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Urease Inhibition of Fixed Oils and Fractions of Caralluma tuberculata: Component Identification by GC-MS.

Byline: Murad Ali Khan, Haroon Khan, Muhammad Saeed, Abdul Rauf, Tayaba Basharat and Afsar Khan

Summary: The in vitro urease inhibitory activity of fixed oil and various organic fractions of Caralluma tuberculata followed by GC-MS analysis of the fixed oil are described in this research article. The fixed oil caused marked attenuation of jack bean urease with half-maximal inhibitory concentration (IC50) of 2.97 mg/ml. The similar urease inhibitory profile was observed for chloroform and ethyl acetate fractions with IC50 values of 3.36 and 4.90 mg/ml, respectively. GC- MS analysis led to the identification of 20 different constituents of the fixed oil by their m/z ratio and retention time in comparison with the standard compounds. The major constituents were methyl linoleate (30.97%) followed by methyl octadecadienoate (19.16%), ethyl linolenate (13.70 %), and methyl palmitate (9.86 %). The fixed oil and organic fractions of C. tuberculata exhibited marked urease inhibition and thus provide scientific background for use of the plant as antiulcer agent.

Keywords: Caralluma tuberculata, Fixed oil, Urease inhibitory activity, GC-MS.

Introduction

The genus Caralluma belongs to family Asclepiadaceae and consists of approximately 260 plant species which are commonly found in Spain, India, Africa, Saudi Arabia, Middle East, and Pakistan. Ethnobotanically, some species of the genus Caralluma have been used as traditional and modern dietary ingredients to suppress appetite [1]. Extracts of various Caralluma species are being used as nutraceuticals to combat obesity [2].

Two species, Caralluma tuberculata and Caralluma edulis, of this genus are found in Pakistan [3]. C. tuberculata is a leaess, succulent, and angular wild growing plant. In Pakistan, traditional healers use it as a food in the treatment of inflammation such as rheumatoid arthritis, irritation and swelling, pain and fever, leprosy, diabetes, and snake and scorpion bites [4, 5]. In Saudi Arabia, it is used in the treatment of ulcers, diabetes, and juice of the plant as ear drops for inflammation and pain [6]. Its chemical investigation showed the isolation of various glycosides; mostly flavone [7] and pregnane [8, 9].

The current study was planned to find out the antiulcer potential of the fixed oil (n-hexane fraction) and CHCl3, EtOAc, and H2O fractions of C. tuberculata as well as component identification of fixed oil by GC-MS.

Experimental

Plant Material

Fresh plants of Caralluma tuberculata were purchased from local market in 2013. A plant taxonomist at the Department of Plant Sciences, Kohat University of Science and Technology, Kohat, Pakistan determined the taxonomic identity of the plant.

Extract Preparation

Air-dried and coarsely powdered plant material was extracted three times with CH3OH. The methanol extract, evaporated under reduced pressure, resulted into the crude extract. The crude extract was subjected to solvent-solvent extraction; suspended in H2O and fractionated with n-hexane, CHCl3, and EtOAc in order to get their respective fractions. The crude plant extract and resulting solvent fractions were then stored at 4 C for future use.

Chemicals and Reagents

10% methanolic BF3 solution was obtained from Fluka Chemie (Buchs, Switzerland). Methanolic NaOH solution (0.5 N) and NaCl (analytical grade) were provided by Merck (Darmstadt, Germany) while CH3OH and n-hexane (HPLC grade) were purchased from Fischer Scientific (Leicestershire, UK). Helium gas (99.9999%) was procured from Pakistan Gas (United Arab Emirates).

Methyl tridecanoate and fatty acid methyl esters (FAMEs), and 37-component standard mix (methyl hexanoate, methyl caprylate, methyl caproate, methyl undecanoate, methyl laurate, methyl tridecanoate, methyl myristate, methyl myristoleate, methyl pentadecanoate, methyl pentdecenoate, methyl palmitate, methyl palmitoleate, methyl margarate, methyl heptadecenoate, methyl stearate, methyl oleate, methyl elaidate, methyl octadecenoate, methyl linoleate, methyl octadecadienoate, methyl Ylinolenate, methyl linolenate, methyl arachidate, methyl eicosenoate, methyl eicosadienoate, methyl 8,11,14-eicosatrienoate, methyl heneicosanoate, methyl arachidonate, methyl eicosatrienoate, metyl eicosapentaenoate, methyl behenate, methyl erucate, methyl docosadienoate (22:2), methyl tricosanoate, methyl tetracosanoate, methyl docosahexaenoate, and methyl tetracosenoate) were acquired from Accu Standard (New Haven, Connecticut, USA).

Preparation of FAMEs

Methylation of fatty acids in the oil was carried out prior to GC-MS analysis using methanolic BF3 as a derivatizing reagent [10]. AOAC standard method was used for derivatization [11]. Briefly, sample (25 mg fat equivalent) was added to 0.1 ml internal standard (1.37 mg) and 1.5 ml of 0.5 N methanolic NaOH solutions, sealed and heated for 5 min in boiling water bath. The hydrolyzed material was cooled and 2.5 ml of 10% methanolic BF3 solution was added to it. The solution was again sealed and heated in boiling water bath for 30 min and then cooled. 5 ml saturated NaCl solution was added to this esterified solution followed by extraction with n-hexane (1 ml) two times. The n-hexane soluble components were filtered through 0.45 m membrane and 1 l from the filtrate was subjected to GC-MS using auto injector.

Chromatographic Separation of FAMEs

The GC-MS instrument used was of the same model as used by Qureshi et al. [12] and we also followed the same conditions which were used by them; only the solvent cut time was 1.6 min instead of 1.8 min. Briefly, gas chromatograph (Shimadzu) equipped with AOC-20S auto-sampler and AOC-20i auto-injector, and hyphenated to a mass spectrometer QP2010 plus, was used for fatty acid analysis. The following conditions were used for GC analysis. Carrier gas was Helium.

A capillary column TRB-FFAP (length: 30 m; i. d.: 0.35 mm; thickness: 0.25 m) pre-treated with polyethylene glycol was used for chromatographic separations. Other GC-MS conditions were: ion source temperature (EI): 250 C, interface temperature: 240 C, pressure: 100 KPa, solvent cut time; 1.6 min. 1 l of each of the sample and the standard were injected to the GC. The operating condition for injector was split mode with a split ratio 1:50 at 240 C. The column temperature program began at 50 C for 1 min and increased to 150 C at the rate of 15 C/min. The temperature was changed to 175 C at the rate of 2.5 C/min and held for 5 min., it was followed by an increase to 220 C at the rate of 2.5 C/min and kept constant for 5 min. Total elution time was 45 min. MS scanning was performed from m/z 85 to 380. GC- MS Solutions software of the supplier was employed for system control and data collection.

The mass spectra of the compounds obtained were identified by peak matching with those of standard mass spectra from the NIST library (NIST05).

In vitro Urease Inhibition Assay

For urease inhibition assay the procedure described by Tariq et al. was followed [13]. The aliquot was taken after 15 min and incubated once more with 55 l of buffers having 100 mM urea for 15 min at 30 C. The production of ammonia was calculated as urease activity by indophenol method as described earlier [14]. The final volumes were maintained as 200 l by using the same procedure as described by Tariq et al. [13] but at pH 8.2. The results (change in absorbance per min) were calculated spectrometrically at various concentrations of the samples in the absence and presence of ascorbic acid. The standard drug used was thiourea. The percentage inhibitions were calculated as follows:

Equation

The IC50 values were calculated using statistical software, GraphPad PRISM 6.

Statistical Analysis

The experimental findings were expressed as the mean S.E.M. of three independent assays in each group. One-way analysis of variance (one-way ANOVA) was performed for determining the differences between the groups, using the least significant difference (LSD) test at Pless than 0.5.

Results and Discussion

Urease Inhibitory Effect of n-Hexane Fraction (Oil)

The result of n-hexane fraction of C. tuberculata against urease is depicted in Fig. 1. It had profound dose dependent attenuation of urease at various test concentrations. The maximum inhibition (83.48%) was observed at a dose of 10 mg/ml. The half maximum concentration (IC50) value of the fixed oil was 2.97 mg/ml (Table-1).

Table-1: The estimated IC50 values of fixed oil and fractions of Caralluma tuberculata against urease.

###S. No.###Extract/fractions###IC50S.E.M. (g/ml)a

###1###Fixed oil (n-hexane fraction)###2.970.01

###2###Chloroform fraction###3.360.10

###3###Ethyl acetate fraction###4.900.09

###4###Aqueous fraction###4.000.07

###b

###5###Thiourea###3.330.05

Urease Inhibitory Effect of Chloroform Fraction

The effect of chloroform fraction of C. tuberculata against urease at various test concentrations is illustrated in Fig. 2. The fraction demonstrated marked inhibition of urease activity with maximum activity of 71.01% at a dose of 10 mg/ml. The estimated IC50 value of chloroform fraction was 3.36 mg/ml (Table-1).

Urease Inhibitory Effect of Ethyl Acetate Fraction

The urease inhibitory profile of EtOAc fraction of C. tuberculata is shown in Fig. 3. In a dose dependent manner, it evoked significant urease inhibition with a maximum activity of 62.44% at a dose of 10 mg/ml. The estimated IC50 value of ethyl acetate fraction was 4.90 mg/ml (Table-1).

Urease Inhibitory Effect of Aqueous Fraction

The results of aqueous fraction of C. tuberculata against urease at various concentrations are displayed in Fig. 4. The fraction showed urease inhibition effect in a dose dependent manner with maximum effect of 43.65% at 10 mg/ml. The estimated IC50 value of aqueous fraction was 4.00 mg/ml (Table-1).

Components Identified from GC-MS Analysis of Fixed Oil

The results of GC-MS analysis of fixed oil of C. tuberculata are presented in Table-2. These led to the identification of 20 different compounds. In terms of concentration, the highest amount of methyl linoleate (30.97%) was followed by methyl octadecadienoate (19.16%) and ethyl linolenate (13.70 %).

Urease (urea amidohydrolase) mostly occurs in unicellular organisms and plants. Ammonia and carbamate are produced by the hydrolysis of urea in the presence of urease in living organisms [15, 16]. The natural decay of carbamate leads to another ammonia molecule. These reactions may be a reason for the significant increase in pH and are, therefore, accountable for the unwanted effects of urease actions in humans and agriculture [17, 18].

Research has revealed that infections produced by bacteria (Helicobacter pylori and Proteus mirabilis) typically occurred due to over urease activity. Urease is vital to H. pylori metabolism and pathogenicity. The colonization of H. pylori in gastric mucosa is dependent on urease activity [19, 20]. It is employed in the diagnosis and follow-up after treatment. It provides an attractive replica for metalloenzyme studies. The humans were considered to generate "gastric urease", before the discovery of H. pylori, but now it is well-established that the foundation of this prominent protein is actually H. pylori, which forms colonies in the human gastric mucosa [21]. It implicates in urinary tract and gastrointestinal infections, probably augmenting the severity of pathological conditions such as peptic ulcer and stomach cancer. Urease is also concerned in the development of several human and animal pathogenicities of urinary system [22, 23].

As one of the primary virulence in the pathophysiology of multiple human and animal disorders, targeting urease for treating pathogenic disorders caused by urease-producing-bacteria has already opened a new line for the treatment of infections caused by such bacteria. In this regard, urease inhibitors have gained unbelievable attention in recent times and, therefore, resulted into the discovery of several inhibitors [13, 16, 20, 23].

GC-MS analysis of the fixed oil was carried out by using the standard procedure. Table-2 shows the results obtained from GC-MS analysis which exhibited the relative concentrations of individual esterified fatty acids based on the external standard and the standard deviation values (STD) in each case. The quantification of esterified acids was performed using three points calibration curve with R2 value less than 0.99 in each case. Fig. 5 is the GC chromatogram of Caralluma tuberculata fixed oil with properly labeled signals of analytes detected. Both the saturated and unsaturated fatty acids were found in the sample under investigation.

Methyl linoleate (30.97%) was the major constituent of the fixed oil. Methyl linoleate appears to often mediate antioxidant and urease inhibition properties [24, 25]. The second saturated fatty acid with higher concentration was methyl octadecadienoate with a concentration of 19.16% while the next major constituents were ethyl linolenate (13.70 %) and methyl palmitate (9.86 %), respectively. Amounts of rest of the fatty acids were less than 1% (Table-2).

Table-2: Chemical composition of fixed oil of Caralluma tuberculata.

###ID ####Name of Compound###tR (min.)###Area###Conc. (%)###Std. Dev.

###1###C6:0; Methyl hexanoate###3.031###11608###0.030###0.000

###2###C8:0; Methyl caprylate###3.995###4022###0.045###0.002

###3###C10:0; Methyl caprate###5.784###6617###0.051###0.002

###5###C12:0; Methyl laurate###8.409###65430###0.402###0.001

###6###C14:1c; Methyl myristoleate###9.581###1052###1.121###0.002

###8###C15:0; Methyl pentadecanoate###10.961###60683###0.571###0.012

###10###C16:0; Methyl palmitate###12.582###50076###9.868###0.000

###11###C16:1c; Methyl palmitoleate###14.603###2892301###1.308###0.020

###12###C17:0; Methyl margarate###16.005###9650###0.648###0.002

###14###C18:0; Methyl stearate###17.013###89336###2.040###0.001

###15###C18:1c; Methyl oleate###18.003###6535###0.414###0.001

###16###C18:1n9T; Methyl elaidate###19.862###232562###0.594###0.003

###17###C18:ln8T; Ethyl octadecenoate###20.372###383836###0.054###0.003

###18###C18:2c; Methyl linoleate###20.519###26030###30.971###0.001

###19###C18:2t; Ethyl octadecadienoate###21.697###1427339###19.165###0.002

###21###C18:3n3; Ethyl linolenate###23.995###81835###13.70###0.002

###22###C20:0; Methyl arachidate###26.948###71770###0.468###0.005

###24###C20:2; Methyl eicosadienoate###31.035###30052###1.017###0.001

###30###C22:0; Methyl behenate###34.674###139782###0.634###0.001

###34###C24:0; Methyl tetracosanoate###38.002###88528###0.334###0.000

The results of the current study demonstrated marked blockade of urease activity by the fixed oil (n-hexane fraction) and other organic fractions of C. tuberculata at various test concentrations in comparison with thiourea as a standard urease inhibitor (IC50 3.33 mg/ml). The fixed oil was the most potent among the tested samples followed by CHCl3 and EtOAc fractions, respectively. The GC-MS analysis of the fixed oil of the plant led to the identification of 20 known compounds. The order of major components was methyl linoleate followed by methyl octadecadienoate and ethyl linolenate. The current findings could be attributed to these constituents. However, the isolation of individual components will tell us the real chemical background of this study.

Conclusion

The fixed oil and other organic fractions of C. tuberculata are the potent source of urease inhibition and, therefore, rationalize the traditional use of the plant as anti-ulcer agent by blocking urease activity. The GC-MS analysis of the fixed oil resulted into the identification of some of the chemical constituents but the isolation of pure secondary metabolites is suggested to explore comprehensive chemical background.

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