Bioactivity-guided isolation of compounds from Sophora flavescens with antibacterial activity against Acinetobacter baumannii

Abstract Bioactivity-guided fraction of an extract of Sophora flavescens to identify antibacterial compounds against Acinetobacter baumannii, led to the isolation of two new compounds, (2″R)-5-methoxy-7-hydroxy-8-lavandulylchromone (13) and (2S,βS)-(-)-sophobiflavonoid CE (19), and 18 known flavonoids, (6aR,11aR)-(-)-maackiain (1), (2S)-(-)-8-prenylnaringenin (2), (2S)-(-)-exiguaflavanone K (3), (2S)-(-)-sophoraflavanone G (4), (2S)-(-)-leachianone A (5), (2S)-(-)-kushenol E (6), (2S)-(-)-leachianone G (7), (±)-kushenol F (8), (2S)-(-)-kurarinone (9), (2S)-(-)-kurarinol (10), (2 R,3R)- (+)-3,7,4'-trihydroxy-5-methoxy-8-prenylflavanone (11), (2S)-(-)-isoxanthohumol (12), (2S)-(-)-2'-methoxykurarinone (14), (2 R,3R)-(+)-kushenol I (15), calycosin (16), kuraridin (17), (2S)-(-)-kushenol A (18), and trifolirhizin (20). Their structures were elucidated based on NMR, MS, and CD spectroscopic analysis. Among them, 1, 2, 5, and 15 exerted modest antibacterial activity against A. baumannii, with MIC95 of 128-256 μg/mL for 2 and 256-512 μg/mL for 1, 5 and 15. Graphical Abstract


Introduction
Acinetobacter baumannii is a Gram-negative bacterium and an opportunistic human pathogen, which can cause a broad range of infections, including respiratory tract infections, pneumonia, septicaemia, infections of the skin and soft tissues, and urinary tract infections. It is becoming increasingly important as a cause of hospital-acquired infections (Harding et al. 2018). Carbapenem-resistant A. baumannii is at the top of the World Health Organization's list of resistant pathogens for which new antimicrobial discovery is a critical need (Willyard 2017). Plants have been used as remedies in traditional medicine for thousands of years, yet compounds from these plants are still an underutilized source of antimicrobials.
Sophora flavescens Ait. (Fabaceae) is a medicinal plant distributed mainly in China, Korea and Japan (Huang et al. 2017). The dried root of S. flavescens (Chinese name "KuShen") has heat-clearing and damp-drying functions in traditional medicine. It has been used in China and some other Asian countries for thousands of years in the treatment of fever, diarrhea, jaundice, eczema, vaginal itching with leukorrhagia, anuresis, gastrointestinal hemorrhage, inflammatory disorders, and microbial infections (Chinese Pharmacopoeia Commission 2015; He et al. 2015). Flavonoids and alkaloids are the two principal chemical classes in S. flavescens (He et al. 2015). Du et al. (2010) found that the total flavonoid extract from S. flavescens was more potent than the total alkaloid extract in inhibiting Gram-positive bacteria including Staphylococcus species and Gram-negative bacteria including Pseudomonas aeruginosa, Escherichia coli and Shigella flexneri. Huang et al. (2016) also demonstrated that the total flavonoid extract was more potent than the total alkaloid extract against S. aureus and E. coli, with MICs of 0.001-0.004 mg/L and 0.001-0.004 mg/L, respectively for the flavonoid extract and 0.005-0.032 mg/L and 0.008-0.032 mg/L, respectively for the alkaloid extract. Some flavonoids isolated from S. flavescens have been reported to inhibit S. aureus, B. subtilis, S. epidermidis, E. coli, Propionibacterium acnes, and Salmonella typhimurium (Kuroyanagi et al. 1999;Sohn et al. 2004;Huang et al. 2016).
In our previous anti-bacterial screening of 50 traditional Chinese medicines, it was first found that an 80% ethanolic ultrasonic extract of S. flavescens displayed anti-bacterial activity against A. baumannii, with a MIC 95 value of 512 mg/mL ( Figure S1). While this activity was only weak, we hypothesized that individual compounds within this extract may display improved activity against this critical priority pathogen. In this study, we undertook bioactivity-guided isolation of the compounds from S. flavescens and testing of purified compounds against A. baumanni.

Results and discussion
2.1. Bioactivity-guided fraction of the 80% ethanol extract of S. flavescens The bioactivity-guided fraction of the 80% ethanol extract of S. flavescens ( Figure S1) showed that both the 80% ethanol extract (SF1) and its water-insoluble fraction (SF-3) dose-dependently inhibited A. baumannii, with 82.9 ± 3.4% and 80.2 ± 5.3% inhibition at 512 lg/mL. Further column chromatography showed that the fraction eluted with EtOAc (SF5) was the most active fraction in SF3. Further fractionation of SF5 afforded 9 sub-fractions. As shown in Figure S2,  showed some anti-bacterial activity (MIC 95 of 256-512 lg/mL), and the other eight fractions showed partial inhibitory effects, with inhibition rates ranging from 40.7-76.2% at 512 lg/mL.

Plant material
The root pieces of S. flavescens were bought from Jianlian Pharmacy (Jinan, China), and identified by Prof. Lan Xiang (one author). A voucher specimen (No. 20171201) was deposited in the Institute of Pharmacognosy, School of Pharmaceutical Sciences, Shandong University.

Conclusion
Bioactivity-guided fraction of anti-bacterial components from S. flavescens against A. baumannii led to the isolation of two new constituents (13, 19) and 18 known flavonoids, and discovery of four anti-bacterial flavonoids maackiain (1), 8-prenylnaringenin (2), leachianone A (5) and kushenol I (15). This study provides evidence contributing to the scientific rationale for the traditional uses of S. flavescens and its future development as natural anti-infectious agent.