Antibacterial Activity of a Phenylpropanoid from the Root Extract of Carduus leptacanthus Fresen

Background The emergence and rapid spread of antimicrobial drug-resistance microorganisms exacerbate the treatment of infectious diseases, underscoring the importance of finding new, safe, and effective drugs. In Ethiopia, the roots of Carduus leptacanthus have traditionally been employed to treat microbial infectious diseases The aim of this study was to evaluate the antibacterial activity of the root extract and its primary components against six bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia). Methods The extraction involved maceration of air-dried and powdered roots of C. leptacanthus with 80% methanol. The compound was isolated from the root extract using silica gel column chromatography and recrystallization in CHCl3/MeOH (9 : 1) and was characterized using ESI-MS and 1D-NMR spectroscopy. Antibacterial activity of the extract was assessed using agar well diffusion and broth microdilution methods. Results Syringin, a phenylpropanoid, was isolated and characterized from the extract of C. leptacanthus. The extract showed the most substantial efficacy against S. epidermidis (MIC = 5.33 mg/ml and inhibition zone diameter of 24 mm at 200 mg/m). Syringin also elicited antibacterial activity against S. aureus (MIC = 13.33 mg/ml), S. epidermidis (MIC = 16 mg/ml), and S. agalactiae (MIC = 16 mg/ml). Despite being tested up to a maximum concentration of 16 mg/ml, syringin did not exhibit antibacterial activity against the Gram-negative bacteria (P. aeruginosa, E. coli, and K. pneumonia). Conclusions In conclusion, the findings suggest that syringin exhibits partial involvement in the root extract's antibacterial activity, thereby potentially supporting the traditional medicinal use of the plant.


Background
Infectious diseases are the main threats that lead to morbidity and mortality in the world, particularly in the developing countries [1].Antimicrobial drug resistance and side efects have become major sources of morbidity and mortality worldwide.Tus, searching for new, efective, and safe antimicrobial agents to mitigate the wide spread of drug resistance has become indispensable.Tus, natural products, including secondary metabolites from medicinal plants, are sources of a new drug.Utilization of medicinal plants for the variety of ailments by the human beings was the oldest one as they contain diferent class of compounds, such as tannins, alkaloids, carbohydrates, terpenoids, steroids, and favonoids [2,3].
Te genus Carduus (family Asteraceae) comprises about 90 species [4,5] that occurs mainly in Europe, Central Asia, West Asia, Eurasia, Africa, Northern Africa, and the Mediterranean region [5][6][7].Carduus leptacanthus is locally known as Guccino (in Afan Oromo), and its root parts are traditionally used for the treatment of infectious diseases and pain in Ethiopia.It was also reported that the powdered dry stem of C. leptacanthus is used for the treatment of ascariasis and hemorrhoids [2,[8][9][10].However, the roots of C. leptacanthus have not been previously subjected to any chemical and biological investigations.Te present study has been conducted to evaluate the antibacterial activity of 80% methanol root extract of C. leptacanthus and its major constituents.

2.2.
Instruments.Organic solvents were removed using a rotary evaporator R-200 (Buchi, Switzerland).UV cabinet (CAMAG, Muttenz, Switzerland) was used to view the TLC chromatograms. 1 H-NMR and 13 C-NMR spectra were recorded at room temperature on a Bruker Avance DMX400 FT-NMR spectrometer (Bruker, Billerica, MA, USA), operating at 400 MHz for 1 H and 100 MHz for 13 C at room temperature using deuterated MeOD-d 4 , with tetramethylsilane (TMS) as the internal standard.Spin multiplicities were reported as following: s (singlet), d (doublet), dd (doublet of doublets), and dt (doublet of triplets).ESI-MS were recorded on an Ultimate 3000LC-MS.Te measurement was carried out by an electrospray ionization method in a positive mode with the source voltage and temperature being fxed at 3 kV and 250 °C.

Plant Materials. Te roots of C. leptacanthus
Fresen were collected from Machakel Woreda, East Gojjam Zone, Amhara Regional State, around 327 km North of Addis Ababa, Ethiopia.Authentication of the plant was conducted by Mr. Melaku Wondaferash, at the Department of Biology, National Herbarium, Ethiopia, where the specimens were deposited giving a voucher number (AD004).

Extraction.
Te roots were cleaned of dust and debris, washed gently with water, and air-dried under shade for two weeks.Ten, the dried roots were pulverized with a grinder to reduce to an appropriate size.Dried and powdered roots (200 g) were macerated in 80% methanol at room temperature with occasional shaking for 72 h.It was then frst fltered using nylon cloth, followed by Whatman flter paper no. 1. Te residue was remacerated for another 72 h twice.Ten, the combined fltrates were evaporated under reduced pressure using a rotary evaporator.Te extract was further concentrated to dryness with oven at 40 °C.Ten, the root extract was weighed (24.8 g) and stored in a refrigerator in air tight plastic containers until used.

Compound Isolation.
Column chromatography was employed for compound isolation.Te column was initially packed with slurry, which was prepared by mixing silica gel (75 g) in chloroform (100 ml).Te root extract (3.3 g) was adsorbed on silica gel by adding root extract and silica gel (2.5 g) in methanol.Te mixture was then concentrated with a rotary evaporator until dried.Te adsorbed sample was then loaded on the top of the column and eluted with gradual increase of methanol in chloroform.A total of 162 fractions were collected, of which fractions 121-162 eluted with CHCl 3 / MeOH (9 :1; 400 ml) resulted in a white-plate crystal (30 mg, coded as CL-1).Te purity of the compound was monitored by TLC when viewed under ultraviolet (UV) light of wavelengths 254 and 366 nm.For the TLC analysis, the process began by taking an individual sample weighting 1 mg and dissolved it in 0.5 ml of methanol.Te solution (10 μl) was then carefully spotted onto TLC plate using a TLC applicator.Te plate was then transferred to a TLC chamber, which contained a mixture of CHCl 3 /MeOH in a ratio of 9 :1 as a mobile solvent system.

Media and Inoculum Preparation.
MHA was used to subculture the bacteria [11].Well-isolated colonies (3-5) of the same morphological type from an agar plate culture were selected [12].Te colonies of bacteria were transferred to the broth using loop.For this, a UV spectrophotometer was used to adjust the bacterial suspension by measuring its absorbance with 1 cm path length at the wavelength of 625 nm.So, the absorbance should be in the range from 0.08 to 0.10 which is proportional to 1 × 10 8 CFU/ml bacteria [13,14].Tis inoculum suspension was diluted in 1 : 10 to get 1 × 10 7 CFU/ml to determine the agar well difusion assay [12].

Agar Well Difusion Method.
A volume of the microbial inoculum was spread over the entire agar surface.After solidifying, a sterile cork borer aseptically punched the streaked plate to form holes with the diameter of 8 mm. 100 µL of the standard drug, the negative control, and extract solution at desired concentrations were introduced into the wells.Te plates were incubated at 37 °C for 18-24 h, at 28 °C for 48 h.Te zones of inhibition diameters in mm were measured using a ruler, and the average values were calculated [15,16].Te antibacterial activity of the extract was evaluated by comparing its inhibition diameter with the standards [17].Ciprofoxacin 5 µg was used as a positive control, while 1% DMSO was used as a negative control.Each assay was carried out in triplicate.

Determination of Minimum Inhibitory Concentration.
Te minimum inhibitory concentration (MIC) of the crude extracts and the isolated compound were evaluated by the 2 Journal of Tropical Medicine broth microdilution method using 96-well plates according to the Clinical and Laboratory Standards Institute [18].A stock solution of the respective plant extract (128 mg/ml) and the isolated compound (32 mg/ml) were prepared by dissolving in 1% DMSO.Two-fold serial dilutions with multichannel micropipettes were made down the column from 64 mg/ml to 0.05 mg/ml (extract) and 16 mg/ml to 0.05 mg/ml (compound).Te bacterial suspension containing approximately 5 × 10 5 CFU/ml was prepared from a refreshed culture.From this suspension, 100 μl was inoculated into each well and incubated.Sterility control was put on the last column.After incubation, 40 μl of a 0.2 mg/ml solution of 2,3,5-triphenyltetrazolium chloride was added to each well as an indicator of microbial growth and incubated at 37 °C for 30 min.After incubation, the MIC values were visually determined by observing the presence or absence of pink color.Te lowest concentration of each extract displaying no visible pink color was recorded as the MIC.MIC values were determined in triplicate [19,20].
2.8.Data Analysis.Te data were analyzed using SPSS (Statistical package for social science) software version 25.0.Te results were presented as the mean ± standard error of the mean.One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare diferences in mean among the treatment and control groups.p values <0.01 were considered statistically signifcant.  1C-NMR spectrum of compound 1.Analysis of the spectral data of compound 1 (see below for complete assignments of both 1 H and 13 C signals) and comparison to previously reported data led to the identifcation of compound 1 as syringin (Figure 1), in agreement Yang [21].

Results and Discussion
CL-1: white-plate crystal (yield � 0.91% from the root extract); R f value of 0.

Antibacterial Activity of the Extract.
In Ethiopia, the root parts of C. leptacanthus have been traditionally used to treat infectious diseases.To assess its traditional medicinal claim, the root extract was tested against six bacterial strains using the agar well difusion method to determine the inhibition zone diameter (Table 1).Te root extract demonstrated antibacterial activity against most of the tested bacterial strains at a concentration of 100 mg/ml (Table 1), with S. aureus (zone diameter inhibition of 16.67 ± 0.33 mm) being the most susceptible strain, followed by S. epidermidis (with a zone diameter inhibition of 16.00 ± 0.58 mm).S. aureus can lead to various serious ailments of the human skin and even life-threatening infections, while S. epidermidis is a frequently occurring species that can cause infective endocarditis [22].However, the root extract did not exhibit antibacterial activity against S. agalactiae at 100 mg/ ml, but increased concentration up to 200 mg/ml resulted in improved activity.
Te root extract showed inhibitory activity against S. epidermidis and S. agalactiae at 200 mg/ml, with the mean inhibition zone diameter of 24.00 ± 0.58 and 10.67 ± 0.33 mm, respectively.A previous study conducted by Muhaisen [23] on C. marianium L. also showed that the methanolic extract produced a clear inhibition zone diameter of 13-18 mm against S. aureus, but the aqueous extract was inefective against S. aureus.Te root extract at 100 and 200 mg/ml did not have any efect on Gramnegative bacteria (P.aeruginosa, E. coli, and K. pneumonia) as it was evident by an equal level of zone inhibition with the negative control.However, in other studies, the root extract of C. macracanthus suppressed the growth E. coli at a concentration of 50 mg/mL [24].Notably, Journal of Tropical Medicine diferent studies have reported varying efects of C. leptacanthus extracts on bacteria, which may be attributed to diferences in their constituents.E. coli has been reported as one of the predominant pathogens associated with hospital-acquired neonatal ocular infections, as well as causing various diseases ranging from less severe diarrhea to a grave condition termed hemolytic uremic syndrome [25].
According to the data presented in Table 1, the MIC values of the root extract against the six tested bacterial strains ranged from 5.33 to 32 mg/ml.Of these strains, S. epidermidis was the most susceptible to the root extract with an MIC value of 5.33 mg/ml, while E. coli demonstrated the least susceptible with an MIC value of 32 mg/ml.Strong susceptibility (p < 0.01) to the root extract was observed in S. epidermidis (MIC � 5.33 ± 1.33 mg/ml) and S. agalactiae (MIC � 6.67 ± 1.33 mg/ml) compared to the isolated compound, syringin.Tis disparity may be due to the presence of some other additional compounds in the root extract or synergetic efect between constituents.MIC values are used to determine the efcacy of an extract against a particular bacterial species, and the cut-of value varies depending on the strain tested.Tere is no universally accepted cut-of value for antibacterial activity of plant extracts.Te MIC value of ≤8000 μg/mL is generally considered as an indicator of antibacterial activity, while a value of ≥8000 μg/mL indicates weak or no activity [26].Tus, in this study, the root extract exhibited antibacterial activity against S. epidermidis, S. agalactiae, and S. aureus, while syringin displayed weak antibacterial activity against these same strains.Nevertheless, there was no notable diference observed between the root extract and syringin when tested against E. coli, P. aeruginosa, and K. pneumonia.Tese pathogenic strains are commonly associated with nosocomial infections, such as bloodstream infections, chronic obstructive pulmonary disease (COPD), cystic fbrosis, and ventilator-associated pneumonia (VAP) [25,27].
For the frst time, this study identifed syringin, a phenylpropanoid glycoside, in the root of C. leptacanthus.Syringin demonstrated antibacterial activity against S. aureus (MIC � 13.33 mg/ml), S. epidermidis (MIC � 16 mg/ml), and S. agalactiae (MIC � 16 mg/ml).Pan et al. [28] suggested that syringin's phenolic di-methoxy group may contribute to the antibacterial activity of the compound.Furthermore, the inhibition of antibiotic efux pump and bacterial cell wall synthesis could be the mechanism by syringin exerts its antibacterial activity [29].However, when tested against Gram-negative bacteria (P.aeruginosa, E. coli, and K. pneumonia), syringin did not demonstrate any antibacterial activity.Generally, Grampositive bacteria are more sensitive to antimicrobial substances due to the structural component of their cell wall [30].As such, both the root extract and syringin were more efective against Gram-positive strains than Gram-negative strains.

Conclusions
Te current research involved an examination of the phytochemicals present in the root extract of C. leptacanthus, resulting in the identifcation of syringin for the frst time.
Te root extract possessed greater efectiveness against the Gram-positive bacterial strains than the Gram-negative bacterial strains.Within the Gram-positive strains, the root extract demonstrated particularly strong antibacterial activity against S. aureus, followed by S. epidermidis.While syringin elicited a moderate antibacterial activity against S. aureus, S. epidermidis, and S. agalactiae, it was less potent than the root extract.Nevertheless, these fndings provide evidence that the presence of syringin partially accounts for the antibacterial efects of the root extract.We suggest that future studies should attempt to isolate additional compounds given the comparatively lower activity of syringin.
Overall, this study advances the traditional claims regarding the plant's ability to infectious diseases.

Table 1 :
[40] of inhibition and minimum inhibitory concentrations of root extract and compound isolated from Carduus leptacanthus.Values are presented as the mean ± SEM; n � 3; means followed by a diferent letter indicate signifcant diferences between diferent doses of the same treatments and negative control and positive control in the same row (p < 0.01) for DZI and MIC separately; NA � not active up to the concentration of 16 mg/ml.4JournalofTropicalMedicineplants,includingFraxinusrhynchophylla[21], Musa paradisiaca[39], Centaurea bella Trautv, and many other species[40].Syringin isolated from Stevia rebaudiana (Bert.)demonstrated efcacy against various bacterial species, including B. cereus, B. megaterium, B. aureus, Sarcina lutea, E. coli, Salmonella paratyphi, Salmonella typhi, Shigella dysenteriae, and Vibrio parahaemolyticus.However, it was inefective against B. subtilis, P. aeruginosa, Shigella boydii, and Vibrio mimicus Note.