Triterpene hexahydroxydiphenoyl ester and phenol glucosides from the leaves of Castanopsis eyrie (Champ. ex Benth.) Hutch

Abstract As a part of our systematic study on Castanopsis genus (Fagaceae), one new triterpene hydrolysable tannin (1) and two new phenol glucosides (2 and 3) were isolated from the leaves of Castanopsis eyrei (Champ. ex Benth.) Hutch. Compound 1 was identified as a triterpene hexahydroxydiphenoyl (HHDP) ester. This type of compounds has only been isolated from Castanopsis genus. The structures of 1-3 were elucidated by the combination of spectroscopic analysis (MS, 1 D and 2 D NMR) and chemical evidence. Graphical Abstract


Introduction
The Castanopsis (Fagaceae) genus, with approximately 120 species, is widely distributed in tropical and subtropical Asia (CAS, Flora Republicae Popularis Sinicae 1998).Some Castanopsis species are used as folk medicine to treat a variety of diseases, such as chronic ulcers, diarrhea, headache, and wounds (Khan et al. 2001).Numerous bioactive natural products have been isolated from this genus, such as polyphenols (Wang et al. 2022), ellagitannins (Huang et al. 2012), triterpene hexahydroxydiphenoyl (HHDP) esters (Wang et al. 2018).Moreover, triterpene HHDP esters, as chemotaxonomic markers for Castanopsis genus, have only been isolated from C. cuspidata var.sieboldii (Ageta et al. 1988), C. hystrix (Chen et al. 1993), C. fissa (Huang et al. 2011), C. fargesii (Wang et al. 2018) and C. sieboldii (Kim et al. 2020).Our previous study on C. eyrie led to the isolation of 42 known polyphenols and flavonoids (Pang et al. 2018(Pang et al. , 2019(Pang et al. , 2020)).In order to find more characteristic metabolites and to enrich the chemical composition from C. eyrie, further investigation was carried out for the leaves of C. eyrie, which led to the isolation of one new triterpene HHDP esters (1) with chemical taxonomic significance and two new phenol glucosides (2 and 3).This paper shows the isolation and structure elucidation of these new compounds (Figure 1).

Results and discussion
Compound 1 was isolated as a white amorphous powder.The molecular formula was determined as C 57 H 68 O 23 by the data of 13 C NMR and HR-ESI-MS spectrum, which showed a [M þ Na] þ peak at m/z 1143.4086(calcd.for C 57 H 68 O 23 Na þ , 1143.4044).The 1 H NMR spectrum of 1 showed a two-proton singlet at d H 7.12, combining with seven carbon signals at d C 167.2, 145.9, 138.8 (2 C), 121.4, 109.9 (2 C) in the 13 C NMR spectrum, which deduced the presence of a galloyl group.In addition, two one-proton singlets at d H 6.63 and 6.56, which suggested the presence of a hexahydroxydiphenoyl (HHDP) ester group in the molecule, and more suggestion was that showing a dark blue coloration after to the FeCl 3 reagent (Huang et al. 2011).According to the 13 C NMR spectrum, with the aid of the HSQC experiment, HHDP group was ascribed to the signals at d C 169.6, 169.0, 145.3, 145.2, 144.5, 144.3, 136.2, 135.3, 126.7, 126.4,115.3, 113.8, 108.0, 107.9.Moreover, the 1 H NMR showed an anomeric proton signal at d H 5.44 (1H, d, J ¼ 8.2 Hz) suggested that 1 had a b-glucopyranose moiety, and this was supported by carbon signals at d C 94.9, 73.3, 77.5, 70.8, 75.3, and 64.4 (Huang et al. 2017).Furthermore, the 1 H NMR spectrum showed the presences of two hydroxy methine proton at d H 3.82 (1H,dd,J ¼ 7.6,13.3 Hz),4.88 (1H,m), two hydroxy methylene [d H 4.13 (d,J ¼ 10.9 Hz),4.41 (d,J ¼ 10.9 Hz),and 3.98 (d,J ¼ 11.3 Hz),5.40 (d,J ¼ 11.3 Hz), an olefinic proton at d H 5.23 (1H, t, J ¼ 3.2 Hz), three singlets at d H 0.74 (3H), 0.88 (3H), 1.06 (3H), and two doublet at d H 0.83 (3H, d, J ¼ 6.4 Hz), 0.90 (3H, d, J ¼ 6.4 Hz) for two secondary methyl groups.All these protons indicated a presence of an urs-12-ene skeleton, particularly the two secondary methyl groups (Ntchatcho et al. 2009).Meanwhile, the signals in 13 C NMR spectrum at d C 126.7 and 138.5, which were characteristic for the C-12/C-13 double bond in the ursane-type structure, further confirmed the urs-12-ene skeleton.Based on the experiments of HSQC and HMBC, the HHDP group was assigned to C-3 and C-24 on the basis of the correlations between H-3, H-24 with the HHDP ester group.The glucose was assigned to C-28 of the ursane skeleton according to the correlations between glc-H-1 and the carboxyl carbon (d C 176.8).The above-mentioned data were similar to those of castanopsinins E, which was previously obtained from the leaves of C. cuspidate (Ageta et al. 1988).The difference between 1 and Castanopsinins E was the presence of an additional galloyl group, which was determined by the HMBC correlations of glc-H-6a (d H 4.41)/C-galloyl-7 (d C 167.2) and glc-H-6b (d H 4.29)/C-galloyl-7 (d C 167.2), inferring the galloyl ester was bond with the glucose C-6 hydroxyl group.Thus, the structure of compound 1 was determined as shown and named as eyrein A.
Compound 2 was obtained as a yellowish amorphous powder and a characteristic dark blue coloration with ferric chloride reagent suggested the presence of a pyrogallol ring in the molecule.The molecular formula C 20 H 22 O 12 was established on the HR-ESI-MS spectrum, which showed the  64.5, 75.0, 71.1, 76.8, 74.2, 103.7), and an aromatic ring with a 3,4,5-trihydoxy-substitution system (d C 110.1, 112.1, 129.4, 133.4, 146.1, 146.6), which was closely related to that of castamollissin (Feng et al. 1988).The coupling constant of the anomeric sugar proton was 7.7 Hz, indicating that the sugar moiety was the b-configuration.The galloyl group is shown be located at the glucose C-6 by the low field shift of the glucose H-6 [d H 4.39 (dd, J ¼ 6.8, 11.9 Hz), 4.64 (dd, J ¼ 1.7, 11.9 Hz)], and the HMBC correlation between glucose H-6 and the carboxyl carbon of galloyl group ester d C 167.1.The location of the glucose group at the C-1 and methyl group at the C-5 was confirmed by the HMBC correlation between glucose H-1 (d H 4.77, d, J ¼ 7.7 Hz) and C-1 (d C 146.6), d H 1.96 (-CH 3 ) and C-6 (d C 110.1), C-4 (d C 112.1), respectively.Based on these spectroscopic results, the structure of 2 was established to be as shown and was named eyrein B.
Compound 3 was obtained as a yellowish-brown amorphous powder, and its molecular formula C 34 H 30 O 22 was determined by HR-ESI-MS analysis, which showed

Materials
The leaves of C. eyrei were collected at Bajiaozhai, Ziyuan, Guilin, Guangxi, China, in March 2017, and authenticated by Prof. Tao Ding, Guangxi Institute of Botany, China.The voucher specimen (20170301) was deposited in the Guangxi Key Laboratory of Plant Functional Phytochemicals and Sustainable Utilization, Guangxi Institute of Botany, China.