Infectious diseases have long been known as a major problem in developing countries. Development of antibiotic resistant bacteria is due to inadequate antibiotic use in human and animal  health  and  to  their  continued use as growth promoters in poultry and livestock production (Elisha et al., 2017). Two-thirds of deaths from infections in 2010 were reported to be caused by around 20 species, mainly bacteria and viruses (Dye, 2014). Novel, effective and affordable drugs are needed to combat infectious diseases especially in developing countries of the world; where up to one-half of deaths are due to infectious diseases (Awouafack et al., 2013; Srivastava et al., 2013).

Oxidative stress has been considered as a major contributing factor in the development of numerous life-threatening complications. The antioxidant potential of plants has received a great consideration as they play an important therapeutic role in human disease (Kasote et al., 2015).

Sudan harbour a high diversity of medicinal plants that play important role in health care system and consequently represent an integral part of life in Sudan. People in different regions of the Sudan use medicinal plants for the treatment of various diseases due to lack of medical doctors and exorbitant prices of pharmaceutical products. Hence, evaluation of their claimed pharmacological potential efficacy and safety is warranted (Issa et al., 2018).

Pistacia lentiscus L. (family Anacardiaceae) and Cinnamomum verum J. Presl (family Lauraceae) are widely used spice and have many applications in perfumery, flavoring and pharmaceutical industries (Mbaveng and Kuete, 2017; Bozorgi et al., 2013). Phyllanthus anderssonii Müll.Arg. (family Phyllanthaceae) is used in Sudan to treat uterus infections, female infertility, and broken bones.

The aim of this study was to evaluate the in vitro antimicrobial and antioxidant activities of the ethanolic (80%) extracts from leaf and stem of P.  lentiscus, P.  anderssonii and leaf of C. verum and their derived fractions.

Plant

Leaves and stems of P. lentiscus and leaves of C. verum were collected from the district of Shambat-Khartoum North, Sudan. Leaves and stems of P. anderssonii were collected from Kasala State, Sudan. Botanical identification and authentication were performed and voucher specimens (No. 2015/4PL for P. lentiscus, No. 2015/4PA for P.  anderssonii and No. 2015/4CV for C. verum) have been deposited in the herbarium of Al-Neelain University.

Preparation of extracts and fractions

The  dried,   powdered   materials   (500 g  each)   were   separately extracted thrice in 80% ethanol (EtOH) (v/v) at room temperature for 24 h, followed by filtration using filter paper. The three percolates were mixed, concentrated and subsequently freeze-dried. The freeze-dried sample was sequentially extracted with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (BuOH). Extracts were concentrated under vacuum by rotary evaporator and aqueous fractions were freeze-dried.

Preliminary phytochemical screening

The extracts and fractions were subjected to phytochemical analysis using standard phytochemical methods (Trease and Evans, 2002).

Antimicrobial activity

Test strains and culture media

Standard strains of microorganism were used in this study and were obtained from Medicinal and Aromatic Institute of Research, National Research Center, Khartoum. The bacterial species used were the Gram-negative bacteria: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) and the Gram-positive bacteria: Bacillus subtilis (NCTC 8236) and Staphylococcus aureus (ATCC 25923). Fungal species were Candida albicans (ATCC 7596) and Aspergillus niger (ATCC 9763). Bacteria were grown in Mueller Hinton Agar and fungi were grown in Sabouraud Dextrose Agar. The concentration of bacterial suspensions was adjusted to 10⁸ cells/mL and that of fungal suspensions to 10⁷ cells/ml.

Antibacterial assay

Antibacterial activity of extracts and fractions was evaluated by the disc diffusion method (Kil et al., 2009). Extracts/Fractions solutions (20 mg/ml) were prepared by diluting with 5% dimethyl sulfoxide (DMSO). The test microorganisms were seeded into respective medium by spread plate method. After solidification, filter paper discs with a diameter of 6.0 mm were impregnated with 10 μl of crude extracts/fractions followed by drying off. DMSO was used as a negative control, while gentamicin (10 μg/disc) was used as a positive control. Antibacterial discs were dispensed onto the surface of the inoculated agar plates and Petri plates were incubated for 24 h at 37°C. Experiment was done in triplicate. Diameters of clear zone of inhibition produced around the discs were measured and recorded.

Antifungal assay

Antifungal activity was also evaluated by the disc diffusion method (Mothana and Lindequist, 2005).  Paper discs were impregnated with 10 μl of extracts at 20 mg/ml followed by drying off. DMSO was used as a negative control, while nystatin (10 μg/disc) was used as a positive control. Antifungal discs were dispensed onto the surface of the inoculated agar plates, after which the plates were incubated at 27°C for 48 h. Experiment was done in triplicate. After the colonies grew, the zones of inhibition around the discs were measured and recorded.

Antioxidant activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay

Antioxidant activity of the extracts was estimated using DPPH in vitro method (Mensor et al., 2001). Test samples were dissolved separately in 5% DMSO to get test solution of 1 mg/ml. Assay was performed in 96-well, microtiter plate. 140 µL of 0.6 × 10^-6 mol/l DPPH were added to each well containing 70 µl of sample. The mixture was shaken gently and left to stand for 30 min in dark at room temperature. The absorbance was measured spectrophotometrically at 517 nm using Cecil-Elect Spectrophotometer. Blank was done in the same way using 5% DMSO and sample without DPPH and control was done in the same way but using DPPH and 5% DMSO without sample. Ascorbic acid was used as reference antioxidant compound.  All analyses were done in triplicate. The ability to scavenge DPPH radical was calculated by the following equation:

DPPH radical scavenging activity (%) = 100 - [(Abs_sample - Abs_blank) × 100] / (Abs_control)]

where Abs_sample is the absorbance of DPPH radical + sample; Abs_blank is the absorbance of sample+ 5% DMSO; and Abs_control is the absorbance of DPPH radical + 5% DMSO.

Extraction yield and phytochemical screening

Results depicted in Table 1 show a higher extractive yield in the leaf crude extract (34.1%) of P.  lentiscus as compared to the stem (24.5%) and in the leaf crude extract (39.1%) of P. anderssonii compared to the stem (13.1%). Crude leaf extract of C. verum revealed an extractive yield of 29.5%. Fractions extractive yields were variable and it was observed that all aqueous fractions (except that of P. anderssonii stem) showed high extractive yield after different solvent-solvent fractionation.

Phytochemical screening of crude ethanolic (80%) and different fractions of P.  lentiscus, P. anderssonii and C. verum revealed the presence of different classes of secondary metabolites like flavonoids, sterols, triterpenes, coumarins. Tannins were only detected in crude ethanolic (80%) extracts of the three plants and in their butanol and aqueous fractions. Saponins were present in crude ethanolic (80%) extracts as well as their aqueous fractions. Alkaloid was only detected in the ethyl acetate and butanol fractions of P. anderssonii stem. Anthraquinone glycosides and cardiac glycosides were not detected in the investigated plants (Table 1).

Antimicrobial activity

Crude ethanolic (80%) leaf and stem extracts of P.  lentiscus,   P.  anderssonii    and    C. verum    and    their fractions were tested for their antimicrobial activity. Results are shown in Table 2.

P. lentiscus crude extracts and fractions from stem and leaf

The crude ethanolic (80%) extract of P. lentiscus stem displayed higher antibacterial activity than that obtained from the crude ethanolic (80%) extract of the leaf against E. coli with inhibition zone of 19 mm. The latter showed the highest antifungal activity with inhibition zones of 18 and 19 mm against A. niger and C. albican, respectively. A remarkable increase in the antibacterial activity of P. lentiscus leaf was observed upon fractionation where the ethyl acetate fraction showed higher inhibition zones against the four tested bacteria species than those obtained from the crude ethanolic extract. Moreover, the remained aqueous fraction after fractionation possessed higher activity against S. aureus (21 mm) than that obtained from the crude extract and all fractions. The antifungal activity of the leaf was not altered after fractionation and the ethyl acetate fraction displayed the same inhibition zone value as the crude extract.

Fractionation of the stem ethanolic (80%) extract of P. lentiscus increased generally the antimicrobial activity where the highest activity was observed in the ethyl acetate fraction against B. subtilis (19 mm), P. aeruginosa (18 mm) and C. albicans (20 mm) and in the butanol extract against B. subtilis (19 mm). The aqueous extract displayed high and similar inhibition diameter against B. subtilis as those obtained by the ethyl acetate and butanol fractions.

P. anderssonii crude extracts and fractions from stem and leaf

Both leaf and stem ethanolic (80%) extracts of P. anderssonii exhibited good antimicrobial activity against all tested microorganisms where the leaf displayed an activity in the range of 20 to 22 mm and the stem in the range of 18 to 21 mm. However, fractionation reduced the antimicrobial activity of crude extracts of both organs except aqueous fraction of the stem. The observed increase in antibacterial activity of aqueous fraction was against S. aureus (20 mm) and E. coli (21 mm).

C. verum crude extracts and fractions from leaf

Crude ethanolic (80%) extract of C. verum leaf displayed good antimicrobial activity against all tested microorganisims except in the case of A. niger which was not susceptible to the crude extract. However, fractionation resulted in a pronounced antifungal activity where the ethyl acetate fraction displayed inhibition zone of  20 mm  against  A. niger  and  increased   the   activity against C. albican from 18 to 21 mm. Also, the aqueous fraction displayed a remarkable antifungal activity against A. niger (19 mm). For other microorganisms, a slight increase in the antibacterial activity against B. subtilis, E. coli and P. aeruginosa was observed in the ethyl acetate fraction.

Antioxidant activity

Crude ethanolic (80%) leaf and stem extracts of P.  lentiscus, P.  anderssonii and C.  verum leaf and their fractions were evaluated for their in vitro antioxidant activity using DPPH method and results are shown in Table 3. Crude ethanolic (80%) extracts of the three investigated plants showed high DPPH scavenging capacity with ranking order of activity (in terms of % inhibition) as leaf of P. lentiscus (95%) > stem of P.  anderssonii (93%) > stem of P. lentiscus (84%) > leaf of P. anderssonii (76%) > leaf of C.  verum (8%). Fractionation of the crude leaf extract of P. lentiscus did not influence the scavenging capacity where butanol fraction displayed the same activity (95%) while others fractions showed minor reduction (93%) in their activity. The scavenging activity of the stem was increased upon fractionation by 7, 6, 11 and 13% in the petroleum, chloroform, ethyl acetate and butanol fractions, respectively. Fractionation of crude leaf and stem extracts of P. anderssonii reduced their scavenging potentiality except for the ethyl acetate fractions. The scavenging activity of the ethyl acetate fraction of the leaf increased by 25% when compared with the crude extract and that of the stem was comparable to its corresponding crude extract. The scavenging activity of the aqueous fraction of the leaf demonstrated good scavenging potential and showed 21% increase in the scavenging activity when compared with the crude leaf extract. A remarkable increase of the scavenging activity of the crude leaf extract of C. verum was obtained upon fractionation by 262, 475, 857 and 600% in the petroleum, chloroform, ethyl acetate and butanol fractions respectively. 

Generally, the leaf gave higher yield of extract than the stem and the aqueous fractions gave the highest yield after fractionation followed by the butanol fractions. It was reported that ethanol and water extracts showed higher amount of extracted compounds in comparison with ethyl acetate extract (Tuberoso et al., 2010). Moreover, polar solvents extracted large molecules like glycosides, proteins, saponins and tannins (Tan et al., 2013).

The phytochemical analysis revealed the presence of different classes of secondary metabolites. As shown in Table 1, each extract or fraction contained at least five types of  secondary metabolites and some of the extracts and fractions displayed high antimicrobial and antioxidant activities (Tables 2 and 3). It is known that secondary metabolites such as flavonoids, terpenoids, steroids, phenols, saponins, alkaloids and tannins have good antimicrobial properties (Dorman and Deans, 2000; Kuete and Efferth, 2010; Biswas et al., 2013). Flavanoids and phenols form complexes with extra cellular and soluble proteins of bacterial cell walls leading to the death of the bacteria (Cowan, 1999). Terpenoids are known to have antibacterial property by affecting the synthesis of cell membranes components, prenylation of proteins and the use of carbon source (Nayak et al., 2010). Saponins have been found to have inhibitory effects on Gram-positive organism, S. aureus (Biswas et al., 2013).

Crude ethanol extracts and fractions of the three plants exhibited varying degrees of antibacterial activity against the tested microorganisms. It is well known that using different solvents may extract different compounds and consequently could lead to different extract potentials (Aleksic and Knezevic, 2014). Fractionation increased mainly the antibacterial activity of P. lentiscus leaf; the ethyl acetate fraction showed higher activity against all tested bacteria than that obtained from the crude ethanolic extract and the aqueous fraction displayed highest activity against S. aureus (Table 2). Furthermore, fractionation improved   mainly   the   antimicrobial   activity   of the stem against B. subtilis, P. aeruginosa and C. albicans. Fractionation of P. anderssonii stem ameliorated only the antibacterial activity against S. aureus and E. coli where the aqueous fraction exhibited the highest activity. Although crude leaf extract of C. verum did not show antifungal activity against A. niger, upon fractionation, the ethyl acetate and aqueous fractions displayed high antifungal activity; also it improved its activity against C. albicans (ethyl acetate fraction) and B. subtilis (aqueous fraction). These results suggested the existence of compounds with an antagonistic  effect  in  the  crude   extract.   Fractionation reduced the antimicrobial activity of both the leaf and stem of P. anderssonii; this could be probably due to synergistic activities of compounds present together in the crude extract but separated from each other upon fractionation. Petroleum ether and chloroform fractions of the investigated plants had no or poor activity against all tested microorganisms suggesting that the observed antimicrobial activity of the studied plants could mainly be to the presence of compounds of hydrophilic nature.

Results of antioxidant activity showed that leaf of P. lentiscus and stem of P.  anderssonii displayed the highest DPPH scavenging activity. Fractionation improved mainly the antioxidant potentiality of C. verum leaf where the ethyl acetate showed the highest activity. Also, fractionation improved the DPPH scavenging activity of P.  anderssonii leaf and slightly improved that of P. lentiscus stem. However, fractionation (except aqueous fraction) slightly alter the DPPH scavenging activity of P. lentiscus leaf and generally all fractions showed high DPPH scavenging activity indicated that the activity might be attributed to the presence of several antioxidant agents with polar and non-polar nature.

Previous studies on P. lentiscus were mainly reported for essential oil and gum where they were found to possess remarkable antimicrobial (Hayder et al., 2005; Koutsoudaki et al., 2005; Miyamoto et al., 2014) and antioxidant (Benhammou et al., 2008; Chryssavgi et al., 2008; Bampouli et al.,2014) activities. Essential oil and oleoresins (acetone extract) from C. verum leaf displayed high antioxidant, antibacterial and fungal activity (Singh et al., 2007). Moreover, it is worth mentioning that this is the first phytochemical study and biological activity evaluation of Phyllanthus anderssonii.

Data obtained in the present study showed that crude extracts of P.  lentiscus, P.  anderssonii and C.  verum demonstrate good antimicrobial and DPPH scavenging potential. Fractionation with solvents of different polarities yielded different fractions with different chemical composition and thus displayed different levels of antimicrobial and antioxidant activity. This study provides scientific insight to further determine the antimicrobial and antioxidant principles in the three studied plants.

The authors have not declared any conflict of interests.

The authors would like to acknowledge Prof. Maha Kordofani (Botany Department, Faculty of Science, University of Khartoum) for the identification of the plants.