2.3.1 Qualitative analysis of phytochemicals

Major constituents of both the M/C and aq. extracts were screened qualitatively as per standard procedures described by Jamil et al. (2012a). Major constituents analyzed were alkaloids, tannins, terpenoids, saponins, anthraquinones, cardiac glycosides and coumarins.

2.3.2 Quantitative analysis of phytochemicals

2.4.1 Antibacterial activity

Antibacterial activity of the extracts was determined against six bacterial strains, two gram positive i.e., Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538) and four gram negative i.e., Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 15224), Bordetella bronchiseptica (ATCC 4617) and Enterobacter aerogens (ATCC 13048) using the disc diffusion method as reported earlier (Jamil et al., 2012b). Samples (100 μg/disc), two positive controls (Roxithromycin and Cefixime-USP, one for each) and negative control (DMSO) were used on each plate. Experiments were performed in triplicate and mean inhibitory zone was calculated with standard error.

2.4.2 Antifungal activity

Antifungal activity against five fungal strains (Aspergillus flavus, Fusarium solani, Aspergillus fumigatus, Mucor spp. and Aspergillus niger) was determined by the disc diffusion method (Abbasi et al., 2013). Sample concentration was 100 μg/disc and terbinafine (same concentration) was used as positive control. Petri dishes were incubated at 28 °C for 72 h and zones of inhibition were measured.

2.5.1 Reducing power

The reducing power assay was performed according to the method described by Jafri et al. (2017). Concentration of each sample was 100 μg/mL and absorbance was measured at 630 nm using microplate reader (Biotech, Elx-800, USA). The reducing power of each sample was expressed as ascorbic acid (vit. C) equivalent.

2.5.2 Total antioxidant capacity (phosphomolybdenum method)

Total antioxidant capacity was determined by phosphomolybdenum method (Prieto et al., 1999). 0.1 mL of sample was combined with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Mixture was incubated at 95 °C for 90 min and then cooled to room temperature Absorbance was measured at 695 nm. Antioxidant capacity of each sample was expressed as ascorbic acid equivalent.

2.5.3 DPPH (2,2-diphenyl1-1-picryl-hydrazyl radical) free radical scavenging

Free radical scavenging activities were determined as described by Bibi et al. (2011) with few modifications. 180 μl of DPPH solution (in methanol) was added to 20 μl of sample solution in DMSO (final concentration 100 μg/mL). Samples were incubated in dark at 37 °C for 15 min and absorbance was measured at 517 nm using microplate reader.

3.1.1 Qualitative analysis of phytochemicals

Local inhabitant knowledge and literature about the curative properties helped for the selection of the plants under study. Detection of alkaloids, tannins and terpenoids in several extracts indicates that these were major secondary metabolites in these as shown in Table 1. These compounds are known to exhibit bioactive properties as triterpenoids display analgesic and anticancer properties (Ali et al., 2008). Saponins are reported to have hypocholesterolemic and antidiabetic properties, while triterpenoids display analgesic and anticancer properties (Ali et al., 2008). So these secondary metabolites contribute to potent use of plants in pharmacological industries. Differences were observed in current and previous studies. In current study, terpenoids, tannins, coumarins, saponins and cardiac glycosides were present in both M/C and aq. extracts of Punica granatum while alkaloids and quinones were not found. In previous studies alkaloids were found to be present in aq. extracts of the Punica granatum while terpenoids were not detected (Wadood et al., 2013). Similarly, in current study terpenoids and tannins were detected in Ficus microcarpa while in previous studies alkaloids and steroids have also been reported (Shripad et al., 2012). The differences were might be due to variation in genetic makeup, weather and geographic location of the plants and extraction procedure used or their phytochemicals. In our study M/C (1:1) proved to be a better solvent system for extraction of wide range of metabolites from these plants.

3.1.2 Quantitative analysis of phytochemicals

3.2.1 Antibacterial activity

The plant extracts showed varying degree of antibacterial potential due to different chemical composition (Table 3). In general M/C extracts inhibited bacterial growth more effectively as compared to aq. extracts but interestingly aq. extracts of Pinus roxburghii, Mentha piperita, Skimmia laureola and Morus nigra inhibited the growth of E. aerogens more efficiently than their M/C extracts. It is also noteworthy that extracts of Pinus roxburghii, Mentha piperita, Skimmia laureola, Mallotus philippensis, Phyllanthus emblica and Grewia asiatica demonstrated broad spectrum antibacterial activity in both solvent systems. Data revealed that maximum inhibition in the growth of S. aureus was caused by the M/C extract of Ageratum conyzoides and aq. extract of Grewia asiatica. Datura innoxia (M/C extract) and Pinus roxburghii (aq. extract) significantly inhibited the growth of B. subtilis. Datura innoxia (M/C extract) also efficiently inhibited the growth of E. coli. Mentha piperita (M/C extract) was very active against E. aerogens and its aq. extract was very also potent against E. coli, S. typhi, E. aerogens and B. bronchiseptica (all gram negative bacteria). Our results indicated that S. typhi, E. aerogens and B. bronchiseptica were resistant to most of the extracts. This could be explained on the basis of presence of an extra outer membrane in their cell wall which resulted in selective permeability of samples but we also observed that extracts of Pinus roxburghii, Centorea calcitropa, Mentha piperata, Skimmia laureola, Fagonia cretica, Mallotus philippensis, Coleobrookea oppositifolia, Phyllanthus emblica, Datura innoxia, Grewia asiatica and Ficus microcarpa had ability to inhibit the above mentioned gram negative bacteria due to the presence of active compounds which can act by inhibiting the bacterial growth without necessarily penetrating into the bacterial cell itself (Mulaudzi et al., 2011). We noticed that alkaloids, saponins and flavonoids were detected in all extracts exhibiting antibacterial activities. Thus we deduce that these metabolites are responsible for their antibacterial activities. This is in agreement with reports of Jaberian et al. (2013) that plant extracts have antibacterial activities may be due to the presence of potent compounds such as flavonoids, alkaloids, tannins, etc. However, the absence of activities in extracts does not mean complete lack of bioactive compounds but might be due to the lower amount of these compounds or their actions were antagonized by the presence of other compounds. Roxithromycin (control 1) was found to be active against B. subtilis, E. coli and Bordetella bronchiseptica while cefixime (control 2) was active against all the strains except E. coli.

3.2.2 Antifungal activity

Results of the antifungal investigations are shown in Table 4. Phyllanthus emblica and Anagallis arvensis very strongly repressed the growth of all fungal strains used, so these plants are proposed for the isolation of compounds with remarkable antifungal properties. It is also worth noting that aq. extracts of Azadirachta indica and Morus nigra significantly inhibited the growth of Mucor spp. which is the major cause of mucormycosis. The results support the use of water extracts in traditional medicine as reported by Mulaudzi et al. (2011). Extracts of Anethum graveolens vr. white flower, Peganum harmala, Mentha piperita, Cedrela toona, Justicia adhatoda, Citrus limon and Anethum graveolens vr. yellow flower also expressed antifungal properties against few fungal strains. Wide variety of secondary metabolites are reported to exhibit antifungal properties especially flavonoids (Varghese et al., 2009). These metabolites were also detected in our samples exhibiting antifungal activity. Terbinafine was found to be very active against all the fungal strains with maximum zone of inhibition against F. solani (35.5 mm).

3.3.1 Reducing power (RP)

Plants displayed a large variation in RP both in M/C and aq. extracts (Table 2) and the values ranged from 6.57 to 114 mg vit. C equivalent (eq.)/g DW in M/C extracts, with average value of 35.7 mg vit. C eq./g DW. The RP values for the aq. extracts ranged from 4.9 to 123.9 mg vit. C eq./g DW, with mean value 35.9 mg vit. C eq./g DW. Different RP values of plants and variable behavior of same plant in different solvents suggest that nature of plant and solvent system used for the extraction are very crucial to study antioxidant activity as observed by Qader et al. (2011) and Katalinic et al. (2006). Plants demonstrating remarkably high antioxidant activities in both the extracts include Punica granatum, Phyllanthus emblica, Mentha piperita, Syzygium cumini, Ficus microcarpa, Rosa indica. M/C extract of Berberis lycium, Sambucus nigra and Mallotus philippensis and aq. extract of Pinus roxburghii also showed remarkable RP. Due to their tremendous reduction capacity we recommend these plants for the extraction of antioxidant compounds for medicinal and commercial use. Aqueous extracts are mostly non-toxic and hence further isolation of antioxidant compounds is not necessary. In addition, due to synergistic effects of their phytochemical, health benefits might be increased using plant infusions (Liu, 2003). Aq. extracts of most plants showed better RP than M/C extracts. Hence, it can be concluded that the compounds with reducing capacity are more efficiently extracted out by water and this agrees with the previous study of Wong et al. (2006) presenting the significance of aq. solvent for extracting reducing compounds.

3.3.2 Total antioxidant capacity (TAC)

Samples exhibited wide range of TAC values, expressed as the number of equivalents of ascorbic acid, from 9.4 to 88.6 mg vit. C eq./g DW in M/C extracts and 5.8–56.70 mg vit. C eq./g DW in aq. extract as shown in Table 2. Ficus microcarpa showed maximum TAC (88.64 μg vit. C eq./mg DW). None of aq. extract of any plant rose to category of highly significant TAC. This might be due to less extraction of phenolic compounds by aq. solvent which leads to decline in efficiency of these extracts to reduce Mo, this is in agreement with the report of Shon et al. (2004) who proposed a positive correlation between plant phenolic compounds and their antioxidant character.

3.3.3 DPPH free radical scavenging activity

The DPPH free radical scavenging activities were expressed as a percentage of inhibition and results are shown in Table 2. The results varied from 8 to 90% for M/C extracts and 3.9 to 89% for aq. extracts. Top plants exhibiting great antioxidant effects in both of the extracts were Punica granatum, Syzygium cumini, Mentha piperita, Jasminum officinale, Ficus microcarpa, Dodonaea viscosa, Phyllanthus emblica, Rosa indica, and Mallotus philippensis. It was noticed that the M/C and aq. extracts of Punica granatum showed the highest percentage scavenging i.e. 91% and 89%, respectively. From distribution of antioxidant activities, it appeared that most of the plant extracts have moderate DPPH values, either because of low concentration of the antioxidants and/or because of antagonistic behavior of the active compounds thus inhibiting the antioxidant effects. This is in accordance with the previous finding of Surveswaran et al. (2007) while working on 133 Indian medicinal plants.

Extracts with scavenging percentage more than fifty were selected for the determination of IC₅₀ values. The IC₅₀ value is defined as the concentration of plant extract with 50% free radical scavenging potential. Using three fold dilutions IC50 value of the M/C extract of Phyllanthus emblica was minimum among all the extracts while the M/C extract of Punica granatum also exhibited good IC₅₀. IC₅₀ values of aq. extracts were found to be greater than M/C extracts with minimum value observed for Syzygium cumini i.e. 12.8 μg/ml.