Isolation and purification of terpenoid compounds from Ferula haussknechtii and evaluation of their antibacterial effects

Abstract The roots of F. haussknechtii are used by local people in order to treat wounds and urinary infections. Ferula species are rich in bioactive compounds with biological effects. In line with our previous studies about screening antibacterial natural products, five terpenoid derivatives were purified from Ferula haussknechtii. The separation and purification were performed by column chromatography. Their structures were determined by 1 D and 2 D NMR as hawraman 8-p-hydroxybenzoyl-tovarol (1), ferutinin (2), lancerotriol 6-(p-hydroxybenzoate) (3), chimganin (4), and chimgin (5). Then, the antibacterial effects of the purified compounds were evaluated by measuring their MIC values against Gram-positive and Gram-negative bacteria. The results showed that compound (1) had the most antibacterial effect on Bacillus cereus (MIC = 16 µg/mL). The antibacterial effects of F. haussknechtii compounds are in line with their local application and it is suggested that further studies should be conducted to determine their mechanism of action. Graphical Abstract


Introduction
After cardiovascular diseases, infectious diseases are the second leading cause of death in the world (Angeli et al. 2018). Increased bacterial resistance, especially in nosocomial pathogens which require antibiotics for treatment, is a major concern. Drug resistance necessitates the discovery of new antimicrobial agents and antibiotics (Butler and Buss 2006). Resistance to various antibiotics such as penicillin, cephalosporins, carbapenems, and vancomycin is increasing all over the world (Gould 2008).
Natural products can be used to make new medicines because they contain many active compounds and have fewer side effects due to their greater compatibility with the human body (Kerdar et al. 2019). Studies have shown that more than 20% of various extracts of traditional Australian medicinal plants are active especially against Gram-positive bacteria (Palombo and Semple 2001). In a previous study, several extracts from 35 plants in the Turkish flora were evaluated for in vitro antibacterial activity, revealing that these extracts were active against at least one microorganism (Sokmen et al. 1999).
The genus Ferula from the Umbelliferae family is widely distributed in Asia. 185 species of this genus have been identified so far of which about 30 species are endemic to Iran (Mohammadhosseini and Nekoei 2014;Dastan et al. 2022;). Different metabolites of the genus Ferula have various antiparasitic, antibacterial, anti-Alzheimer, anticancer, radiosensitizer, and estrogenic activities (Zhou et al. 2017;Bamehr et al. 2019;Mohammadhosseini et al. 2019;Abdali et al. 2020;Dastan et al. 2020;Ebadi et al. 2021). Sesquiterpene compounds isolated from extracts of F. kuhistanica and sesqui-and disesquiterpene coumarins of F. pseudalliacea have shown significant antibacterial activity against different bacterial species (Tamemoto et al. 2001;Dastan et al. 2014Dastan et al. , 2016. F. haussknechtii H. Wolff ex Rech. f. is found in the middle of Asia especially in Iran and Turkey. In our preliminary study, essential oils from the aerial parts and roots of F. haussknechtii showed antibacterial activity against Bacillus pumilus, Staphylococcus epidermidis, and Staphylococcus aureus (Arjmand and Dastan 2020). A previous study in Turkey on F. haussknechtii showed that the dichloromethane extract of its roots contained ten new sesquiterpene esters (Miski et al. 1987a). As part of a phytochemical project of the endemic Iranian plants and their screening for new antibacterial natural products, we investigated an n-hexane and methanolic extracts from the roots of F. haussknechtii. The terpenoid compounds from F. haussknechtii ( Figure 1) were purified and identified and finally their antibacterial effects on Gram-positive and Gram-negative bacteria were measured (Akaberi et al. 2020;Mohammadhosseini et al. 2021).

Structural elucidation of the new compound
The n-hexane and methanolic extracts of F. haussknechti roots were separated by normal phase chromatography to give one new (1) and four known terpenoid derivatives (2-5) (Figure 1). Their structures were elucidated on the basis of 1 D-and 2 D-NMR and HR-ESI-MS techniques and were compared with the data previously reported in the literature. The known compounds were elucidated as ferutinin (2), lancerotriol 6-(phydroxybenzoate) (3), chimganin (4), and chimgin (5) (Kadyrov and Nikonov 1972;Fraga et al. 1985).
Compound 1 was isolated as colorless crystals. Diagnostic signals in the 1 HNMR, HSQC, and HMBC spectra suggested a sesquiterpene benzoate. The HSQC and HMBC spectra of compound 1 displayed 22 carbon signals (Table S1). Seven signals indicated a hydroxybenzoate moiety, while the remaining 15 signals suggested a sesquiterpene residue. Resonances at d 7.91 (2H, d) and 6.89 (2H, d) in the 1 H NMR spectrum corroborated the para-hydroxybenzoate moiety.
According to literature, the signals in the 1 HNMR, HSQC and HMBC spectra suggested a germacrane-type sesquiterpenoid. The attachments of the para-hydroxybenzoate and germacrane moieties were established by the HMBC spectra ( Figure S1). The 1 HNMR spectrum showed a two olefinic methylene proton at dH 5.11 (2H, d) and 5.36 (1H, d), and four methyl protons, two of 3H singlet at dH 1.73 and 1.51 and two of each 3H doublets at dH 1.17 and 1.06, respectively. The HSQC and HMBC spectra of compound 1 showed 22 carbon signals consisting of 11 sp 3 carbon signals from d C 16.2-75 ppm, 10 sp 2 carbon signals, and 1 carbonyl moiety (C ¼ O) at d C 168.4 ppm. 13 C NMR included one ester carbonyl carbon at dC 168.4 and six aromatic carbons at dC 121 (C-2 0 ), 132.1 (C-3 0 ), 115.5 (C-4 0 ), 162 (C-5 0 ), 115.5 (C-6 0 ), and 132.1 (C-7 0 ). The other 15 carbons contained four olefinic carbons at dC 129.5 (C-3), 132.1 (C-4), 133.7 (C-7), and 132.7 (C-8) and four methyls at dC 21.2 (C-11), 16.2 (C-12), 23.5 (C-14), and 21.2 (C-15) were attributable to a sesquiterpene moiety. The positions of double bonds and hydroxyl groups were established by HMBC and COSY spectra. Other important HMBC and COSY correlations are given in Figure S1. The relative configuration of stereocenters at C-1, C-9, and C-10 was determined according to reported germacrane ester compounds and NOESY spectrum (NOESY cross peaks between H-1 and H-13). According to the literature, compound 1 has the same constitution and relative configuration as 8-p-hydroxybenzoyl-tovarol. The novelty of compound 1 was determined according to the difference between the specific rotation of compound 1 (þ24.0) and 8-p-hydroxybenzoyl-tovarol (-54.2). Compound 1 (RRR) and 8-p-hydroxybenzoyl-tovarol (SSS) are enantiomers with the same NMR, MS, and IR data. 8-p-Hydroxybenzoyltovarol was extracted from F. orientalis roots by Miski, Mabry, and Saya (1987b) from the Agri province of Eastern Anatolia, Turkey. Differences in natural habitat, environmental conditions, and biosynthetic pathway enzymes of F. haussknechtii and F. orientalis cause the biosynthesis of specific isomers in each of them. On the other hand, there are similarities between the two species that can be used to study the chemotaxonomy of the genus Ferula.

Antibacterial assay
The antibacterial activity of compounds 1-5 were measured against seven pathogenic bacterial strains including three Gram-positive and four Gram-negative bacteria (Table  S2). According to the results, the overall inhibitory activities of the compounds were higher against the tested Gram-positive bacteria than the Gram-negative bacteria.
Hawraman 8-p-hydroxybenzoyl-tovarol (1) had the widest antibacterial spectrum among these compounds. All these 5 compounds had a significant effect on S. aureus but a moderate effect on K. pneumonia and P. aeruginosa. The inhibited growth of S. aureus occurred at a concentration of 64 lg/mL, whereas the MIC for chloramphenicol was 4 lg/mL and clarithromycin was inactive. Among these compounds, compound 1 had the greatest antibacterial effect on B. cereus with the MIC of 16 lg/mL. Two daucane sesquiterpenes with a similar structure to compounds 2 and 3 had an IC 50 of 1.5 and 3.5 lg/mL against S. aureus (Galal et al. 2001). The inhibition of bacterial growth is one of the important parameters in the treatment of wounds and urinary infections which is in line with the traditional use of F. haussknechtii roots.

General experimental procedures
Melting points were obtained on a Stuart melting point apparatus. Optical rotations were measured on an Electrothermal polarimeter. UV spectra were recorded on a Shimadzu UV spectrometer. IR spectra were measured on a Bruker FT-IR spectrometer. NMR spectra were measured on a Bruker 500 MHz spectrometer. Silica gel (70-230 and 230-400 mesh, Merck) was used for column chromatography. Silica TLC was performed on Merck F254 silica gel plates. HR-ESIMS spectra were acquired on a Bruker MicroTOF ESI-MS system.

Plant material
The roots of F. haussknechtii were collected at an altitude of 2100 m from the Hawraman mountains of Kurdistan, Iran, in June 2017. The samples were kept at the Herbarium of Hamadan University of Medical Sciences after Hiva Ghaderi distinguished and confirmed them according to code 402. The plant parts were wiped free of dust and kept under shadow and room temperature (25 C).

Antibacterial assay
The in vitro antibacterial activity of the isolated compounds was evaluated by determining their MIC values against S. aureus ATCC 25923, B. cereus PTCC 1015, P. aeruginosa PTCC 1430, E. coli ATCC 25922, clinical isolate of H. pylori, b-lactamase producing clinical strain of K. pneumonia and clinical isolate of vancomycin resistant E. faecium. The antibacterial activity was tested in triplicate as described in our previous study (Dastan et al. 2016).

Conclusion
Finding new medicinal compounds is a great challenge at the present time. Meanwhile, new antibacterial compounds have particular importance due to the spread of bacterial resistance and the side effects of current antibiotics. Five terpenoids were isolated from F. haussknechtii extracts and one of them was a new germacrane-type sesquiterpenoid. These compounds were assayed for antibacterial activity and the results showed that all of them were generally more active against S. aureus compared with other species. The experimental results confirmed the traditional use of F. haussknechtii by local people for treating wounds and urinary infections. In addition, the results of the study suggest that the terpenoids of F. haussknechtii may be appropriate natural antibacterial agents and potent lead compounds. Due to the special ring structure and the presence of hydroxyl groups in these compounds, they can have various antifungal, antiviral, acetylcholinesterase inhibitory, antioxidant, and cytotoxicity effects. Therefore, further investigations may be conducted on these compounds to investigate their other effects.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
We are grateful to Vice-chancellor for Research and Technology, Hamadan University of Medical Sciences (Grant No. 9603231789) for financial support of this work.