Chemical constituents from Centaurothamnus maximus and their antimicrobial activity

Abstract A new sesquiterpene lactone, 3β,10α-dihydroxy-10β-(hydroxymethyl)-8α-(4-hydroxymethacrylate)-1αH,5αH,6βH,7αH-guai-4(15), 11(13)-dien-6,12-olide (1), along with twenty-one known compounds, were identified from the aerial parts of Centaurothamnus maximus. The structures of the isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. Compounds (2, 3, 5‒13 and 15‒22) were identified from C. maximus for the first time. Antibacterial and antifungal activities of the isolated compounds were tested using the agar disc diffusion method. Compounds that demonstrated promising antimicrobial activity were evaluated for their minimum inhibitory concentration (MIC). The results showed that compounds 3 and 7 were the most effective antibacterial compounds against B. subtilis ATCC 6633, S. aureus ATCC 25923 and S. pyogenes ATCC 27736, with MIC estimates between 8 and 32 mg/mL. In addition, compound 2 exhibited the strongest antifungal activity against C. albicans ATCC 14243 and C. krusei ATCC 14243 with MIC 8 mg/mL. Graphical Abstract HIGHLIGHTS Twenty-two compounds were first isolated from Centaurothamnus maximus The structure of the new sesquiterpene lactone, thamnolide (1), was established. Antibacterial and antifungal activities were tested for the isolated compounds. Compounds 3 and 7 were the strongest antibacterial compounds whilst 2 exhibited the strongest antifungal activity.


Introduction
Genus Centaurothamnus Wagentz and Dittri (Asteraceae), is a monotypic genus from Centaurea comprises the one and only species Centaurothamnus maximus, (Hussein et al. 2021). It is native to the Arabian Peninsula, particularly the South western regions of Saudi Arabia and Yemen (Collenette 1999). It is described as a component of the vegetation subcommunity of juniperus procera stands, found in the upper part of the escarpment of Asir region, between the elevation of 2100 to 3000 m above sea level. The plants of C. maximus grow as leafy branched shrubs with silvery lower surface and green upper surface leaves. The flowers are tubular purple, and fluffy with faint sweet fragrant (Chaudhary and Al-Jowaid 1999). Literature review didn't reveal any known folkloric use of this plant, probably due to its limited distribution and the difficulty to collect it from the high mountains (Basahi et al. 2010). However, investigating the plant phytochemistry and exploring the possible biological activities of its extracts and isolated compounds will be encouraging to propagate the plant in other areas. The few published researches about this species, reported the isolation of oxygenated homoditerpenoid, with potent antibacterial activity against Gram-positive bacteria (Alam et al. 2015), cytotoxic sesquiterpene guainolide lactones (Muhammad et al. 2003), chlorinated guainolide lactones (Hussein et al. 2021), flavonoids (Hussein et al. 2021, aliphatic esters (Siddiqui et al. 2015a), acyclic diterpenic alcohols (Siddiqui et al. 2015b).
Mothana et al. reported excellent cytotoxic activities of C. maximus with IC 50 <50 lg mL À1 against several cancer cell lines. It also revealed notable antimicrobial properties against Gram-positive multi-resistant bacteria with MIC values < 500 lgmL À1 (Mothana et al. 2009). Notable antioxidant activity was reported for the ethanolic extract of C. maximus, probably due to the presence of flavonoids such As part of our ongoing study to search for new bioactive constituents from natural sources, in particular those found in plants grown in Saudi Arabia, we had the chance to work on the aerial parts of C. maximus to investigate its bioactive compounds and their potential biological activity. The phytochemical investigation of aerial parts of C. maximus led to the isolation of twenty-two compounds (1-22, Figures 1 and 2), including a new compound thamnolide (1), and twenty-one known ones (2-22). Their structures were identified via extensive NMR and HRESIMS. All the isolated compounds were tested on a panel of pathogenic microbial strains for antimicrobial activities using the standard disc diffusion method described by Bauer et al. (Bauer et al. 1966).

Isolation and identification of the compounds
The air-dried aerial parts and fruits of C. maximus were extracted with 96% ethanol by maceration at room temperature. The ethanolic extract (60 g, aerial parts) and (5 g, fruits) were further separated by different organic solvent extraction, various chromatographic methods including column chromatography, and preparative chromatotron to obtain compounds (1-22) (Figures 1 and 2). Their structures were identified by IR, UV-visible, NMR spectra, and HRESIMS analyses.
Nineteen carbons were detected through the 13 C NMR spectrum (Table S1 and Figure S3). The classification of these carbons was inferred from the DEPT analyses ( Figure S4) as; seven methylenes comprising two aliphatic at d C 37.5 and 39.6, two oxygenated at d C 61.7 and 68.9, and three olefinic at d C 108.4, 120.4 and 125.9; six methines including three aliphatic at d C 46.3, 49.5 and 50.3, three oxygenated at d C 73.1, 81.8 and 82.6; and six quaternary carbons comprising one oxygenated at d C 84.9, three olefinic at d C 140.5, 141.7 and 154.3, and two carbonyl carbons at d C 166.3 and 171.2 (Table S1).

Biological activities
Primary screening of twenty-two compounds for in vitro antimicrobial activity was performed against variety of standard microorganisms using the disc-diffusion method (BSAC 2020). Standard strains used for antimicrobial activity studies were including Gram-positive bacteria (S. aureus, B. subtilis and S. pyogenes), Gram-negative bacteria (P. aeruginosa, K. pneumonia and E. coli), as well as unicellular fungi (C. albicans and C. krusei). The results of the biological activity including the average zone of inhibition (mm) and MIC values (mg/mL) are presented in Tables S2 and S3. Generally, tested compounds were exhibited variable antimicrobial activity against tested strains. Some of these compounds are inactive and the others exhibited strong activity against Gram-positive and Candida strains. The pronounced antimicrobial activity was observed for compounds (1, 3, 7, 10 and 14) against S. aureus, B. subtilis and S. pyogenes with zones of inhibition wider than 15 mm (Table S2) and the MIC estimates ranged between 8 and 32 mg/mL (Table S3).
The strongest activity was observed with compounds 3 and 7 against B. subtilis ATCC 6633, S. aureus ATCC 25923 and S. pyogenes ATCC 27736, with MIC estimates between 8 and 16 mg/mL. Notably, the evaluated antimicrobial effects of the isolated compounds, in particular sesquiterpene lactones, confirmed and explained the previously reported pronounced antimicrobial activity of the methanolic extract C. maximus reported by Mothana et al. (Mothana et al. 2009) against Gram-positive bacteria including some multi-resistant Staphylococcus strains.
On the other hand, all tested compounds express weak antibacterial effect against Gram-negative strains including P. aeruginosa, E. coli and K. pneumonia. For Candida species, the compounds of (1-3, 5, 10 and 14) were the most active compounds with MIC values ranged from 8 and 32 mg/mL. The most promising compound against Candida species was compound 2 with MIC of 8 mg/mL. Although the compound 4 had promising antibacterial effect against strains of Gram-positive bacteria, it had no antifungal activity against C. albicans and C. krusei. The current findings are in agreement with the previously reported antifungal activity of guaianolide sesquiterpene lactones from C. maximus against C. albicans (Muhammad et al. 2003). As a results, these outcomes support the view that some of the isolated compounds from C. maximus may represent the potential new antibacterial and antifungal agents.

Conclusion
Phytochemical study of the aerial parts of C. maximus afforded one new guaianolide sesquiterpene lactone, thamnolide (1), in addition to the total of twenty-one diverse known bioactive compounds, including four guaianolide sesquiterpenes lactones (2-5), one megastigman (6), six flavonoids (7-12), one phenylpropanoid glycoside (13), five steroids (14-18) and four triterpenes (19)(20)(21)(22). Compounds (2,3, 5-13 and 15-22) were identified from C. maximus for the first time. The structures of isolated compounds were confirmed by intensive (1 D and 2 D) NMR studies and mass spectrometry. Compounds (3 and 7) demonstrated strong antibacterial activity against Gram-positive bacteria B. subtilis ATCC 6633, S. aureus ATCC 25923 and S. pyogenes ATCC 27736, while the strongest antifungal activity was revealed by compound 2 against C. albicans ATCC 14243 and C. krusei ATCC 14243. None of the isolated compounds exhibited a significant inhibitory activity against Gram-negative strains. These findings are considered an additional effort for the discovery and development of new anti-infective drugs.

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