MATERIALS AND METHODS

Study area: All the experiments were performed during June-December, 2020 in the Microbiology Laboratory, Major of General Science, Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, Thailand.

Chemicals and reagents: Hexane, dichloromethane, ethyl acetate, ethanol and methanol were purchased from QRëC^™ (Republic of New Zealand), Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), Nutrient Broth (NB) and Bacterial Agar were purchased from HiMedia (HiMedia Laboratories Pvt. Ltd., India).

Plant materials and extraction: The Cissus quadrangularis were collected from Ban That subdistrict, Phen District, Udon Thani Province, Thailand. The plant sample was washed 3 times using tap water and cut into a small piece before dried using a hot air oven (POL-EKO-APARATURA company, Wodzisław Śląski, Poland) at 50°C for 48 hrs. The dried plant sample was grounded into powder form using a grinding machine. Twenty grams of plant powder were extracted with 100 mL of different solvent (ethanol, methanol hexane, ethyl acetate and dichloromethane) by shaking at room temperature for 3 hrs. The extracts solution was filtered through Whatman Filter paper No. 1 and was evaporated using a rotary vacuum evaporator (BÜCHI Labortechnik AG, Switzerland). The percentage yield was calculated¹¹. Dimethyl sulfoxide (DMSO, Sigma) was added into each plant extract to the final concentration at 500 mg mL^–1 before used:

Test human pathogenic bacteria: Acinetobacter baumannii, Stenotrophomonas maltophilia, Enterococcus faecalis, Burkholderia pseudomallei, Proteus mirabilis, MDR Klebsiella pneumoniae (MDR-KP), Colistin resistant Pseudomonas aeruginosa (CoR-PA) were obtained from the Department of Clinical microbiology, Roi Et Hospital, Roi Et, Thailand. Active bacterial cultures were generated by inoculating a single colony of each bacteria in separate 50 mL Nutrient Broth (NB) and incubating with shaking at 37°C for overnight. The bacterial cells were harvested by centrifugation at 10000 rpm for 30 sec and were adjusted the cell concentration at OD₆₀₀ to 0.1 before use.

Screening of antibacterial activity of Cissus quadrangularis extracts using disc diffusion assay: The antibacterial activity of C. quadrangularis extracts was primary screened using disc diffusion assay²⁴. One hundred microliters of each human pathogenic bacteria were spread onto Nutrient Agar (NA) and a sterile paper dish (0.6 mm) was placed on NA. Ten microliters of each C. quadrangularis extract were dropped onto the centre of the paper disc. The DMSO was used as a negative control. The C. quadrangularis extracts were allowed to diffuse for 15 min at room temperature before the plates were incubated at 37°C for 24 hrs. The inhibition zone formation around the paper disc was measured.

In vitro antibacterial activity determination using a microdilution assay: The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of C. quadrangularis extracts were determined using a microdilution assay²⁵. The C. quadrangularis extracts which presented the inhibition zone from primary screening were twofold serial diluted in a 96-well plate containing NB to obtain various concentrations. One hundred microliters of the overnight human pathogenic bacterial inoculum (OD₆₀₀ = 0.1) were added into each well. DMSO was used as a negative control. The 96-well plates were incubated at 37°C for 24 hrs. The colorimetric assay was done by using Iodonitrotetrazolium Chloride (INT) as an indicator of bacterial growth. Fifty microliters of INT were added into each well of the 96-well plate and were incubated at 37°C for 1 hr. The well containing the bacterial growth turned into pink colour whereas the well without bacterial growth remained yellow colour. The MIC was referred to as the lowest concentration of the C. quadrangularis extract that inhibits bacterial growth. The MBC was considered as the lowest concentration of C. quadrangularis extract that eliminates the bacteria that did not produce a colour change after the addition of INT²⁶.

Data analysis: In this study, it was used experimental design followed by descriptive analysis.

RESULTS AND DISCUSSION

C. quadrangularis extraction yields: Five extraction solvents were used for C. quadrangularis extraction. The extraction was done by shaking for 3 hrs of the mixer the C. quadrangularis powder and each extraction solvents. The result indicated that the highest extraction yield at 7.25% was obtained from methanolic extraction, follow by ethyl ethanol, acetate and dichloromethane extraction was at 5.44, 1.55 and 1.52%. The lowest extraction yield at 1.28% was presented when extracted with hexane (Table 1). The extraction yield of ethanolic extraction from this research was lower than previously reported by Alain et al.²⁷ that presented the extraction yield of C. quadrangularis was at 6.8%. The suitable solvents for antibacterial reagent extraction from C. quadrangularis were methanol and ethanol.

Disc diffusion assay: Disc diffusion assay was used for the primary screening of antibacterial activity of C. quadrangularis extracts. The results showed that the highest diameter inhibition zone at 15 mm were presented when C. quadrangularis was extracted using ethanol against E. faecalis and CoR-PA (Table 2). The result was similar to Almawlah et al.²⁸ which presented that 100 mg mL^–1 of Allium cepa bulbs extract has the inhibition zone at 14-30 mm against multidrug resistant P. aeruginosa isolated from burn wound. The inhibition zone from this research was larger than the report from Duailibe et al.²⁹ which found that Bixa orellana L. extract did not present the antibacterial activity against E. faecalis ATCC 292012 and E. faecalis 44 AB. Rachuonyo et al.³⁰ reported that extracts of Tagetes minuta, Aloe secundiflora, Bulbine frutescens and Vernonia lasiopus were presented as the zone of inhibition at 18.67, 17.76, 18.50 and 18.99 mm, respectively.

The smallest inhibition zone from this research at 7 mm was obtained from methanolic extraction against S. maltophilia and E. faecalis and ethanolic extraction against A. baumannii. This research presented the antibacterial activity against S. maltophilia lower than the report from Shah and Shelar⁹.

They reported that Piper betel leaf extract using 70% ethanol showed the zone of inhibition against S. maltophilia at 27 mm. Uğur et al.³¹ was reported the Senecio sandrasicus extracted with ethanol had an inhibition zone at 13 mm against S. maltophilia MU 23. Kidane et al.³² also reported about the aqueous extract of Lannea fruticosa showed the highest activity against P. mirabilis which was 19.5 mm of inhibition zone. No inhibitions were observed when C. quadrangularis was extracted using hexane, dichloromethane and ethyl acetate.

MIC and MBC values: The MIC and MBC values C. quadrangularis extracts were determined using broth microdilution assay. The results indicated that the lowest MIC value at 0.19 mg mL^–1 was presented in ethanol extraction against A. baumannii. The highest MIC values at 6.25 mg mL^–1 were observed when extracted using ethanol against CoR-PA (Table 3). The MIC value of C. quadrangularis extracts against A. baumannii from this research was lower than the MICs value of Rosa rugosa extracts, Terminalia chebula extracts which reported by Miyasaki et al.³³. Montagu et al.³⁴ were measured the MIC value using eugenol, carvacrol and cinnamaldehyde against A. baumannii. The result indicated that eugenol, carvacrol and cinnamaldehyde had MIC value at 1.25, 0.31 and 0.31 mg mL^–1, respectively. Zhang et al.³⁵ reported the Mentha arvensis extract had MIC value against A. baumannii at 23.5 μg mL^–1. The methanolic C. quadrangularis extracts from this research had a MIC value lower than Barringtonia acutangula (L.) extract (4 mg mL^–1) which reported by Panomket et al.³⁶. The MIC values of aqueous extracts of Lannea fruticosa against P. mirabilis was at 1.953 mg mL^–1 which lower than methanolic C. quadrangularis extracts³². The lowest MBC values at 3.13 mg mL^–1 were obtained from methanolic and ethanolic extracts against S. maltophilia and E. faecalis. The highest MBC value at 12.5 mg mL^–1 was presented in ethanolic extract against CoR-PA (Table 4). Zhang et al.¹² were reported 0.125% Litsea cubeba oil can be inhibited the growth of S. maltophilia. The MBC values of C. quadrangularis extracts from this research was lower than Tanacetum vulgare and Bidens sulphurea extracts which presented MIC values against E. faecalis at 62.50 and 31.25 mg mL^–1 which reported by Chiavari-Frederico et al.¹³. This research demonstrated that C. quadrangularis presents anti-human pathogenic bacteria activity that was never reported before and should be used for new natural drug development or combined with antibiotics used for treating bacterial infectious diseases. C. quadrangularis is not widely cultivated so the imperative to promote the cultivation of this plant is require.

CONCLUSION

The C. quadrangularis was extracted using 5 solvents. The suitable extraction solvents were methanol and ethanol. The lowest MIC value at 0.19 mg mL^–1 was obtained from ethanol extraction against A. baumannii. The lowest MBC values at 3.13 mg mL^–1 were obtained from methanol and ethanol extraction against S. maltophilia and E. faecalis. The new information from this research gives a novel report on antihuman pathogenic bacterial activity profile of C. quadrangularis extracts against infectious pathogenic bacteria in humans. This research is useful and beneficial for new antibiotic drug development and can be combined with antibiotics to increase the efficiency of the disease treatment.

SIGNIFICANCE STATEMENT

This study discovers the anti-human pathogenic bacterial activity of C. quadrangularis extracts that can be beneficial for drug development. This study will help the researcher to uncover the critical areas of the antibacterial profile of C. quadrangularis extracts against human pathogenic bacteria that many researchers were not able to explore. Thus, a new application using the C. quadrangularis extracts for treating the bacterial infection may be arrived at.

ACKNOWLEDGMENT

This research project is supported by Roi Et Rajabhat University grant No. 2557A15062001.