Kinetics of urease inhibition by different fractions of Cassia fistula

SAJB-02086; No of Pages 6 South African Journal of Botany xxx (2018) xxx–xxx

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Kinetics of urease inhibition by different fractions of Cassia fistula N. Sikri a, S. Dhanda b, S. Dalal a,⁎ a b

Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India

a r t i c l e

i n f o

Article history: Received 9 January 2018 Received in revised form 13 April 2018 Accepted 5 July 2018 Available online xxxx Edited by KRR Rengasamy Keywords: Cassia fistula Urease Line-weaver Burk plot Inhibition kinetics Mome-inositol

a b s t r a c t Urease serves as the basis for Helicobacter pylori infection of stomach as well as urinary tract infection by Proteus, Klebsiella, Pseudomonas and Staphylococus species. Ureolytic activity by these pathogens causes various gastric and urinary diseases. Urease inhibitors can be used as potential approach for treatment of these infections. Cassia fistula, an ayurvedic medicinal plant also named as Aragvadha (a disease killer), was explored as source of urease inhibitors. In the present study, we evaluated the five different extracts viz. aqueous, methanol, hexane, chloroform and ethyl acetate for their urease inhibition potential. Except chloroform extract, all four samples exhibited more than 50% urease inhibitory activity. The IC50 of ethyl acetate sample was 0.27 mg/ml lower than standard thiourea. The inhibition kinetics of all four samples was studied. Line weaver Burk plot and Dixon plots revealed the ethyl acetate sub fraction as most potent inhibitor as it exhibited competitive mode of inhibition with Ki of 280 μg/ml. All the other samples exhibited mixed and uncompetitive mode of inhibition. Further characterization of bioactive compounds in the ethyl acetate sample was performed using GC-MS. The analysis identified 26 compounds in the sample. Mome-inositol, a derived sugar was found to be major compound (80.83%). From the present observation it can be concluded that C. fistula plant needs to be explored further for identifying the lead molecule to treat various ureolysis based diseases. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction The global prevalence of Helicobacter pylori infection indicates more than half the world's population at target (Hooi et al., 2017). The infection induces gastric inflammation and increases the risk of duodenal and gastric ulcers, gastric adenocarcinoma and gastric lymphoma (Algood and Cover, 2006). The virulence factor for this human pathogen is urease, present in cytoplasm or bound on the surface of microorganism. The urease released in human gut at a concentration of 3 mM cause ureolysis, which is accompanied by the release of ammonia. This increases the pH of medium and further supports survival of H. pylori. The human urine contains approximately 0.5 mM urea, the ureolytic activity by species of Proteus, Klebsiella, Pseudomonas and Staphylococus acts as an important virulence factor in urinary tract infection. Urease induced alkalization of urine leads to precipitation of available polyvalent cations and anions producing struvite (NH4MgPO4·6H20) and carbonate apatite (Ca10(PO4)6Co3, the major components of urinary stones (Burne and Chen, 2000). The elevated levels of ammonia are toxic and induce inflammation in fibroblasts, uroepithelium and immune cells. Such condition leads to serious diseases, like urinary stones, urolithiasis, pyelonephritis and hepatic encephalopathy etc. The conventional line of therapy for the above mentioned diseases is adversely affected by ⁎ Corresponding author at: Department of Biotechnology, Kurukshetra University Kurukshetra, Haryana, India. E-mail address: [email protected] (S. Dalal).

development of resistance to various antimicrobial agents. The urease inhibitors might be effective therapeutic agents for treatment of diseases caused by urease dependent pathogenic microorganisms. Some urease inhibitors viz. phosphoroamidates, hydroxamic acid derivatives and imidazoles, are widely used clinically. Though they were found to be effective, but usually cause toxicity and also have less hydrolytic stability. Some studies exhibited severe side effects, such as teratogenicity, psychoneurological and musculo-integumentary symptoms (Ibrar et al., 2013). Keeping aforesaid points, efforts have been made to identify more effective and specific urease inhibitors, identified with application of natural sources. Due to chemical diversity, natural materials have always been widely recognized as potential chemical resources in drug screening. Therefore, urease inhibitors from a reputed gastrointestinal curing ayurvedic plant, “Aragvadha” (disease killer), the Cassia fistula (Amaltas) were studied for urease inhibitory studies. C. fistula belongs to the family Fabaceae and is native to the south-east Asia and also found in upper regions of Himalayas. It is traditionally used against the various diseases like skin diseases, diabetes, leucoderma, constipation, fever and intestinal disorders (Alper, 1993). The extracts of various parts of this plant exhibited wide range of beneficial activities viz. ethanol extract (80%) of inflorescence of C. fistula had antioxidant Goldson et al. (2016), antiulcer (Karthikeyan and Gobianand, 2010), ethyl acetate extract of fruit pulp/seeds exhibited anticancer Irshad et al. (2014) and ethanol extract of roots exhibited anti-diabetic activity (Balraj et al., 2016). C. fistula contains various bioactive compounds

https://doi.org/10.1016/j.sajb.2018.07.016 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Sikri, N., et al., Kinetics of urease inhibition by different fractions of Cassia fistula, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.07.016

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N. Sikri et al. / South African Journal of Botany xxx (2018) xxx–xxx

2. Materials and methods

Table 1 The IC50 values by percent inhibition of urease to the different samples. Samples

% inhibition at 0.50 mg/ml

IC50 ± SD (mg/ml)

Aqueous extract Methanol extract Hexane sub fraction Chloroform sub fraction Ethyl acetate sub fraction Thiourea (standard)

62.46 ± 0.91 70.35 ± 0.04 58.92 ± 0.02 35.43 ± 1.73 76.08 ± 0.30 71.47 ± 0.08

0.54 ± 0.57 0.38 ± 0.94 0.70 ± 0.68 N.A. 0.27 ± 0.18 0.34 ± 0.23

2.1. Chemicals Urease (Type IX from Canavalia ensiformis (Jack Bean), Specific activity: 50,000–100,000 units/g solid) was purchased from Sigma Aldrich, of which one unit liberated 1.0 mol of NH3 from urea per minute at pH 7.0 at 25 °C. One unit is equivalent to 1.0 I.U. Other chemicals like urea, thiourea and phenol were obtained from Hi-Media. All reagents were of analytical grade.

like phenolic metabolites, flavonoids like rotenoids, pterocarpans, two anthraquinone derivatives:1,4 dihydroxy 2 metylanthraquinone and 6,7,11 trihydroxynaphthacene 5,12 dione along with β sitosterol Koushik et al. (2015) as well as Amentoflavone (biflavonoid) (Srividhya et al., 2017). These bioactive compounds exhibit the antioxidant and anticancer activity. The phenolic metabolites anthraquinone, flavonoids and favan 3 ol derivatives also contribute to the antioxidant property of the plant (Bahorun et al., 2005). There is no report about kinetics of urease inhibition by any extracts/fractions of C. fistula. Hence, the aim of the present study was to investigate bioactive molecule present in C. fistula that acts as potential competitive inhibitor and then further be explored as therapeutic lead.

2.2. Plant material and preparation of extract Leaves of the C. fistula were collected from campus of Kurukshetra University in themonth of October (29° 57′ 31.353″ N, 76° 48′5 2.128″ E; mean temperature, 24 °C). Thematerial was identified by Prof. B.D. Vashistha at Department of Botany, Kurukshetra University, Kurukshetra. A voucher specimen (Herbarium/BOT.KU/BIOTECH-1-2017) of the plant was deposited at herbarium, Department of Botany, Kurukshetra University, Kurukshetra. The leaves were washed under tap water followed by distilled water, shade dried for 7 days. The dried leaves were crushed and then milled to a coarse powder using mechanical grinder and the powder was stored till the further use.

3.5

(a)

3

1/V(1/IU)

2.5 2 Control

1.5 0.5

1

0.25

0.5 0 -10

-5

0

5

10

-0.5

15

20

25

1/[S](mM)

2.5

(b)

1/V(1/IU)

2 1.5

Control

1 0.5 0.25

0.5

0 -5

0 -0.5

5

10

15

20

25

1/[S](mM)

Fig. 1. The Lineweaver-Burk plots of kinetic analysis of urease at three different concentrations of plant extracts (0.00 mg/ml, 0.25 mg/ml, 0.50 mg/ml) in aqueous extract (a), methanol extract (b), hexane fraction (c) and ethyl acetate fraction (d).

Please cite this article as: Sikri, N., et al., Kinetics of urease inhibition by different fractions of Cassia fistula, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.07.016

N. Sikri et al. / South African Journal of Botany xxx (2018) xxx–xxx

3

1.2

(c) 1

1/V(1/IU)

0.8 0.6

Control 0.5

0.4 0.25

0.2 0 -5

0

5

10

15

20

25

1/[S](mM)

2.5

(d)

1/V(1/IU)

2

1.5 control

1

0.5mM 0.25mM

0.5

0 -5

0

5

10

15

20

25

1/[S](mM)

Fig. 1 (continued).

The powder of dried leaves (10 g) of C. fistula was separately soaked in distilled water and 99% methanol (100 ml each) in a reagent bottle covered with a lid at 37 °C for 24 h. Each soaked powder was packed into soxhlet column for 48 h. The water and methanol extracts were evaporated and concentrated to dryness using the rotatory evaporator at 50 °C. 4.2 g of methanol crude extract was dissolved in methanol– water (5:5, v/v), followed by liquid-liquid fractionation using n hexane, chloroform and ethyl acetate respectively. The fractions were dried and stored at −4 °C for further use. 2.3. Measurement of urease inhibitory activity Reaction mixture comprising 1.2 ml of phosphate buffer solution (10 mM potassium phosphate, 10 mM lithium chloride and 1 mM ethylene diamine tetra acetic acid, pH 8.2 at 37 °C), 0.2 ml (1 mg/5 ml) of urease enzyme solution and 0.1 ml of test compound was subjected to 5 min incubation. 0.5 ml (400 μM) of substrate was added. After 20 min. of incubation at 27 °C, 1 ml of solution A (contained 0.5 g phenol and 2.5 mg of sodium nitroprusside in 50 ml of distilled water) and 1 ml of solution B (contained of 250 mg sodium hydroxide and 820 μl of sodium hypochlorite 5% in 50 ml of distilled water) was added. Urease activity was determined by measuring the ammonia released during the reaction at 640 nm, modified method described by Weatherburn (1967). Thiourea was taken as standard inhibitor. I% ¼ 100 − ðT=C  100Þ

where I (%) is the inhibition of the enzyme, T (test) is the absorbance of the tested sample in the presence of enzyme C (control) is the absorbance of the solvent in the presence of enzyme.

2.4. Statistical analysis All the assays were done in triplicates to test the reproducibility. The results are presented as mean ± S.E.M. SPSS 15.0 was used for statistical analysis. The values of p b 0.05 were considered statistically significant. Correlations among data obtained were calculated using Pearson's coefficient (r).

2.5. Determination of kinetics parameters The concentration of sample that inhibited the hydrolysis of substrates by 50% (IC50) was determined by monitoring the inhibition effect of various concentrations of sample in the assay. Line weaver Burk plots of 1/V versus 1/[S] were used to determine the type of enzyme inhibition. Urease inhibition was measured by varying the concentration of urea in the presence of different concentrations of extracts or fractions. Inhibitory constants (Ki) were determined as the intersection on the x-axis of the plots of 1/V max approx. vs. different concentrations of sample from the Dixon plots. All experiments were conducted in triplicate.

Please cite this article as: Sikri, N., et al., Kinetics of urease inhibition by different fractions of Cassia fistula, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.07.016

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2.6. GC–MS analysis GC–MS technique was used for identifying bioactive compounds in the urease inhibitory sample of C. fistula Ethyl acetate extracts. The sample was analyzed using Shimadzu Mass Spectrometer-QP 2010 Plus equipped with a split injector and a PE Auto system XL gas

chromatograph interfaced with a Turbo-mass spectrometric mass selective detector system. The sample (1 μl) was injected in GC–MS and MS was operated in EI mode (70 eV) with helium as carrier gas at a flow rate of 1.21 ml/min. The analytical column viz. Rtx-5 capillary column (length − 60 m × 0.25 mm i.d., 0.25 μm film thickness) was used for separation of compounds. The column head pressure was

Sample Information Analyzed by Analyzed Sample Type Sample Name Method File

: $Admn.$ : 10/26/2017 11:03:56 PM : $Organic$ :1 : D:\GCMS Method\Method\Extract.qgm

Chromatogram D:\GCMS DATA\GC-MS Data\KUK\Neha\1-.qgd

43.354 43.808

41.538

42.607

40.467

38.929

38.210

37.770

30.382

27.223

26.625

28.340

TIC*1.00

26. 904

25.827

24.60025.028

20.142

18.734

39.357

37.024

34.520 34.927

35.793

33.195 31.816

2 3 . 4 9 6

4,000,000

20.0

30.0

40.0

47.0 min

Fig. 2. GC–MS chromatogram for ethyl acetate extract of C. fistula leaves.

Please cite this article as: Sikri, N., et al., Kinetics of urease inhibition by different fractions of Cassia fistula, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.07.016

N. Sikri et al. / South African Journal of Botany xxx (2018) xxx–xxx Table 2 Mode of inhibition and Ki values of different samples. Sample

Mode of inhibition

Ki values

Aqueous extract Methanol extract Hexane fraction Ethyl acetate fraction

Uncompetitive inhibition Mixed inhibition Uncompetitive inhibition Competitive inhibition

410 μg/ml 350 μg/ml 520 μg/ml 280 μg/ml

adjusted to 73.3 kPa. Column temperature was programmed from 60 °C to 300 °C at withhold time 2 and 28 min respectively. A solvent cut time of 8 min was adjusted. The injector temperature was set at 270 °C and GC–MS interface was maintained at 280 °C. The MS was operated in the ACQ mode scanning from m/z 40 to 700. In the full scan mode, electron ionization (EI) mass spectra in the range of 40–700 (m/z) was recorded at electron energy of 70 eV. Compounds were identified by comparing mass spectra with library of the National Institute of Standard and Technology (NIST), USA/Wiley.

5

inhibitory potential of methanol extract of leaves of Acacia nilotica, another Fabaceae family plant (Bai et al., 2014). Recent report, by Khan et al. (2017) has scientifically validated the traditional application of C. fistula in gastrointestinal disorders. 3.2. Urease kinetic studies Based upon % inhibition and IC50, the samples exhibiting strong urease inhibition were studied to understand the inhibitory mechanism and to characterize the inhibition process. Determination of the inhibition type is important in understanding the mechanism of inhibition. The potential of these compounds to inhibit urease was determined by the Line weaver-Burk kinetic analysis technique. The Lineweaver– Burk plot of 1/V versus 1/[S] in the presence of different concentrations of samples resulted in a series of straight lines as shown in Fig. 1. The Ki was calculated using Lineweaver–Burk plot (1/V vs. 1/[S]) at different concentrations of plant extracts. 3.3. GC–MS analysis

3. Results and discussion 3.1. In vitro urease inhibition Aqueous and methanol extracts of C. fistula were tested for inhibitory potential against urease. The methanol extract showed a significant activity of more than 70%, close to standard. So sub fractions were generated from methanol extract, as already detailed. The highest inhibitory potential was shown by ethyl acetate fraction of C. fistula, 76.08 ± 0.30%. This was higher than the standard thiourea. Inhibition potential was also compared on the basis of their IC50 values. Ethyl acetate fractions exhibited highest inhibitory potential with lowest IC50 value of 0.27 mg/ml (Table 1). Various studies have revealed that plants belonging to Fabaceae family exhibits high efficacy as urease inhibitors. Polyphenols from the bark of Acacia decurrens, a plant of Fabaceae family is reported to control the urease activity in soil (Fernando and Roberts, 1976). Another report by Suescun et al. (2012) showed reduction in urease activity in soil by ethanolic extract of root, bark and water extract of leaves of Acacia caven. Previous work from our laboratory identified significant urease

GC–MS analysis of the ethyl acetate fraction has led to identification of twenty-six compounds by comparison of their retention time and molecular weight pattern with those stored on GC–MS computer library (Fig. 2, Table 3). The main constituent identified was a derived sugar named Mome-inositol. A number of alkanes have also been identified; they were Pentacosane, n-Tetracontane, Dotriacontane and Heneicosane. Phytosterol named Stigmasterol was present in traces. Literature reports validate the presence of Mome-Inositol in whole plant ethanolic extract of C. fistula (Kulkarni et al., 2015). The various biological activities of Mome-inositol are anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic, sweetener (Kumar et al., 2012). In the previous studies, the different parts of Cassia fistula were also reported to contain secondary metabolites, namely flavan 3 ol derivatives epiafzelechin and epicatechin (Kashiwada et al., 1996). Out of the twenty eight tropical trees of the CASSIA species section FISTULA, six Indian species contain kaempferol and mixture of anthraquinones (rhein, chrysophanol and physcion) (Mahesh et al., 1984). Urease inhibition has been significantly attributed in alleviating various stomach and kidney ailments. Combining already established

Table 3 GC–MS spectral analysis of Ethyl acetate fraction of C. fistula leaves. S. no.

RT

AREA%

Name

Molecular formula

Molecular weight

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

18.734 20.142 23.496 24.600 25.028 25.827 26.625 26.904 27.223 28.340 30.382 31.816 33.195 34.520 34.927 35.793 37.024 37.770 38.210 38.929 39.357 40.467 41.538 42.607 43.354 43.808

1.46 0.17 80.83 0.08 0.31 0.50 0.43 0.09 0.09 0.05 0.57 1.35 2.24 2.53 0.29 2.41 1.94 0.14 1.49 0.10 0.98 0.75 0.63 0.30 0.15 0.12

1,6 Anhydro .beta. D glucopyranose 1,2 Diethylcyclooctane Mome inositol 1,3 Butadiene 1,2 Benzenedicarboxylic acid, bis(2 methylpropyl) ester 1,2 Benzenedicarboxylic acid, bis(2 methylpropyl) ester Dibutyl phthalate 1,2 Benzenedicarboxylic acid, bis(2 methoxyethyl) ester 1 Octadecanol 1,1,1,3,5,7,7,7 Octamethyl 3,5 bis(trimethylsiloxy)tetrasiloxane n Heneicosane n Heneicosane n Pentacosane n Pentacosane 1,2 Benzenedicarboxylic acid n Pentacosane n Tetracontane Dotriacontane, 1 iodo n Tetracontane Dotriacontane, 1 iodo Tetracontane Dotriacontane Dotriacontane Dotriacontane Stigmasterol Octacosane, 1 iodo

C6H10O5 C12H24 C7H14O6 C20H38 C16H22O4 C16H22O4 C16H22O4 C14H18O6 C18H38O C14H42O5Si6 C21H44 C21H44 C25H52 C25H52 C14H18O6 C25H52 C40H82 C32H65I C40H82 C32H65I C40H82 C32H66 C32H66 C32H66 C29H48O C28H57I

162 168 194 278 278 278 278 282 270 458 296 296 352 352 282 352 562 576 562 576 562 450 450 450 412 528

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therapy with urease inhibitors is of high interest to researchers to treat both H. pylori and urinal tract infections. Several herbal extracts as sources of polyphenolic urease inhibitors have been considered to be complementary as compared to first choice therapeutic approaches but their restrictions are reduced due to inappropriate pharmacokinetic profiles. Large number of studies has been done for traditional use of plants for the treatment of ulcers, where urease inhibitory activity studies has only been attested by IC50 and percent inhibition. Ki and the type of inhibition caused by different samples was measured at different concentrations of studied samples and results are summarized in Table 2. It is evident from Line weaver Burk plot that ethyl acetate sample competitively inhibited the enzyme urease with lowest Ki of 280 μg/ml whereas aqueous and hexane samples caused uncompetitive inhibition. The Line weaver Burk plot for methanol extract at different concentration neither intersects on any axis nor is their slope same, thus suggesting to be a mixed type of inhibitor. Urease serve as basis for H. pylori and urinary tract infection, so any drug that competitively inhibits this enzyme will have a significant effect on treating these diseases. The exact mechanism by which these samples inhibit the enzyme urease needs to be elucidated and principal active factor inhibiting the enzyme is to be identified. The GC–MS analysis of the ethyl acetate fraction of C. fistula shows the presence of 26 bioactive compounds and it seems that Mome -inositol is the key factor responsible for the urease inhibition or if different components work in concerted manner is a subject to study. The most plausible explanation is that ethyl acetate sub fraction components are eliminating the binding of substrate to urease. Identification of active and inhibitory component will prove promising for therapeutic use. Uncompetitive inhibition is not desirable while developing the drugs because rate of isolated enzymes in vitro is determined by the concentration chosen by the experimenter and increase in concentration of substrate. Mixed inhibition is a mixture of competitive (if inhibitor binds to free enzyme and Km increases) and uncompetitive inhibition (if inhibitor binds to ES-complex and Km decreases) but in both cases reaction rate decrease. Even allosteric inhibitor may result in mixed inhibition. So, the ethyl acetate sub fraction that result in competitive inhibition has to be explored further for identifying the active compound/s for developing a potential inhibitor against the enzyme urease. 4. Conclusion This is the first kinetics analysis of the plant C. fistula with the enzyme urease. Though IC50 values have been calculated to indicate the functional strength of a sample but Ki helps to get an insight about affinity for enzyme and Line weaver Burk plots tells about the type of inhibition. Further the study of structure – activity relationship will help us to develop some better therapeutic agents. Acknowledgement The authors greatly acknowledge the Haryana State Council for Science & Technology (HSCST) for financial support for the project entitled “Urease Inhibitors from Medicinal plants of Haryana in context

to agricultural soil protection and therapy against clinically significant virulence factor”(Endst. No. HSCST/R&D/2015/2383). Declaration of interest The authors of this article have no conflict of interest to declare. References Algood, H.M.S., Cover, T.L., 2006. Helicobacter pylori persistence. An overview of interactions between H. pylori and host immune defenses. Clinical Microbiology Reviews 19, 597–613. Alper, J., 1993. Ulcers as an infectious disease. Science 260, 159–160. Bahorun, T., Neergheen, V.S., Aruoma, O.I., 2005. Phytochemical constituents of Cassia fistula. African Journal of Biotechnology 4, 1530–1540. Bai, S., Bharti, P., Seasotiya, L., Malik, A., Dalal, S., 2014. In vitro screening and evaluation of some Indian medicinal plants for their potential to inhibit Jack bean and bacterial ureases causing urinary infections. Pharmaceutical Biology 326–333. Balraj, S., Indumathy, R., Jayshree, N., Abirami, S.M., 2016. Evaluation of in vitro anti-diabetic activity of various root extract of Cassia fistula L. Imperial Journal of Interdisciplinary Research (IJIR) 2 (2454-1362). Burne, R.A., Chen, Y.M., 2000. Bacterial ureases in infectious diseases. Microbes and Infection 2, 533–542. Fernando, V., Roberts, G.R., 1976. The partial inhibition of soil urease by naturally occurring polyphenols. Plant and Soil 44, 81–86. Goldson, B.A., Reid, R., Warren, D., 2016. Antioxidant activity, total phenolics and fatty acid profile of Delonix regia, Cassia fistula, Spathodea campanulata, Senna siamea and Tibouchina granulosa. Journal of Analytical & Pharmaceutical Research 3, 1–7. Hooi, J.K.Y., Lai, W.Y., Ng, W.K., Suen, M.M.Y., 2017. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology 153, 420–429. Ibrar, A., Khan, I., Abbas, N., 2013. Structurally diversified heterocycles and related privileged scaffolds as potential urease inhibitors: a brief overview. Arch Pharm Chemistry in Life Sciences 346, 423–446. Irshad, M., Mehdi, S.J., Atheer, A., Fatlawi, al., Zafaryab, M., Ali, A., Ahmad, I., Sing, M., Rizvi, M.M.A., 2014. Phytochemical composition of Cassia fistula fruit extracts and its anticancer activity against human cancer cell lines. Journal of Biologically Active Products from Nature 4, 158–170. Karthikeyan, S., Gobianand, K., 2010. Antiulcer activity of ethanol leaf extract of Cassia fistula. Pharmaceutical Biology 48, 869–877. Kashiwada, Y., Toshika, K., Chen, R., Nonaka, G., Nishioka, I., 1996. Tannins and related compounds. XCIII. Occurrence of enantiomeric proanthocyanidins in the Leguminosae plants, Cassia fistula L.; Cassia Javanica L. Chemical & Pharmaceutical Bulletin 38, 888–893. Khan, A.B., Akhtar, N., Khan, H., Mustafa, G., Niazi, R.Z., Menna, F., 2017. Urease inhibitory activity of Hippophae rhamnoids and Cassia fistula. Pakistan Journal of Pharmaceutical Sciences 30, 1779–1781. Koushik, S., Fatima, A.M., Habib, M.A., Syeda, T.A., Shahin, A., 2015. Isolation and characterization of two anthraquinone derivatives from the roots of Cassia fistula Linn. International Journal Pharmaceutical and Phytopharmacological Research 5, 31–34. Kulkarni, A., Govindappa, M., Ramachandra, Y.L., Koka, P., 2015. GC-MS analysis of methanol extract of Cassia fistula and it's in vitro anticancer activity on human prostate cancer cell line. Indo American Journal of Pharmaceutical Research 5, 937–944. Kumar, N.R., Reddy, J.S., Gopikrishna, G., Solomon, K.A., 2012. GC-MS determination of bioactive constituents of Cycas beddomei cones. International Journal of Pharma and Bio Sciences 3, 344–350. Mahesh, V.K., Sharma, R., Singh, R.S., 1984. Anthraquinones and kaempferol from Cassia fistula species. Journal of Natural Products 47, 733–751. Srividhya, M., Hridya, H., Shanthi, V., Ramanathan, K., 2017. Bioactive Amento flavone isolated from Cassia fistula L. leaves exhibits therapeutic efficacy. 3 Biotech 7, 1–5. Suescun, F., Paulino, L., Zagal, E., Ovalle, C., Muñoz, C., 2012. Plant extracts from the Mediterranean zone of Chile potentially affect soil microbial activity related to N transformations: a laboratory experiment. Acta Agriculturae Scandinavica Section B Soil and Plant Science 62, 556–564. Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry 39, 971–974.

Please cite this article as: Sikri, N., et al., Kinetics of urease inhibition by different fractions of Cassia fistula, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.07.016