Benzophenone Rhamnosides and Chromones from Hypericum seniawinii Maxim.

Two new benzophenone glycosides, hypersens A and B, along with four known compounds, (S)-(+)-5,7-dihydroxy-2-(1-methylpropyl) chromone (3), 5,7-dihydroxy-2-isopropylchromone (4), urachromone B (5), and 3-8′′ bisapigenin (6), were isolated from Hypericum seniawinii. The structures of new compounds (1 and 2) were elucidated according to comprehensive spectroscopic data analyses. The absolute configurations of 1 and 2 were determined by electronic circular dichroism (ECD) calculations. All isolated compounds were evaluated for their neuroprotective effect using corticosterone-induced PC12 cell injury. In addition, compounds 1–6 were evaluated for their anti-inflammatory activity in lipopolysaccharide-induced RAW 264.7 cells. Compound 6 was a biflavonoid and significantly inhibited the production of nitric oxide with an IC50 value of 11.48 ± 1.23 μM.


Introduction
Hypericum seniawinii Maxim. (Hypericaceae) is a perennial herbaceous plant, and widely distributed in temperate regions [1]. It has been used as a folk medicine for the treatment of inflammation and infectious diseases in China [2]. Previous studies on the chemical constituent and bioactivities of this herb are minimal. However, the genus Hypericum, consisting of about 500 species, is an important resource of medicinal and cosmetic plants [3]. The genus Hypericum has been reported to contain phloroglucinol derivatives, flavonoids and xanthonoids [4][5][6][7], and other miscellaneous compounds [8] which exhibit various pharmacological activities, such as antioxidative, antitumor, antiviral, anti-inflammatory, and antifungal activities [9,10].
As a part of our investigation on bioactive compounds from natural sources [11][12][13], phytochemical investigation on this species was conducted. As a result, two new benzophenone glycosides (1 and 2), along with four known compounds (3)(4)(5)(6), were isolated and characterized from H. seniawinii. Benzophenones glycosides isolated from the genus Hypericum have been reported to show various activities, such as antibacterial and antiinflammatory activities [14]. The anti-inflammatory and neuroprotective activities of all compounds were investigated. Compounds 1-6 exhibited neuroprotective effects against corticosterone-induced PC12 cell injury. Moreover, all compounds reduced the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) at the concentration of 10 µM. Among these, 6 showed a significant inhibitory effect with an IC 50 value of 11.48 ± 1.23 µM.
The anti-inflammatory activity of all isolated compounds in LPS-induced RAW 264.7 cells was investigated. The cytotoxicity of compounds 1-6 against RAW 264.7 cells was evaluated-using the Cell Counting Kit-8 (CCK-8) assay [20]. None of the compounds were cytotoxic to RAW 264.7 cells at a concentration of 50 µM. As depicted in Table 2, all compounds (10 µM) exhibited inhibitory effects on NO production in LPS-induced RAW 264.7 cells. Among these, compound 6 exhibited the strongest inhibitory effect with an IC 50 value of 11.48 ± 1.23 µM. Therefore, the effect of compound 6 on tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) production in LPS-induced RAW 264.7 cells was detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the effect of compound 6 on mRNA expression levels of TNF-α, IL-1β, and IL-6 in LPS-induced RAW 264.7 cells was also measured. As shown in Figure 5, compound 6 inhibited the production of TNF-α, IL-1β and IL-6 by down-regulating the mRNA expressions of TNF-α, IL-1β, and IL-6. The extracts from the genus Hypericum have previously been used for the treatment of depression [19]. All isolates were evaluated for their protective effects on corticosterone-induced PC12 cell injury. As shown in Figure 4, compounds 1-6 (10 μM) exhibited neuroprotective activity with cell viabilities of 79.27 ± 1.70%, 78.92 ± 2.09%, 82.02 ± 2.87%, 81.35 ± 2.90%, 83.35 ± 1.62%, and 70.91 ± 5.06%, respectively (62.00 ± 1.92% for the model). The anti-inflammatory activity of all isolated compounds in LPS-induced RAW 264.7 cells was investigated. The cytotoxicity of compounds 1-6 against RAW 264.7 cells was evaluated-using the Cell Counting Kit-8 (CCK-8) assay [20]. None of the compounds were cytotoxic to RAW 264.7 cells at a concentration of 50 μM. As depicted in Table 2, all compounds (10 μM) exhibited inhibitory effects on NO production in LPS-induced RAW 264.7 cells. Among these, compound 6 exhibited the strongest inhibitory effect with an IC50 value of 11.48 ± 1.23 μM. Therefore, the effect of compound 6 on tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) production in LPS-induced RAW 264.7 cells was detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the effect of compound 6 on mRNA expression levels of TNF-α, IL-1β, and IL-6 in LPS-induced RAW 264.7 cells was also measured. As shown in Figure 5, compound 6 inhibited the production of TNF-α, IL-1β and IL-6 by down-regulating the mRNA expressions of TNF-α, IL-1β, and IL-6.

General Experimental Procedures
UV data were collected using a Shimadzu UV-1600PC spectrophotometer (Shimadzu

Plant Material
The air-dried aerial parts of Hypericum seniawinii Maxim. (Hypericaceae) were collected from Yuexi county, Anqing city, Anhui Province, China, in June 2018. A voucher specimen (No. XJ201806) has been deposited in the School of Pharmacy, Anhui Medical University.

Extraction and Isolation
The air-dried aerial parts of H. seniawinii (500 g) were powdered and extracted with CH 2 Cl 2 . After removing the solvent, the CH 2 Cl 2 extract (20 g) was eluted by a gradient of Petroleum ether/EtOAc

ECD Calculations
According to the relative configuration of each compound deduced from the coupling constant and ROESY spectrum, systematic conformational searches were performed with Confab [21]. The initial conformations were optimized and re-optimized with the Molclus program (version 1.9.9.5) [22] by invoking the xtb program (version 6.4) [23,24] and ORCA-5.0 [25,26] at the B97-3c level. The programORCA-5.0 was used to calculate the ECD spectra at the PBE0/def2-SV(P) level with a CPCM solvent model (methanol).

Hydrolysis of Compounds 1 and 2
The compound (0.5 mg) was treated with 3 M hydrochloric acid (0.5 mL) at 90 • C for 2 h. After neutralization with 3 M ammonium hydroxide, the reactants were dried by evaporation of the solvent. L-cysteine methyl ester (0.5 mg) and pyridine (0.2 mL) were added, and then stirred at 60 • C for 1 h. Finally, phenyl isothiocyanate solution (0.5 mL) was added and stirred at 60 • C for 1 h. [27]. The residue of each sample was subjected to analytical HPLC (with MeCN/H 2 O (25:75,1.0 mL/min) using a C18 RP column (4.6 mm × 250 mm, 5 µm, Thermo Fisher Scientific Inc., Waltham, MA, USA). The configuration of the sugars was determined by comparing their retention times with that of derived L-rhamnose (t R = 20.2 min).

NO Production
RAW 264.7 macrophages were seeded in 48-well plates, pretreated with compounds (10 µM) for 1 h, and subsequently incubated with LPS (1.0 µg/mL) (Escherichia coli, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Griess reagent (Beyotime, Shanghai, China) was used to measure NO production. The absorbance was determined at 540 nm using a BioTek Synergy HTX multimode reader.

Determination of IL-6, TNF-α, and IL-1β
The concentrations of IL-1β, IL-6, and TNF-α in the supernatants of RAW 264.7 cells were investigated by ELISA (ELISA LAB, Wuhan, China) according to the manufacturer's protocols.
3.6.6. Statistical Analysis IBM SPSS 25.0 software (Armonk, NY, USA) was used for the statistical analysis. Data are presented as mean ± SD (n = 3).

Conclusions
In summary, two new benzophenone glycosides (1 and 2), along with four known compounds (3)(4)(5)(6) were isolated from the aerial parts of H. seniawinii. The absolute configurations of the sugar moiety of benzophenone glycosides were determined by hydrolysis and the calculation of the ECD spectrum. The neuroprotective and anti-inflammatory activities of these compounds were evaluated. Based on the results, compound 6 possessed a significant inhibitory effect on the production of NO, TNF-α, IL-1β, and IL-6 in LPSinduced RAW 264.7 cells. This study will enrich the chemical diversity of H. seniawinii and facilitate the development of inflammatory inhibitors and neuroprotective agents.