Plants have long been used as a source of antibiotics but the potential of plants as sources for new drugs is still generally unexplored. Plant originated drugs also have the advantage of being less hazardous to environmental health (Bajwa et al. 2008; Shafique et al. 2011). Thus, the plant kingdom is being screened for new and effective chemotherapeutic agents (Shafique et al. 2013). Despite all efforts made by researchers, only a few plant species have been phytochemically investigated until now (Sabir et al. 2007). Plant extracts of many higher plants have been reported to exhibit antibacterial, antifungal and insecticidal properties under laboratory trials (Akram et al. 2013; Bouamama et al. 2006). Among these plants, Alstonia scholaris is an evergreen tropical tree having latex rich in alkaloids (Hussain et al. 2010).

Another medicinally important plant, Millettia pinnata (Fabaceae), contains oils and juices with strong antiseptic and antimicrobial properties (Baswa et al. 2001). Considering plants as great sources of antibiotic agents, an investigation was performed to study the local flora for comparative phytochemistry, antibacterial and antifungal activity.

Leaves of Alstonia and Milletia plant species were collected from University of the Punjab, Lahore, Pakistan (31°17′34″N 74°10′29″E), in December 2012. Leaves of both plants were washed, dried and blended into fine powder separately. Aqueous, methanol, ethanol, n-hexane and chloroform extracts were prepared using the method of Azu and Onyeagba (2007). Extraction of plant biochemicals was performed at room temperature (25 ± 2 °C) using solvents for plant biochemicals. Solvents of all extracts were removed using vacuum drier at 40 °C.

Different extracts were phytochemically analysed using the method of Brain and Turner (1975). Briefly, Mayer’s reagent was used for alkaloids analysis; magnesium and acetone solutions were used for anthraquinones test; concentrated HCl and magnesium solutions were used to determine flavonoids; ferric chloride was used for phenols; thionyl chloride crystals were used in terpenoids test; lead acetate was used for determination of tannins and water was used to perceive the presence of saponins and formation of foam.

Bacterial cultures of Bacillus subtilis (FCBP-0174), Bacillus fortis (FCBP-0162), Staphylococcus aureus (FBT-0014), Escherichia coli (FCBP-0257), Pseudomonas syringe (FCBP-0275), Pseudomonas putida (FBT-0032), Erwinia amylovora (FBT-0053), Burkholderia gladioli (FBT-0034) and fungal cultures of Alternaria alternata (FCBP-0003), Alternaria solani (FCBP-0831) and Fusarium oxysporum (FCBP-0866) were obtained from the Culture Conservatory of the Institute of Agricultural Sciences, University of the Punjab, Lahore Pakistan. Bacteria were cultured upon nutrient broth (NB) at 37 °C and fungal cultures were maintained on potato dextrose agar (PDA) at 28 °C.

Bacterial inoculum (106 cells/mL) was prepared by recording its absorbance at 550 nm (Sutton 2011) and fungal inoculum of 106 spores/mL was also prepared using a haemocytometer (Valgas et al. 2007). Both of the inocula were used in antibacterial and antifungal assays performed by disc diffusion method. Filter paper discs (0.5 cm diameter) were impregnated with 100 μg of 300, 200 and 100 μg/mL concentrations of extracts in a way that each disc contained 30, 20 and 10 μg extract, respectively. Discs impregnated with DMSO and Nystatin 100 μg were used as negative and positive controls, respectively. Activity was determined after 5 days of incubation.

Eight phytochemical classes (i.e. alkaloids, flavonoids, phenolics, steroids, tannins anthraquinone, saponins and terpenoids) were extracted using methanol as extraction solvent. Four different classes of biochemicals were observed in ethanol extract (Table 1), whereas five different phytochemical classes were observed in aqueous extract. Moreover, chloroform extract showed the presence of anthraquinones, flavonoids, phenolics, saponins and terpenoids. Presence of flavonoids, phenolics, saponins, steroids, tannins and terpenoides were observed in n-hexane extract (Table 1).

Table 1 Observations of phytochemical screening
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Maximum number of phytochemicals was observed in the methanol extract of M. pinnata; only steroids being absent. Aqueous extract contained anthraquinones, alkaloids, phenolics and tannins. Minimum number of phytochemical classes was observed in ethanol and chloroform extracts (Table 1).

All of the extracts presented varying antibacterial activity for tested bacterial strains at different concentrations. Leaf extracts of A. scholaris had antibacterial effects against different bacterial strains (Table 2). Methanol extract of A. scholaris presented the highest antibacterial activity. Methanol extract was able to inhibit growth of the most bacterial strains even at the lowest concentration of 100 μg/mL. However, P. putida and E. amylovora were found resistant at the lower concentration of methanol extract. Ethanol extract was least effective against bacterial strains of both gram positive and gram negative types. Even at a concentration of 200 μg/mL, S. aureus, E. coli and B. gladioli were resistant against ethanol extract of A. scholaris. Maximum inhibitory zone diameter (34 mm) was observed in the aqueous extract of A. scholaris against S. aureus at the concentration of 300 μg/mL. The same bacterial strain was found to be resistant against n-hexane extract at the concentration of 100 μg/mL.

Table 2 Antibacterial activity of extracts of A. scholaris
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Methanol extract of M. pinnata was found effective against bacterial strains of both gram negative and gram positive types (Table 3). Three bacterial strains (i.e. E. coli, P. putida and B. gladioli) were found to be resistant against lower concentrations of ethanol extract. One bacterial species, E. coli, was found to be resistant against n-hexane extract at the concentration of 100 and 200 μg/mL. Bacterial species, P. putida, was also found resistant against chloroform extract of M. pinnata at lower concentrations but susceptible at higher concentrations. All other extracts represented average antibacterial activity at different concentrations.

Table 3 Antibacterial activity of extracts of M. pinnata
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Table 4 Antifungal activity of extracts of A. scholaris and M. pinnata
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Appreciable antifungal activity was observed in methanol extract of M. pinnata in which all fungal pathogens showed significant zones of inhibition at 100 μg/mL concentration. Radius of inhibition zone directly increased with the increased concentration of plant extract which clearly showed that fungal growth inhibition was strictly concentration dependent. It was closely followed by methanol extract of A. scholaris for which the minimum concentration also significantly inhibited fungal growth on synthetic medium (Table 4).

Aqueous extract of M. pinnata had the second highest antifungal activity. However, F. oxysporum showed resistance to that extract and a very little inhibition zone (3 mm) was observed at the highest concentration. However, both species of Alternaria were efficiently controlled by a lower concentration of that extract. Every adjacent higher concentration brought significant increase in antifungal activity. Zones of inhibition produced by aqueous extract of M. pinnatta against A. solani were 9, 16 and 27 mm at the concentrations of 100, 200 and 300 μg/mL, respectively. Two fungal species (F. oxysporum and A. solani) were resistant against aqueous extract of A. scholaris at 100 μg/mL concentration. However, at 200 μg/mL concentration, the extract controlled all three fungi efficiently.

Chloroform extract exhibited efficient antifungal activity at 200 μg/mL. At the lowest concentration, most of the fungal species were resistant to the chloroform extract. At 100 μg/mL concentration of both plant species, F. oxysporum was resistant, while A. alternata and A. solani were resistant to least concentrations of A. scholaris and M. pinnata, respectively. Increased concentrations of these extracts significantly inhibited fungal growth up to 32 mm zone of inhibition in case of A.alternata with 300 μg/mL of M. pinnata extracts.

Antifungal potential was decreased from n-Hexane to ethanol extract. Only one fungal species was resistant to 200 μg/mL n-Hexane plant extract but this number was doubled in 200 μg/mL of ethanol extract of the same plants. A concentration dependent trend was also found in both n-Hexane and ethanol extracts.

In the qualitative phytochemical studies, it was observed that secondary metabolites of leaves of both plant species were more soluble in methanol suggesting that methanol is a good solvent for recovery of most of the phytochemicals from plants. Among both types of plants, ethanol extract was least effective against both bacteria and fungi. This can be attributed to this solvent being unable to dissolve many types of phytochemical groups.

These phytochemical constituents are valuable sources of antimicrobial agents (Bashir et al. 2013). Scalbert (1991) and Aboaba et al. (2006) reported antimicrobial properties of tannins in their investigations. Other compounds such as alkaloids (Damintoti et al. 2005), saponins (Hostettman and Nakanishi 1979), terpenoids and flavonoids (Leven et al. 1979) have also been investigated for their antimicrobial properties. This study indicates groups of metabolites extracted through different solvents, and it will help researchers for isolation of specific biochemical groups from plant material.

In this investigation we used the strains of fungi and bacteria of both gram positive and gram negative genera. They are important microorganisms due to their pathogenicity towards plants and humans. Methanol extract of leaves of both plant species comprised more antimicrobial properties as compared to other solvents (Ahmad et al. 2014). Therefore, methanol can be used to extract antimicrobial material from plants but cannot be encouraged for isolation of specific biochemical groups. The present study also justifies the use of phytochemicals against plant pathogenic fungi. All of the test fungal strains were susceptible to leaf extracts of both plant species. Based on all these results, use of leaf extracts of both these plant species as natural antibacterial agents is justified.