Plant material and extraction
In this study, four plants (Chrozophora hierosolymitana Spreng, Chrysanthemum leucanthemum L., Ephedra gerardiana Wall. ex Stapf and Quercus dilatata L.) were collected from different regions of Pakistan. These were identified following by Professor Dr. Mir Ajab Khan and voucher specimens were deposited at the Herbarium of Pakistan, Quaid i Azam University, Islamabad. The plant tissues were macerated at room temperature for two weeks with methanol and filtered. The solvent was removed by rotary evaporator under reduced pressure and low temperature. Extraction of each plant part (leaf, stem and root) was carried out separately and all necessary precautions were adopted to avoid cross contamination. Seven crude extracts of these plants (leaf, stem and root extracts of C. hierosolymitana, aerial parts of C. leucanthemum, stem and root extracts of E. gerardiana and aerial parts of Q. dilatata) were prepared. All the extracts were stored at –20°C.
Antifungal assay
Antifungal activity against seven fungal strains (Fusarium moniliformes, Fusarium solani, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Alternaria sp. and Mucor sp.) was determined using agar tube dilution method as reported earlier [22]. All fungal strains were grown on 6.5% SDA (Sabouraud dextrose agar, pH 5.7) at 28°C and preserved at 4°C in refrigerator. Screw capped test tubes containing Sabouraud dextrose agar (SDA) medium (4ml) were autoclaved at 121°C for 15 minutes. The tubes were allowed to cool at 50°C and non solidified SDA was loaded with 66.6μl of plant extracts pipeted from the stock solution (24mg/ml in DMSO) to make 400μg/ml final concentration. Tubes were then allowed to solidify at room temperature in slanting position. Each slant was inoculated with equal amount of fungal culture of size 4mm diameter and incubated at 28°C for 7-10 days. The media supplemented with DMSO and Turbinafine (200μg/ml) were used as negative and positive control respectively. The fungal growth was determined by measuring linear growth (mm) and compared with negative control to get the % age inhibition by using the following formula.
(1)
Antibacterial assay
Antibacterial assay was carried out as reported earlier [23]. All plant extracts and fractions were tested against three gram-positive bacterial strains i.e., Bacillus subtilis (ATCC 6633), Micrococcus leuteus (ATCC 10240), Staphylococcus aureus (ATCC 6538)] and three gram negative ones i.e., Escherichia coli (ATTCC 1522), Salmonella setubal (ATCC 19196) and Bordetella bronchiseptica (ATCC 4617).
Agar well diffusion method
The agar well diffusion method, was applied for the determination of inhibition zones and minimum inhibitory concentration (MIC) of plant extracts and partitioned fractions. Briefly, 0.75ml of the broth culture containing 108 colony forming units (CFU) per ml of the test strain was added to 75ml of nutrient agar medium at 45°C, mixed well, and then poured into a 14cm sterile petriplate. The medium was allowed to solidify, and 8-mm wells were dug with a sterile metallic borer. Eight concentrations of extract, two solutions for positive control (Roxithromycin and Cefixime-USP, one for each) and one for negative control (DMSO) were applied to each petriplate. These plates were incubated at 37°C. After 24 hrs of incubation the diameter of the clear zones, showing no bacterial growth, around each well was measured. Triplicate plates were prepared for each extract. Mean clear zone of these plates was calculated with standard error. The minimum inhibitory concentration (MIC) was determined by agar well diffusion [24] method. Serial dilutions of each extract (1mg/ml) in DMSO were prepared to obtain a 0.1–0.9mg/ml concentration range. A 100 μL of each dilution of the extract was introduced into wells in nutrient agar plates pre inoculated with test bacterial strains. The extracts were allowed to diffuse at room temperature before incubation at 37°C for 24 h.
Disc diffusion method
Disc diffusion method was applied for the determination of inhibition zones and minimum inhibitory concentration (MIC) of fractions. Filter paper discs of 6 mm diameter containing only one concentration of each fraction i.e., 1mg/mL in methanol were tested against the six bacterial strains. A 20μL of the prepared concentration of each fraction was applied to a disc. The solvent was evaporated and the disc was placed on nutrient agar plates pre inoculated with test bacterial strains.
Fraction preparation
Solvent partitioning
The crude extract of the most active antibacterial plant was subjected to bio-guided fractionation by solubilisation in water and sequential partition with hexane (5 × 400mL), ethyl acetate (5 × 400mL), chloroform (3 × 400mL), acetone (5 × 400mL), ethanol (5 × 400mL) and 50% methanol (3 × 400mL) as indicated in Figure 1. Each fraction thus obtained, including the final hydromethanol fraction, was evaporated to dryness and subjected to antibacterial assay.
HPLC analysis of ethanol fraction
The highly active partitioned fraction (ethanol) was analyzed by HPLC system consisting of Agilent 1200 series preparative pump coupled with UV diode array detector. Optimization of mobile phase and wave length for RP- HPLC analysis of ethanol fraction of (A) Q. dilatata was carried out using Zorbax SB-C18 analytical column (4.6 × 150mm, 5 μm particle size, Agilent, Germany). Sample was prepared in methanol at concentration of 25 mg/mL. The sample was filtered quickly through a 0.2μm syringe filter. The injection volume was 20μL. Different mobile phases used for optimization with the isocratic flow kept constant at 1mL/min were as follows:
-
a)
Methanol: water: acetonitrile: acetic acid (90:100:10:2).
-
b)
Acetonitrile: water (85: 15)
-
c)
Acetonitrile: methanol: water (75: 10: 15)
-
d)
Methanol: acetonitrile: acetic acid (90: 10: 1)
-
e)
Acetonitrile: methanol: ethyl acetate: toluene (59: 30: 10: 1) with 0.1% triethylamine.
-
f)
Acetonitrile: water (60: 40)
-
g)
Acetonitrile: methanol (70: 30)
A UV diode array detector was set at five different wave lengths i.e. 230nm, 235nm, 240nm, 245nm and 250nm. Acetonitrile: methanol (70: 30) and 230nm wavelength was selected for the preparative HPLC on the basis of results of analytical HPLC.
Preparative HPLC
RP- HPLC analysis of ethanol fraction of (A) Q. dilatata was done using LiChrospher 100 RP-C18 preparative column (25 × 250 mm, 5 μm particle size, Merck, Germany). The sample was prepared in methanol at a concentration of 50mg/mL. The injection volume was 1 mL. Acetonitrile: methanol (70: 30) and 230nm wavelength was used for the preparative HPLC. The isocratic flow was kept constant at 10mL/min.
RP- HPLC analysis of purified fractions was done using Zorbax SB-C18 analytical column (4.6 × 150 mm, 5 μm particle size, Agilent, Germany).
Characterization of the purified active component
Thirty micrograms of the purified active component and each of four standards (Quercitrin, Gallic acid, Rutin and Ascorbic acid) were dissolved in 3mL of methanol to prepare 10μg/mL concentration. Absorption spectra of the purified active component and each of four standards (Quercitrin, Gallic acid, Rutin and Ascorbic acid) were obtained at 210-900nm by using spectrophotometer coupled with UV-diode array detector (DAD).
Phytochemical analysis
Qualitative phytochemical analysis of partitioned fractions was carried out by using standard procedures to identify the constituents as described by Edeoga et al. [25] and Parekh and Chanda [26].
Alkaloids
To identify presence of alkaloids, 4mL of 1% HCl was added to the 0.25g of plant extract and then warmed and filtered. To 1ml filtrate 6 drops of Mayor’s reagents/Dragendroff reagent was added separately. Creamish precipitate/orange precipitate indicated the presence of respective alkaloids.
Saponins: (Frothing test)
To detect saponins, 0.5g of the plant extract was boiled in 5mL of distilled water. After cooling it was shaken vigorously to produce stable persistent froth.
Anthraquinones
To check presence of anthraquinones, 0.5g of the plant extract was boiled with 3mL of 1% HCl and filtered. To filtrate, 2mL of benzene was added and was shaken well. The benzene layer was removed and few drops of 10% NH4OH were added. Formation of pink, violet or red color indicated the presence of anthraquinones.
Couramins
For couramins analysis, 0.5g of moistened plant extract was taken in a test tube and covered with a filter paper moistened with 0.1N NaOH. The test tube was placed, for few minutes, in boiling water. Then the filter paper was removed and examined in UV light for yellow florescence to indicate the presence of couramins.
Terpenoids
(Liebermann-Burchard reaction): To identify presence of terpenoids, 0.5g of the plant extracxt was dissolved in 2mL of chloroform and filtered. To filtrate, equal volume of acetic acid and a drop of conc. H2SO4 were added. Blue-green ring indicated the presence of terpenoids.
Flavonoids, Flavones
To detect flavanoids and flavones, 0.5g of the extract was washed with petroleum ether. The defatted residue was dissolved in 20mL of 80% of ethanol and filtered. The filtrate was used for the following test;
-
a)
About 3ml of the filtrate was mixed with 4ml of 1% AlCl3 in MeOH in a test tube. Formation of yellow colour indicated the presence of flavanols, flavones.
-
b)
About 3ml of the filtrate was mixed with 4ml of 1% KOH. A dark yellow colour indicated the presence of flavonoids.
Tannins
To test tannins, 0.25g of plant extract was boiled in 10mL of distilled water and filterd. Then 1% FeCl3 was added to the filtrate. Browrish green or a blue-black colouration indicated the presence of tannins.
Phlobatannins
Deposition of a red precipitate when 0.25g of plant extract was boiled with 5mL of 1% aqueous hydrochloric acid was taken as evidence for the presence of phlobatinnins.
Cardiac Glycosides: (Keller – Kiliani Test)
To detect cardiac glycosides, 2mL of glacial acetic acid and few drops of 1% FeCl3 were added to 0.5g of plant extract. Then it was underlayed with 1mL of conc. H2SO4. Green-blue color indicated the presence of cardiac glycosides.
