Biological and phytochemical characterization of Phragmanthera regularis (Loranthaceae) from Ethiopia

Abstract Phragmanthera regularis is a hemi-parasitic shrub. It is known for treating various health ailments. The aim of this study was to evaluate the antimicrobial activity, toxicity, and chemical characterization of the leaf extracts of P regularis collected from the Schinus molle host plant in Ethiopia. The antimicrobial properties of crude extracts obtained with chloroform, ethyl acetate, methanol, and water solvents were assayed against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The methanol extract significantly inhibited the growth of S. aureus, E. coli and P. aeruginosa were resistant to any of these solvent extracts. The methanol extract was tested at 175, 550, and 2000 mg/kg body weight doses in white mice and did not reveal any toxicity. The LC-MS qTOF analysis detected flavonoids, phenolic acids, and alkaloids in the crude methanol extract. Further study is needed to investigate the effectiveness of these compounds against S. aureus. Graphical Abstract


Introduction
Medicinal plants play a vital role in covering the basic health needs of millions of people in developing countries (Shihabudeen et al. 2010). Resistance to antimicrobial agents has become a pressing global health problem. Thus, the screening of chemical entities from natural products (Sorokina and Steinbeck 2020) attracts interest in developing novel drugs (Faboro et al. 2016).
Despite the fact that different communities recognize the ethnomedicinal use of Loranthaceae species, only a few of these species have been documented in the ethnobotanical studies of Ethiopia. Hence, the biological activities and chemical constituents of these species are not well known in the country. The aim of this study was to determine the phytochemical constituents, antimicrobial potential, and acute toxicity of the leaves of P. regularis collected from the branches of Schinus molle in Ethiopia.

Results and discussion
2.1. Physicochemical properties of P. regularis The extraction of P. regularis using water, chloroform, ethyl acetate, and methanol as solvents yielded 3.84%, 2.05%, 2.90%, and 7.51%, respectively. The total ash, acid-insoluble ash, water-soluble ash, and moisture content of P. regularis were 9.64 ± 0.29 (g, w/w), 2.63 ± 1.13 (g, w/w), 5.32 ± 0.15 (g, w/w), and 25.80 ± 1.04 (%), respectively. The weight of the total ash content was largely derived from the physiological ash of the plant, and thus the non-physiological ash has a minimal contribution to the adulteration of P. regularis (Siraj et al. 2020). The higher moisture content of leaves may initiate microbial growth that degrades plant materials and makes them unsafe for storage (Zhang et al. 2012).

In vitro antibacterial activity
In the disk diffusion assay, the crude methanol leaf extract of P. regularis showed antimicrobial activity against S. aureus, but no effect has been detected on E. coli and P. aeruginosa. The water, chloroform, and ethyl acetate extracts did not show activity against all the tested bacteria. The standard drug produced a significantly (p < 0.05) higher diameter of inhibition zone against S. aureus, followed by 100 mg/ml crude methanol leaf extract (Supplementary information Figure 1S, Table 1S). Previous study showed that S. aureus was sensitive to the methanolic extract of P. regularis and resistant to the water extract (Mothana et al. 2012).
The antimicrobial activity of P. regularis may be associated with the higher solubility of bioactive compounds in more polar solvents such as methanol (Truong et al. 2019), the ability of existing bioactive compounds to diffuse in the bacteriological media used in the assay, and intrinsic bioactivity of the respective compounds (Parekh and Chanda 2007). In addition, the significantly lower inhibition diameter of the crude methanolic extract relative to the standard drug may be associated with the lower quality and quantity of bioactive constituents of compounds responsible for growth inhibition of S. aureus. In contrast, the absence of antimicrobial activity of water, chloroform, ethyl acetate, and methanol extracts of P. regularis against E. coli and P. aeruginosa may be due to the presence of a thick layer of lipopolysaccharide outer membrane covering the cell wall in these Gram-negative bacteria rather than Grampositive bacteria (Nikaido 2003).

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC and MBC values of P. regularis methanolic extract against S. aureus were 25 mg/ml and 50 mg/ml, respectively. There was active bacterial growth in the control test tube. In contrast, no color change occurred for the standard antibiotic (i.e. erythromycin) treated test tube ( Figure 2S and Figure 3S). The result showed a significant antimicrobial activity of the plant extract only at high concentrations. Thus, fractionation of the plant extract to obtain pure molecule could have a more significant effect on the selected bacteria as it increases the quality and quantity of bioactive principles (Waltrich et al. 2015).

Phytochemical characterization of P. regularis
The LC-MS qTOF analysis of the P. regularis methanol extract revealed nine flavonoids, three phenolic acids, two alkaloids, and other nine compounds (Table 2S, Figures  4S-26S). The positive ionization mode was more successful in identifying a greater number of metabolites than the negative ionization mode. On the other hand, the negative ionization mode was important for identifying flavonoids and phenolic acids.
The antimicrobial activity of the methanol leaf extract of P. regularis may be due to the individual and/or the synergetic activities of flavonoids, phenolic acids, alkaloids, and other bioactive compounds widely known for their biological activities in the literature. For example, caffeic acid and its derivatives (Stojkovic et al. 2013), quinic and shikimic acids (Bai et al. 2018), quercetin (Amin et al. 2015), and fisetin (Gabor and Eperjessy 1966) have been reported for their antibacterial activity against S. aureus. In addition, compounds such as robinetin (Gorniak et al. 2019), trifolin (Li et al. 2005), and melanoxetin (Lin et al. 2018) have a wide range of pharmacological and healthbeneficial properties.

Acute toxicity study
The acute toxicity test on mice was carried out using a methanol extract that showed antimicrobial activity against S. aureus. Some of the treated mice showed hair erection, isolation, sleep, and burial under wood shaves during the first 3 h of dosing, and afterwards returned to normal activity. The appearance of these physical and behavioral changes during the first four hours of observations has also been reported in the literature (Kefe et al. 2016). No mortality of mice occurred at any dose and observation period. Thus, the LD 50 value of P. regularis is greater than 2000 mg/kg bw. Reports on the other species of Phragmanthera, P. capitata and P. incana showed no deaths of experimental rats up to 3000 mg/kg (Takem et al. 2014) and 5000 mg/kg (Ogunmefun et al. 2013) body weight (bw), respectively.
In the present study, both treated and control mice showed a progressive weight gain during the period of observation. Meanwhile, the weight gain between treated mice with 550 and 2000 mg/kg bw doses and those treated with normal saline and tap water showed a non-significant difference (p > 0.05) during the respective days of observation (Table 3S). In contrast, the 175 mg/kg bw dose resulted in a significantly greater weight gain at the end of the experiment as compared to the first day of administering the extract. On the other hand, there was no significant gain in the relative weight of vital organs (heart, lungs, liver, and kidney) of all treated mice (Table  4S). Macroscopic examination of these vital organs showed the absence of adverse effects of the different extract doses. Therefore, the significant weight gain in the body weight of mice treated with a 175 mg/kg bw dose can be explained by other than the toxicity of the plant extract, such as significant consumption of food and water intake during the course of the experiment (Porwal et al. 2017).

Conclusions
This study showed that the crude methanol leaf extract of P. regularis inhibited the growth of S. aureus (12.33 ± 2.08 at 100 mg/ml) and caused no mortality in mice. The acute oral toxicity result (> 2000 mg/kg bw dose) could categorize the methanol extract of P. regularis under Category 5 of the Globally Harmonized System of Classification and Labeling of Chemicals (UN 2011) that indicates substances with low acute toxicity. To our knowledge, this is the first report on the biological and chemical characterization of P. regularis in Ethiopia and other growing countries. In the future, it is recommended to fractionate the active methanol extract to come up with a more effective bacterial growth inhibitor.