DETERMINATION OF THE OPTIMUM CONDITIONS FOR UREASE INHIBITTION EXTRACTED FROM SOME LOCAL PLANTS

In the current study, four types of plants commonly used namely Soybean, chickpea, bean, pea were obtained and screened for urease activity, among this plants, chickpea was chosen with maximum enzymatic activity, and it had the highest productivity of urease enzyme (1243 U/mg protein). Also sodium acetate buffer (0.2 M, pH 5.0) was chosen as a best extraction buffer with specific activity 1460 U/mg protein. The optimum extraction ratio represented by 1:8 (w:v) after 15 min, it was given 1988 U/mg protein. As well as four types of plants include garlic, red onion, green onion and cabbage were used to select the optimum plant material that inhibited urease enzyme. Cabbage was chosen, it had the highest inhibition activity of the enzyme (41%). Also tris buffer (0.2 M, pH 9) was selected as a best extraction buffer of plants inhibitor with inhibition activity 80%. The optimum extraction ratio represented by 1:8 (w:v) after 60 min, it was given 86% enzyme inhibition activity. Keyword: chickpea, cabbage, plant inhibitor, urease enzyme, optimization ةيقارعلا ةيعارزلا مولعلا ةلجم 2021 : 52 ) 4 :) 802 809 نيسح نورخآو ل ىلثملا فورظلا ديدحت ت بث ي زييرويلا ط صلختسملا هيلحملا تاتابنلا ضعب نم نيسح ميحرا رحس فراع دومحم فلخ يرخف ليسا حماس حلاص رئاث هورم دعاسم سردم دعاسم ذاتسا ثحاب ثحاب ةيئايحلاا تاينقتلا مسق قا رعلا/دادغب ةعماج/مولعلا هيلك صلختسملا تخا مت ةسا ردلا هذه يف اب تابنلا رايتخا ضرغل ةفلتخم ةيتابن عاونأ هعبرا ر ا ميزنلا ردصمك لثملاا ،ايوصلا لوف( تلمش زييرويل ةيلاعف ىلعا كلتما ثيح ،ةبختنملا تاتابنلا تنيب نم لثملأا وه صمحلا تابن نا جئاتنلا تنيب .)ايلا زبلاو ءلاقابلا ،صمحلا ( هيميزنا 1243 ةدحو / نيتورب مغلم ( زيكرتب مويدوصلا تلاخ رفب مادختسا كلذك ، 0.2 ةضومح ةلادبو يرلاوم 5 ئراد لضفأك ةيلاعفب صلاختسلال 1460 ةدحو / ةبسن نا جئاتنلا ترهظأ امك .نيتورب مغلم 1:8 صلاختسلا ةبسن لضفأ يه )مجح :نزو( نم ميزنلاا بختنملا تابنلا دعب 15 ىلا هيميزنلاا ةيلاعفلا تلصو ذا ةقيقد 1988 ةدحو / ضعب ريثأت اضيا سرد .نيتورب مغلم ( ميزنلال يطيبثت ريثات ربكا كلتما هناهللا تابن نا جئاتنلا تنيب ثيح زيرويلا ميزنا طيبثت يف هيلحملا تاتابنلا 41 هنراقم )% و ضيبلاا لصبلا( تاتابنلا هيقبب ا لصبلا يزنلاا طبثملل ىلثملا فورظلا هسا رد دنعو )هناهللاو موثلاو رمحلاا ىلعا نا دجو يم تناك هيطيبثت هيلاعف 86 زيكرتب يدعاقلا سرتلا رفب مادختسا دنع % 0.2 هضومح هلادبو يرلاوم 9 نب صلاختسا هبس 1:8 .صلاختسلاا نم ةدحاو هعاس دعب ،ةناهل ، صمح :هيحاتفم تاملك ىلثم فورظ ،ةيتابن تاطبثم Received:23/6/2020, Accepted:27/9/2020 Iraqi Journal of Agricultural Sciences –2021:52(4):802-809 Hussein & et al. 803 INTRODUCTION Urease (EC 3.5.1.5; urea amidohydrolase) is a nickel-dependent enzyme that catalyzes the hydrolysis of urea to ammonia and carbamate; the latter decomposes spontaneously, producing another molecule of ammonia and carbon dioxide (15). It’s a key enzyme benefiting bacteria H. pylori and Proteus mirabilis through making its persistence possible in the acidic environment of the stomach and as a result, cause gastrointestinal diseases, in particular gastritis, duodenal, peptic ulcer, and gastric cancer (3). Additionally, urease activity leads to other disease like urinary stones, pyelonephritis (24). Urease is produced by a wide variety of plants, fungi, bacteria and invertebrates, enabling them to utilize urea as a nitrogen source (18). Research has indicated that urease of H. pylori and Proteus mirabilis is located in the cytoplasm in freshly prepared cultures and in the outer membrane in older cultures (11). In addition to pathogenicity from these bacteria, evidence indicates that ammonia generated by urease can cause injury to the gastroduodenal mucosa (23). Specific inhibition of urease activity has been proposed as a possible strategy to inhibit this microorganism (20). Many naturally occurring compounds found in dietary and medicinal plants, herbs, and fruit extracts have been shown to possess antimicrobial activities (25). According to the literature, most of the prescribed medicines and antibiotics for the mentioned disease treatment, not only evince adverse effects but also the bacteria grow resistance against (6). Furthermore, application of some compounds and especially synthetic ones for controlling urease function has been banned due to their toxicity and low chemical and physical stability in the natural environment (4). Medicinal plants have long been applied as remedies to cure diseases which nowadays are known as viral infections. This study has focused on monitoring and evaluating the ability of 4 herbs for their possible inhibitory activity against plant urease. And determination the optimum extraction condition for both enzyme and inhibitor from some local plants. MATERIALS AND METHODS Plants: The plants used throughout this study were locally available in the market. Glycine max (Soybean), Cicer arietinum (chickpea), Vicia faba (bean), Pisum sativum (pea) were used as a source of material to screen for urease enzyme activity and Allium sativum (garlic), Allium cepa (red onion), Allium cepa (green onion) and Brassica oleracea var. capitata (Cabbage) were used as a source of material to screen for urease inhibitor activity. Extraction and recovery of urease enzyme One gm of each plant were homogenizing separately with 8 ml of 0.2 M of phosphate buffer pH 7.0, the mixing was done by using mortar for 15min at room temperature. The slurry was centrifuged at 10000 rpm for 10 min and filtered through a whatman No.1 filter paper for removing any cell debris that remains in the preparation (12). The clear supernatant obtained represented the crude extract, and was assayed for urease enzyme. Estimation the standard curve of NH4Cl In order to prepare NH4Cl standard curve for the urease assay, serial concentrations (100500 μM) from stock solution of NH4Cl (0.5 mM) were prepared in triplicate. The standard curve of NH4Cl was plotted between the ammonium chloride concentration (μM) and the corresponding absorbance of standard ammonium chloride at 625 nm as shown in (Fig. 1). Determination urease assay The activity of urease enzyme was measured by modified Berthelot reaction (14) which depend on standard curve of NH4Cl Iraqi Journal of Agricultural Sciences –2021:52(4):802-809 Hussein & et al. 804 throughout liberated of ammonia by the enzyme. All glassware sterilized should be washed with warm dilute hydrochloric acid and rinsed thoroughly with distilled and deionized water. The reaction mixture (11 ml) contained 1 ml of crude enzyme with 10 ml of substrate [5 ml from 0.01 M of reagent A (5gm of phenol and 25 mg of sodium nitropruside) with 5 ml from 0.01 M of reagent B (2.5gm of sodium hydroxide with 8.4 ml of sodium hypochloride)] in 500 ml distilled water for each, for 1 hr. in water bath at 37 °C, reaction was stopped with heating at 80 o C for 5 min. Urease activity was detected by measuring the absorbance increase at 625 nm. Enzymatic activity unit known as the amount of enzyme liberated 1 μmole from ammonia in one minute under optimum conditions as follows: Urease Activity = Ab


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
. It's a key enzyme benefiting bacteria H. pylori and Proteus mirabilis through making its persistence possible in the acidic environment of the stomach and as a result, cause gastrointestinal diseases, in particular gastritis, duodenal, peptic ulcer, and gastric cancer (3). Additionally, urease activity leads to other disease like urinary stones, pyelonephritis (24). Urease is produced by a wide variety of plants, fungi, bacteria and invertebrates, enabling them to utilize urea as a nitrogen source (18). Research has indicated that urease of H. pylori and Proteus mirabilis is located in the cytoplasm in freshly prepared cultures and in the outer membrane in older cultures (11). In addition to pathogenicity from these bacteria, evidence indicates that ammonia generated by urease can cause injury to the gastroduodenal mucosa (23). Specific inhibition of urease activity has been proposed as a possible strategy to inhibit this microorganism (20). Many naturally occurring compounds found in dietary and medicinal plants, herbs, and fruit extracts have been shown to possess antimicrobial activities (25). According to the literature, most of the prescribed medicines and antibiotics for the mentioned disease treatment, not only evince adverse effects but also the bacteria grow resistance against (6). Furthermore, application of some compounds and especially synthetic ones for controlling urease function has been banned due to their toxicity and low chemical and physical stability in the natural environment (4). Medicinal plants have long been applied as remedies to cure diseases which nowadays are known as viral infections. This study has focused on monitoring and evaluating the ability of 4 herbs for their possible inhibitory activity against plant urease. And determination the optimum extraction condition for both enzyme and inhibitor from some local plants.

MATERIALS AND METHODS Plants:
The plants used throughout this study were locally available in the market. Glycine max (Soybean), Cicer arietinum (chickpea), Vicia faba (bean), Pisum sativum (pea) were used as a source of material to screen for urease enzyme activity and Allium sativum (garlic), Allium cepa (red onion), Allium cepa (green onion) and Brassica oleracea var. capitata (Cabbage) were used as a source of material to screen for urease inhibitor activity. Extraction and recovery of urease enzyme One gm of each plant were homogenizing separately with 8 ml of 0.2 M of phosphate buffer pH 7.0, the mixing was done by using mortar for 15min at room temperature. The slurry was centrifuged at 10000 rpm for 10 min and filtered through a whatman No.1 filter paper for removing any cell debris that remains in the preparation (12). The clear supernatant obtained represented the crude extract, and was assayed for urease enzyme.

Estimation the standard curve of NH 4 Cl
In order to prepare NH 4 Cl standard curve for the urease assay, serial concentrations (100-500 µM) from stock solution of NH 4 Cl (0.5 mM) were prepared in triplicate. The standard curve of NH 4 Cl was plotted between the ammonium chloride concentration (µM) and the corresponding absorbance of standard ammonium chloride at 625 nm as shown in (Fig. 1).

Determination urease assay
The activity of urease enzyme was measured by modified Berthelot reaction (14) which depend on standard curve of NH 4 Cl throughout liberated of ammonia by the enzyme. All glassware sterilized should be washed with warm dilute hydrochloric acid and rinsed thoroughly with distilled and deionized water. The reaction mixture (11 ml) contained 1 ml of crude enzyme with 10 ml of substrate [5 ml from 0.01 M of reagent A (5gm of phenol and 25 mg of sodium nitropruside) with 5 ml from 0.01 M of reagent B (2.5gm of sodium hydroxide with 8.4 ml of sodium hypochloride)] in 500 ml distilled water for each, for 1 hr. in water bath at 37 °C, reaction was stopped with heating at 80 o C for 5 min. Urease activity was detected by measuring the absorbance increase at 625 nm. Enzymatic activity unit known as the amount of enzyme liberated 1 μmole from ammonia in one minute under optimum conditions as follows:

Extraction time
In order to determine the optimum extraction time for urease inhibitor, the time course for extraction was (5, 15, 30 and 60) min by mortar, then centrifugation at 10000 rpm for 10 min and filtered through a whatman No.1 filter paper. The clear supernatant obtained represented the crude extract and was assayed for urease inhibitor activity.

RESULTS AND DISCUSSION Optimum conditions for urease extraction
Different bioprocess conditions that effect on urease extraction from some plants were optimized for maximum enzyme production, a large number of factors affect the extraction of urease such as types of plant material, types of buffer, extraction ratio and extraction time etc. Hence, optimization of these conditions helps to reduce extraction cost and to obtain a high yield of urease enzyme.

Type of plant material
The influence of plant type on the enzyme extraction was determined by four types of plants commonly used soybean, chickpea, bean and pea by using 0.2 M of phosphate buffer pH 7.0, it was observed from the results that urease extraction was found to be maximum in chickpea with specific activity of 1243 U/mg, while the specific activity of urease in bean, pea and soybean were 32.6, 505 and 595 U/mg respectively (Fig. 2). El-Hefnawy et al., (5) founded the specific activity of urease extracted from Pisum Sativum L. seeds was reached to 454.5 U/mg protein.

Type of extraction buffer
The specific activity of urease was estimated after extraction using different buffers, and the results were illustrated in (Fig. 3). These results show that sodium acetate buffer (0.2 M, pH 5.0) was best extraction buffer with specific activity 1460 U/mg protein. While other buffer with different pH were given low specific activity. pH effected of enzymatic extraction also activity and stability by the fact that protein structure of an enzyme molecule is influenced by the acidity and alkalinity of the solution because of the differences in ionization state of various amino acid residues through changing the charge state of the solute. If the pH of the solution is such that a particular molecule carries no net electric charge, the solute often has minimal solubility and precipitates out of the solution (19).The pH of enzyme environment affects the activity of the enzyme in several ways. Firstly each enzyme has its own optimum pH, at which the maximum enzyme activity, but the enzyme is stable within certain limits under and above the optimum. Secondly, enzyme stability is influenced by environmental pH, at extremes acidity or alkalinity the enzyme may be denatured. Thirdly, the reaction mixture pH may effects on association of substrate with the enzyme (9).There are many studies that used different buffers with different pH values for urease extraction from different sources, Narjis (21) pointed to extraction of urease from Proteus mirabilis by phosphate buffer (20 Mm, pH 7.5).

Extraction ratio
Five extraction ratio were chosen 1:4, 1:8, 1:12, 1:15 and 1:20 (w:v) to determine the best extraction ratio of urease by using sodium acetate (0.2 M, pH 5.0). The highest specific activity was measured for crude extract at 1:8 ratio, it was reached to 1864 U/mg protein, while other ratio were gave the following specific activities 1089.6, 1759, 1210 and 856.7 U/mg protein respectively (Fig. 4). Variation in native extract ratio can result in a variable amount of herbal material used in an extract. In some instances, variation in the equivalent dry weight of a herb used in herbal preparation. Where a large proportion of extractable material is obtained from a herbal material, the native extract ratio will be low. For example, a low native extract ratio of 2:1 indicates that 50 percent of the extractable matter obtained from the herb is represented in the final extract. However, when only a small amount of extractable material is obtained using a particular extraction profile, the native extract ratio will be high e.g. a native extract ratio of 20:1 indicates that only 5 percent of extractable components are obtained (2).

Extraction time
Four extraction periods were chosen (5,15,30 and 60) min to determine the best extraction time of urease by using sodium acetate (0.2 M, pH 5.0). The highest specific activity was measured for crude extract after 15 min, it was reached to 1988.5 U/mg protein, compared to the lower of specific activity after 5, 30 and 60 min 710, 926 and 900 U/mg protein respectively (Fig. 5). Granick (8) found that the extraction time for plant urease was varies between 10 and 15 minutes.

Optimum conditions for urease inhibitor extraction Type of plant material
Four types of plants, commonly used in this study, namely (garlic, red onion, green onion and cabbage) were obtained and screened for their enzymatic inhibition activity. Herein, urease inhibitory activity of 4 natural extracts were evaluated among which 2 extracts were elucidated as the most potent ones including cabbage and red onion. As it is presented in (fig. 6). Natural therapy has recently absorbed many attentions to itself. Although, herbs have always been applied for the treatment of a vast variety of diseases throughout the history, but the drawbacks of the synthetized medicines, especially the side effects coming along their consumption, has again arisen a significant interest among scientists to more precisely monitor and extract herbal active compounds pharmacological properties from these appropriate sources of active chemicals to be used as templates for designing and/or developing more effective compounds, preferably with less side effects (10). Gastrointestinal disorders, particularly gastritis, duodenal, peptic ulcer, and gastric cancer are mainly caused as a result of H. pylori infection. This bacterium agitates human pathogenic state and causes diseases from which the most common ones are urinary stone formation, peptic ulcer, pyelonephritis, and hepatic coma. H. pylori habitance in the acidic medium of the stomach is highly dependence on the urease enzyme activity. A unique feature of H. pylori infection is its persistence as a result of urease enzyme buffering activity. The enzyme changes the stomach medium to an endurable environment for the bacteria via neutralizing gastric acid through hydrolysis of urea to form carbon dioxide (CO 2 ) and ammonia (NH 3 ) (16).

Type of extraction buffer
The inhibition activity of urease enzyme was estimated after extraction, using different buffer, and the results were illustrated in (Fig  7).The higher urease inhibition value was obtained at acidic and alkalinatic buffering solution with maximum inhibitory effect (80%) at pH 9 using 0.2 M of tris buffer. While in neutral pH the urease inhibitor activity was reached to minimum value (50 and 41) %. Shi with coworkers (22) extracted 12 chines herbs as sources of urease inhibitor by using 95% ethanol or water (400 mL each). Ten grams of different ratios of oregano/cranberry powder mixture using as urease inhibitor were extracted by 90 ml of water to make concentrated stock extracts and stored at 4°C (17). Eighty grams of peat was extracted with 800 ml of alkaline potassium hydroxide (KOH) at a concentration of 0.1 mol L-1, determining a mass/ volume (m/v) ratio of 1:10 for urease inhibitor extraction. The material was left for a two-hour rest and then centrifuged, extraction was performed at room temperature (25-30°C) (7).

Extraction Time
Four extraction periods were chosen (5,15,30 and 60) min to determine the best extraction time on extraction the urease inhibitor by using tris buffer (0.2 M, pH 9). The highest inhibition activity of cabbage was measured in 60 min; it was reached to 86 %, compared to the lower of inhibition activity in 5, 15 and 30 min (68, 70 and 73) % respectively (Fig. 9). Shi with coworkers (22) extracted 12 chines herbs as sources of urease inhibitor by using 95% ethanol or water (400 mL each) by refluxing for 4 h.