A new macrocyclic spermidine alkaloid from the aerial part of Hyssopus cuspidatus Boriss.

Abstract Hyssopus cuspidatus Boriss. grows in Xinjiang, China. A new macrocyclic spermidine alkaloid, namely hyssopusizine (1), along with sixteen known compounds were isolated and identified from the aerial parts of H. cuspidatus. Their structures were elucidated on the basis of spectroscopic data and comparison with the literature. Among them, fifteen compounds were isolated from H. cuspidatus for the first time. The absolute configuration of compound 1 was established by comparing the calculated and experimental ECD spectroscopic data. All isolated compounds were tested for their antioxidant and antimicrobial activities. Among them, compound 10 exhibited significant effects on ABTS free-radical scavenging activity with an IC50 value of 15.6 μM. Compounds 5–7 exhibited potent antioxidant activities against ABTS and DPPH. Most compounds exhibited moderate antimicrobial activities. Hyssopusizine (1) is the first macrocyclic spermidine alkaloid discovered from the Hyssopus genus. Graphical Abstract


Introduction
Plants of Hyssopus genus are perennial herbs or subshrubs mainly distributed in East Mediterranean to Central Asia (Abduwaki et al. 2014). H. cuspidatus has generally been used as a traditional medicine to treat fever, pneumonia and bronchus asthma and aromatic vegetable or spice among the local people in Xinjiang, China (Zhao et al. 2013;Shomirzoeva et al. 2020). Pharmacological research in recent years revealed that the extracts of H. cuspidatus showed anti-bacterial, anti-oxidation and anti-tumor bioactivities (Zhao et al. 2013).

General experimental procedures
The optical rotation was recorded on a Rudolph RS Autopol VI automatic polarimeter (Rudolph Research Analytical, Flanders, NJ, USA) in CH 3 OH at 25 C. Analytical HPLC was carried out on DIONEX Ultimate 3000 system (Thermo-Fisher, USA), combined with the XBridge RP C18 (5 lm, 4.6 mm Â 250 mm). The NMR spectra were recorded on a 400 or 600 NMR spectrometer (Varian, CA, USA). HRMS spectra were measured on a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc., USA). Silica gel  column chromatography (CC). Semi-preparative HPLC was conducted on a Shimadzu LC-20AT instrument (Shimadzu Corporation, Kyoto, Japan). Analytical grade solvents including ethanol, petroleum ether, ethyl acetate, n-butanol, methanol and HPLC grade solvents including acetonitrile and methanol were purchased from Shanghai Titan Scientific Co. Ltd (Shanghai, China) and Merck Company (Germany).

Plant material
The aerial parts of H. cuspidatus were collected from the Tacheng region of Xinjiang on September of 2019. The plant was identified by Chunfang Lu of the Xinjiang Technical Institute of Physics and Chemistry, CAS, and the voucher specimen (WY01503) has been deposited in the same institute.

Extraction and isolation
The dried and grounded aerial parts of H. cuspidatus were extracted with 95% ethanol at room temperature. The ethanol crude extract was suspended in water and successively partitioned with petroleum ether, ethyl acetate, and n-butanol, respectively. The obtained ethyl acetate extract was separated using silica gel column chromatography and eluted with a stepwise gradient of CH 2 Cl 2 /MeOH solvent system (100:0-0:1) to provide thirteen subfractions (Fr.1-Fr.13). Fr

Quantum chemical ECD calculation method
The ECD calculations were conducted by the Turbomole program package in version 3.3. The geometry optimization was performed by the time-dependent density functional theory (TDDFT) method at the pbe0m4/def-TZVP level to obtain the minimized energy confirmation.

Antioxidant activity
Compounds 1-17 were evaluated for their antioxidant activity against DPPH and ABTS radical scavenging activities. The screening method was based on the literature (Shataer et al. 2021) and results are shown in

Antimicrobial activity
Antimicrobial activities against E. coli, C. albicans and S. aureus were based on the method described by (Shataer et al. 2021). The results are shown in Table 1 and  , 5, 6, 8, 9, 16 and 17 exhibited inhibitory effects against the proliferation of C. albicans with inhibitory zone diameters were ranging from 7.5 to 12.0 mm. Compounds 8, 13 and 14 exhibited inhibitory effects against the proliferation of E. coli with inhibitory zone diameters were ranging from 7.5 to 8.5 mm. Compounds 3, 10, 13 and 14 exhibited inhibitory effects against the proliferation of S. aureus with inhibitory zone diameters were ranging from 7.5 to 9.0 mm.

Conclusion
A new macrocyclic spermidine alkaloid, hyssopusizine (1), along with sixteen known compounds were isolated from the aerial part of H. cuspidatus. Compounds 5-7 exhibited potent antioxidant activity against ABTS and DPPH. Most of compounds exhibited moderate antimicrobial activities. This is the first report on the discovery of macrocyclic spermidine alkaloid from Hyssopus genus.