Glycosides with galloyl groups from Balakata baccata and their antineuroinflammatory activities

Abstract Seven new glycosides (1 − 7) with galloyl groups and two known kaempferol glycosides (8 and 9) were obtained from the overground parts of Balakata baccata. The structures of the new compounds were determined by comprehensive spectroscopic analyses. The rarely seen allene moiety in compounds 6 and 7 were described by detailed analysis of 1D and 2D NMR data. The antineuroinflammatory effect of all the isolates was assessed through inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Compounds 1, 2, 6, and 7 showed potent inhibitory activities with IC50 values of 25.7, 17.2, 15.5 and 24.4 μM, respectively, compared with the positive control minocycline (IC50 = 16.1 μM). Graphical Abstract


Introduction
Sapium baccatum, a plant of the genus Sapium in the Euphorbiaceae family, has been revised as Balakata baccata.It is an arbor and wildly distributed in southern Yunnan of China as well as some countries in south and southeast Asia (Ma and Tseng 1997).The overground parts of B. baccata have been used for the treatment of scapulohumeral periarthritis, cerebral infarction, and gouty arthritis as one of the ingredients of Chinese herbal prescriptions (Li et al. 2019).
Currently, the frequency of central nervous system pathologies increased year by year, among which the most common are neurodegenerative and neuroinflammatory diseases such as Alzheimer's disease and multiple sclerosis (Gogoleva et al. 2019;Bagheri-Mohammadi 2021).During our previous research, it was found that some plants of this genus, such as Triadica sebifera (Yu et al. 2020), Triadica rotundifolia (Zhao et al. 2021b), Neoshirakia japonica (Zhao et al. 2021c), and Sapium discolor (Zhang et al. 2019) contained potential antineuroinflammatory components.
Phytochemical research on B. baccata revealed that it contained a wide array of coumarins, tannins, terpenes, and steroids, many of which showed significant anti-inflammatory, anti-bacterial, and anti-fungal activities (Al Muqarrabun et al. 2014;Vu et al. 2017;Li et al. 2019).In order to search for natural products with potential antineuroinflammatory components from B. baccata, a chemical investigation and the evaluation of potential antineuroinflammatory activity on the overground parts of B. baccata were conducted.
Balakatoside E (5) shared the same molecular formula as 4 based on their HRESIMS and 13 C NMR data.Their similar uV, IR, 1 H NMR, and 13 C NMR data (Table S2) implied that they possess the identical structural scaffold and the same substituent groups.Analysis of its 2D NMR data proved the above conclusion, which also explained that C-8′ of α-terpineol group was attached at C-1 via ether bond based on the significant HMBC correlation of H-1/C-8′ as described in Figure S1.Thus, compound 5 was  In its 2D NMR spectra, the key 1 H-1 H COSY and HMBC correlations not only explained the existence of a sugar unit and two galloyl groups but also explained that the attachment of a sugar unit and two galloyl groups in 6 was identical with that of 2. Subsequently, the 1 H-1 H COSY correlations of H-1′/H-2′ and HMBC correlations from H 3 -13′ to C-2′, 3′, 4′ ascertained the presence of C-1′-C-4′ unit as described in Figure S2.The attachment of C-1 and C-1′ via ether bond was ensured based on the significant HMBC correlation of H-1/C-1′.Then, the typical chemical shift values (δ C 92.3, 205.2, 92.7) implied the existence of an allene unit (Ohigashi et al. 1972;Zhang et al. 2010).According to the significant 1 H-1 H COSY correlations of H 2 -6′/H 2 -7′, H 2 -7′/H 2 -8′, H 2 -8′/H-9′, and H-11′/H 2 -12′ as well as the key HMBC correlations from H 2 -8′ to C-9′, 10′ and from H 2 -12′ to C-10′, 11′ determined the attachment of C-5′-C-12′ unit as shown in Figure S2.The attachment of C-4′ and C-5′ via ester bond was confirmed based on the HMBC correlations from H 2 -4′/H 2 -6′ to C-5′.Finally, based on the NOESY correlations of H 2 -4′ (δ H 4.41 and 4.37) and H-2′ (δ H 5.46), the trans relative configuration of the double bond between C-2′ and C-3′ was ascertained.Therefore, compound 6 was named balakatoside F.
Balakatoside G (7) was isolated as a white amorphous powder.Its molecular formula was deduced as C 33 H 36 O 15 according to its HRESIMS ion at m/z 695.1948 [M + Na] + (calcd for C 33 H 36 O 15 Na + , 695.1946) and 13 C NMR data (Table S2).Its 1 H and 13 C NMR data (Table S2) suggested that the structure of 7 was closely similar to that of 6.Further analysis of its 2D NMR data showed the presence of a β-glucopyranosyl group with two galloyl units at C-2 and C-6.The HMBC correlations from H 3 -13′ to C-4′, 5′, 6′, from H 3 -11′, 12′ to C-1′, 2′, 6′, and from H-4′ to C-2′, 3′ determined the attachment from C-1′ to C-6′ as described in Figure S2.The attachment of C-1 and C-9′ via ether bond was confirmed based on the HMBC correlation from H-9′ to C-1.The molecular formula and the significant chemical shifts of C-6′ (δ C 116.2), C-7′ (δ C 205.3), and C-8′ (δ C 101.3) explained the existence of an allene group as shown in Figure S2 (Zhang et al. 2010).Consequently, the structure of 7 was elucidated and named balakatoside G.
The antineuroinflammatory effect of compounds 1 − 9 were tested by Griess assay in LPS-induced BV-2 microglial cells, when the isolates and a known neuroinflammatory inhibitor, minocycline, indicated no cytotoxicity at the tested concentrations (Table S3).The inhibition rates of NO production were almost zero in the negative control group (cells without treatment) and 100% in LPS-induced control group.The results of anti-inflammatory activity showed that compared with the positive control minocycline (IC 50 = 16.1 ± 0.1 μM), compounds 3 − 5, 8, and 9 were inactive (IC 50 > 50 μM), but to a certain extent compounds 1, 2, 6, and 7 displayed inhibitory activities with IC 50 values of 25.7, 17.2, 15.5, and 24.4 μM, respectively (Table S4).Preliminary structure-activity relationship analysis suggested that the glycosides with 2,6-di-O-gralloyl-β-D-glucopyranosyl group have better anti-inflammatory activity.

General experimental procedures
High resolution electrospray ionization mass spectroscopy (HRESIMS) data of all the isolates were recorded at Agilent 6545 Q-TOF LC-MS apparatus.Nuclear magnetic resonance (NMR) spectra were acquired on 400 MHz or 600 MHz Bruker spectrometer.Chemical shifts were expressed in δ (ppm) and referenced to the solvent residual peak.Electronic circular dichroism (ECD) spectra and optical rotations were measured on JASCO J-1500 spectropolarimeter and JASCO P-2000 polarimeter, respectively.The other testing instruments and the materials used in the isolation and purification of compounds were the same as previous (Pan et al. 2015;Jiang et al. 2017).The inhibitory effect of NO production in LPS-induced BV-2 microglial cells, using to evaluate antineuroinflammatory activity, was assessed by Griess reaction, as described in the previous study (Li et al. 2017;Zhang et al. 2019).

Plant material
The overground parts of B. baccata, obtained from Xishuangbanna, Yunnan province, China, in June 2019, were authenticated by Prof. Shao-Qing Tang (College of Life Science, Guangxi Normal university).A voucher specimen (No.BB-201906) has been deposited at the Collaborative Innovation Center for Guangxi Ethnic Medicine, Guangxi Normal university.