Metabolites Profiling of An Anti-Urease and Free Radical Scavenging Fraction from the Acetone Leaves Extract of Microsorum Pustulatum (G. Frost) Copel (Polypodiaceae)

Many of the plants within the family (Polypodiaceae) have been used traditionally for the treatment of wounds, ulcer, urinary tract disorder, viral, bacterial, fungal, antioxidant and in inflammatory states. As a follow-up to an earlier report on the anti-urease and free radical scavenging potentials of the leaves of the epiphytic fern Microsorum pustulatum,1 this study is reporting the metabolites profile of an active chromatography fraction using Gas Chromatography- Mass Spectrometry (GC-MS). Urease is a big enzyme that is within amidohydrolase family.2 Commonly found in bacteria, fungi, algae, plants, some invertebrates and as soil enzyme. Urease catalyzes the hydrolysis of urea to form ammonia and carbon dioxide, hence automatically alters the pH of the surroundings to basicity level as a result of high concentration of ammonia produced. This resultant pH elevation is of diverse clinical and agronomic significance.3 Urease is a virulence factor for Helicobacter pyroli, Proteus mirabilis, S. saprophyticus and Mycobacterium tuberculosis among others that are associated with certain diseases in human and animals. It has been associated with the pathogenesis of kidney stones in acute pyelonephritis and other urinary tract infections. It is also implicated in arthritis, gastric intestinal infections and peptic ulcer.4 Its soil activity may damage crop yield as the urea on the surface of the soil transfers ammonia away from rhizosphere during the hydrolysis process because of its volatile nature. This has negative impact on the environment due to eventual acid rain or nitrous oxide pollutant.5 On the other hand, in the management of oxidative stress, free radical scavenging compounds(antioxidants) helps to keep reactive oxygen (ROS) and reactive nitrogen (RNS) species in check, without being destabilized themselves. They help to promote longevity and good wellbeing.

MATERIALS AND METHODS

Materials

Reagents and solvents used in this study were of analytical grade and are products of JHD & Sigma Alderich Chemicals. Thiourea as reference standard and Urease test kits (Agape diagnostics Switzerland GmbH), 2,2-diphenyl-1-picrylhydrazyl(DPPH) and ascorbic acid as reference standard.

Sample collection and extraction

The leaves of Microsorum pustulatum was collected from the palm tree trunk in Rivers State University at Diobu in Obiakpor Local Government Area of Rivers State, Nigeria.  The sample was identified by the Taxonomist with voucher specimen deposited within the herbarium in the Department of Pharmacognosy and Phytotherapy University of Port Harcourt. The plant material was air-dried for two weeks at room temperature and pulverized. A 200g quantity of the pulverized sample was defatted by cold macerated in n-hexane for 96 hours with intermittent agitation and fresh replacement of solvent after every 48 hours as earlier reported.1 The defatted marc was then extracted similarly with acetone and the combined acetone filtrate concentrated using a rotary evaporator to afford the acetone extract as earlier reported by Olebunne at al., and was used in this study.

Fractionation of the acetone extract

The most active acetone extract was partitioned into a water soluble (polar phase) and chloroform soluble (organic phase) fractions using separating funnel as reported earlier.1 The organic phase (chloroform soluble fraction) coded “AOP” which was previously reported to be the more active was further fractionated using column chromatography. Briefly, approximately 1.3g of the AOP was pre-adsorbed on silica gel (Mesh 200-400) to form a homogeneous paste and allow to dry in a fume closet. The pre-adsorbed AOP was introduced into the normal phase column packed with the same silica gel (Mesh 200-400) as adsorbent to a height of 50cm and cotton wool was placed above it to protect it. The elution process was done using a gradient (300ml each) of n-hexane:chloroform: methanol (4:0:0, 3:1:0, 2:2:0, 1:3:0, 0:4:0, 0:3:1, o:2:2, 0:1:3, 0:0:4).The eluates were collected at 50ml intervals and pooled based on observed R_f spots and color reaction with chromogenic spray reagent from TLC and three pooled fractions (A₁, A₂ and A₃) were obtained and assessed for urease and free radical scavenging inhibitory activity.

Urease inhibitory analysis of the three extracts

The test was done using Phenol hypochlorite technique.6 Thiourea a known urease inhibitor was used as reference standard. The enzyme activity was measured through absorbance in the presence or absence of an inhibitor at 625nm using the spectrophotometer. The pooled chromatography fractions were assayed at 1 mg/ml. The reaction mixture (in the test tubes) containing BUN enzyme solution (1ml), sample solution (1ml) and urea (10 μl) were mixed and then incubated for 10mins at 37⁰c, followed by the addition of 1 ml of a color developer.  The mixture was further incubated for another 10 minutes, then 1ml of distilled water was added and the absorbance was taken. The percentage of urease inhibition was calculated using the formula:

% Inhibition of Urease = Absorbance of control - Absorbance of test sample x 100

Absorbance of control

The A₁ fractions was found to be more active and different concentrations of the A1 fraction (0.1-1mg/ml) were examined and its median or half maximal inhibition concentration(IC₅₀) determined from regression analysis both for the urease and free radical scavenging inhibition activity.

Free radical scavenging assays

The DPPH radical scavenging assay was employed as modified.7 Briefly, a 1 ml aliquot of a 1mg/ml solution of the chromatography fractions A1, A2 and A3 were separately assayed for DPPH radical scavenging activity by adding 1ml of DPPH (0.05g/100ml of ethanol). The mixture was left in the dark for 30 min before reading at 517nm with ethanol as blank. The negative control contains ethanol in place of extract and ascorbic acid as reference standard. Radical scavenging activity was expressed as percentage and calculated using the formula:

% Scavenging = Absorbance of negative control – Absorbance of sample x 100

Absorbance of control

The A₁ fractions was found to be more active and different concentrations of the A1 fraction (0.05 - 1mg/ml) were examined and its median or half maximal inhibition concentration (IC₅₀) determined from regression analysis both for the urease and free radical scavenging inhibition activity.

GC-MS Analysis

The metabolite(s) present in the active pooled chromatography fraction A1 were identified and characterized using GC-MS technique.  2 µL of sample dissolved in chloroform was injected into the column for the analysis. The Instrument: GC (Agilent model 7890B) coupled to a Mass Spectrometer (MS) detector (5977A MSD), was equipped with a DB-5MS capillary column (30m x 0.25 mm) and film thickness 0.25 µm). The oven temperature programming was set initially at 80^oC and increased gradually to 200 ^oC at the rate of 15^oC/minute. The temperature was further increased to 280^oC at the rate of 5^oC/ minute with a hold time of 5 minutes at 280 ^oC. The ion source set at 230^oC. Helium was used as carrier gas at a flow rate of 1ml/minute. Sample injection was done at a split ratio of 110:1. The MS ionization voltage was 70 eV with mass spectra data acquisition scanned form 50-550 m/z ratio. Metabolites assayed were identified from their retention time and their mass spectra fragmentation compared with that from the NIST library (2014) as well as critical review of the fragmentation scheme in line with the principles of mass spectrometry.8,9,10

RESULTS

Figure 1. Urease inhibition activity of the three pooled chromatography fractions; A₁, A₂, A₃ from the AOP fraction of Microsorum pustulatum leaves at 1 mg/ml test concentration

Figure 2. Concentration dependent Urease inhibitory activity of most bioactive chromatographic fraction(A₁) from the organic phase of acetone extract of Microsorum pustulatum leaves.

Figure 3. Half maximum inhibition concentration (IC₅₀) of the referece standard and active chromatography fraction A1 from the leaves of Microsorum pustulatum

Figure 4. DPPH free radical inhibition activity of the three pooled chromatography fractions; A₁, A₂, A₃ from the AOP  fraction of Microsorum pustulatum at 1 mg/ml test concentration

Figure 5. Concentration dependent DPPH free radical inhibition activity of the most active chromatography fraction A₁ from Microsorum pustulatum
Table 1: Compounds characterized from GC-MS analysis of most active chromatography fraction A₁  from Microsorum pustulatum