Activity guided isolation and characterization of antioxidant and antibacterial agents from some local Nigerian plants

1 Department of Biochemistry, Usmanu Danfodiyo University, Sokoto, Nigeria. 2 Medicinal Chemistry Department, Central Institute of Medicinal and Aromatic plants, Lucknow, India. 3 Molecular Bioprospection Department, Biotechnology Division, Central Institute of Medicinal and Aromatic plants, Lucknow, India. 4 Molecular Bioprospection Department, Central Institute of Medicinal and Aromatic plants, Lucknow, India.


INTRODUCTION
Diseases caused by pathogenic bacteria and fungi present critical problem to human health and are one of the main causes of morbidity and mortality worldwide (WHO, 1998). Resistance to antibiotics and the occur-*Corresponding author. E-mail: hassanwara@gmail.com. Tel: +2348036355866. rence of toxicity during prolonged treatment with present day drugs have been the reasons for extended search for newer drugs to treat microbial infections (Fostel and Lartey, 2000). Drug resistance is on the increase and there is need to search for other antimicrobial agents (Sharma and Kumar, 2006;Negi and Dave, 2010). Combination therapy is an alternative approach in the search for novel compounds with ability to deal with antibiotic resistant microorganisms. The combination can be of different plant extracts or plant extracts with standard antibiotics or chemicals. Studies have shown that plant extracts in combination of two or more are yielding effective antimicrobial activity against several microorganisms that even include drug resistant bacteria (Karmegam et al., 2008). Thus, interviews with traditional healers in Sokoto, Nigeria, indicated the use of the six plants in combination of 1:1 in the treatment of bacterial infections without any scientific validations. Plants have been used to treat infectious diseases due to their antimicrobial properties. This is due to the presence of various kinds of phytochemicals including phenolic compounds, alkaloids, terpenoids and essential oils (Lewis and Elvin-Lewis, 1995;Cowan, 1999).
Pergularia tomentosa (PS milk weed) is used in Northern Nigeria for tanning and treatment of skin diseases. Its isolated cardenolides have been shown to cause apoptotic cell death of Kaposi's sarcoma cells (Hamed et al., 2006). The roots have found applications in the treatment of bronchitis, constipation and skin diseases (Hammiche and Maize, 2006). It is well known that free radicals cause cell damage through mechanism of covalent binding and lipid peroxidation with subsequent tissue injury (Osawa et al., 1990). Antioxidant agents of natural origin have attracted special interest because they can protect human body from free radicals. Antioxidant properties of certain flavonoids of plant origin have already been established (Di Carlo et al., 1999). Ficus sycomorus is used locally for antimicrobial treatment in Nigeria and has been reported to have antimicrobial activities (Hassan et al., 2007). In the present work, we evaluated the synergistic antibacterial properties of the combined mixture of plants (CMP) and isolated and characterized the bioactive principles of the CMP and P. tomentosa. The pure compounds were also screened for antibacterial and antioxidant properties. To the best of our knowledge these have not been reported so far. Therefore, it is worthwhile in this study to present the activity guided fractionation, isolation and characterization of antioxidants and antibacterial agents from the CMP and P. tomentosa.

Plant material
The leaves, roots and stems of the selected plants were collected from the adjoining area of Usmanu Danfodiyo University (UDU), Sokoto, Nigeria. After proper taxonomic identification of all the plants (before combination) by the Taxonomist of Botany Unit (U.D.U.), the plant parts (leaf, root and stem) were open air-dried under the shade and pulverized into a moderately coarse powder.

Chemicals
DPPH, ascorbic acid, quercetin and FeCl 3 were purchased from Sigma Chemical Co. (St. Lois, MO, USA). Vanillin from BDH, Follin Ciocalteu's phenol reagent and sodium carbonate were from Merck Chemical supplies (Darmstadt, Germany). All the chemicals and solvents used were of analytical grade.

Microbial organisms
The microbial organisms used were available in the Molecular and Bio-prospection Unit, of Central Institute of Medicinal and Aromatic Plants, Lucknow, India. The bacterial isolates were maintained on nutrient agar medium.

General experimental procedures
The 1 H and 13 C NMR spectra were recorded on a Bruker Avance 300 (300 MHz). Column chromatography was performed with silica gel (60 to 120 mesh). TLCs were run on ready-made aluminum sheets (silica gel 60 F254, 0.25 mm, 20 × 20 cm, Merck, Germany) while preparative TLCs were run on glass plates (silica gel 60 F254, 0.5 mm, glass plates 20 × 20 cm) from Merck, Germany. Spots on the TLC plates were visualized by spraying with vanillin sulfuric acid and heating the plate in oven for 5 min at 100°C. Vacuum liquid chromatographic (VLC) separation was run over silica gel H (average particle size approximately 10 μm). The powdered leaf of P. tomentosa (PT) and the parts of the combined mixture of plants (1:1) were extracted with methanol separately and each residues obtained were dissolved in water separately and each were further fractionated with hexane, petroleum ether and n-butanol. The following fractions were obtained: P. tomentosa (methanolic leaf) extract = 28.9 g → HF (g), EF (g), BF (g), Combined mixture of plants = 40.0g → HF (g), EF (g), BF (g).

Isolation of bioactive compounds from the ethyl acetate fraction (EF) of combined mixture of plants (CMP)
The ethyl acetate fraction (EF) of combined mixture of plants that showed remarkable antibacterial and antioxidant activities was further fractionated. Eight grams (8 g) of this fraction was subjected to vacuum liquid chromatographic (VLC) separation over silica gel H (average particle size approximately 10 μm). Stepwise gradient elution was carried out with hexane, hexane-chloroform, chloroform, chloroform-methanol and methanol. A total of 249 fractions were collected. The fractions were pooled on the basis of their TLC profile as follows: Fractions 14 to 62 (270 mg), fractions 63 to 73 (308 mg), fractions 152 to 184 (325 mg), fractions 201 to 214 (524 mg), fractions 215 to 230 (495 mg), fractions 231 to 246 (1000 mg).

Isolation of bioactive compounds from the hexane fraction of Pergularia tomentosa
Separately, activity guided separation of hexane fraction of P. tomentosa which showed antibacterial activity was carried out. After series of chromatographic separation, a total of 114 ddfractions were collected. The fractions were pooled on the basis of their TLC profiles and the hexane fraction resulted in the isolation of lupeol acetate ( Figure 6) and its antibacterial activity was determined.

Free radical scavenging activity
It was measured using the modified method of Blois (1985). DPPH (50 μL of 0.1 mM dissolved in methanol) was added to the tested compounds at different concentrations (1, 5, 10, 25, 50 and 100 μg) and 40 μL of Tris-HCl were also added. Equal volume of methanol, Tris-HCl and DPPH were added in the control test. The mixture was shaken vigorously and incubated at 37°C for 20 min. The absorbance at 517 nm was measured spectrophotometrically. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The percentage of scavenging of DPPH was calculated using the following equation: AO -A1 DPPH scavenging effect (%) = × 100 AO Where, AO is the absorbance of the control reaction, A1 is the absorbance in the presence of the sample.

Total phenolics estimation
The amount of total phenolics was determined by Folin-Ciocalteu's colorimetric method (Wolf et al., 2003). Briefly, the concentration of the compounds (1, 5, 10, 25, 50 and 100 μg) were mixed with 50 μL of distilled water and 250 μL of Folin-Ciocalteu's reagent were added and mixed properly. A 250 μL of sodium carbonate was then added. The mixture was incubated at 37°C for 90 min and the absorbance was measured at 765 nm by a XPLORER XP2001 spectrophotometer. Gallic acid was used as a standard and total phenolics were expressed as grams of Gallic acid equivalent (g of GAE) per 100 g of fresh weight.

Nitric oxide scavenging activity
Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH, interacts with oxygen to produce nitrite ions which were measured by Griess reaction (Green et al., 1982;Marcocci et al., 1994). The reaction mixture containing 100 μL of sodium nitroprusside (10 mM) in phosphate buffered saline (PBS) and the compounds (1, 5, 10, 25, 50 and 100 μg) were incubated at room temperature for 30 min. After incubation, 50 μL of incubated reaction mixture were added to 100 μL of Griess reagent (1:1 sulfanilamide: naphthylethylene diaminehydrochloride). The absorbance of the chromophore formed was measured at 546 nm. The percentage of nitric oxide scavenging activity was calculated using the following equation: Where, AO is the absorbance of the control reaction, A1 is the absorbance in the presence of the sample.

Total antioxidant capacity
The assay was based on the reduction of molybdenum (VI) to molybdenum (V) by the compounds and the subsequent formation of a green phosphate Mo (V) complex to acid pH (Priesto et al., 1999). Compounds (1, 5, 10, 25, 50 and 100 μg) were combined with 1 ml of total antioxidant capacity (TAC) reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95°C for 90 min and cooled down to room temperature. The absorbance was measured at 695 nm against reagent blank. The total antioxidant capacity was expressed as the number of equivalent of ascorbic acid (mg/g of dry mass).

Reducing power
The reducing power of the extract/compound was determined according to the method of Oyaizu (1986). Different concentrations of the compounds (1, 5, 10, 25, 50 and 100 μg) were mixed with 250 μL phosphate buffer (pH 6.6, 0.2 M) and 250 μL (1%) potassium ferricyanide. The mixture was incubated at 50°C for 20 min. A 250 μL of trichloroacetic acid (10%) was added to the mixture and centrifuged at 5000 rpm for 3 min. Then 250 μL of the supernatant was mixed with 250 μL of distilled water and 50 μL of FeCl 3 (0.1%). The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated reducing power.

Total flavonoids
Estimation of total flavonoids was done according to the method of Ordon Ez et al. (2006). To 50 μL of the compounds (1, 5, 10, 25, 50 and 100 μg), 150 μL of methanol, 10 μL of AlCl 3 , 10 μL of potassium acetate and 280 μL of distilled water were added. The mixture was incubated at room temperature (25 to 37°C) for 30 min. The absorbance of the reaction mixture was measured at 415 nm. A yellow color indicated the presence of flavonoids content. Total flavonoids content was calculated as quercetin (mg/g).

Disc diffusion assay
The CMP and P. tomentosa extracts and pure compounds were screened for antibacterial activity against the following organisms: Staphylococcus aureus (MTTC 96), Staphylococcus aureus (MTTC 2940), Escherichia coli (MTTC 739), Micrococcus luteus (MTCC 2470), Bacillus subtilis (MTCC 121), Streptococcus mutants (MTCC 890), Raoultella planticola (MTCC 530), Klebsiella pneumoniae and Salmonella typhimurium. Strains were grown overnight at 36°C in nutrient broth medium. Inoculums for the assays were prepared by diluting cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5 (1.5 × 10 8 CFU/ml) and prepared nutrient agar plates were seeded with 1.5 × 10 8 CFU/ml suspensions of test bacteria. The antibacterial activity of culture was determined using disc diffusion assay according to the Clinical and Laboratory Standard Institute (CLSI, formerly NCCLS). Absorbent disc (5 mm) were impregnated with 5 μL of the CMP and P. tomentosa extracts (100 Table 1. Antibacterial activity of ethyl acetate fraction of combined mixture of plants and the isolated compounds. mg/ml) and pure compounds (10 mg/ml) and placed onto the surface of inoculated agar plates. Plates were incubated at 37°C for 24 h. Positive control discs of kanamycin and ampicillin was included. Antibacterial activity was expressed as the diameter of the inhibition zone (mm) produced by the extracts (Mellou et al., 2005).

Minimum inhibitory concentration
The CMP and P. tomentosa extracts that showed some activity were subjected to MIC test. MIC test was carried out according to the method of Ellof (1998), using Muller-Hinton Broth on a tissue culture test plate (96 wells). The stock solutions of extracts were transferred into the first well, and serial dilutions were performed in order to have concentrations in the range of 1000 to 7.81 μg/ml. Inoculums for the assays were prepared by diluting cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5, added to all wells and incubated at 36°C for 24 h. MIC was defined as the lowest concentration of the extracts that inhibited visible growth.

RESULTS AND DISCUSSION
The results of antibacterial activity are presented in Table  1, which shows that only ethyl acetate fraction ( Our findings are consistent with the study on synergistic activity of six plants that showed activity against pathogenic bacteria by Karmegam et al. (2008). All the pure compounds isolated from the CMP extract did not show antibacterial activity. The antibacterial activity of the ethylacetate fraction of CMP was due to combination of all the constituents in the CMP rather than the individual compounds isolated. A series of chromatographic separation of ethylacetate fraction of CMP (as outlined in the experimental section) resulted in the isolation and characterization of bioactive constituents, Gallic acid (Figure 1), 3β-Hydroxy-α-amyrin (Figure 2), 5,7,3'.4',5'pentahydroxy-3-O-glucophyranoside flavones (Figure 3), 5,7,3',4' tetrahydroxy-3-O-glucopyranoside flavones (Figure 4), 3,5,7,3',4'-pentahydroxy flavone ( Figure 5). All the compounds isolated were characterized with the help of ESI-MS, IR, 1 H C 13 , HMBC/HSQC and COSYNMR. Chemical analysis has indicated that some complex compounds elaborated by natural organisms may hardly be synthesized by chemical processes (Azas et al., 2002). However, the bacterial resistance to chemical treatment still remained important. Natural products isolated from the plants in the present study may be potential sources of new antioxidant drugs.
Activity guided separation of hexane fraction of P. tomentosa was also carried out. After series of chromatographic separation, a total of 114 fractions were collected. The fractions were pooled on the basis of their TLC profile and the hexane fraction resulting in the isolation of bioactive constituent ( Figure 6) lupeol acetate (LA). LA was screened for its antibacterial activity, which showed marginal but selective activity against M. luteus (Table 1). This confirms that the lack of antibacterial activity of hexane extract of P. tomentosa was due to combination of all the constituents rather than LA alone.  Results of antioxidant studies are presented in Figures 7 to 13. In Figure 7, the amount of DPPH reduced was quantified by measuring increases in absorbance at 517 nm. The DPPH scavenging ability of the tested compounds may be attributed to their hydrogen donating ability. Non DPPH scavenging activity was observed for 73 to 91 LPE ( Figure 6) and 45 to 46 cmp fractions ( Figure 2). The fractions 126 to 141 (Figure 4), 128 to 157 cmp (Figure 3), cp87 to 112 ( Figure 5) and 79 to 83 (Figure 1) have showed appreciable DPPH scavenging activity in different manner and for Figure 1 the activity decreases at 50 and 100 μg; this indicates that at higher concentration the activity was inhibited. This order was reversed in 126 to 141 fractions (Figure 4), in which the DPPH scavenging activity increases with increasing concentration of the compound but reduction was observed at 100 μg. Hence, the DPPH scavenging activity of the compounds may be represented as: cp87 -112 > 126 -141 > 128 -157 cmp > 79 -83 Nitric oxides (NO) are potent inhibitors of physiological processes such as smooth muscle relaxation, neuronal signaling, platelet aggregation and regulation of cell mediated toxicity (Hagerman et al., 1998). In Figure 8, a non NO scavenging activities were observed for fractions 73 to 91 LPE ( Figure 6). The nitric oxide (NO) scavenging activity of all the compounds was low with maximum of 22% inhibition for fractions 126 to 141 (Figure 4) at 25 μg but with no activity at 50 and 100 μg. The compound 45 to 46 cmp ( Figure 2) has little NO scavenging activity but the activity increased as the concentration increased; however, the activity dropped drastically at 100 μg.   In Figure 10, little amount of total flavonoids (TF) were observed for 73 to 91 LPE fractions ( Figure 6). The total flavonoids contents were also found to increase in a dose-dependent manner for 128 to 157 cmp ( Figure 3) and 79 to 83 (Figure 1) compounds. It dropped slightly at 100 μg for compound 126 to 141 ( Figure 4) and at 50 to 100 μg for 45 to 46 cmp ( Figure 2). The total phenolics (TP) were increased in dose dependant manner with exception of 45 to 46 cmp fractions ( Figure 2) and 73 to 91 LPE ( Figure 6) that showed little or no activity ( Figure  11). The antioxidant activity of polyphenolic compounds is mainly due to their redox properties which play an important role in adsorbing to and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Zheng and Wang, 2001). Phytochemicals like polyphenols possess significant antioxidant  capacities that are associated with lower mortality and rate of diseases (Anderson et al., 2001;Djeridane et al., 2006). The pharmacological effect demonstrated by the ethylacetate fraction of the combined plants mixture suggests that the phenolics have some pharmacological effects and could be attributed to these valuable constituents. All the results of total phenolic estimation were expressed as Gallic acid equivalent ( Figure 11) and are represented as follows:   The effects of the isolated compounds in the present study is due to their phenolic acids and flavonoids nature and have been demonstrated to exhibit antioxidant activity (Sliva et al., 2006;Kasture et al., 2009).

Conclusion
These findings, suggest that the extracts/pure compounds possess antibacterial and antioxidant properties.
The pharmacological effects demonstrated by the extracts could be attributed to their phytocompounds. Further screenings for in vitro antimalarial and anticancer activities of the compounds isolated are recommended.