Carbonic Anhydrase and Urease Inhibition Potential

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Mar 1, 2017 - The EtOAc fraction from both fungal strains Screlotium rolfsii and Aspergillus ... Nigrospora spp, Verticillium spp, Aspergillus spp, etc. The S.

Bashir Ahmad et al.,

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Carbonic Anhydrase and Urease Inhibition Potential of Soil Borne Fungi Screlotium Rolfsii and Aspergillus Flavus 1

Bashir Ahmad, 1Muhammad Rizwan, 2Abdur Rauf*, 3Saifullah Mehsud, 4 Muhammad Saleem, 5Umar Farooq and 5Ajmal Khan** 1 Center of Biotechnology and Microbiology, University of Peshawar, Peshawar-KPK-25120, KPK, Pakistan. 2 Department of Chemistry, University of Swabi, Ambar-23561, Khyber Pakhtunkhwa, Pakistan. 3 Department of Pharmaceutical Sciences, Abbottabad University of Sci. & Tech. Havelian, KPK, Pakistan. 4 Department of Chemistry, Ghazi University, Dera Ghazi Khan, Punjab Pakistan. 5 Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan. [email protected]*; [email protected]** (Received on 20th June 2016, accepted in revised form 1st March 2017) Summary: The current study was aimed to evaluate the inhibitory activity of ethyl acetate and nhexane fractions of Screlotium rolfsii and Aspergillus flavus against carbonic anhydrase-II and urease enzymes. The EtOAc fraction from both fungal strains Screlotium rolfsii and Aspergillus flavus were found significantly active against carbonic anhydrase-II with IC50 values of 45.40 ± 0.75 and 59.89 ± 1.65 respectively. Similarly the n-hexane fraction of both fungi also showed significant result against carbonic anhydrase having IC50 values of 52.77 ± 0.81 and 61.3±1.75 respectively. The ethyl acetate and n-hexane fraction of both fungal strain did not shows significant results against urease enzyme inhibition studies and showed % inhibition less than 50. In conclusion, the crude extract and its fractions exhibited remarkable inhibition against carbonic anhydrase-II.

Keywords: Screlotium rolfsii, Aspergillus flavus, Extract, Urease inhibition, Carbonic anhydrase-II activities. Introduction Zinc metallo-enzymes carbonic anhydrases (CAs, EC 4.2.1.1) are present in a variety of organisms [1, 2]. Many physiological processes, such as homeostasis in pH, transportation of carbon dioxide from lungs to the metabolizing tissue and some other physiological reactions are catalyzed by these enzymes [3, 4]. Based on protein structure and amino acid sequence CAs are categorized into five distinct classes, α, β, γ, δ and ζ, [5]. In mammals, there are 16 different CA iso-zymes or CA related proteins (CARP) are present. Two important tumorassociated membrane carbonic anhydrase iso-zymes CA-IX and CA-XII are documented in literature. The role of CA-IX in tumour physiology has been reported, such as the control of tumour pH and the influence in the cell microenvironment that promote cell proliferation, invasion, and metastasis [6, 7]. There are many effective inhibitors of CAs, most of classical sulfonamides/sulfamates CA inhibitors and their derivatives, such as ethoxzolamide (EZA), acetazolamide (AZA), do not have a good selectivity of CA-IX, over the sulfonamide-avid against isozyme CA-II [8-10]. Inhibition of CAs iso-zymes by aromatic/heterocyclic sulfonamides has been used clinically for the treatment of a variety of diseases, such as epilepsy, glaucoma, mountain sickness, *

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gastric duodenal ulcers, and congestive heart failure etc [11]. Urease (urea amidohydrolase) is also found in different organism such as fungi, algae, bacteria and plants. Urease catalyses the conversion of urea to ammonia and carbamate during nitrogen metabolism in living organism [12]. Carbamate rapidly decomposes, produce another molecule of ammonia, which causes increase in pH that are negative effect urease activity in human health and agriculture [13, 14]. Medially it is reported that Helicobacter pylori and Proteus mirabilis have high urease activity and are also involved in different animals and human diseases i.e. gastric ulcer, urolithiasis, hepatic encephalopathy, pyelonephritis, urinary catheter encrustation and hepaticcoma [15-17]. The fungi kingdom is one of the most important kingdoms of microorganism in the biosphere [18]. Until now 99,000 species of fungi have been discovered and new species are described at the rate of approximately 1200 per year [19]. Microbial activity is the general term used to indicate the vast range of activities carried out by soil microorganisms. Fungi performing a wide range of function in soil by secreting low molecular weight secondary metabolites. Secondary metabolites are not

Bashir Ahmad et al.,

essential to the common metabolic pathways of the fungi and are often only produced when these fungi are stressed condition. Until now, approximately 50,000 microbial metabolites have been discovered. S. rolfsii and A. flavus are soil borne phytopathogenic fungi cause different diseases in plants. Besides their pathogenicity these fungi also produce a wide range of secondary compound. The present study belongs to evaluate the enzyme inhibition potentials of different fractions of secondary metabolites S. rolfsii and A. flavus. Experimental Soil Samples Collection Soil samples were collected from different localities of district Malakand, Khyber Pakhtunkhwa Pakistan. The soil samples were collected in sterilized bags and transferred to Laboratory. Isolation, Identification and Preservation of Fungi The fungi were identified by morphologically and microscopically at plant pathology department Agricultural University Peshawar Pakistan. By using serial dilution, the samples were inoculated using different selective fungal media. Different fungi were isolated such as Nigrospora spp, Verticillium spp, Aspergillus spp, etc. The S. rolfsii and A. flavus were selected for the present study. Extraction of Crude Metabolites Optimization of different growth parameter such as temperature, pH, incubation period and nutritional requirements were carried out. The fresh fungal strains were cultured for production of secondary metabolites. Czapek Yeast-extract Broth (CYB), were prepared and sterilized at 121 0C for 20 min. The 5 days old cultured were inoculated in each erlenmeyer flask containing media. The flask were incubated, at 25 0C at 150 rpm in shaking incubator. After incubation period, 40% HCl were added to each flask, which helps in separating components of media. After vigorously mixing, equal volume of ethyl acetate was added in each flask. Mycelial biomass was filtered using cheese cloth. The process was repeated three times. Ethyl acetate fraction were separated using separating funnel. Anhydrous sulphate Na2SO4 were added for dehydration of organics layer and then filtered. The ethyl acetate portion were filtered using whatman filter paper. The extract were then concentrated at 45 0C in rotary evaporator. Fractionation The crude EtOAc extract of S. rolfsii and A. flavus were suspended in distilled water and

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partitioned with n-hexane to obtained n-hexane fraction. Then the water is filtered out to get ethyl acetate fraction. Carbonic anhydrase-II inhibition Carbonic anhydrase-II inhibition assay was performed according to ‘standard procedure [20], with slight modification. Total mixture volume was 200 µL in a well contained 140 µL mM HEPES (bioworld: cat#40820000-1) Tris (Invitrogen: cat# 15504-020) buffer of pH 7.4), 20 µL of enzyme (sigma Aldrich, C2624, PCode: 1001584424) (0.1 0.2 mg ̸ mL in deionized water), 20 µL (0.5 mg ̸mL in DMSO) of test extract was mixed and incubated at 25º C for 15 minutes. After incubation pre-read was taken at 400 nm and 20 µL of substrate (4nitrophenyl acetate, sigma Aldrich, N-8130, lot#BCBK4587V) (0.7 mM in methanol) was added and the reaction was run at same condition for 30 minutes and final read was taken at 400 nm by the spectrophotometer (Synergy HT Bio Tek USA microplate reader). Acetazolamide was used as positive control. IC50 values were calculated by EZfit enzyme kinetics software. % Inhibition = 100-(absorbance of test compound ÷ absorbance of control) 100 Urease Inhibition Assay The urease inhibitory activity was performed according to the standard procedure [21] with minor modification. A solution containing 25 µl of Jack bean Urease, 55 µl and 100 mM urea of buffer, were incubated with 5 µL (0.5 mM) of the test compounds for 15 min at 30⁰ C in a 96 well microtiter plate. For the determination of urease inhibition activity, release of ammonia was measured using indophenol method. To each well the alkali reagents (70 µL, 0.5% w/v sodium hydroxide and 0.1% NaOCl) and phenol reagents (45 µL, 1 % w/v phenol and 0.005 % w/v sodium nitroprusside) were added. After 50 minutes a microplate reader ((Synergy HT Bio Tek USA microplate reader) was used to measure the increasing absorbance at 630 nm. The results were processed using Soft-Max Pro software after measuring change in absorbance per minute. The experiments were performed in triplicate at pH 8.2 (0.01 M K2HPO4.3H2O, 1.0 mM EDTA and 0.01 M LiCl2). The standard inhibitor thiourea was used as positive control. The percent inhibition activity was calculated using formula: % Inhibition = 100-(absorbance of test compound ÷ absorbance of control) 100

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Table-1: Carbonic anhydrase-II inhibition of Srelotium rolfsii and Aspergillus flavus. NO 1 2. 3.

Fungi S. rolfsii A. Flavus Acetazolamide

Ethyl acetate fraction % inhibition IC50 [µM] 58.67 45.40 ± 0.75 66.71 59.89 ± 1.65 89.44 18.36 ± 1.14

n-Hexane fraction % inhibition IC50 [µg/mL] 62.54 63.91

52.77 ± 0.81 61.3±1.75

Table-2: Urease inhibition of Srelotium rolfsii and Aspergillus flavus NO. 1 2 3

Fungi S. rolfsii A. flavus Thiourea

Ethyl acetate fraction (% inhibition) 6.58 13.56 98.12

Results and Discussion Over-expression of urease and carbonic anhydrase enzymes leads to a number of disorder. Urease produced by Helicobactor pylori is one of the major causes of the pathogenesis of gastric and peptic ulcers and related cancer. The discovery and development of enzymes inhibitors are very important in order to treat related diseases [22, 23]. The secondary metabolites produced by different fungi perform a wide range of function. These metabolites are used in different industry such as food, agricultural and pharmaceuticals. Some secondary metabolites are used as a shield for fungi [24]. Fungi are most important component of the soil microbial community, typically constituting more of the soil biomass than bacteria. In the present study two fungal strains S. rolfsii and A. flavus were isolated from collected soil samples.

n-hexane fraction(% inhibition) 22.58 28.51 98.12

metabolites, majority of them are phenolic in nature [27, 28]. In vitro phenolic compound against various were investigated. Among these enzyme, phenolic compound were active against urease. In the present research, the fraction of both fungi were not showed good activity against urease enzyme, which may be due to a limited number of phenolic compound. Conclusion It was concluded from the present study that the fraction of the crude metabolites of S. rolfsii and A. flavus have a potential to inhibit the carbonic anhydrase, while the fraction shows insignificant result against Urease. Furthermore, from these results pure compounds of biological relevance can be isolated, which can increase the chemical space of organic compounds. Acknowledgements

These two fungi extracts were evaluated for carbonic anhydrase and urease inhibition activity. Both extract of S. rolfsii and A. flavus showed excellent activity against carbonic anhydrase. Both isolated fractions; ethyl acetate and n-hexane were found active against CAs (58.67 and 66.71 % at 0.2 mg/ml respectively) with IC50 values of (45.40 ± 0.75 and 59.89 ± 1.65 µg/mL respectively). Similarly the n-hexane fraction of both fungi also showed significant result against carbonic anhydrase (62.54 and 63.91% at 0.2 mg/ml respectively) with IC50 values (52.77 and 61.3 ± 1.75 µg/mL respectively) Table-1. The secondary metabolites of pathogenic fungi, candida albicans and Cryptococcus neoformans i.e. oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and acetate showed inhibitory activity against carbonic anhydrase [25]. S. rolfsii also produces oxalate, which work in synergistic process to attack and destroy different plant tissue [26]. It is also evaluated that phenolic compounds inhibited both α-CAs, β-CAs. Lichens (symbiotic relationship between fungi and photosynthetic organism such as algae or Cyanobacteria) possess a wide range of biological functions due to their ability to produce secondary

The authors are grateful to Higher Education Commission of Pakistan for award of research start up grant No 21:619/SRGP/R&D/HEC/2014. Conflicts of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References 1. 2.

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