Tamarixetin 3-O-β-d-Glucopyranoside from Azadirachta indica Leaves

Article pubs.acs.org/jnp

Tamarixetin 3‑O‑β‑D‑Glucopyranoside from Azadirachta indica Leaves: Gastroprotective Role through Inhibition of Matrix Metalloproteinase‑9 Activity in Mice Dharmendra K. Yadav,† Yogesh P. Bharitkar,‡ Abhijit Hazra,‡ Uttam Pal,§ Sugreev Verma,† Sayantan Jana,† Umesh P. Singh,⊥ Nakul C. Maiti,§ Nirup B. Mondal,∥ and Snehasikta Swarnakar*,† †

Cancer Biology and Inflammatory Disorder Division, §Structural Biology and Bioinformatics Division, ⊥Central Instrumentation Division, and ∥Organic and Medicinal Chemistry Division, CSIR−Indian Institute of Chemical Biology, Kolkata 700032, WB, India ‡ Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata 700032, WB, India S Supporting Information *

ABSTRACT: Neem (Azadirachta indica) is a well-known medicinal and insecticidal plant. Although previous studies have reported the antiulcer activity of neem leaf extract, the lead compound is still unidentified. The present study reports tamarixetin 3-O-β-D-glucopyranoside (1) from a methanol extract of neem leaves and its gastroprotective activity in an animal model. Compound 1 showed significant protection against indomethacin-induced gastric ulceration in mice in a dose-dependent manner. Moreover, ex vivo and circular dichroism studies confirmed that 1 inhibited the enzyme matrix metalloproteinase-9 (MMP-9) activity with an IC50 value of ca. 50 μM. Molecular docking and dynamics showed the binding of 1 into the pocket of the active site of MMP-9, forming a coordination complex with the catalytic zinc, thus leading to inhibition of MMP-9 activity. constituents include several flavonoids, phenolic substances, isoprenoids, and polysaccharides,9−11 which are effective as remedies for wounds, skin diseases, and fungal infections.10,12 Neem compounds are also reported to have roles in preventing cancer, diabetes, and microbial infections.10,13,14 Moreover, crude neem leaf extracts were reported to inhibit ulceration in murine models;15 however the bioactive compound and mode of action for the antiulcer response are still unknown. The present study reports the identification of the previously known compound tamarixetin 3-O-β-D-glucopyranoside (1) from a methanol extract of neem leaf, which shows protection against gastric ulceration.

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astric ulcer is a complex multicausal disease that develops by different aggressive factors, including Helicobacter pylori infection, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, alcohol, and stress. The disease affects ∼4% of the population worldwide, and almost 10% of all humans develop ulcers at some point of their life.1 The pathophysiology of gastric ulceration involves mucosal damage, induction of inflammatory signals, increased oxidative stress, and cellular damage.1 Altered responses of a family of proteolytic enzymes, namely, the matrix metalloproteinases (MMPs), are associated with the gastric ulceration process owing to degradation of extracellular matrix (ECM).2 Previous studies have established the ulcer-inducing function of MMP-9 in NSAID- and alcohol-mediated in vivo models.2,3 Especially, the role of MMP-9 in the development of gastric ulceration was found to be important through its involvement in ECM degradation for mucosal damage, cellular remodeling, and infiltration of inflammatory cells.4 Therefore, targeting MMP-9 activity can offer a new therapeutic avenue for multiregulated pathways of gastric ulcers. Medicinal plants are rich sources of diverse small molecules with various therapeutic properties. Among these plants, neem (Azadirachta indica A. Juss.; Meliaceae) is a well-known natural source of several bioactive compounds and used in traditional medicine in the Indian subcontinent.5,6 Neem leaf and its bioactive constituents play an important role in scavenging free radicals and inhibiting inflammatory responses.7,8 These © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

A methanol-soluble extract of fresh neem leaves was prepared and purified by silica gel column chromatography with gradient mixtures of CHCl3−MeOH. Compound 1 was isolated as a yellow solid powder with an mp of 202−204 °C. This compound was identified as tamarixetin 3-O-β-D-glucopyranoside by interpretation of its spectroscopic data (IR, NMR, ESIMS; Table S1, Figures S1−S10, Supporting Information) and comparing with literature values.16 Compound 1 was indentified from A. indica leaves for the first time in this investigation (Figure 1). Received: October 17, 2016 Published: May 11, 2017 1347

DOI: 10.1021/acs.jnatprod.6b00957 J. Nat. Prod. 2017, 80, 1347−1353

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Figure 1. (A) Structure of compound 1. (B) Absorbance and emission spectra of 1.

found to be 8 ± 3.5 and 5 ± 2.5, respectively, for 15 and 25 mg/kg b.w. doses (Figure 2B). To examine cellular homeostasis of the gastric tissues, formalin-fixed tissues were sectioned and stained with hematoxylin and eosin. Histological inspection of the tissue indicated that indomethacin caused exfoliation of the gastric epithelial cells along with disruption of the mucosal layer of stomach (shown by the black arrowhead) compared with that of the control (Figure 3A and B). The extent of the gastric mucosal injury was evaluated from the percentage of the length of microscopically identified mucosal injury, which comprised hemorrhage and disruption of gastric epithelial cells, to the length of the circumference of the gastric corpus. Higher magnification of the submucosal part showed infiltrated inflammatory cells in the ulcerated gastric tissues (shown by red asterisks). Pretreatment with 1 rescued gastric damage and restored the intact epithelial layer as well as continuous mucosal and submucosal layers. The histological damage indices were reduced to approximately 10 ± 4 and 5 ± 3 for doses of 15 and 25 mg/kg b.w., respectively, of treatment with 1, as compared to 40 ± 7 for ulcerated sections (Figure 3A and B). In addition, 1 treatment inhibited infiltration of inflammatory cells at the submucosal layer significantly (Figure 3). Previous studies have established the role of MMP-9 in the inflammatory responses of gastric tissues, whereupon inhibition of MMP-9 pathogenesis was ameliorated.2−4 Thus, in order to propose a mechanism of action for 1, MMP-9 activities were assessed from the experimental gastric tissues. As shown in Figure 4, indomethacin increased proMMP-9 activity in the gastric tissues significantly. Indomethacin caused ∼2-fold induction of proMMP-9 during gastric ulceration, while 1 treatment prior to indomethacin treatment reduced the proMMP-9 activity almost to control values (Figure 4A and

A neem leaf crude extract has been shown to have antiulcerative properties (Figure S11, Supporting Information),15 but the active principle is unknown. Therefore, the antiulcerative property of 1 was evaluated in a murine model using an indomethacin-induced gastric ulceration procedure in BALB/C mice.17 Indomethacin at a dose of 80 mg/kg b.w. was fed orally to overnight-fasted mice to develop gastric ulcers. Compound 1 was administered orally 30 min prior to indomethacin at two different doses (15 and 25 mg/kg b.w.) to evaluate the gastroprotective efficacy. Gastric lesions in the fundic mucosa were scored and expressed as the ulcer index as described in the Experimental Section. In comparison to the control group, mice treated with indomethacin showed severe gastric lesions with an ulcer index of 35 ± 7.5 (Figure 2A and

Figure 2. Gastroprotective action of compound 1 against indomethacin-induced gastric ulceration. (A) Macroscopic image of gastric tissues of control, indomethacin, and indomethacin + 1 treated mice (in each group, n = 4). (B) Histogram for ulcer index of the gastric mucosa from experimental groups (***p < 0.001).

B). Visible gastric hemorrhagic lesions in the fundic stomach were decreased significantly in a dose-dependent manner in 1pretreated mice. The ulcer indices for the 1-treated groups were

Figure 3. Histological analysis for protective role of compound 1 against indomethacin-induced gastric ulceration. (A) Hematoxylin and eosin staining for stomach tissue sections of the control (i, ii), indomethacin (iii, iv), and indomethacin + 1 pretreated mice using 15 mg/kg b.w. (v, vi) and 25 mg/kg b.w. (vii, viii) doses. Red asterisks indicate the infiltrating cells present at the submucosal part. The black arrowhead indicates the mucosal damage. (B) Histogram for histological damage index of the gastric mucosa from control, indomethacin, and indomethacin + 1 pretreated mice (***p < 0.001). 1348

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Figure 4. Gastroprotective roles of compound 1 by inhibition of MMP-9 activity. (A) Gelatin zymography of gastric tissues from control, indomethacin, and indomethacin + 1 pretreated mice. (B) Histogram for gelatinolytic activity of proMMP-9 (***p < 0.001, NS p > 0.05).

Figure 5. Ex vivo study of compound 1 and MMP-9 binding. (A) Gelatin zymography of full-length human MMP-9 activity in the presence of different doses of 1. (B) Graphical plot of differences in MMP-9 activity versus 1 concentration at the IC50 value (50 μM). (C) Circular dichroism spectrum for binding of different doses of 1 and MMP-9. (D) Double reciprocal plot for Δθ of 1 versus concentration of MMP-9 for Kd value (100 nM).

compound were found to be decreased with the addition of protein, suggesting interaction (Figure 5C and D). From the change in the CD spectrum as a function of MMP-9 concentration, the binding constant (Kd at half-saturation) was calculated and was found to be ca. 100 nM (Figure 5D). Physicochemical data for 1 revealed that there is direct interaction with MMP-9. Therefore, molecular docking analysis was performed to obtain a deeper view of the structural relationships.19 Molecular docking analysis by AutoDock 4.2 showed that 1 did bind into the active site of MMP-9 (crystal structure of MMP-9 PDB ID:2OW0 obtained from the Protein Data Bank) and the binding was thermodynamically favorable with a binding energy of −7.35 kcal/mol. In addition, a cluster analysis18,20 suggested that the binding was highly specific in nature (Figure S13, Supporting Information). Figure 6A shows the lowest energy binding mode as obtained by molecular docking simulation with MMP-9 and 1 in a surface representation in frontal and lateral views. The compound was found to form hydrogen bonds with Ala191 and Tyr420 of MMP-9. Docking results also suggested a coordination complex formation with the catalytic zinc of MMP-9. The catalytic zinc remained bound to three histidine residues, two of which (His401, His405) formed pi-stacking interactions with 1 (Figure 6B,C). Apart from these specific interactions, hydrophobic residues inside the binding cavity of MMP-9 played a

B). It is to be noted that only 1 administration did not show any alteration to gastric tissues (data not shown). Furthermore, 1 dose dependently reduced proMMP-9 activity in indomethacin-treated gastric tissues (Figure 4A and B). Since MMP-9 activity is suppressed by 1 during protection against gastric ulceration, whether or not MMP-9 activity is directly modulated by binding with 1 was investigated. Purified human MMP-9 was incubated with increasing concentrations of 1 (0−500 μM) for 1 h in vitro at room temperature.18 Gelatin zymography was performed using these samples to assess the MMP-9 activity. A gradual decrease in the activity of MMP-9 was observed with increasing doses of 1. The MMP-9 activity was reduced to ca. 60%, 45%, and 40% with treatments of 60, 125, and 250 μM 1, respectively, as compared to no treatment samples (Figures 5A and S12, Supporting Information). From the change in the MMP-9 activity (values for no treatment − values for 1 treatment) with the respective 1 concentration, the IC50 value was determined by considering the saturation point as 100%. The half-maximal inhibitory concentration (IC50) of 1 on MMP-9 activity was found to be ca. 50 μM in the zymography assay (Figure 5A and B). The molecule also showed a circular dichroism (CD) spectrum over this range with two positive Cotton effects (260 and 330 nm) and a negative Cotton effect at 420 nm. Therefore, the CD spectrum of the compound was monitored in the presence of increasing concentrations of MMP-9. The Cotton effects of the 1349

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Figure 6. Interaction of compound 1 with MMP-9 by molecular docking simulation. (A) Binding of 1 (green) into the active site groove of MMP-9 is shown in the frontal and lateral views. Protein and the ligand (1) are shown in surface representation, and the site and the specificity loop of MMP-9 are marked. (B) Orientation of the functional groups of 1 inside the active site. The protein backbone is shown in ribbon diagram. Compound 1 and some interacting residues are shown as a ball-and-stick rendition. The C atoms in 1 are colored green. The active site Zn as bound to three histidines (His401, 405, and 411) is shown. The two amino acid residues (Tyr420, Ala191) with which 1 formed hydrogen bonds are also presented. Hydrogen bond and coordinate bond (between ring O of 1 and catalytic Zn) distances are shown. (C) Detailed interaction diagram obtained by molecular docking. Green, hydrophobic; cyan, polar; pink arrow, hydrogen bonding; green line, pi-stacking; gray line, metal coordination.

significant role in the complex formation via gain of entropy due to solvent exclusion.18 Molecular dynamics simulation was further performed to investigate the stability of the MMP-9 complex obtained by molecular docking simulation and to understand the role of solvent molecules in this interaction.21 The complex was perturbed in water at a normal temperature and pressure for 40 ns of chemical time. The dynamics simulation was converged after about 30 ns, suggesting a stable conformation of the complex (Figure 7A). Contributions of hydrogen-bonding, ionic, and hydrophobic interactions and water bridges were monitored over the course of simulation time. During the molecular dynamics some of the interactions observed in the docking were lost, but new interactions were found to form. For example, hydrogen bonding with Ala191 and Tyr420 was lost, whereas hydrogen bonding with nearby residues such as Ala 189 and Tyr423 was observed. Three hydrogen-bonding interactions were very prominent with Glu111, Gly186, and Leu188, respectively. Interaction with His190 was also observed, and it was mostly through water bridges (Figure 7B). Fluctuations of the protein backbone were observed near the specificity loop, although 1 made contacts mostly with the less fluctuating regions of MMP-9 (Figure S14, Supporting Information). The secondary structural organization of MMP-9 remained intact over the simulation time with a little decrease in the helical and strand content (Figure S14, Supporting Information). In addition, torsions over the 12 freely rotatable bonds of 1 were observed over the simulation, and it was found that the two ring systems became perpendicular to each other in the protein cavity (Figure S15, Supporting Information), in contrast to the planar geometry in solution. Fluctuations in the ligand properties such as the geometry, radius of gyration, and the molecular-, polar-, and solvent-accessible surface area

Figure 7. Molecular dynamics study between compound 1 and MMP9. (A) Changes in the complex over 40 ns molecular dynamics simulation. (B) Interactions observed during the molecular dynamics simulation.

equalized after 30 ns of simulation, indicating the accommodation of 1 inside the binding cavity of MMP-9 (Figure S16, Supporting Information). An overall decrease in the solvent accessibility was also observed (Figure S16, Supporting Information). There are side effects and recurrence of gastric ulcer, even after use of well-known drugs such as proton pump inhibitors and H2 receptor inhibitors.22 Thus, the development of reliable, cheap, and novel nontoxic antiulcer drugs is clearly 1350

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values.16 The purity of the compound was estimated to be greater than 95%, as determined by its 1H NMR spectrum. Animal Experiments. Male Balb/c mice (25−30 g), bred in-house with free access to food and water, were used for all experiments. The mice were kept in 12 h light/dark cycles and housed at 25 °C, room temperature. The experiments were designed to minimize animal suffering and to use the minimum number associated with valid statistical evaluation. On completion of the study, the animals were anesthetized by ketamine (12 mg kg−1 body weight) followed by cervical dislocation for killing. The animal experiments were conducted with approval (protocol number 2015/SSN-10) of the Animal Ethics Committee of the CSIR−Indian Institute of Chemical Biology (IICB/AEC/Meeting/July 2015). Mice (n = 4/group) were fed orally with indomethacin at 80 mg/kg b.w. to induce a maximum level of acute ulcers. The control group received the vehicle only, while the experimental group received indomethacin to cause gastric ulceration. After 4 h, the animals were sacrificed, and gastric lesions in the fundic mucosa were scored in a blind manner and expressed as the ulcer index as follows: 0, no pathology; 10, small pinhead ulcer; and 20−50, lesions of 2−5 mm length. The histological extent of the gastric mucosal injury was evaluated in a blind manner, as the percentage of the length of microscopically identified mucosal injury, which comprised hemorrhage and disruption of gastric epithelial cells, to the length of the circumference of the gastric corpus. The sum of the total scores divided by the number of animals indicated the mean ulcer index. Compound 1, at doses of 15 and 25 mg/kg b.w., was administered orally 30 min prior to indomethacin treatment to test the gastroprotective effect. Indomethacin was dissolved in distilled water with a minimum amount of NaOH. Tissue Extraction. Gastric tissues were suspended in PBS containing protease inhibitors, minced at 4 °C. The suspension was centrifuged at 12000g for 15 min, and the supernatant was collected as PBS extracts. The pellet was further extracted in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and protease inhibitors) and centrifuged at 12000g for 15 min to obtain Triton X100 extracts. Total protein was estimated using Lowry’s method. Hematoxylin and Eosin Staining. Tissues were sectioned into 2−3 mm 2 pieces. The tissue samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin wax. Approximately 5 μm thick serial sections were rehydrated in descending alcohol series and stained with hematoxylin and eosin. Fixation, permeabilization, and staining runs were carried out in exact parallel to ensure comparative significance among groups. Images were captured with an Olympus microscope using Camedia software (Chicago, IL, USA) and processed using Adobe Photoshop version 7.0. Gelatin Zymography. For the assay of MMP-2,-9 activity, PBS extracts (70 μg protein/lane) were electrophoresed in 8% SDS− polyacrylamide gel containing 1 mg/mL gelatin under nonreducing conditions. The gels were washed twice in 2.5% Triton X-100 and then incubated in calcium assay buffer (40 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 10 mM CaCl2) for 18 h at 37 °C. Gels were stained with 0.1% Coomassie blue followed by destaining. The zones of gelatinolytic activities appeared as negative staining. Quantification of zymographic bands was done using densitometry linked to the appropriate software (Lab Image, Kapelan Gmbh, Leipzig, Germany). Measurement of Half-Maximal Inhibitory Concentration (IC50) of 1 on MMP-9 Activity. To evaluate the effect of 1 on the activity of commercially available full-length human MMP-9, an equal amount (∼5 μg) of recombinant protein was incubated with different concentrations (0−500 μM) of 1 before they were electrophoresed in 8% SDS-PAGE containing 1 mg/mL gelatin under nonreducing conditions. The gels were washed twice in 2.5% Triton X-100 and then incubated in calcium assay buffer (40 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM CaCl2) for 18 h at 37 °C. Gels were stained with 0.1% Coomassie blue followed by destaining. The zones of gelatinolytic activity presented as negative staining. Quantification of zymographic bands was performed using densitometry linked to the proper software (Lab Image; KapelanGmbh, Leipzig, Germany). The data for MMP-9 activity were plotted on the y-axis using the delta MMP-9 value

needed. The next-generation drugs should possess minimal side effects and act through additional mechanisms. The present study identified 1 (tamarixetin 3-O-β-D-glucopyranoside) from neem leaf as a lead compound against gastric ulceration. Compound 1 successfully inhibited MMP-9 activity with an IC50 value of ca. 50 μM. Also, an in silico study confirmed that 1 binds to the active site of MMP-9 and interferes with catalytic zinc binding to two histidine (His401, His405) residues, thus inhibiting the enzyme activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was determined in a capillary tube and is uncorrected. The IR spectrum was recorded as a KBr pellet using a Bruker Tensor 27 FTIR spectrometer. The NMR spectra were recorded using a Bruker 600 DPX spectrometer (Coventry, UK) operating at 600 MHz for 1H and 150 MHz for 13C in pyridine-d5 with tetramethylsilane as internal standard, and the chemical shifts are reported in δ units. The mass spectra (positive mode) were obtained on an LC-ESI-Q-TOF micro mass spectrometer in the electrospray ionization mode. Thin-layer chromatography was performed on precoated silica gel 60F254 aluminum sheets (E. Merck, Darmstadt, Germany) using various solvent systems (5%, 10%, 15%, and 20% MeOH in CHCl3), and spots were developed using UV irradiation, iodine, and Liebermann− Burchard reagent. Full-length MMP-9, gelatin, and indomethcin were procured from Sigma-Aldrich (St. Louis, MO, USA). Silica gel and other chemicals and solvents were of analytical grade and procured from Merck India Ltd. All other solvents and chromatographic absorbents were procured from Merck (Darmstadt, Germany) and Sisco Research Laboratories Ltd. (Mumbai, India). Plant Material. The mature fresh leaves of neem (Azadirachta indica) were collected by Indian Association for the Cultivation of Science, Kolkata, in November 2014. The plant material was identified by Dr. R. Gogoi, Botanical Survey of India, Central National Herbarium, Botanic Garden, West Bengal, India. A voucher specimen (DKY-01) has been deposited in the herbarium of the Botanical Survey of India. Extraction and Isolation. The leaves of A. indica were cut into small pieces and air-dried at room temperature (24−27 °C). The dried leaves (2 kg) were defatted with petroleum ether for 24 h and extracted three times with methanol (10 × 3 L) for 48 h each time at ambient temperature. The methanol extract was filtered, and the solvent was dried under vacuum at 40−45 °C to afford 440 g of crude extract (yield 22%). The methanol extract was partitioned between nBuOH and water-saturated n-BuOH. The organic layer was further washed with water for complete removal of inorganic impurities, free sugars, and other water-soluble residues and then evaporated to dryness under reduced pressure using a rotary evaporator to yield a dark brown residue. The extract was chromatographed on a silica gel column (on a 60−120 mesh). Gradient elution was carried out with chloroform followed by various mixtures of CHCl3−MeOH (19:1, 9:1, 8:2, 7:3, and 1:1). Altogether, 106 fractions (400 mL each) were collected, and fractions giving similar spots on TLC were combined. Fractions (48−54) eluted with 10% MeOH in CHCl3 were collected, combined, and then purified by chromatography over a column of silica gel (100−200 mesh). Gradient elution was carried out with CHCl3 followed by various mixtures of CHCl3−MeOH (19:1, 9:1, 20:3, and 8:2). A total of 75 fractions each (100 mL) were collected, and each fraction was monitored by TLC. Fractions 18−47, eluted by CHCl3−MeOH (19:1), were further purified by rechromatography over a silica gel column (100−200 mesh). The column was then eluted successively with CHCl3 and CHCl3−MeOH mixtures (19:1, 9:1). TLC examination revealed that fractions 8−25 were homogeneous and were combined and crystallized with MeOH to yield compound 1 (58 mg). The compound was identified as tamarixetin 3-O-β-Dglucopyranoside by comparison of its physical and spectroscopic data (IR, 1H and 13C NMR with DEPT-90 and DEPT-135, 2D NMR (COSY, HMBC, HSQC, and NOESY), and ESIMS) with literature 1351

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(changes in values of no treatment and 1-treated value) for corresponding 1 concentrations on the x-axis, and the IC50 value was determined considering the saturation point as 100%. Circular Dichroism. CD spectra over the near-UV and visible range were measured on a JASCO J-810 spectrometer using a 1.0 mm quartz cell under constant nitrogen flow conditions and at room temperature. The CD spectra of compound 1 were recorded in the absence and presence of the enzyme within a wavelength range of 240−700 nm. A 10 mM stock solution of 1 was prepared in methanol, and the CD experiment was performed in aqueous buffer. The concentration of 1 was kept at 100 μM, and the MMP-9 concentration was varied from 0 to 333 nM. The CD results were represented in terms of ellipticity (θ). Binding constant (Kd) was determined from a double reciprocal plot using the equation θ0/Δθ = (θ0Kd/Δθmax) × 1/ [MMP-9] + θ0/Δθmax. Molecular Docking. Molecular docking experiments were performed using AutoDock 4.2. MMP-9 structural information was obtained from the Protein Data Bank. The ligand structures were drawn in Avogadro, and the geometry was optimized in vacuo using the steepest descent followed by conjugate gradient algorithms in the UFF force field, as implemented in Avogadro. AutoDockTools was used to prepare the ligand and proteins. For the docking in AutoDock 4.2 polar hydrogen atoms and Gasteiger charges were added to the proteins and the ligand. All the rotatable bonds in the ligand were set free. No flexibility was added to the protein side chains. The whole protein was placed in the center of a simulation box. Grid point spacing of the genetic algorithm was run (ga_run) 100 times to generate a statistically significant number of docked poses. All the other parameters were kept constant. Docking results were rendered in PyMOL and MGLTools. System states were clustered using binding free energy and standard deviation cutoffs of 0.5 kcal mol−1 and 2 Å, respectively. The binding state from the high-frequency and the least energy cluster was chosen for further molecular dynamics analysis. An interaction diagram of the docked complex was produced using the Schrödinger Maestro Molecular Modeling environment (Academic Release 2015-4). Molecular Dynamics. Molecular dynamics (MD) analysis was carried out in a Schrödinger Maestro Molecular Modeling environment (Academic Release 2015-4). In this, 40 ns dynamics were carried out for the protein ligand complex in an SPC water environment using the Desmond molecular dynamics program implemented in Schrodinger Maestro. The protein ligand complex was placed in the center of the simulation box, with periodic boundary conditions, and the whole system was charge neutralized using sodium ions. MD were run in the OPLS 2005 force field. A five-step relaxation protocol was used starting with Brownian dynamics for 100 ps with restraints on solute heavy atoms at NVT (with T = 10 K) followed by 12 ps of dynamics with restraints at NVT (T = 10 K) and then at NPT (T = 10 K) using the Berendsen method. Then, the temperature was raised to 300 K for 12 ps, followed by a 24 ps relaxation step, without restraints on the solute heavy atoms. The production MD were run at NPT with T = 300 K for 40 000 ps. The molecular dynamics output was rendered in Schrodinger Maestro Suite. Statistical Analysis. Data were fitted using Sigma plot (version 11.0, GmbH, Germany) represented as means ± SEM. The statistical analysis of the data was done using GraphPad Instat-3 (version 3.06, San Diego, CA, USA) software. Comparison between groups was performed using one-way analysis of variance (ANOVA) followed by Student−Newman−Keuls t test. For this study, p < 0.05 was accepted as the level of significance; ***very highly significant, p < 0.001; **highly significant, p < 0.01; *significant, p < 0.05; NS, not significant for p > 0.05.

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Experimental procedures for isolation, characterization HRESIMS, FTIR, 1H NMR, 2D NMR spectra (Table S1, Figures S1−S10), and details of in vivo (Figure S11), ex vivo (Figure S12), in silico assays (Figures S13−S16) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel (S. Swarnakar): 91-33-2473-0492, ext. 759. Fax: 91-332473-5197. E-mail: [email protected]. ORCID

Nakul C. Maiti: 0000-0002-8498-6502 Snehasikta Swarnakar: 0000-0003-4739-2980 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.K.Y. is the recipient of a CSIR-SRF fellowship; Y.P.B. is the recipient of a DST-National Post-Doctoral fellowship (PDF/ 2016/000088); A.H. is the recipient of a DST-Young Scientist Grant (YSS/2015/001141); U.P. is the recipient of a DSTINSPIRE fellowship. N.B.M. acknowledges an Emeritus Scientist grant [21(0197)/12/EMR-II] from CSIR. The authors are thankful to Mr. J. Mandal, Central Instrumentation Devision, CSIR-IICB, for helping in CD experiments. S.S. and S.J. acknowledge CSIR network projects INDEPTH (BSC0111) and HUM (BSC0119) for funding.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00957. 1352

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DOI: 10.1021/acs.jnatprod.6b00957 J. Nat. Prod. 2017, 80, 1347−1353