In vitro antimicrobial activity
Microbial infections still have a great public issue, and there is a dramatic increase in the microbial resistance to the existence antimicrobial agents (Collins and Lyne 1985). Most medicinal plant extracts showed a synergistic antimicrobial activity (Co-activity), which may be attributed to the collaborative action between their mixed constituents like flavonoids, anthraquinones, coumarins, and phenolic acids (Murugan et al. 2013).
Reviewing the literature revealed that the antibacterial activity of three solvent extracts (methanol, dichloromethane, and petroleum ether) of E. camaldulensis leaves growing in Nigeria was evaluated against six microbial species, namely Klebsiella spp., Salmonella typhi, Yersinia enterocolitica, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis; for the methanolic extract, it showed strong activity with inhibition zones of 14, 16, 14, 15, 15, and 16 mm while for dichloromethane were 15, 15, 13, 13, 14, and 14 mm, respectively, for the abovementioned species, and there is no any activity was recorded with petroleum ether (Saad et al. 2017). Moreover, the in vitro antimicrobial activity of the Nigerian E. camaldulensis was evaluated against six strains of Helicobacter pylori (Adeniyi et al. 2009). Previous studies stated that polyphenolic compounds are responsible for the antimicrobial activity of the plant extracts (Funatogawa et al. 2004; Buzzini et al. 2008; Min et al. 2008). To sum up, E. camaldulensis exhibited noticeable antimicrobial potentials which may be return to their unique chemical profile.
Toxicity to Schistosoma mansoni larvae
Recently, there has been an interest to control schistosomes infection within snails rather than killing these intermediate hosts. This method has many advantages including maintenance of natural biodiversity in aquatic habitats and minimizing the environmental impacts related to application of chemical molluscicides (Mossalem et al. 2018). Plants rich with antioxidant molecules such as flavonoids and phenolic acids compounds are great targets for exogenous source of antioxidants that can maintain the balance between oxidative stress result from infection or other stressors and antioxidant system within organisms (Ghareeb et al. 2018b). Indeed, exposure of B. alexandrina to 90% defatted methanol extract from Punica granatum peels reduced the infection rates of snails with S. mansoni to 20% compared to 95% in infected unexposed snails (Mossalem et al. 2017). The same reduction in infection rate was obtained when snails exposed to ethyl acetate extract from E. camaldulensis leaves (Mossalem et al. 2018).
The results obtained from testing the larvicidal properties of fraction 5 n-hexane/EtOAc isolated from ethyl acetate extract of E. camaldulensis leaves indicate that this fraction possesses a potent toxic effect to miracidia and cercariae of S. mansoni. At concentration of 200 mg/L from fraction 5 n-hexane/EtOAc (20: 80 v/v), 100% mortality was recorded in miracidia and cercariae after 20 and 30 min of continuous exposure, respectively. Mossalem et al. (Mossalem et al. 2018) showed that ethyl acetate extract of E. camaldulensis possesses a powerful antioxidant activity and its administration to B. alexandrina snails prior and during pre-patent infection with S. mansoni resulted in a significant decrease in the infection rate of snails and increased the survival rate of snails. In the same context, Al-Sayed et al. (Al-Sayed et al. 2014) reported a potent molluscicidal activity of 80% MeOH extract of E. globulus against B. alexandrina snails and also showed strong miracidicidal and cercaricidal activity (80% and 100% mortality, respectively) after a 2 h exposure. A wide range of plants were proven to have potent schistosomicidal prosperities. The seeds of Nigella sativa either in the form of powder or as extracts had larvicidal potency against S. mansoni miracidia and cercariae. LC50 values of this drug were 1 and 2 mg/L for miracidia and cercariae, respectively (Mansour et al. 2002; Mohamed et al. 2005). Studies of the effect of this plant on adult female and male worms suggested that the possible mechanism of its action against S. mansoni parasite is mediated by inhibiting important antioxidant enzymes in the worms (Mohamed et al. 2005). The plants Allium sativum and Allium cepa (powder) at concentrations of 50 and 100 mg/L caused 50% mortalities to S. mansoni miracidia, respectively (Mantawy et al. 2012). Also, tannins extracted from P. granatum at a concentration as low as 0.39 mg/L killed 100% of miracidia after 50–150 min and 50% of miracidia within 25.1–48.3 min (Abozeid et al. 2012).
Reports from other countries on testing different plant species for their larvicidal effect showed their potential as miracicidal and cercaricidal agents. Extracts of the leaves and fruits of Piper marginatum, Protium heptaphyllum, and Capsicum annuum from Brazil show a remarkable effect on the cercariae of S. mansoni. In the case of the oils of Piper marginatum and Capsicum annuum, 90–96% of the cercariae of S. mansoni were killed within 15 min (Frischkorn et al. 1978). Moreover, extracts from Phytolacca dodecandra, Tamarindus indica, Acacia nilotica, Hibiscus sabdariffa and Tacca leontopetaloides from Sudan were toxic to miracidia and cercariae at concentration from 50 to 100 mg/L (Elsheikh et al. 1990).
The toxic effect of n-hexane/EtOAc fraction isolated from ethyl acetate extract of E. camaldulensis leaves may be exerted in a different mode of action that differ from the abovementioned plants. Since E. camaldulensis has a powerful antioxidant activity and is rich with bioactive molecules such as gallic acid, taxifolin, quercetin, luteolin, and hesperidin. One possible explanation is that exposure of the larvae to 200 mg/L of the fraction led to prod-oxidative acts or their interference with critical reactive oxygen species required for maintenance of cellular functions and physiological processes (Bouayed and Bohn 2010).
Structural elucidation of the isolated compounds from the ethyl acetate extract
Compound 1 was isolated as off white powder, m.p. 250–251 °C. On paper chromatography (PC), it showed violet and deep violet spots under long and short UV light respectively as well as positive result with FeCl3. Rf values are 0.82 (BAW) and 0.52 (15% AcOH). 1H-NMR spectra (400 MHZ, DMSO-d6) showed a characteristic signal in the aromatic region for two identical aromatic protons at δH 7.12 ppm (2H, s, H-2,6). The 1H-NMR and chemical data were in agreement with the reported data of 3,4,5-trihydroxybenzoic acid (gallic acid) (Chanwitheesuk et al. 2007).
Compound 2 was isolated as a yellow powder, m.p. 230–232 °C. On PC, it showed a yellow fluorescence colored spot under long UV light turned to bright yellow with ammonia vapors. Rf values are PC 0.76 (BAW) and 0.07 (15% AcOH), an indication of its nature as aglycone.1H-NMR spectra (400 MHZ, DMSO-d6) revealed the presence of three aromatic protons at B-ring was resonated at δH 6.93 (1H, d, J = 1.25 Hz, H-2′), 6.87 (1H, dd, J = 8.0, 1.5 Hz, H-6′), and 6.80 ppm (1H, d, J = 8.0 Hz, H-5′). In addition, the presence of two meta-coupled protons at A-ring appeared at δH 5.89 (1H, d, J = 1.25 Hz, H-6) and 5.95 ppm (1H, d, J = 1.25 Hz, H-8), while the C-ring protons were resonated at δH 4.89 (1H, d, J = 11.5 Hz, H-2) and 4.52 ppm (1H, d, J = 11.5 Hz, H-3). Based on the abovementioned spectral and chromatographic data, this compound could be identified as 3,5,7,3′,4′-pentahydroxy-flavanone (dihydroquercetin or taxifolin) (Kuspradini et al. 2009; Usman et al. 2016).
Compound 3 was isolated as off white powder, m.p. 200–202 °C. On PC, it showed violet and deep violet spots under long and short UV light respectively. Rf values are 0.65 (BAW) and 0.70 (15% AcOH) and positive results with FeCl3. 1H-NMR spectra (400 MHZ, DMSO-d6) showed two sets of protons, the first one for the methoxy protons at δH 3.78 ppm (3H, s, –OCH3) and the second one for the two aromatic protons at δH7.14 ppm (2H, s, H-2, 6). The 1H-NMR and chemical data were in agreement with that of methyl gallate (Choi et al. 2014).
Compound 4 was isolated as yellow powder, m.p. 311–313 °C. On PC, it showed a yellow fluorescence colored spot under long UV light turned to bright yellow with ammonia vapors and green color with FeCl3. Rf values are 0.67 (BAW) and 0.07 (15% AcOH) which were in the range of aglycones. 1H-NMR spectra (400 MHZ, DMSO-d6) showed different sets of resonances; two meta-coupled aromatic protons at A-ring were appeared at δH 6.19 (1H, d, J = 2.1 Hz, H-6) and 6.41 ppm (1H, d, J = 2.1 Hz, H-8). Three aromatic protons located at B-ring appeared at δH 7.53 (1H, d, J = 2.1 Hz, H-2′), 6.88 (1H, d, J = 8.0 Hz, H-5′), and 7.68 ppm (1H, dd, J = 8.0, 2.1 Hz, H-6′). The most downfield protonappeared at δH 12.5 ppm (1H, s, 5-OH). On the basis of 1H-NMR spectra and chromatic data, the compound could be identified as 5,7,3′,4′-flavon-3-ol (quercetin) (Huang et al. 2013).
Compound 5 was isolated as pale yellow powder, m.p. 320–322 °C. On PC, it showed a dark purple florescence spot under long UV light. Rf values are 0.74 (BAW) and 0.07 (15% AcOH) which were in the range of aglycones. 1H-NMR spectra (400 MHZ, DMSO-d6) showed different sets of aromatic protons; two meta-coupled protons at A-ring were appeared at δH 6.25 (1H, d, J = 2.1 Hz, H-6) and 6.54 ppm (1H, d, J = 2.1 Hz, H-8). Another characteristic signal for proton in position 3 at C-ring was appeared at δH 6.73 ppm (1H, s, H-3). Moreover, a characteristic pattern for three protons at B-ring appeared at δH 7.0 (1H, d, J = 8.1 Hz, H-5′), 7.49 (1H, dd, J = 8.0, 2.1 Hz, H-6′), and 7.98 ppm (1H, d, J = 2.1 Hz, H-2′). A most down field proton appeared at δH 13.04 ppm (1H, s, 5-OH) due to the effect of intermolecular hydrogen bond with adjacent carbonyl group at C-ring. Based on 1H-NMR and chromatographic data, the compound could be identified as 5,7,3′,4′-tetrahydroxy-flavone (luteolin) (Tshikalange et al. 2005; Sato et al. 2000).
Compound 6 was isolated as pale yellow powder, m.p. 250–252 °C. On PC, it showed a yellowish green florescence spot under long UV light. Rf values are 0.56 (BAW) and 0.78 (15% AcOH) indicating to its glycosidic nature. 1H-NMR spectra (400 MHZ, DMSO-d6) revealed the presence of set of resonances were appeared at δH 11.97 ppm (1H, br s, 5-OH) for the most downfield proton at position 5 at A-ring. Three aromatic protons of 1,3,4-trisubstituted B-ring were resonated at δH 6.95 (1H, d, J = 2.1 Hz, H-2′), 6.85 (1H, J = 8.2 Hz, H-5′), and 6.81 ppm (1H, dd, J = 8.2, 2.1 Hz, H-6′). Also, two meta-coupled aromatic protons were resonated at δH 6.13 (1H, d, J = 2.1 Hz, H-8) and 6.10 ppm (1H, d, J = 2.1 Hz, H-6). C-ring protons appeared at δH 5.35 (1H, dd, J = 11.2, 5.1 Hz, H-2), 3.15 (1H, dd, J = 16.0, 11.0 Hz, H-3a), and 2.51 ppm (1H, dd, J = 16.0, 5.0 Hz, H-3b). Moreover, two characteristic anomeric protons of sugar moieties were located at δH 5.07 (1H, d, J = 7.5 Hz, H-1″) and 4.57 ppm (1H, br s, H-1″′). Methoxy and methyl protons were resonated at δH 3.78 (3H, s, 4-OCH3) and 1.09 ppm (3H, d, J = 6.0 Hz, H-6), respectively. Accordingly, on the basis of 1H-NMR spectral data and chromatographic features, the compound could be identified as 3′,5,7-trihydroxy-4′-methoxyflavanone-7-O-rutinoside (hesperidin or hesperetin 7-O-rutinoside) (Areias et al. 2001; Cuyckens et al. 2001).