Two truxinate derivatives and a phenyldilactone from the leaves of Castanopsis eyrei

Abstract Phytochemical investigation of the Leaves of Castanopsis eyrei led to the isolation of two new natural truxinate derivatives and a new phenyldilactone. The structures of the new natural compounds were determined by spectroscopic methods and chemical evidence as 3,3',4,4'-tetrahydroxy-β-truxillic acid (1), 3,3',4,4'-tetrahydroxy-δ-truxillic acid (2), 3'-hydroxymaysedilactone A (3). Establishment of a Caenorhabditis elegans lipid metabolism model using GFP and mCherry fluorescently labeled lipid droplets to screen compound 3 for its activity in reducing lipid accumulation. Graphical Abstract


Introduction
The genun Castanopsis (D. Don) Spach., belongs to Fagaceae family, is represented by over 120 species in southern and southeastern Asia (Flora Republicae Popularis Sinicae 1998).There are about 63 species in China, and the resources are very rich.According to the existing literature, the main chemical constituents of Castanopsis genus include gallotannins, ellagitannins, phenolic acids, flavonoids and triterpenoids ellagitannins.

Results and discussion
The fresh leaves of C. eyrei were extracted with 80% (25 L) aqueous methanol, and the extracts were repeated column chromatography over Sephadex LH-20, Diaion HP 20SS, Chromatorex ODS, Toyopearl Butyl-650C, MCI gel CHP 20P and Toyopearl HW-40F to yield three new natural compounds.The structures of 1-3 are shown in Figure 1.
Compound 1 was obtained as brown-yellow amorphous powder.Its molecular formula C 18 H 16 O 8 was established on the basis of deprotonated molecule [M À H] À at m/ z 359.0759 (calcd 359.0772) in the negative mode, with a calculated unsaturation number of 11 supported by the NMR spectroscopic data.The small number of resonances in both the 1 H and 13 C NMR spectra (Table S1) coupled with the MS data indicated that 1 was a symmetrical molecule containing eleven degrees of unsaturation.Compound 1 reacts with ethanolic FeCl 3 to give a dark blue coloration, indicating the presence of the phenolic group in the molecule.The 1 H NMR spectrum in acetone-d 6 showed six aromatic proton signals at d H 6.67 (dd, J ¼ 2.0, 8.2 Hz), 6.75 (d, J ¼ 8.2 Hz) , 6.84 (d, J ¼ 2.0 Hz) revealing the presence of two 1,3,4-trisubstituted aromatic rings.The 13 C NMR spectrum displayed 18 carbon signals, including two carbonyl (d C 174.1), 12 aromatic carbons for two aromatic rings and four methine carbons [d C 41.8, 47.9].It was evident from the unsaturation number that a cyclobutane ring was present in the molecule of 1 (Morrison et al. 1991).The NOESY correlations between H-2 and H-8 (H-2 0 and H-8 0 ) , and H-6 and H-8 (H-6 0 and H-8 0 ) suggest that H-7/H-8 and H-7 0 /H-8 0 are in trans configuration, then the structure of compound 2 is one of four ring arrangements: a-truxinic, d-truxinic, e-truxillic, or b-truxillic (Fujiwara et al. 2016).The negative ESI-MS showed ions at m/z 243 in addition to that measured at m/z 179, suggested the molecule to be a head-to-head dimer (Fujiwara et al. 2016).It has been confirmed that the chemical shift of the H-8/H-8 0 atom of the b-truxinic structure is different from that of the H-8/H-8 0 of the d-truxinic structure, since it falls within the deshielding zone created by the aromatic ring, while the H-8/H-8 0 atoms of the d-truxinic structure are subject to their shielding effect.According to published data 1 H-NMR data that all H-8/H-8 0 chemical shifts of b-truxinic structures with acylated C-8/C-8 0 atoms have been observed between 3.75 ppm and 4.23 ppm (Sudo et al. 2000;Krauze-Baranowska et al. 2013;Fujiwara et al. 2016), while H-8/H-8 0 chemical shifts of d-truxinic structures have been observed between 3.24 ppm and 3.40 ppm (Montaudo and Caccamese 1973;Wang et al. 2014).The chemical shifts of the H-8/H-8 0 atoms of 1, 3.75 ppm, indicated that the ring arrangement of 1 was of the b-truxinic type.
Compounds 2 was obtained as brown-yellow amorphous power with [a] 26 D ¼ À 72 (c 0.1, MeOH).According to HRESIMS, compounds 2 has the same molecular formula as 1.Comparison of the 1 H NMR data in acetone-d 6 of 2 with 1 showed a major chemical shift difference for H-8/H-8 0 [d H 3.75 (1); d H 3.22 (2)] and H-7/H-7 0 [d H 4.18 (1); d H 3.42 (2)] .These differences have been proposed to be due to the anisotropic effects of the aromatic rings attached to C-7 and C-7 0 (Fujiwara et al. 2016).The key correlations of H-7 with C-9/C-9 0 (d C 174.1), H-7 0 with C-9/C-9 0 (d C 174.1) in the HMBC spectrum of 1 and the correlations of H-7 with C-9 (d C 174.8), H-7 0 with C-9 0 (d C 174.8) in the HMBC spectrum of 2 (Supplementary data, Figure S1) suggested that the ring arrangement of 2 was of the d-truxinic type.
Compound 3 was obtained as brown-yellow amorphous powder with [a] 26 D ¼ þ 62 (c 0.1, MeOH).The molecular formula of 3 was deduced as C 15 H 16 O 9 based on the HR-ESI-MS deprotonated molecule [M-H] À at m/z 339.0711, with a calculated unsaturation number of 8 supported by the NMR spectroscopic data.The 1 H NMR spectrum of 3 (Table S2) showed aromatic signals of an ABX system at d H 6.88 (d, J ¼ 2.1 Hz), d H 6.77 (dd, J ¼ 2.1, 8.2 Hz) and d H 6.72 (d, J ¼ 8.2 Hz) integrating for protons assigned to H-2 0 ', H-6 0 ' and H-5 0 ', respectively.This is also confirmed by the six aromatic carbon signals in the 13 C NMR spectroscopic data of 3. DEPT spectroscopic data of 3 showed the presence of 9 carbon signals comprising two carbonyl carbons, one oxygenated quaternary carbon, a primary carbon atom, three oxy-methine carbons, one methylene and one oxy-methylene carbon in addition to six aromatic carbons.The above carbon signal and number of unsaturation led us to propose a phenyldilactone skeleton (Okoye et al. 2014).Based on the observed correlations of H-4 with C-1" and C-2"/C-6" (HMBC), the attachment position of the hydroxyphenyl group of the dilactone was speculated to be C-4.The relative stereochemistry at C-4, C-4a, C-7a, and C-7 was determined by ROESY correlations of protons H-7a, H-7, and H-4 while lack of ROESY correlation between the proton of the hydroxyl group attached to C-4a.The coupling constant values of J 7a,7 ¼ 8.7 Hz suggested a cis confifiguration of protons H-7a and H-7 and HCCH dihedral angle of about 30 or less.The cis configurations of H-7 and H-2 0 and the HCCH dihedral angle of about 60 can be inferred from the observed coupling constant J 7,2 0 ¼ 2.3 Hz.On the basis of the above spectral data and by comparison with literature values (Okoye et al. 2014), Compound 3 was thus deduced as 7-(1,2-dihydroxyethyl)-4a-hydroxy-4-(3,4-dihydroxyphenyl)tetrahydro-2H-furo [3,4b]pyran-2,5 (3H)-dione which we named 3 0 -hydroxymaysedilactone A.
Lipids play a biological role in almost all aspects of life, including growth, development, reproduction, aging, and longevity, and are also closely related to lipid metabolism diseases.Polyphenols are involved in regulating the body's physiological processes such as cholesterol absorption, triglyceride synthesis and secretion, and plasma low-density lipoprotein oxidation in various ways, and play an important role in maintaining or improving the body's lipid metabolism balance.In this paper, compound 3 has been proved not to directly inhibit the synthesis of fat or increase the consumption of fat through C. elegans fat-reducing model test.After three repeated experiments, the results showed that the total fluorescence intensity of C. elegans increased by 18.8%, and the average fluorescence intensity increased by 31.3% compared with the control group (Table S3).The increase of the average fluorescence intensity was higher than that of the total fluorescence intensity, indicating that the C. elegans were smaller and the lipid droplets were larger and larger, and compound 3 did not directly inhibit the synthesis of fat or increase the consumption of fat.Combined with DIC pictures, the difference in volume corresponds to the difference in the total area of lipid droplets (including non-lipid droplets that emit weak fluorescence), so compound 3 reduces the total area of lipid droplets and affects the growth rate of C. elegans.The growth process of C. elegans is complex, and the mechanism of compound 3 affecting the growth of C. elegans will be further studied.

Conclusions
Truxinate derivative are of wide occurrence among plants, but are reported for the first time from the genus Castanopsis.Phenyldilactone have been shown to possess significant bioactivities.In this research, compound 3 did not directly inhibit the synthesis of fat or increase the consumption of fat, but reduced the total area of lipid droplets and affects the growth rate of Caenorhabditis elegans.Future studies will continue to focus on the impact of compound 3 on lipid metabolism.
have been obtained from the C. fissa, C. carlesii, C. sclerophylla, C. fargesii, C. fordii, and C. tibetana Hance in our previous work.Two new natural truxinate derivatives and a new phenyldilactone (Figure 1) were isolated from C. eyrei methanol extract in our continuing search for other biologically active natural products from the Castanopsis (D. Don) Spach.Herein, the isolation and structural elucidation of the three new natural compounds (1-3), along with the evaluation of the ability of compound 3 to reduce lipid accumulation in Caenorhabditis elegans, are described.