Research Article Urease Inhibitory Activity of Aerial...
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Research Article Urease Inhibitory Activity of Aerial Parts of Artemisia scoparia: Exploration in an In Vitro Study Murad Ali Khan, 1 Haroon Khan, 2 Shafiq Ahmad Tariq, 3 and Samreen Pervez 4 1 Department of Chemistry, Kohat University of Science and Technology, Kohat 26000, Pakistan 2 Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan 3 Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25210, Pakistan 4 Department of Pharmacy, University of Peshawar, Peshawar 25210, Pakistan Correspondence should be addressed to Haroon Khan; [email protected] Received 18 October 2013; Revised 30 November 2013; Accepted 17 December 2013; Published 16 January 2014 Academic Editor: Takafumi Ando Copyright © 2014 Murad Ali Khan et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Artimisia scoparia has been used in the treatment of different disorders including ulcers. e current study was therefore designed to investigate the aerial parts of Artemisia scoparia (crude extract, total sterol and flavonoidal contents, and aqueous fraction) for its urease inhibitory potential. e crude of the plant evoked marked attenuation on urease activity, when tested in various concentrations with IC 50 values of 4.06 mg/ml. e inhibitory potential was further augmented in the aqueous fraction (IC 50 : 2.30 mg/ml) of the plant. When the total sterol and flavonoidal contents were challenged against urease, both showed concentration dependent activity; the latter showed maximum potency with IC 50 values of 8.04 and 2.10 mg/ml, respectively. In short, the aerial parts of the plant demonstrated marked antagonism on urease and thus our study validated the traditional use of Artemisia scoparia in the treatment of ulcer. 1. Introduction Urease (urea amidohydrolase) is usually found in different bacteria, fungi, algae, and plants, an enzyme that catalyzes the hydrolysis of urea to ammonia and carbamate, which is the final step of nitrogen metabolism in living organisms [1]. Carbamate rapidly and spontaneously decomposes, yielding a second molecule of ammonia. ese reactions may cause significant increase in pH and are responsible for negative effects of urease activity in human health and agriculture [2, 3]. From the medical viewpoint, infections produced by these bacteria such as Helicobacter pylori and Proteus mirabilis usually have a high urease activity. Urease is central to H. pylori metabolism and virulence, is necessary for its colo- nization of the gastric mucosa, and is a potent immunogen that elicits a vigorous immune response. is enzyme is used for taxonomic identification and for diagnosis and followup aſter treatment and is a vaccine candidate. Urease represents an interesting model for metalloenzyme studies. Before the discovery of H. pylori, humans were thought to produce “gastric urease.” It is now known that the source of this notable protein is this bacterium, which colonizes the gastric mucosa of humans. It contributes in urinary tract and gastrointesti- nal infections, probably augmenting the severity of several pathological conditions like peptic ulcers and stomach cancer as in the case of H. pylori. Ureases are also involved in the development of different human and animal pathogenicity of urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation [4–6]. Being involved 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 of treatment for infections caused by such bacteria. Indeed more effective and potent compounds are required with a whole new level of safety and specificity. In this regard, urease inhibitors have gained tremendous attention over the years which resulted into the discovery of numerous inhibitors [5, 7–11]. Hindawi Publishing Corporation Ulcers Volume 2014, Article ID 184736, 5 pages http://dx.doi.org/10.1155/2014/184736
Transcript of Research Article Urease Inhibitory Activity of Aerial...
Research ArticleUrease Inhibitory Activity of Aerial Parts of Artemisia scoparia:Exploration in an In Vitro Study
Murad Ali Khan,1 Haroon Khan,2 Shafiq Ahmad Tariq,3 and Samreen Pervez4
1 Department of Chemistry, Kohat University of Science and Technology, Kohat 26000, Pakistan2Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan3 Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25210, Pakistan4Department of Pharmacy, University of Peshawar, Peshawar 25210, Pakistan
Correspondence should be addressed to Haroon Khan; [email protected]
Received 18 October 2013; Revised 30 November 2013; Accepted 17 December 2013; Published 16 January 2014
Academic Editor: Takafumi Ando
Copyright © 2014 Murad Ali Khan et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Artimisia scoparia has been used in the treatment of different disorders including ulcers. The current study was therefore designedto investigate the aerial parts of Artemisia scoparia (crude extract, total sterol and flavonoidal contents, and aqueous fraction)for its urease inhibitory potential. The crude of the plant evoked marked attenuation on urease activity, when tested in variousconcentrations with IC
50values of 4.06mg/ml. The inhibitory potential was further augmented in the aqueous fraction (IC
50:
2.30mg/ml) of the plant.When the total sterol and flavonoidal contents were challenged against urease, both showed concentrationdependent activity; the latter showed maximum potency with IC
50values of 8.04 and 2.10mg/ml, respectively. In short, the aerial
parts of the plant demonstratedmarked antagonism on urease and thus our study validated the traditional use ofArtemisia scopariain the treatment of ulcer.
1. Introduction
Urease (urea amidohydrolase) is usually found in differentbacteria, fungi, algae, and plants, an enzyme that catalyzesthe hydrolysis of urea to ammonia and carbamate, which isthe final step of nitrogen metabolism in living organisms [1].Carbamate rapidly and spontaneously decomposes, yieldinga second molecule of ammonia. These reactions may causesignificant increase in pH and are responsible for negativeeffects of urease activity in human health and agriculture[2, 3].
From the medical viewpoint, infections produced bythese bacteria such asHelicobacter pylori andProteusmirabilisusually have a high urease activity. Urease is central to H.pylori metabolism and virulence, is necessary for its colo-nization of the gastric mucosa, and is a potent immunogenthat elicits a vigorous immune response. This enzyme is usedfor taxonomic identification and for diagnosis and followupafter treatment and is a vaccine candidate. Urease representsan interesting model for metalloenzyme studies. Before the
discovery of H. pylori, humans were thought to produce“gastric urease.” It is nowknown that the source of this notableprotein is this bacterium, which colonizes the gastric mucosaof humans. It contributes in urinary tract and gastrointesti-nal infections, probably augmenting the severity of severalpathological conditions like peptic ulcers and stomach canceras in the case of H. pylori. Ureases are also involved in thedevelopment of different human and animal pathogenicity ofurolithiasis, pyelonephritis, hepatic encephalopathy, hepaticcoma, and urinary catheter encrustation [4–6].
Being involved in the pathophysiology ofmultiple humanand animal disorders, targeting urease for treating pathogenicdisorders caused by urease-producing bacteria has alreadyopened a new line of treatment for infections caused by suchbacteria. Indeed more effective and potent compounds arerequired with a whole new level of safety and specificity.In this regard, urease inhibitors have gained tremendousattention over the years which resulted into the discovery ofnumerous inhibitors [5, 7–11].
Hindawi Publishing CorporationUlcersVolume 2014, Article ID 184736, 5 pageshttp://dx.doi.org/10.1155/2014/184736
2 Ulcers
The genus Artemisia L. consists of a approximately 522of species found throughout northern half of the world [12].The aerial parts of the plant have been widely used fortheir hypoglycemic, hypolipidaemic, diuretic, antiulcer andanti-inflammatory activities [13] and antiseptic, antibacterial,cholagogue, diuretic, purgative, and vasodilator properties[14, 15]. The essential oils of A. scoparia showed stronginsecticidal activity against stored-product insects. Simi-larly, the aerial parts of plant exhibited significant anti-malarial, free radical scavenging, and insecticidal activities[16].
Phytochemical analysis of the genus led to the isolationof several coumarins, flavonoids, phenylpropanoids, sterolsand terpenoids (specially sesquiterpenes andmonoterpenes),and their glycosides [16]. Keeping in view the antiulcer useof the plant, the current study was designed to investigateurease inhibitory properties of crudemethanolic extract, totalflavonoidal and sterol contents, and aqueous fraction of aerialparts of the plant in vitro.
2. Materials and Methods
2.1. Plant Material. Fresh plant of Artemisia scoparia wascollected from Parachinar Valley, Pakistan. The taxonomicidentity of the plant was determined by a qualified planttaxonomist at Department of Botany Kohat University ofScience and Technology, Pakistan. The plant was washed 2-3times with running tap water followed by shade-drying. Theplant material was powdered for extraction.
2.2. Chemicals Used. Urea (Sigma-Aldrich), Sodium Nitro-prusside, Phenol Red (BDH Chemicals Ltd, England),Thiourea, Sodium Dihydrogen Phosphate, (Merck, Ger-many), Urease Jack beans (Avonchem Ltd, UK), SodiumHypochlorite (HC Haq Chemicals, Pakistan), and DimethylSulfoxide (UNI-Chem).
2.3. Preparation of Solvent Extraction. 2 kg of the shade-driedpowder of plant materials was soaked separately in methanolfor 10 days, extracted three times at room temperature in thesame solvent, and then filtered.The diluted extracts were con-centrated on the vacuumed rotary evaporator under reducedpressure at a temperature of 46∘C to give a residue (extract),which was further suspended in water and partitioned to getaqueous fraction. All the extracts were concentrated usingrotary flash evaporator. After complete solvent evaporation,each of these extract was weighed and stored in airtightbottles for further use.
2.4. Extraction of Total Flavonoid Contents. Flavonoids con-tent of the crude extract of the aerial parts of A. scoparia wasestimated using our previously reported method [17]. Briefly,10 g was extracted repeatedly with 10mL of the 80% aqueousmethanol at room temperature. The resulting solutions werefiltered through Whatman filter paper No. 42 (125mm). Thefiltered was then transferred into a crucible and evaporatedat water bath and weighted which account for total flavonoidcontents.
2.5. Extraction of Total Sterol Contents. For the determinationof total sterol contents, powdered plant material of aerialparts of the A. scoparia was extracted with methanol 3times and was concentrated. Then it was suspended in 5%methanol and filtered. Aqueous extract was exhaustivelyextracted with hexane [18]. The resulting hexane solubleextract was evaporated and dried which represents the totalsterol contents.
2.6. Urease Assay. Exactly 25𝜇L of enzyme (Jack BeanUrease) solution and 5 𝜇L of test compounds (0.5mMconcentration) were incubated for 15min at 30∘C [9]. Thealiquot was taken after 15min and again incubated with55 𝜇L of buffers containing 100mM urea for 15min at 30∘C.Ammonia production was measured as a urease activity byindophenol method as described earlier [19]. Final volumeswere maintained as 200𝜇L by adding 45 𝜇L phenol reagent(1% w/v phenol and 0.005% w/v sodium nitroprusside) and70 𝜇L of alkali reagent (0.5% w/v NaOH and 0.1% activechloride NaOCl). The increase in absorbance was measuredat 630 nm after 50min at pH 8.2. The results (change inabsorbance per min) were calculated spectrometrically ondifferent concentrations of drugs in the absence and presenceof ascorbic acid. Thiourea was used as the standard inhibitorand percentage inhibitions were calculated as follows:
%Inhibition = 100 − (ODtestwellODcontrol
) × 100. (1)
The IC50
values were calculated using statistical software,GraphPad PRISM 6.
2.7. Statistical Analysis. The resulting data were expressedas the mean ± SEM (𝑛 = 3) in each group. To determinethe differences between groups, one way analysis of variance(ANOVA) was performed (GraphPad PRISM 6, San Diego,CA, USA) using the least significant difference (LSD) test at𝑃 < 0.5 or 𝑃 < 0.01.
3. Results
3.1. Effect of Total Sterol and Flavonoidal Contents. As shownin Figure 1, the crude extract contained significant quantityof both sterols and flavonoidal contents 12.67 and 14.99%,respectively.
3.2. Effect of Crude Extract of A. scoparia on Urease Inhibition.The effect of urease inhibition on the crude extract of theaerial parts of A. scoparia is present in Figure 2. The ureaseinhibition was observed on various concentrations of theextract with maximum antagonism (60.65%) at 10mg/mL.The estimated IC
50value of crude form of the aerial parts of
the plant was 4.06mg/mL (Table 1).
3.3. Effect of Total Sterol Contents of A. scoparia on UreaseInhibition. As shown in Figure 3, the total sterol contentsof A. scoparia provoked concentration dependent ureaseinhibition. The maximum activity (54.13%) was observed
Ulcers 3C
onte
nts (%
)
0
5
10
15
20
Sterols Flavonoids
Figure 1:The total sterols and flavonoidal contents (%) in the crudeextract of Artemisia scoparia.
0 2 4 6 8 10
0
25
50
75
(%of
cont
rol)
Figure 2: The percent effect of crude extract of Artemisia scopariaagainst urease. The resulting data were shown as mean ± SEMof three independent assays. One way one analysis of variance(ANOVA) was carried out for the determination of differencebetween groups. 𝑃 < 0.5 was considered as significant.
Table 1: The estimated IC50 values of tested drugs.
S. No. Drugs IC50 (mg/mL)1 Crude methanolic extract 4.06 ± 0.06
2 Sterols 8.04 ± 0.09
3 Flavonoids 2.10 ± 0.01
4 Aqueous 2.30 ± 0.03
The resulting data were shown as mean ± SEM of three independent assays.
at a dose of 100mg/mL. It had a significant IC50
value of8.04mg/mL (Table 1).
3.4. Effect of Total Flavonoidal Contents of A. scoparia onUrease Inhibition. The results of urease inhibition of total of
(mg/mL)0 2 4 6 8 10
0
25
50
75
(%of
cont
rol)
Figure 3: The percent effect of total sterol contents of Artemisiascoparia against urease. The resulting data were shown as mean ±SEM of three independent assays. One way one analysis of variance(ANOVA) was carried out for the determination of differencebetween groups. 𝑃 < 0.5 was considered as significant.
flavonoidal contents of aerial parts of A. scoparia are shownin Figure 4. It had attenuated urease in a concentrationsdependent manner. The maximum antagonism (86.17%) wasobtained at the concentration of 10mg/mL, while the IC
50
value was calculated as 2.10mg/mL (Table 1).
3.5. Effect of Total Aqueous Fraction of A. scoparia on UreaseInhibition. Urease inhibition caused by the aqueous fractionof A. scoparia is illustrated in Figure 5. It demonstratedmarked attenuation of urease in a concentration dependentmanner with maximum inhibition of 81% at 100mg/mL.Theestimated IC
50value for aqueous was 2.30mg/mL (Table 1).
The standard used in assay, Thiourea, showed dose depen-dent activity with maximum inhibition 91% at 100𝜇g/mL(Figure 6).
4. Discussion
The results of our study showed profound inhibition ofurease (Jack Bean) of the extract, total flavonoidal andsterol contents, and aqueous fraction of A. scoparia in aconcentration dependent manner. The results indicated thatthe total flavonoidal contents were most potent followed byaqueous fraction of the plant. It could, therefore, be assumedthat the flavonoids of the plant were responsible for thaturease inhibitory activity. However, further details on theisolation of active constituents from the plant can explain realchemical background of the current show.
In terms of inhibitory potency, flavonoidal contents werefollowed by the aqueous fraction and crude extract of theplant, respectively. However, the sterol contents were least
4 Ulcers
0 2 4 6 8 10
0
25
50
75
100
(%of
cont
rol)
Figure 4: The percent effect of total flavonoidal contents ofArtemisia scoparia against urease. The resulting data were shownas mean ± SEM of three independent assays. One way one analysisof variance (ANOVA) was carried out for the determination ofdifference between groups. 𝑃 < 0.5 was considered as significant.
0 2 4 6 8 10
0
25
50
75
100
(%of
cont
rol)
Figure 5: The percent effect of aqueous fraction of Artemisiascoparia against urease. The resulting data were shown as mean ±SEM of three independent assays. One way one analysis of variance(ANOVA) was carried out for the determination of differencebetween groups. 𝑃 < 0.5 was considered as significant.
potent in urease inhibition. It reflects that polar constituentsof the plant were responsible for the current urease inhibitoryeffect.
In summary, the aerial parts of A. scoparia possessedpotent urease inhibitory constituents that could be possiblyflavonoids in nature. Thus the study provided scientific
0 20 40 60 80 100
0
25
50
75
100
(%of
cont
rol)
Figure 6: The percent effect of standard of Artemisia scopariaagainst urease. The resulting data were shown as mean ± SEMof three independent assays. One way one analysis of variance(ANOVA) was carried out for the determination of differencebetween groups. 𝑃 < 0.5 was considered as significant.
evidence to the traditional uses of the plant in the treatmentof ulcers.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
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