LC-MS profile, in vitro acetylcholinesterase inhibitory, antibacterial and hemostatic properties of Ranunculus bullatus extract

Abstract The ethanol extract from aerial parts of Ranunculus were investigated for its chemical composition by LC-ESI-MS/MS technique, which allowed to identify a series of glycosylated flavonoids and one phenolic acid. RBEE extract showed acetyl-cholinesterase inhibition higher than the reference compound Galantamine at a concentrationof 200 µg/mL. A moderate antibacterial activity of the extract was also obtained against Staphylococcus aureus ATCC43300, Citrobacter freundii ATCC8090, and Proteus vulgaris ATCC29905 at a concentration 100 µg. Additionally, a good reduction in plasma coagulation time at 200 μL was also observed for RBEE. Graphical Abstract


Introduction
The genus Ranunculus comprises approximately 600 species (Tamura 1995). This genus has been used in traditional medicine for the treatment of skin diseases (Ugurlu and Sec¸men 2008), antihemoroid (G€ urhan and Ezer 2004), antirheumatic (Honda et al. 1996) and wound healing (Sezik et al. 2001). Several biological studies showed that Ranunculus species exert potent effects, in particular, anti-inflammatory (Cao et al. 1992;Marrelli et al. 2021), antibacterial (Bissa and Bohra 2012), antioxidant (Bhatti et al. 2015), anticancer (Yin et al. 2008), cardiovascular (Cai and Li 2004) and cytotoxic (Abdullah et al. 2021). Phenols and terpenes are the major identified constituents in this genus (Gluchoff-Fiasson et al. 1997;Zhao et al. 2008;Boroomand et al. 2018). In Algeria, this genus is represented by 50 species including R. bullatus (known under the names 'Zerhlil' and 'Ouden Helouf'), which is an endemic plant in North Africa where is used to treat sterility problems and also as a solution to weight gain (Ouarghidi et al. 2013). In the present study, chemical composition, acetylcholinesterase inhibition, antibacterial and hemostatic activities of R. bullatus ethanolic extract (RBEE) were investigated for the first time.
According to the obtained results in Table 1, RBEE exhibited potent AChE inhibition with an IC 50 value of 39.48 ± 3.83 mg/mL, along with increasing of the activity from the lowest concentration to the highest one (3.125; 200 mg/mL, respectively). RBEE displayed a lower activity than the standard Galantamine at low concentrations, while potent activity (96.32 ± 1.85 mg/mL) was observed at 200 mg/mL, comparable Galantamine (94.77 ± 0.34 mg/mL). This can be related to the presence of flavonols (Khan et al. 2018), in particular, quercetin (Jung and Park 2007) and also caffeoylquinic acid (Trendafilova et al. 2020) derivatives.
Three different bacteria strains (Staphylococcus aureus, Citrobacter freundii and Proteus vulgaris) were tested by using the disc diffusion method. RBEE exhibited activity against all of them, with the maximum effect against C. freundii for all concentrations used (25, 50 and 100 mg/mL) with values of 7, 10 and 13 mm, respectively (Table  S2). This activity can be related directly to the presence of some antibacterial agents like quercetin (Cushnie and Lamb 2005), isorhamnetin (Gong et al. 2020) and laricitrin (Taheri et al. 2020) derivatives.
Astringent activity promotes vasoconstriction which is an important parameter in hemostasis, due to the presence of tannins in the plants (Dandjesso et al. 2012) and flavonoids in some cases, especially quercetin and isorhamnetin derivatives (Furusawa et al. 2003;Kim and Yun-Choi 2008;Ku et al. 2013). This could be the case of RBEE, containing some glycosylated derivatives of quercetin and isorhamnetin. No response was observed at doses 10 and 50 lL, while a decrease in plasma coagulation time was noticed at higher doses (100 and 200 lL) (Table S3). It is a dose-dependent decrease, which means that RBEE is characterized by hemostatic and astringent activity.

Conclusion
The present study has revealed that the ethanolic extract of Ranunculus bullatus exert potent antibacterial activity against S. aureus, C. freundii and P. vulgaris. Moreover,