Bioactive neolignan, iridoid and flavonoid glycosides from the leaves of Vaccinium bracteatum

Abstract Two neolignan glycosides including a new one (1), along with seven iridoid glycosides (3 − 9) and nine flavonoid glycosides (10 − 18), were isolated from the leaves of Vaccinium bracteatum. Their structures were established mainly on the basis of 1D/2D NMR and ESIMS analyses, as well as comparison to known compounds in the literature. The structure of 1 with absolute stereochemistry was also confirmed by chemical degradation and ECD calculation. Selective compounds showed antiradical activity against ABTS and/or DPPH. Moreover, several isolates also suppressed the production of ROS in RAW264.7 cells and exerted neuroprotective effect toward PC12 cells.


Introduction
Vaccinium bracteatum Thunb.(known as Wu Fan Shu in Chinese) is an evergreen shrub or small arbor tree of family Ericaceae [1].As an indigenous species, it is widely distributed in the subtropical and tropical regions of China, and meanwhile, it also grows in Korea, Japan, and most Southeast Asian countries [2].As a medicinal herb with dietary values, the leaves of V. bracteatum (VBL) have been used as both functional food and natural medicine to nourish body and enhance longevity, with antioxidant, antidepressant, hypoglycemic, immunomodulatory and anti-inflammatory effects [3][4][5].Previous phytochemical investigations into this species have revealed the existence of flavonoids, polyphenols, and polysaccharides as the main chemical components, as well as iridoid glycosides as the minor constituents [6][7][8].
All the isolated compounds were first tested for their antioxidant effect, and compounds 3, 8, 12, 13 and 18 exhibited significant activity by scavenging ABTS radicals with IC 50 values in the range of 12.43 to 17.52 lM, while 11, 12, 13, 15 and 18 exerted better scavenging activity against DPPH radicals with IC 50 values in the range of 6.52 to 14.94 lM (Table 1).Several compounds displayed stronger activity than the positive control ascorbic acid (vitamin C).Moreover, the suppression of them on LPS induced ROS release in RAW264.7 macrophages was also evaluated.As shown in Figure 3, compounds 1, 2, 3, 8, 12, 13, 15 and 18 all exhibited decent inhibition against the ROS production, and 18 showed the best activity comparable to the reference compound vitamin C (VC).In addition, the protection of them toward PC12 cells was also tested, which revealed that compounds 8, 12, 15, 16 and 17 exerted apparent protective effect against 6-hdyroxydopamine (6-OHDA) induced cell injury at 50 lM (Figure 4).

Plant materials
The leaves of Vaccinium bracteatum were collected in July 2020 in Wuxi, Jiangsu province, China, and were authenticated by Prof. Guo-hua Ye from Shandong College of Traditional Chinese Medicine.The voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-058).

ECD calculation
The ECD calculation for compound 1 was performed as previously described [30].

Acid hydrolysis of 1 and determination of the sugar absolute configuration
The absolute configuration of xylose was determined according to a reported procedure [31].A solution of 1 (2.0 mg) in 1.0 N HCl (3.0 ml) was stirred at 90 � C for 4 h in oil bath.After removal of excess HCl under reduced pressure, the residual aqueous mixture was filtered to eliminate the aglycone.The remaining solution was evaporated in vacuum to afford the monosaccharide which was acetylated with acetic anhydride in pyridine at room temperature for 10 h.Standard samples of D and Lxyloses were also acetylated using the same method.Then chiral HPLC analyses were performed on an Chiral MZ-RH 5m column (FLM Inc., Guangzhou, China) for these acetylated products (60% MeCN-H 2 O, 1.00 ml/min) (Figure S9, Supplementary data).

Detection reactive oxygen species (ROS)
RAW264.7 macrophages were cultured according to the protocol described by Wei et al. [10] The suppression of compounds on the generation of ROS induced by lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, U.S.A.) in RAW264.7 cells was investigated by a commercial assay kit (Beyotime Biotechnology, Shanghai, China).

Cell protective assay
The cell protective effect of compounds with decent radical scavenging activity was evaluated in 6-OHDA stimulated PC12 cells as we reported previously [8], and the initial screening concentration was set as 50 lM.

Figure 1 .
Figure 1.Structures of the chemical constituents from V. bracteatum.

Figure 3 .
Figure 3.Effect of the isolated glycosides on ROS release in LPS stimulated RAW264.7 cells (A, B, C); Quantified results of relative ROS level from three independent experiments (D).�� p < 0.01, ��� p < 0.001, compared with the LPS treated group.

Table 1 .
The free radical scavenging activity of the constituents from V. bracteatum.a a IC 50 values were presented as 'mean ± SD' (n ¼ 3) in mM.NA: Not active.