New lignans and phenylethanoid with antioxidant activity from aerial parts of Forsythia suspensa (Thunb.) Vahl

Abstract Two new lignans, phillyroside A(1) and phillyroside B(2), together with three new phenylethanoid glycoside, forsythoside K(3), forsythoside L(5) and forsythol L (4), while compounds 4 was an aglycon of forsythoside L(5), were isolated from the aerial parts of Forsythia suspensa (Thunb.) Vahl. Their structures were elucidated by comprehensive analyses of standard spectroscopic data (MS, IR, and NMR) and the in vitro antioxidant activity of five new compounds were evaluated in DPPH and ABTS radical scavenging experiment and ferric reducing ability of plasma (FRAP) experiment. Compounds 4 and 5 exhibited antioxidant activity with IC50 values ranging from 112.49 to 153.58 μM in DPPH experiment and 45.43 to 64.09 μM in ABTS experiment. Graphical Abstract


Introduction
Forsythia suspensa (Thunb.) Vahl. is a plant belonging to the genus Forsythia in the Oleaceae family, mainly from Shanxi, Shaanxi, Hebei, Henan Provinces and other regions of China (Editorial Board of Flora of China 1978). Its dried fruit is a Traditional Chinese Medicine (Pharmacopoeia Commission of PRC 2015). It is widely used for acute wind-heat, carbuncle and sore toxins, lymph node tuberculosis, urinary tract infections and other diseases (Pharmacopoeia Commission of PRC 2015). Chemical studies have shown that Forsythia plants mainly contain phenethyl alcohol glycosides (Kuang et al. 2011), lignans (Chang et al. 2008), diterpenoid (Ma et al. 2020), terpenes (Rouf et al. 2001), cyclohexanols (Yan et al. 2014), cyclohexanones , flavonoids (Wei et al. 2020) and alkaloid (Dai et al. 2009) compounds. Pharmacological activity studies have shown that Forsythia has antioxidant (Guo et al. 2015), antibacterial (Kuang et al. 2011), antiviral (Qu et al. 2016), anti-cancer (Zhao et al. 2015), antiinflammatory , neuroprotective (Chen et al. 2018) and other effects. Studies in recent years show that the chemical components are similar in F. suspensa leaves and F. suspensa fruit, and the content of certain active ingredients such as forsythin and forsythiaside in F. suspensa leaves is much higher than that in fruits. Five new compounds ( Figure 1) were isolated from the aboveground part of F. suspensa in this study. Furthermore, the antioxidant activities of the isolated compounds were evaluated.
The antioxidant activity of five new compounds was measured by three antioxidant evaluation methods (DPPH, ABTS and FRAP). The experimental results are shown in the Tables S4 and S5. Compound 1 and Compound 3 have weak ABTS free radical scavenging ability. The DPPH radical and ABTS radical scavenging ability of Compound 4 are equivalent to L-ascorbic acid. The DPPH radical and ABTS radical scavenging ability of Compound 5 are significantly stronger than L-ascorbic acid. The FRAP iron reduction activity of Compounds 1-5 were weaker than the positive control at the concentration of 40 mM. The antioxidant activities of these five new compounds were weaker than those of known compounds of forsythia such as forsythin and phillygenin (Lu et al. 2010). Other biological activities can be further explored.

Experimental equipment
Column chromatography was performed on silica gel (300-3400 mesh; Qingdao Marine Chemical Factory, China). RP-18 was performed on reverse silica gel (20-45 mm; Mitsubishi Chemical Corporation, Japan). UV spectra were recorded using a UV3600 spectrophotometer (Shanghai Pharmaceutical Machinery Co. Ltd., China). IR spectra were recorded on a Nicolet 200 SXV spectrometer.HR-ESI-MS data were performed on a Waters Q-TOF Premier mass spectrometer (Waters, USA). NMR spectra were obtained by an AC-E200 400 MHz digital NMR spectrometer (Bruker Corporation, Karlsruhe, German), using TMS as the internal standard. The microplate reader used in antioxidant activity experiment was SparkTM 10 M (Tecan Inc., Switzerland).

Plant material
The F. suspensa (Thunb.) Vahl was collected in July 2019 in Shangluo, Shaanxi Province, China and were identified by associate professor Jianzhong Wang. The specimen (No. FT20200212) was deposited at the Department of Medicinal Natural Products, West China School of Pharmacy, Sichuan University.

Antioxidant activity
DPPH (Goupy et al. 2003) and ABTS (Santos and Silva 2020) radical scavenging experiment and ferric reducing ability of plasma (FRAP) (Santos and Silva 2020) were used to measure the antioxidant activity of five new compounds.
Mix 100 mL DPPH ethanol solution (150 mM) and 100 mL sample ethanol solution (12.5, 25, 50, 100, 200, and 400 mM) in a 96-well plate, react for 30 minutes under dark conditions at room temperature, and measure the UV absorbance at 517 nm wavelength of the mixture with microplate reader. Anhydrous ethanol was used as a blank control, and ascorbic acid was used as a positive control. Perform three parallel experiments. Calculate the IC 50 value of the sample to scavenge DPPH free radicals. Oneway analysis of variance ANOVA was used to analyze the IC 50 value of test compounds and positive control to judge whether there was a significant difference.
Mix 100 lL ABTS ethanol solution (125 lM) and 100 lL sample ethanol solution (12.5, 25, 50, 100, 200, and 400 lM) in a 96-well plate, react for 30 minutes under dark conditions at room temperature, and measure the UV absorbance of the mixture at 734 nm wavelength with microplate reader. With absolute ethanol as the blank control and ascorbic acid as the positive control. Perform three parallel experiments. Calculate the IC 50 value of the sample to scavenge ABTS free radicals. One-way analysis of variance ANOVA was used to analyze the IC 50 value of test compounds and positive control to judge whether there was a significant difference.
Mix 180 mL TPTZ solution (10 mM) and 20 mL sample water solution (12.5, 25, 50, 100, 200, and 400 mM) in a 96-well plate, react for 5 minutes under dark conditions at room temperature, and measure the UV absorbance at 593 nm wavelength of the mixture with microplate reader. L-ascorbic acid was used as a positive control. Perform three parallel experiments. Taking the gradient concentration of FeSO 4 as the control, the Fe 3þ reducing ability of the sample was expressed as the concentration of FeSO 4 (FRAP value). The larger the value, the stronger the FRAP iron reduction activity of the compound.

Conclusion
In general, five new compounds were isolated from the aboveground part of F. suspensa, including one phenylethanol compound, two phenylethanol glycoside compounds, and two isomers with phenylethanol glycoside skeleton and lignan skeleton. The 1,2,4-trisubstituted benzene ring in compounds 4 and 5 have a para-substituted hydroxyl group, which is uncommon in natural products isolated from forsythia, only Forsythiayanoside C isolated by Yan et al. (2016) has a similar structure. Furthermore, Compounds 4 and Compound 5 exhibited good antioxidant activity in DPPH and ABTS radical scavenging experiment. Further study is needed to discover the applicable medicinal value of these new compounds.