Antibacterial activity of Cathormion umbellatum

The aim of this study was to determine the antibacterial activity of Cathormion umbellatum extracts against seven antibiotic-resistant bacteria. The pods, leaves and branches of C. umbellatum were extracted with ethanol and methanol. The disc diffusion assay was used to screen the antibacterial activity and broth microdilution and colorimetric assay were used to measure the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. The result indicated that the highest inhibition zone (11 mm) was presented in ethanolic pods extract against multidrug resistance Klebsiella pneumoniae. The lowest MIC value of 0.05 mg/mL was obtained from branch extracted with ethanol against colistin resistant Pseudomonas aeruginosa. The lowest MBC values of 1.56 mg/mL were obtained when using C. umbellatum leaves extracted with methanol against all test antibiotic-resistant bacteria. This is the first report presented C. umbellatum extracts have the potential to eliminate antibiotic-resistant bacteria in patients. These findings show the antibacterial effect of C. umbellatum.


Abstract
The aim of this study was to determine the antibacterial activity of Cathormion umbellatum extracts against seven antibiotic-resistant bacteria. The pods, leaves and branches of C. umbellatum were extracted with ethanol and methanol. The disc diffusion assay was used to screen the antibacterial activity and broth microdilution and colorimetric assay were used to measure the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. The result indicated that the highest inhibition zone (11 mm) was presented in ethanolic pods extract against multidrug resistance Klebsiella pneumoniae. The lowest MIC value of 0.1 mg/mL was obtained from branch extracted with ethanol against colistin resistant Pseudomonas aeruginosa. The lowest MBC values of 1.6 mg/mL were obtained when using C. umbellatum leaves extracted with methanol against all test antibiotic-resistant bacteria. This is the first report presented C. umbellatum extracts have the potential to eliminate antibiotic-resistant bacteria in patients. These findings show the antibacterial effect of C. umbellatum.
Lannea fruticosa showed the highest inhibition zone activity against both P. aeruginosa and P. mirabilis which was 20 mm and 19.5 mm, respectively (Kidane et al., 2019).
Cathormion umbellatum (Vahl) Kosterm is a flowering plant in the legume family, Fabaceae which belongs to the mimosoid clade of the subfamily Caesalpinioideae. C. umbellatum is Thai mimosaceous plants that contained high antioxidant activity and can be stimulated white blood cell proliferation (Tunsaringkarn et al., 2014). Only antibacterial activity of C. umbellatum extracted with ethanol against E. coli was reported (Ramli, 2010). The determination of antibacterial activity against antibiotic-resistant bacteria has still lacked. Therefore, the aim of this study was to determine the antibacterial activity of C. umbellatum extracts against seven antibiotic-resistant bacteria collected from the Roi Et Hospital, Thailand.

Chemicals and reagents
Ethanol and methanol were purchased from QRëC™ (New Zealand). Dimethyl sulfoxide was purchased from Sigma-Aldrich (USA). Nutrient broth and bacterial agar were purchased from HiMedia (India). Iodonitrotetrazolium chloride was purchased from G-Biosciences (USA).

Plant materials and extraction
The fresh branch, leaves and pods of C. umbellatum were collected from Tha Muang Community, Tha Muang sub district, Selaphum District, Roi Et Province, Thailand. All plant samples were dried using hot air oven (POL-EKO-APARATURA company, Wodzisław Śląski, Poland) at 50ºC for 48 hours before were grounded into powder. The plant powder was extracted with ethanol and methanol with shaking for 3 hours and then filtered and evaporated using a rotary vacuum evaporator (BÜCHI Labortechnik AG, Switzerland). The percent yield was calculated . The plant extracts were adjusted the final concentration to 500 mg/mL using dimethyl sulfoxide.

Antibacterial activity determination
The antibacterial activity of the C. umbellatum extracts was tested against seven antibiotic-resistant bacteria including A. baumannii, S. maltophilia, E. faecalis, B. pseudomallei, P. mirabilis, multidrug resistance K. pneumoniae, colistin resistant P. aeruginosa. The active bacterial cultures were adjusted the cell concentration at OD600 to 0.1 before used.
The antibacterial activity of C. umbellatum extract was primary determined using disc diffusion assay (Boon-gapim et al., 2021;Malaka et al., 2018). Ten microliters of each C. umbellatum extract (500 mg/mL) was dropped onto the center of the paper disc. The dimethyl sulfoxide was used as a negative control. The bacterial culture plates were incubated at 37ºC for 24 hours. The inhibition zone formation around the paper disc indicated as antibacterial activity of C. umbellatum extracts were measured.
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of C. umbellatum extracts were determined using a broth microdilution and colorimetric assay (Rattanasuk and Phiwthong, 2020). The C. umbellatum extracts which presented the inhibition zone from the previous part were 2-fold serial diluted in a 96-well plate containing NB. The 96-well bacterial culture plates were incubated at 37ºC for 24 hours. The iodonitrotetrazolium chloride (4 mg/mL) solution was added into each well of the 96well bacterial culture plate and then incubated at 37ºC for 1 hour. The MIC was referred to as the lowest concentration of the C. umbellatum extract that can inhibit bacterial growth. The MBC was considered as the lowest concentration of C. umbellatum extract that can eliminate the bacteria that did not produce a color change after the addition of iodonitrotetrazolium chloride (Dzotam et al., 2016).

Percent yield and inhibition zone
The result of percent yield indicated that the highest percent yields at 15.9% was obtained when used the C. umbellatum leaves extracted with methanol, followed by pods extracted with methanol (13.3%) and leaves extracted with ethanol (11.7%), respectively. The lowest percent yields at 4.9% was found in branch extracted with ethanol.
The result of disc diffusion assay indicated that the highest inhibition zone at 11 mm was presented in ethanolic pods extract against multidrug resistance K. pneumoniae, followed by pods extracted with ethanol (10 mm), branch extracted with ethanol (9.5 mm) and leave extracted with methanol (9 mm) against B. pseudomallei, P. mirabilis and colistin resistant P. aeruginosa, respectively (Table I).

MIC and MBC values
The results indicated that the lowest MIC value of 0.1 mg/mL against colistin resistant P. aeruginosa was obtained from branch extracted with ethanol followed by 0.1 mg/mL was obtained from leave extracted with methanol against P. mirabilis, pods and leave extracted with methanol against B. pseudomallei (0.4 mg/mL), respectively (Table II). The lowest MBC values of 1.6 mg/mL were obtained when using C. umbellatum leaves extracted with methanol against all test antibioticresistant bacteria, leaves extracted with ethanol against S. maltophilia, B. pseudomallei P. mirabilis and colistin resistant P. aeruginosa, pods extracted with ethanol against A. baumannii, S. maltophilia, E. faecalis and B. pseudomallei, pods extracted with methanol against S. maltophilia, E. faecalis, B. pseudomallei and P. mirabilis. The highest MBC value of 12.5 mg/mL was found in branch extracted with methanol against E. faecalis.

Discussion
C. umbellatum shows antibacterial and antioxidant activity (Tunsaringkarn et al., 2014). It has been found that the ethanolic branch extract was presented that the lowest MIC value of 0.1 mg/mL against CoR-PA and methanolic leave extract was showed the lowest MBC values of 1.6 mg/mL against all test antibiotic-resistant bacteria. The mechanism of action is not clear.
The MBC values of C. umbellatum extract against A.  (Wasihun and Kasa, 2016). Proanthocyanidins and flavonoids glycoside are potential phytochemical groups content of C. umbellatum extracts which act as an antibacterial reagent (Ramli, 2010). Proanthocyanidins are phytochemicals found from C. umbellatum which synthesized from tannin and are presented various biological activities including antioxidant, anticancer, antidiabetic, neuroprotective, and antimicrobial activity (Rauf et al., 2019). The proanthocyanidins of Dalbergia monetaria extracts present the antibacterial activity against methicillin sensitive S. aureus, methicillinresistant S. aureus and P. aeruginosa with MIC values of 64, 64 and 32 μg/mL, respectively (de Moura et al., 2020). The curcuminoids have an antimicrobial activity against E. faecalis with MBC of 50 μg/mL (Suttipalin et al., 2014).

Conclusion
The present study shows the antimicrobial activity of C. umbellatum against antibiotic-resistant bacteria.

Financial Support
This research project was supported by Roi Et Rajabhat University grant No. 2557A15062001