Echinograciolide, a new antibacterial nor-triterpenoid and other constituents from Echinops gracilis O. Hoffm. (Asteraceae)

Abstract Antibacterial resistance is a serious threat against humankind and the search for new therapeutics is needed. This study aims to investigate the antibacterial activity of extracts and compounds from Echinops gracilis O. Hoffm. Standard chromatographic and spectroscopic methods were used to isolate and characterize compounds (1–15) from the methanol extract. The extract, chromatographic fractions and compounds 1–3, 8, 11, 13 and 14 were subjected to in vitro antibacterial assays against Staphylococcus aureus ATCC25923, Salmonella Typhi ATCC6539, Klebsiella pneumoniae 22, and Salmonella Typhi 68, using broth micro-dilution method. As results, a new nor-triterpenoid (1) and fourteen known compounds (2–15) were characterized. The extract and fractions displayed moderate (128 ≤ MIC ≤ 512 μg/mL) and significant (MIC 64 μg/mL) antibacterial activities. Compounds 1 and 14 showed the best anti-staphylococcal and anti-salmonella activity (MIC 16 µg/mL), respectively. These results partially justified the antimicrobial uses of E. gracilis in traditional medicine. Graphical Abstract


Introduction
Increase in infections as a result of emergence of drug-resistant microorganisms and hitherto unknown pathogenic microbes pose enormous public health concerns (Iwu et al. 1999).The situation is most dire in less developed countries, where the uncontrolled use of antibiotics, often coupled with a lack of proper healthcare infrastructure, leads to high morbidity rates (Laxminarayan and Heymann 2012).Therefore, the need of searching and developing new therapeutic agents, capable of overcoming the resistance mechanisms of microbes, remains imperative (Fischbach and Walsh 2009).Historically, plants have provided a good source of anti-infective agents in the fight against microbial infections (Mahady et al. 2008;Nzogong et al. 2018;Nanmeni et al. 2021;Tedonkeu et al. 2022).The genus Echinops, which belongs to the Asteraceae family, comprises over 130 species distributed in the Mediterranean Basin, Central Asia and tropical Africa (Hedberg et al. 2004;S anchez-Jim enez et al. 2010;Bitew and Hymete 2019).Members of this genus are mostly perennial with few annuals (Hedberg et al. 2004;S anchez-Jim enez et al. 2010).Many species of Echinops are used in traditional medicine to treat different infectious diseases such as typhoid, gonorrhea, and ulcerative lymphangitis (Bitew and Hymete 2019;Lah et al. 2020).They are also used in the treatment of different ailments that might be caused by microbial infections including toothache, earache, respiratory diseases, fever, and leucorrhoea (Bitew and Hymete 2019;Lah et al. 2021).Echinops gracilis O. Hoffm is a shrub used traditionally to treat rheumatism (Adjanohoun et al. 1996).Previous work carried out on some species of the genus Echinops led to the isolation of several secondary metabolites such as acetylenic thiophenes (Hymete et al. 2005;Nakano et al. 2011), triterpenoids (Kiyekbayeva et al. 2018;Lah et al. 2020), sesquiterpenes (Abegaz et al. 1991;Li et al. 2010), alkaloids (Chaudhuri 1992;Kiyekbayeva et al. 2017), lignans (Tene et al. 2004;Sandjo et al. 2016), flavonoids (Singh et al. 1990;Singh and Pandey 1990;Khedher et al. 2021;Lah et al. 2021), and hydroxycinnamates (Jaiswal et al. 2011;Lah et al. 2020).Previous biological studies on Echinops gracilis reported anti-inflammatory (Lah et al. 2020), antibacterial and antioxidant (Lah et al. 2021) activities.In our continuous search for bioactive secondary metabolites, such as triterpenoids, from Cameroonian medicinal plants (Tene et al. 2009;Nzogong et al. 2018;Sonfack et al. 2021;Tedonkeu et al. 2022), we report herein the isolation and characterization of a new antibacterial nor-triterpenoid (1), along with fourteen known compounds (2-15), from the whole plant of E. gracilis.

Antibacterial activity of extract, fractions of extract and compounds
The extract, n-hexane and EtOAc fractions of extract and some isolated compounds (1-3, 8, 11, 13 and 14) were tested for their antibacterial activity (Table 1) against four bacteria.As shown in Table 1, and according to the antibacterial activity criteria (Kuete 2010), the extract and fractions of extract displayed antibacterial activity towards 4/4 (100%) of tested bacteria with moderate (100 < MIC 625 lg/mL) and significant (10 < MIC 100 lg/mL) activities.The MeOH extract showed the best and significant antibacterial activity against Salmonella Typhi 68 and Salmonella Typhi ATCC6539 (CMI ¼ 64 lg/mL) and moderate antibacterial activity against Staphylococcus aureus ATCC25923 and Klebsiella pneumoniae 22 (CMI ¼ 256 lg/mL), while fractions of extract only displayed moderate antibacterial activity against the four microorganisms (128 MIC 512 lg/mL) used.The results for the MeOH extract could partially justify the use of the plant material in traditional medicine in the treatment of infectious diseases.Compounds 1, 8, 13 and 14 were the most active samples inhibiting the growth of all four tested bacteria with MICs ranging from 16 ─ 64 mg/mL, 32 ─ 64 mg/mL, 64 ─ 128 mg/mL and 16 ─ 32 mg/mL, respectively.According to literature (Kuete 2010), moderate activity (10 < MIC 100 lg/mL) was observed with compounds 1, 8 and 14 against 4/4 (100.00%), and 13 against 2/4 (50.00%) tested bacterial species (Table 1).The best antibacterial activity (MIC ¼ 16 mg/mL; MBC ¼ 32 mg/mL) was recorded with compound 1 against S. aureus ATCC25923 and 14 against S. Typhi ATCC6539.As shown in Table 1, ciprofloxacin used as standard drug was more potent against bacteria than the isolates (1-3, 8, 11, 13 and 14).Our data showed that the sensitivity of bacteria to the test samples varied from one microorganism to the other.Compounds 1 and 8 are friedelane-type triterpenoids.The difference in their inhibitory activity was observed only against S. aureus ATCC25923 (MIC ¼ 16 mg/mL for 1 and 32 mg/mL for 8), but compound 1 was two times more bactericidal than 8 against K. pneumoniae 22 (MBC ¼ 32 mg/mL for 1 and 64 mg/mL for 8) and S. Typhi ATCC6539 (MBC ¼ 64 mg/mL for 1 and 128 mg/mL for 8).The presence of the lactone ring (27,15a-olide function) in compound 1 instead of 27-carboxylic function in compound 8 could justify the difference in their activity against the tested bacteria.Other triterpenoids, lupeol (3) and its acetate (2), are of lupane-type.The latter was two times less active than the former against S. aureus ATCC25923.The difference in the anti-staphylococcal activity could be attributed to the additional acetyl group at position 3 of lupeol.Also, compounds 13 (apigenin-7-O-b-D-Glc) and 14 [(4 00trans-p-hydroxycinnamoyl)-apigenin-7-O-b-D-Glc] are substituted flavonoids.However, the activity of 14 was two or four times higher than that of 13 (Table 1).This can be attributed to the additional trans-p-hydroxycinnamoyl group at position 4 of the glucopyranosyl moiety in 13.
In all the cases, a bactericidal effect with MBC/MIC ratio 4 was noted for the tested compounds suggesting their lethal effect.The known antimicrobial mechanisms associated to the group of chemicals to which the isolated compounds belong may explain the antibacterial potency of the extracts.Membrane disruption has been suggested as one of the likely mechanisms of action (Cowan 1999;Shah et al. 2004).This might also explain the antibacterial activities of isolated triterpenoids and flavonoids (Cowan 1999;Shah et al. 2004).Triterpenoids and flavonoids, two classes of compounds widely distributed in nature have shown diverse biological activities, including antibacterial properties (Tene et al. 2009;Tamokou et al. 2011;Ndjateu et al. 2014;Nzogong et al. 2018;Nago et al. 2021;Nanmeni et al. 2021;Tedonkeu et al. 2022).

General experimental procedures
Optical rotations were measured with a JASCO P-1020 digital polarimeter.Nuclear magnetic resonance (NMR) experiments (1 D and 2 D) were performed on Bruker AV-400, Bruker DRX-500 or Jeol ECZ 400 NMR spectrometers.The chemical shifts (d) were reported in parts per million (ppm) with reference to TMS (d ¼ 0) and coupling constants (J) given in Hz.Deuterated pyridine (C 5 D 5 N) was used as solvent for the NMR experiments of compound 1.EIMS and HREIMS were carried out on an API Qstar timeof-flight spectrometer.Column chromatography (CC) was performed with silica gel 60 F 254 (70-230 mesh; Merck, Germany) and gel permeation with Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA).TLC was carried out with precoated silica gel Kieselgel 60 F 254 plates (0.25 mm thick, Merck, Germany), and spots were detected with UV light (254 and 366 nm) and further sprayed with 20% H 2 SO 4 reagent followed by heating at 100 C. Bulk quantities of solvents (MeOH, n-hexane and EtOAc) for extraction and purification were distilled prior to use.

Plant material
The whole plant of Echinops gracilis O. Hoffm.(Asteraceae) was collected in Mount Bamboutos (GPS coordinates of the centre of the zone of collection: Latitude 5 43 0 60 00 N, Longitude 10 04 0 0 00 E), West Region of Cameroon, in January 2019.Authentication was performed by Mr. Ngansop, a botanist of the Cameroon National Herbarium in Yaounde, where the specimen was deposited under the voucher number 42365/HNC.

Extraction and isolation
The dried and powdered plant material (3 kg) was macerated three times (72 h each time) in MeOH (15 L) at room temperature to afford 145 g (4.8% yield) of a crude extract, after filtration and removal of the solvent using a rotary evaporator, under reduced pressure (water bath at 50 C).A part (135 g) of this extract was defatted with n-hexane (n-Hex) to give 94 g of fraction of extract.The insoluble portion was partially dissolved in ethyl acetate (EtOAc) to yield 13 g of fraction of extract.92 g of the n-hexane fraction of extract was subjected to silica gel column chromatography (CC) eluting with a mixture of n-hexane/EtOAc of increasing polarity.A total of 85 fractions of 500 mL were collected and concentrated under rotary evaporator.They were thus combined, based on their TLC profiles, into four major fractions: A [30 g, n-Hex/EtOAc (1:0 to 9:1, v/v)], B [22 g, n-Hex/EtOAc (4:1 to 7:3, v/v)], C [13.0 g, n-Hex/EtOAc (3:2 to 1:1, v/v)], and D [15 g, n-Hex/EtOAc (1:1 to 0:1, v/v)].A part (12 g) of the EtOAc fraction of extract was also subjected to silica gel CC eluting with a mixture of n-hexane/EtOAc (1:1) followed by EtOAc/MeOH in increasing polarity.100 fractions of 250 mL each were collected and combined, based on their TLC profiles, into four major fractions: E and H [1.9 g, EtOAc/MeOH (9:1 to 4:1, v/v)] (Figure S11).

Bacteria
Strains and Clinical isolates of bacteria taken from the research unit of microbiology and antimicrobial substances collection (Cameroon), were tested for their susceptibility to the extracts and compounds.Among the clinical isolates, Klebsiella pneumoniae 22 is multi-drug resistant (MDR) to ofloxacin, aztreonam, minocycline, nalidixic acid, cephalexin, fosfomycin, ceftriaxone and erythromycin.The other clinical isolate used in this study was Salmonella Typhi 68 which is multi-drug resistant to levofloxacin, amoxicillin, ampicillin, ceftriaxone, pefloxacin and amikacin.The American Type Culture Collection (ATCC) strains, Staphylococcus aureus ATCC25923 and Salmonella Typhi ATCC6539 were used for quality controls.The conservation of bacterial species was done at 4 C on nutrient agar (NA, Conda, Madrid, Spain), prior to any antibacterial test.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The antibacterial activity was evaluated by determining the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs).MICs of extracts and compounds were determined using the broth microdilution method as previously described (Nyaa et al. 2009).The tested extract/compound was dissolved in 99.9% pure dimethylsulfoxide (DMSO, Fisher chemicals) to give a stock solution.The latter was twofold serially diluted in Mueller-Hinton Broth (MHB Conda, Madrid, Spain) in order to obtain a final concentration range of 2048 to 16 mg/mL for extracts and 256 to 0.25 mg/mL for compounds.The determination of MIC values was done by using a 0.2 mg/mL p-iodonitrotetrazolium (INT, 95%, Sigma-Aldrich) violet solution.Viable bacteria reduced the yellow dye to a pink color.MIC values were defined as the lowest sample concentrations that prevented this change in color indicating a complete inhibition of bacterial growth.The lowest concentration that yielded no growth after the sub-culturing was taken as the MBCs.Ciprofloxacin (CIP, !98%, Sigma-Aldrich, Steinheim, Germany) was used as positive control whereas broth with 20 mL of DMSO was used as negative control.The assay was repeated in triplicate.

Conclusion
The phytochemical study of the whole plant of E. gracilis afforded one undescribed nor-friedelane triterpenoid (1) together with fourteen known compounds (2 ─ 15), including six triterpenoids, four flavonoids, three steroids and one fatty acid.Compounds 2 ─ 8, 11 and 15 are here reported for the first time from E. gracilis.Moderate and significant antibacterial activities were obtained for the MeOH extract and chromatographic fractions (n-hexane and EtOAc fractions of extract) against the tested microorganisms.The best antibacterial activity (MIC ¼ 16 mg/mL; MBC ¼ 32 mg/ mL) recorded with echinograciolide (1) against Staphylococcus aureus ATCC25923, and [(4"-trans-p-hydroxycinnamoyl)-apigenin-7-O-b-D-glucopyranoside] (14) against Salmonella Typhi ATCC6539 could be valuable in further investigations for the search of secondary metabolites with similar structures that may have good activity and would be useful as antibacterial agents against the two strains.The obtained results partially justified the use of E. gracilis in traditional medicine in the treatment of infectious diseases caused by the tested pathogenic bacteria.

Table 1 .
Antibacterial activity (MIC and MBC in mg/mL) of MeOH extract, fractions of extracts, some isolated compounds and reference antibacterial drug.