molecules Article Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus Manoj Kumar 1, Neha Sikri 1, Sulekha Chahal 1, Jitender Sharma 1, Bhavna Sharma 1, Poonam Yadav 2, Monika Bhardwaj 2, Divya Vashishth 2, Pooja Kadyan 2, Sudhir Kumar Kataria 2 and Sunita Dalal 1,* 1 Department of Biotechnology, Kurukshetra University, Kurukshetra 136119, India; [email protected] (M.K.); [email protected] (N.S.); [email protected] (S.C.); [email protected] (J.S.); [email protected] (B.S.) 2 Department of Zoology, Maharshi Dayanand University, Rohtak 124001, India; [email protected] (P.Y.); [email protected] (M.B.); [email protected] (D.V.); [email protected] (P.K.); [email protected] (S.K.K.) * Correspondence: [email protected] Abstract: Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Citation: Kumar, M.; Sikri, N.; Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Chahal, S.; Sharma, J.; Sharma, B.; Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of Yadav, P.; Bhardwaj, M.; Vashishth, D.; C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. Kadyan, P.; Kataria, S.K.; et al. Urease The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC–MS) analysis of hexane Cinnamomum Genus. Molecules 2021, fraction revealed the contribution of various terpenes. The present study targets terpenes as a new 26, 3803. https://doi.org/10.3390/ class of inhibitors that have potential therapeutic value for further development as novel drugs. molecules26133803 Keywords: urease inhibition; Cinnamomum; camphene; cuminaldehyde Academic Editors: Elisa Ovidi and Antonio Tiezzi Received: 22 April 2021 1. Introduction Accepted: 14 June 2021 Enzyme inhibition studies are a significant area of research in pharmaceutical studies, Published: 22 June 2021 as they result in the discovery of drugs for the treatment of various physiological conditions. Urease is a highly efficient catalyst for the hydrolysis of urea with a rate approximately Publisher’s Note: MDPI stays neutral 1014 times the rate of non-catalyzed reaction. Urease has been exhaustively studied and is with regard to jurisdictional claims in considered the basis of biochemistry as it was the first enzyme to be crystallized. The three- published maps and institutional affil- dimensional structure of this Ni2+-dependent hydrolytic enzyme, from various sources has iations. been elucidated [1]. The similarity in amino acid sequence from various origins suggests a common ancestry of urease [2]. Urease activity is widespread among the prokaryotes. The enhanced activity of ure- ase leads to various conditions such as pathological changes in humans infected with Copyright: © 2021 by the authors. Helicobacter pylori, Proteus mirabilis, Ureoplasma urealyticum, Klebsiella pneumonia, Staphylo- Licensee MDPI, Basel, Switzerland. coccus and Salmonella species. H. pylori prevails in 50% of the global population, with or This article is an open access article without disease symptoms. The urease enzyme in cytoplasm and/or on the surface of distributed under the terms and H. pylori is a major virulence factor and is implicated for its high prevalence in the human conditions of the Creative Commons Attribution (CC BY) license (https:// population [3]. H. pylori infection leads to gastric inflammation, duodenal and gastric creativecommons.org/licenses/by/ ulcers, gastric adenocarcinoma and gastric lymphoma. The ureolytic activity of P. mirabilis, 4.0/). U. urealyticum, K. pneumoniae, Staphylococcus and Salmonella species is a major virulence Molecules 2021, 26, 3803. https://doi.org/10.3390/molecules26133803 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 prevalence in the human population [3]. H. pylori infection leads to gastric inflammation, duodenal and gastric ulcers, gastric adenocarcinoma and gastric lymphoma. The ureolytic activity of P. mirabilis, U. urealyticum, K. pneumoniae, Staphylococcus and Salmonella Molecules 2021, 26, 3803 species is a major virulence factor responsible in the pathogenesis of many other2 of 10clinical conditions such as hepatic coma, pyelonephritis and urinary stones [4,5]. The basic mechanism in all these conditions is urea hydrolysis by ureolytic microorganismsfactor responsible in inthe the stomach, pathogenesis gastrointestinal of many other tract clinical (GI) conditionsas well as suchin the as urinary hepatic tract. Here,coma, the pyelonephritis rapid hydrolytic and urinary decomposition stones [4,5]. of urea occurs, producing ammonia and carbamateThe basicin mechanismthe presence in all theseof urease. conditions The is urea carbamate hydrolysis byeventually ureolytic microor-decomposes spontaneouslyganisms in the into stomach, a second gastrointestinal molecule tract of (GI)ammonia as well asand in thebicarbonate urinary tract. (Scheme Here, the 1). The rapid hydrolytic decomposition of urea occurs, producing ammonia and carbamate in the increase in pH favors various human pathogenic bacteria which exploit urease as a presence of urease. The carbamate eventually decomposes spontaneously into a second virulencemolecule factor of ammonia to further and infect bicarbonate and coloni (Schemeze 1the). Thehost. increase Therefore, in pH the favors slowing various down of ureahuman hydrolysis pathogenic may bacteria reduce whichthe ailments, exploit ureaseammonia as a loss virulence and improves factor to furtherurea utilization infect in agriculturaland colonize soils the as host. well. Therefore, Urea has the been slowing universally down of ureaaccepted hydrolysis as an mayinexpensive reduce the protein sourceailments, for ammoniavarious lossruminants and improves and ureanon-rumina utilizationnts. in agriculturalEnhanced urease soils as well.activity Urea by GI microorganismshas been universally in ruminants accepted as fed an inexpensivewith urea proteincauses sourcedamage for to various GI mucosa, ruminants resulting and in impairednon-ruminants. nutrient Enhanced absorption, urease protein activity spill by GIage microorganisms and decreased in ruminantsgrowth performance fed with urea [6]. causes damage to GI mucosa, resulting in impaired nutrient absorption, protein spillage and decreased growth performance [6]. Scheme 1. The carbamate decomposes spontaneously into a second molecule of ammonia Schemeand bicarbonate. 1. The carbamate decomposes spontaneously into a second molecule of ammonia and bicarbonate. Plants of the Lauraceae family have been widely reported as gastro protective [7,8], antiulcerogenic [9], stomach disorder protectant, in treating kidney infection and kidney stonesPlants [10 ].of Traditionally, the Lauraceae the plantsfamily of have this familybeen widely are used reported to treat gastrointestinal as gastro protective spasms [7,8], antiulcerogenicfor digestive disorders [9], stomach such as disorder indigestion, protecta bloatingnt, gas,in treating etc. kidney infection and kidney stones Crude[10]. Traditionally, extracts and constituents the plants fromof this about family thirty are species used ofto Cinnamomumtreat gastrointestinal displayed spasms forsignificant digestive activities,disorders namely such as antibacterial, indigestion, anticancer,bloating gas, antifungal, etc. antiseptic, antiviral, anti-inflammatory,Crude extracts antipyretic,and constituents antioxidant, from chemopreservative, about thirty species cytotoxic, of Cinnamomum anti-diabetic, displayed an- significanttiulcer, immune activities, stimulant, namely etc. Compoundsantibacterial, such anticancer, as Camphene, antifungal, Cuminaldehyde, antiseptic, Eugenol, antiviral, anti-inflammatory,Proanthocyanidins antipyretic, etc., have been antioxidant, reported to chemopreservative, be responsible for these cytotoxic, activities anti-diabetic, [11–13]. The compounds Camphene and Cuminaldehyde exhibit antibacterial and antifungal antiulcer, immune stimulant, etc. Compounds such as Camphene, Cuminaldehyde, properties [14]. Treatment with Camphene exhibited no toxicity in human hepatic cells; Eugenol,also, it hasProanthocyanidins been reported to have etc., antitumorhave been activities reported [15 to]. be Camphene responsible exhibits for these antiulcer activities [11,12,13].activity and The was compounds found to be Ca helpfulmphene in the and expulsion Cuminaldehyde of urolithiasis exhibit [16]. Aantibacterial binational, and antifungaldouble-blind, properties placebo-controlled [14]. Treatment randomized with Camphene clinical trials exhibited study reports no improvementtoxicity in human hepaticin the bloodcells; sugaralso, ofit peoplehas