Phytochemical analysis of Eucalyptus camaldulensis leaves extracts and testing its antimicrobial and schistosomicidal activities

Collection of plant material, extraction, and fractionation

The fresh leaves of Eucalyptus camaldulensis Dehnh were collected from Giza governorate during June, 2016. The identification and authentication of the plant was carried out by Dr. Therese Labib, Department of Flora and Taxonomy, El-Orman Botanical Garden, Giza, Egypt. A voucher specimen (No. E25/4/3) is kept in the herbarium of the garden. Details of extraction of E. camaldulensis leaves using organic solvents and testing the free radical scavenging activity of the obtained extracts are described in Mossalem et al. (Mossalem et al. 2018).

Instruments, chemicals, and reagents

The ¹H and ¹³C-NMR analyses were recorded on BRUKER 400 (400 and 100 MHz for ¹H and ¹³C-NMR, respectively). For column chromatography, we used the following chemicals and materials: Sephadex LH-20 (Pharmacia Fine Chemicals), Silica gel (60–200 mesh) (S.D. fine-CHEM Ltd.) and Whatmann No. 1 sheet filter papers (Whatmann Ltd., Maidstone, Kent, England). Thin layer chromatography with aluminum sheet (20 × 20) percolated with silica gel 60 F₂₅₄ was obtained from E. Mark (Darmstadt, Germany).

Vacuum liquid chromatography (VLC) of the ethyl acetate extract

Antimicrobial activity

Disc agar plate method has been recognized to assess the antimicrobial activities of organic solvent extracts from E. camaldulensis as well as the selected ethyl acetate VLC fractions (El-Neekety et al. 2016). Four different test microbes, Staphylococcus aureus (G+ve bacterium), Pseudomonas aeruginosa (G-ve bacterium), Candida albicans (yeast), and Aspergillus niger (fungus), were selected to evaluate the antimicrobial activities. The bacterial and yeast test microbes were grown on a nutrient agar medium. On the other hand, the fungal test microbe was cultivated on Czapek-Dox medium. The culture of each test microbe was diluted by distilled water (sterilized) to 10⁷ to 10⁸ colony-forming units (CFU)/mL then 1 mL of each was used to inoculate 1-L Erlenmeyer flask containing 250 mL of solidified agar media. These media were put on the previously sterilized petri dishes (10 cm diameter having 25 mL of solidified media). Filter paper discs (5 mm Ø, Whatmann No. 1 filter paper) loaded with 0.2 mg of each extract. The discs were dried at room temperature under sterilized conditions. The paper discs were placed on agar plates seeded with test microbes and incubated for 24 h, at the appropriate temperature of each test organism. Antimicrobial activities were recorded as the diameter of the clear zones (including the disc itself) that appeared around the discs (Hathout et al. 2016; Madkour et al. 2017).

Effect of E. camaldulensis extracts against Schistosoma mansoni larvae

Miracidia and cercariae of S. mansoni were obtained from Schsitosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute. The selected concentration was 200 mg/L (Mossalem et al. 2018). A double concentration of fraction 5 n-hexane/EtOAc (20: 80 v/v) was prepared in 5 ml volume of water. Then, it was added to a petri dish containing 50 fresh hatched miracidia in 5 mL of water (final volume is 10 mL). Another 10 mL dechlorinated tap water containing 50 fresh hatched miracidia served as control (Mohamed et al. 2012). Following 5, 10, 15, 20, 25, and 30 min, the petri dishes were examined under dissecting microscope and mortalities in miracidia were recorded (the larvae were considered dead when they stopped movement completely for 1 min) (Mahmoud and Ibrahim 2011). The same experiment was repeated with cercariae. Percentages of dead miracidia and cercariae were compared by Chi-square test (Mohamed et al. 2012).

In vitro antimicrobial activity

The in vitro antimicrobial activity of organic solvent leaf extracts of E. camaldulensis was evaluated via disc agar method against four pathogenic strains of microbes, Staphylococcus aureus (G+ve bacteria), Pseudomonas aeruginosa (G-ve bacteria), Candida albicans (yeast), and Aspergillus niger (fungus), and compared to Neomycin and Cyclohexamide as standard antibiotics. The antimicrobial activities of the tested extracts were expressed in clear zone of inhibition (mm). The results in Table 2 and Fig. 1 revealed that water extract showed the highest inhibition zones (13, 11, 11, and 9 mm, for S. aureus, P. aeruginosa, C. albicans, and A. niger, respectively. Ethyl acetate extract exhibited antimicrobial activity very close to the water extract with inhibition zones of 11, 10, 10, and 8 mm for S. aureus, P. aeruginosa, C. albicans, and A. niger, respectively. The other solvent extracts showed antimicrobial activities according to the following order: n-butanol > dichloromethane > petroleum ether > 90% methanol. Further fractionation of ethyl acetate extract using VLC yielded 13 fractions, six of them (fractions 4, 5, 6, 7, 8, and 9) exhibited antimicrobial activities against all test microbes except A. niger. While the fractions (1, 2, 3, 10, 11, 12, and 13) are inferior in the antimicrobial activity, fraction 5 showed the highest antimicrobial activity among the fractions tested. Inhibition zones were 15, 13, and 14 mm against S. aureus, P. aeruginosa, and C. albicans, respectively (Table 3 and Fig. 2).

Solvent extracts	Clear zone (ϴmm)
	Staphylococcus aureus	Pseudomonas aeruginosa	Candida albicans	Aspergillus niger
1. 90% MeOH	–	8	–	–
2. EtOAc	11	10	10	8
3. Water	13	11	11	9
4. Dichloromethane	6	6	–	–
5. n-Butanol	8	8	7	6
6. Petroleum ether	–	–	6	9
Antibiotic
Neomycin1	17	20	21	–
Cyclohexamide2	–	–	–	25

¹Neomycin was used at 200 microgram per disc

²Cyclohexamidewas used at 200 microgram per disc

Ethyl acetate VLC fractions	Clear zone (ϴmm)
	Staphylococcus aureus	Pseudomonas aeruginosa	Candida albicans	Aspergillus niger
1
2	–	–	–	–
3	–	–	6	–
4	8	8	12	–
5	15	13	14	–
6	11	12	11	–
7	7	7	6	–
8	9	10	8	–
9	9	10	10	–
10	–	–	–	–
11	–	–	–	–
12	–	–	–	–
13	–	–	–	–
Antibiotic
Neomycin	18	19	21	–

Miracidicidal and cercaricidal effect

Chromatographic isolation of the ethyl acetate extract

Six phenolic compounds were isolated from the EtOAc extract of E. camaldulensis. Based on ¹H-NMR spectra, chromatographic data, and available data in the literature; their chemical structures were elucidated as gallic acid (1), taxifolin (2), methyl gallate (3), quercetin (4), luteolin (5), and hesperidin (6) (Fig. 3).

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. R_f 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 δ_H 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. R_f 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 δ_H 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 δ_H 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 δ_H 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. R_f 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 δ_H 3.78 ppm (3H, s, –OCH₃) and the second one for the two aromatic protons at δ_H7.14 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. R_f 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 δ_H 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 δ_H 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 δ_H 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. R_f 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 δ_H 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 δ_H 6.73 ppm (1H, s, H-3). Moreover, a characteristic pattern for three protons at B-ring appeared at δ_H 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 δ_H 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. R_f 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 δ_H 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 δ_H 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 δ_H 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 δ_H 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 δ_H 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 δ_H 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).