NMR, Novel Pharmacological and In Silico Docking Studies of Oxyacanthine and Tetrandrine: Bisbenzylisoquinoline Alkaloids Isolated from Berberis glaucocarpa Roots

Urease enzyme is responsible for gastric cancer, peptic ulcer, hepatic coma, and urinary stones in millions of people across the world. So, there is a strong need to develop new and safe antiurease drugs, particularly from natural sources. In search for new and effective drugs from natural sources bioassay-guided fractionation and isolation of Berberis glaucocarpa Stapf roots bark resulted in the isolation and characterization, on the basis of 1D and 2D NMR data, of two bisbenzylisoquinoline alkaloids, oxyacanthine (1) and tetrandrine (2), followed by urease inhibition studies. Crude extract, all the subfractions and the isolated compounds 1 and 2 displayed excellent urease enzyme inhibition properties in vitro. The antiurease nature and possible mode of action for compounds 1 and 2 were verified and explained through their molecular docking studies against jack-bean urease enzyme. Half-maximum inhibitory concentration (IC50) was calculated for compounds 1 and 2. The IC50 value was found to be 6.35 and 5.51 µg/mL for compounds 1 and 2, respectively. Both compounds 1 and 2 have minimal cytotoxicity against THP-1 monocytic cells.


Introduction
e genus Berberis (Berberidaceae) is represented by about 500 species distributed almost all over the world but mainly in Pakistan, India, Japan, China, Central and West Asia, Southeast Asia, Europe, East Africa, and North America [1]. e genus Berberis is known to possess isoquinoline, bisbenzylisoquinoline, protoberberine, aphorphine, benzyl isoquinoline, and protopine alkaloids [2]. e phytochemical screening of the crude extracts of Berberis species have revealed the presence of alkaloids, flavonoids, steroids, glycosides, terpenoids, reducing sugars, and saponins [3]. Recent pharmacological studies have established diverse pharmacological properties for different members of the genus Berberis including antimicrobial, antinociceptive, anti-inflammatory, antihistaminic, anticholinergic, vasodilatory, antipyretic, antibacterial, antifungal, antirheumatic, and alleviating urinary and gastrointestinal discomforts. e presence of protoberberine and bisbenzylisoquinoline alkaloids like berberine, oxyberberine, palmatine, oxyacanthine, berbamine, and tetrandrine are mainly responsible for the diverse pharmacological properties of the genus [4,5].
Urease (urea amidohydrolases) is a major contributor to the pathologies induced by H. pylori. e active sites of urease enzyme are nickel ions (Ni 2+ ) and sulfhydryl groups, and they are responsible for the catalytic effects of the urease enzyme. Urease is responsible for the hydrolysis of urea which produces ammonia which then neutralizes the acid produced in stomach, thus producing a pH environment conducive for the survival and colonization of H. pylori bacterium [6]. Urease causes a variety of human diseases such as peptic and duodenal ulcers and gastric carcinoma. Berberine and palmatine, isoquinoline alkaloids from Berberis, have already shown antiurease properties [7,8]. Bisbenzyl isoquinoline alkaloids have been reported to possess biological properties like antimicrobial, cardiovascular, immunomodulatory, antiblood coagulant, cytotoxic effects, and antituberculosis [9,10]. Tetrandrine and oxyacanthine have been found to have antioxidant, antidiabetic, antimicrobial, anticancer, antihair loss, anti-inflammatory, antiallergic, anticalcium channel blocker, antihypertensive, sleep enhancing, and anticancer effects [11][12][13].
us keeping in mind the broad spectrum of pharmacological properties of Berberis and its bisbenzyl isoquinoline alkaloids, we explored Berberis glaucocarpa crude extract, subfractions, and oxyacanthine (1) and tetrandrine (2) for their in-vitro antiurease enzyme properties against jack-bean urease enzyme and molecular docking studies.

Extraction and Isolation.
Root bark (4 kg) was first dried at room temperature, pulverized, and then extracted with commercial grade methanol. is upon concentration with rotary evaporator yielded a dark brownish-black residue (296 g). e residue was then treated with 5% aqueous HCl solution to afford Fraction A (107 g). e filtrate was then extracted with dichloromethane to afford Fraction B (16 g). e acidic filtrate was then basified with NH 3 to pH 9 and extracted with ethyl acetate to afford Fraction C (59 g). e remainder aqueous solution was termed as Fraction D, which was a mixture of quaternary alkaloids. Fraction A was found to mostly consist of isoquinoline alkaloid berberine and was therefore not pursued further. Fraction B also consists of nonalkaloids and protoberberine alkaloids and hence was not pursued further.
Subfraction 8 CFC (674 mg) was subjected to column chromatography (CC) over silica gel, and the column was eluted with a mixture of chloroform and methanol (90 : 10) to afford 46 minor fraction (8CFC1-8CFC46) which were combined on the basis of comparative thin layer chromatography to 5 major fractions (8CFCM1-8CFCM5).

2.4.
Culture of THP-1 Cells. THP-1 cells were initially cultured in complete RPMI1640 medium. e cells were grown in carbon dioxide incubator in humidified air with 5% CO 2 at 37°C. e cells were added with fresh medium on every third day, and after bulking up, they were harvested for checking the cytotoxicity of compounds 1 and 2 ( Figure 1).

THP-1 Cytotoxicity of Berberis glaucocarpa Stapf.
For the determination of cytotoxicity of the tested alkaloids, THP-1 monocytic cells were used. THP-1 cells were originally obtained from London School of Hygiene and Tropical Medicine. THP-1 cells were maintained in RPMI1640 medium (containing 10% HI-FCS, 2 mM L-glutamine, 1% penicillin, and streptomycin) with 5% CO 2 at 37°C temperature. THP-1 cells with 100 percent viability were centrifuged at 1200 rpm, and the supernatant was removed. Fresh medium was added to the cells, and 50,000 cells/200 µL medium/well were added to flat bottom 96-well plate. For optimization, each compound and the standard (thiourea) were applied in triplicates with four different concentrations (100, 75, 50, and 25 µM).
e plate was then placed in a humidified CO 2 incubator at 37°C. Viability of cells was determined by using the trypan blue exclusion technique after 48 hours via improved neubauer haemocytometer. Viability of cells was determined by trypan blue exclusion analysis before and after treatment with the drugs. An equal volume of 0.4% trypan blue reagent was added to the cells suspension, and the percentage of viable cells was evaluated under dark field microscope. e assay is based on the principle that viable cells are nonpermeable for the dye while the dead cells lost their membrane property and turned blue. Viability was calculated using the following formula: live cell count total cell count × 100.
(1)  Indophenol method was used to determine the urease activity by measuring the production of ammonia [14]. Briefly, 45 μL of each phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. e increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Devices, USA). All reactions were performed in triplicate in a final volume of 200 μL. e results (change in absorbance per min) were processed by using soft Max Pro software (Molecular Devices, USA). e entire assays were performed at pH 6.8 [15]. iourea was used as the standard inhibitor of urease. Percentage inhibitions were calculated by using the following formula: optical density of test well optical density of control well × 100.
(2) 2.7. Docking Simulation. e 3D structure of receptor urease (jack bean) was obtained from protein data bank (PDB) with a four-letter code of 4GY7. e receptor structure energy minimization was carried out by the Swiss pdb viewer v4.1.0 program. e pdb files of compounds 1 and 2 and standard thiourea were prepared by using ChemDraw and Avogadro ' s software. e docking simulation was carried out by using AutoDock Vina [16] and i-GEMDOCK v 2.1 software [17]. Before starting the compounds docking, the method validation of both software was carried out by redocking of the cocrystallized thiourea in receptor structure. AutoDock Vina was connected with PyRex tools. Water molecules were cleaned from the enzyme structure; hydrogens addition and gasteiger charges calculation were carried out. e receptor, compounds 1 and 2, and standard thiourea files (pdb) were uploaded in the PyRex tool. e files were converted into pdbqt format. e grid center was identified by cocrystallized ligand of the receptor with a grid box of center x � 20, y � −55, and z � −20 and size of x � 25Å, y � 25Å, and z � 25Å with an exhaustiveness global search algorithm of 8.
i-GEMDOCK v 2.1 software was also used for docking simulation. e docking study was carried out by setting the software at 70 generations per compound and the population size of 200 random individuals. e best docking conformations were carried out twice implemented by the genetic algorithm. e receptor urease binding pocket was recognized with already cocrystallized metal ions at a distance of 12Å. e scoring function of i-GEMDOCK is composed of fitness � vdW + H-bond + Elec. e terms vdW, H-bond, and Elec. stands for van der Waal energy, hydrogen bonding energy, and electrostatistic energy, respectively. e predicted docked poses of compounds 1 and 2 against urease enzyme were analyzed by Discovery studio visualizer version 4.0 PyMOL version 1.7.2 and LIGPLOT+ version v.1.4.5 software.

Urease Enzyme Inhibition Effects of Alkaloids 1-2.
e urease enzyme inhibition effects of the extract, subfractions, and isolated compounds 1 and 2 are shown in

Docking Studies.
Molecular docking studies of urease enzyme were carried out to calculate the inhibiting potency of compounds 1 and 2 by comparison with the standard thiourea. Docking studies were carried out by using two docking software (AutoDock Vina and i-GEMDOCK). Generally, if a compound displays lesser docking score then that compound has good activity.

Discussion
e current study deals with the chromatographic isolation and spectroscopic characterization of bisbenzylisoquinoline alkaloids 1 and 2 followed by their urease inhibitory effect in vitro. Urease (urea amidohydrolase) is typically observed in different bacteria fungi, algae, and plants. Urease catalyzes the hydrolysis of urea to ammonia and carbamate, which is the final step of nitrogen metabolism in living organisms [20]. Carbamate rapidly and spontaneously decomposes to yield a second molecule of ammonia. ese reactions may cause significant increase in the pH and are responsible for negative effects of urease activity in human health and agriculture [21,22]. Infections induced by bacteria such as Helicobacter pylori and Proteus mirabilis usually have a high urease activity. Urease is vital to H. pylori metabolism and virulence. It is necessary for its colonization 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 follow-up after treatment and is a vaccine candidate. Urease represents an interesting model for metalloenzyme studies. H. pylori contributes in the urinary tract and gastrointestinal infections and probably augments the severity of several pathological conditions like peptic ulcers and stomach cancer. Ureases are also involved in the development of urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation [23,24]. In this regards, targeting urease for treating pathogenic disorders caused by it may open a new line of treatment for infections caused by urease-producing bacteria. e evaluation of oxyacanthine and tetrandrine for their antiurease was inspired from the use of Berberis and its isoquinoline alkaloids, namely, berberine and palmatine against H. pylori. e two tested bisbenzyl isoquinoline alkaloids displayed good antiurease properties like berberine and palmatine. Both compounds 1 and 2 displayed better antiurease enzyme inhibition properties than Berberis glaucocarpa crude extracts and subfractions which may possibly be due their pure nature as compared to the crude extracts and subfractions.
is may be because of the fact that crude extract and subfractions are usually a mixture of different classes of compounds or mixture of different compounds of the same class, and in the mixture form, different compounds may cause to block a certain pharmacological activity of each other. While on the contrary, the antiurease properties of both compounds 1 and 2 were found to be almost similar to each other, which may be due the fact that both of them were pure and are bisbenzyl isoquinoline alkaloids in nature which makes them natural products with broad spectrum of phamacological properties. e minimal cytotoxic values of crude extracts, subfractions, and compounds 1 and 2 against THP-1 monocytic cells are due to the fact that they are natural products (alkaloids) in nature and that is why they are almost perfectly safe for in-vivo use.
is analysis of urease enzyme with compounds 1 and 2 is based on the hydrogen bonding and hydrophobic interactions. Biologically, active compounds have the property of making hydrogen bonding and hydrophobic interactions with the enzyme binding pocket. Both compounds 1 and 2 displayed better docking results than the standard thiourea. e predicted docking poses of compounds 1 and 2, standard thiourea, and cocrystallized Ni atoms of the receptor    and their superimposition are presented in Figure 3. e docking score (Table 4) of compound 1 shows that the binding energy is −6.8 kcal/mol (Autodock vina score) while the total energy is −104 kcal/mol (i-GEMDOCK score). ese interaction energies are lower than those of the standard thiourea. Compound 1 interaction analysis ( Figure 4) revealed that there is only one residue Arg438 involved in the hydrogen bond interaction with a distance of 2.36Å, while seventeen hydrophobic contacts were observed from the residues His406, His408, Ile410, r437, Ala439, r440, r441, His491, His518, Tyr543, His544, Gly549, Meth587, Asp632, Gln634, Ala435, and Meth636. Compound 2 does not show hydrogen bonding interactions, but the residues of active site of urease enzyme were found to be involved in hydrophobic interactions (Figure 4). ese residues include His406, His408, Arg438, Ala439, r441, His491, Glu492, Asp493, Gly549, Gly550, Gly551, Asp632, Ala635, and Meth636. In short, it can be concluded that the docking study and in-vitro results for compounds 1 and 2 were found to be completely supporting their antiurease nature.
e results of our study showed profound inhibition of urease (jack bean) in concentration-dependent manner. erefore, our study provided the scientific basis for the traditional uses of Berberis as antiulcer.

Conflicts of Interest
e authors declare that they have no conflicts of interest.