column chromatography could completely isolate MGDGs (Rf ¼ 0.62) and DGDGs (Rf ¼ 0.17), which were eluted by chloroform: acetone (1:1, v/v) and acetone elution, respectively (Figure S2).Crude MGDGs (378 mg/1.2 g total lipids) and crude DGDGs (185 mg/1.2 g total lipids) were further separated by preparative HPLC-ELSD (Figure S3) to gain 10 peaks.This separation method was simple, time-saving and had higher recovery than the previous method in our laboratory (Zi et al. 2021).Then the substances of 10 peaks were verified again by silica gel thin-layer chromatography (TLC) (Figure S4).No methyl esters were detected in the substances of peaks 4 and 5 by gas chromatography-mass spectrometry (GC-MS), and no fragment ions related to glycolipids existed in the substances of peaks 1, 6, and 7 by LC-MS.Therefore, the substances of peaks 2, 3, 8, 9, and 10 were numbered compounds A (66.8 mg/1.2 g total lipids), B (159.5 mg/1.2 g total lipids), C (68.3 mg/1.2 g total lipids), D (4.2 mg/1.2 g total lipids) and E (24.4 mg/1.2 g total lipids), respectively.The purity of them was more than 95% (Figure S5).The fatty acid compositions of the five galactoglycerolipids are shown in Table S1.The structures of compounds B, C, and E are shown in Figure 1 showing the presence of hexose fragment (m/z 185) and hexose glycerol fragment (m/z 243) (Figure S9).The fragment ion peaks m/z 491 and 519 were due to the loss of octadecatrienoic acid and the loss of hexadecatrienoic acid (Erwan et al. 2013).The loss of R 1 COOH was more abundant than that of R 2 COOH, which suggested that the octadecatrienoic acid and hexadecatrienoic acid were attached to the sn-1 and the sn-2 of the glycerol, respectively (Guella et al. 2003).
The 1 H and 13 C NMR data of compound A revealed the presence of three-peaked proton signal (d H 0.9), methylenes (d H 1.27-1.33),double bonds of fatty acids (d H 5.24-5.37;d C 127.10-132.25), a terminal methyl carbon (d C 14.51) and one sugar (d C 103.92) (Table S2; Figures S11 and S12  D configuration of the galactose residue.The COSY and HSQC spectra showed the coupling between the adjacent hydrogen atoms (Figure S13A) and the coupling between the adjacent C and H atoms (Figure S13B).The HMBC spectrum (Figure S13C) displayed a remote correlation between C-4 and H-2.Moreover, H-3 correlated with C-1 0 and H-1 showed a remote correlation with C-37 (Dai et al. 2001).Thus, the sugar linkage was at the sn-3 of the glycerol.Combined with the COSY and HSQC spectrum, the position and numbers of double bonds in the fatty acids were indicated in the HMBC spectrum, in consistent with the results of GC-MS  S10).The positive ion mode gave the characteristic ions of two hexoses linked fragments (m/z 347), two hexoses and a glycerol linked fragment (m/z 405).The fragment ion peaks m/z 681 and 683 were consistent with the loss of octadecadienoic acid and the loss of octadecatrienoic acid, respectively.The fragment ion peak m/z 799 was due to the loss of a hexose.The fatty acid at sn-1 of the glycerol was eliminated by the action of lipase XI (Guella et al. 2003) and determined to be octadecadienoic acid by GC-MS (m/z 294, methyl octadecadienoate) (Figure S14).According to the MS fragmentation data of compound D in Figure S10 and subsequent NMR data, the octadecatrienoic acid was linked at the sn-2 position.
The 1 H NMR data of compound D showed the existence of two fatty acyl chains characterized by two triplet methyl signals (d H 0.89 and 0.95), an extensive methylene signal (d H 1.27), and several olefinic methine protons (d H 5.29-5.39)(Table S2; Figures S15 and S16).The 13 C NMR spectrum revealed the presence of a terminal methyl carbon (d C 13.08 and 13.3) and two galactoses (d C 99.25 and d C 103.92).H-1 0 signal (d H 4.23, J ¼ 7.8 Hz) and H-1 0 ' signal (d H 4.83, J ¼ 3.9 Hz) indicated the presence of a b and an a-galactopyranoside linkage, respectively.The chemical shift of C-6 0 ' (d C 68.66) was in lower field compared with C-6 0 (d C 62.62), suggesting that two galactoses were connected 1!6 (de Los Reyes et al. 2016).Compound A and compound D were all extracted from perilla.According to the measurement result of compound A, the configuration of galactose residues in compound D should be D. The COSY and HSQC data are shown in Figures S17 and S18.As shown in the HMBC spectrum, H-1 was remotely correlated with C-39, and H-2 was remotely associated with C-4.H-3 was remotely correlated with C-1 0 (Figure S19).According to the chemical shifts of the allylic carbons (d C 24.65-29.36),the geometric configurations of D 9 and D 12 in octadecadienoic acid and D 9 , D 12 , and D 15 in octadecatrienoic acid were defined to be Z.Compound D was therefore identified as 1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Zoctadecatrienoyl-3-O-(b-D-galactopyranosyl-(1 0 !6 0 ')-a-D-galactopyranosyl)-sn-glycerol.
Compounds A-E were tested for their cytotoxicity by MTT assay in mouse macrophage RAW264.7 cells.The results are shown in Figure S20.When the concentrations of galactoglycerolipids were higher than 12.5 lg/mL, compounds A, C and E were cytotoxic to cells.However, when the concentrations of galactoglycerolipids were no more than 12.5 lg/mL, all of the cell viabilities were above 100%, which suggested that low concentrations of galactoglycerolipids had promotion effects during cell growth.
Five compounds were tested through NO and iNOS gene expression inhibition assays in LPS-induced RAW264.7 cells.The results are showed in Table S3.All compounds had significant inhibition effects against NO and iNOS gene expression in a dose-dependent manner.Compound B (C18:3/C18:3) showed significant inhibitory effects compared with compound A (C16:3/C18:3) (p < 0.05).Compound C (C18:3/ C18:3) and compound D (C18:2/C18:3) significantly reduced NO and iNOS gene levels compared with compound E (C16:0/C18:3) (p < 0.05).These results suggested that fatty acid chain length affected the anti-inflammatory activities.Compound C (C18:3/ C18:3) exhibited significant inhibitory effects compared with compound D (C18:2/ C18:3) (p < 0.05), demonstrating that unsaturation degree also influenced the antiinflammatory activities.Our results that fatty acid chain length and unsaturation degree influenced the anti-inflammatory activities were consistent with those (Kiem et al. 2012;Banskota et al. 2013;Leutou et al. 2020).At the same time, compound B showed significant NO and iNOS gene expression inhibition activities compared with compound C (p < 0.05).The differences between them are the number of galactoses and unsaturation bond positions, but what factor is at work that is not sure.Further experiments are needed to determine.

Conclusion
In this study, a simple, time-saving with high recovery separation method for the isolation of galactoglycerolipids from Perilla frutescens (L.) Britton by one-step silica gel column chromatography and preparative HPLC-ELSD was developed.The presence of additional MGDG and DGDG was concluded for the first time in perilla, by LC-MS and NMR.Compounds A-E all exhibited inhibitory activities against NO and iNOS gene expression in LPS-induced RAW264.7 cells, and fatty acid chain length and unsaturation degree affected the anti-inflammatory activities.However, the role of galactoses or unsaturation bond positions in anti-inflammatory activities cannot be sure, and further experiments are needed to determine.

Figure 1 .
Figure 1.Structures of compounds A-E.