Pharmacological basis of the use of the root bark of Zizyphus nummularia Aubrev. (Rhamnaceae) as anti-inflammatory agent

Background The root bark of Zizyphus nummularia (Rhamnaceae) is traditionally used as an anti-inflammatory agent. The current study aimed to explore the anti-inflammatory activity (in vivo) of a crude ethanolic extract (EE) and the pure identified octadecahydro-picene-2,3,14,15-tetranone (IC) in the root bark of Z. nummularia. IC was further subjected to suitable in vitro and in silico studies to find out the mechanistic pharmacology. Methods EE (100 and 200 mg/kg, p.o.) and (IC) (400 and 600 μg/kg, p.o.) were subjected to in vivo anti-inflammatory assays to evaluate the anti-inflammatory activity and predict the probable mechanism(s) of action. Suitable acute (carrageenan-induced paw edema, arachidonic acid-induced ear edema, xylene-induced ear edema) and chronic (cotton pellet granuloma) models were employed to investigate in vivo the anti-inflammatory activity. Based on in vivo observation, IC was further subjected to in vitro assays to estimate the inhibition of nitric oxide (NO), prostaglandin-E2 (PGE-2) and tumor necrosis factor-α (TNF-α) production in PBS stimulated RAW 264.7 cells. Based on the observation of in vitro studies, finally, ADME prediction and molecular docking studies of IC were performed for better understanding of interaction of IC with TNF-α. Results Oral administration of EE (100 and 200 mg/kg) exhibited significant inhibition of carrageenan (p < 0.05) and arachidonic acid (p < 0.05) induced oedema, and the reduced the granuloma tissue formation (p < 0.05) in experimental mice. IC (400 and 600 μg/kg, p.o.) exhibited significant (p < 0.01) inhibition of carrageenan, xylene and arachidonic acid-induced edema, and reduced the granuloma tissue formation. In in vitro assays, IC caused a concentration-dependent inhibition of LPS stimulated NO (up to ~ 67.4 % at 50 μM) and TNF-α (~84.5 % at 50 μM) production. However, the PGE-2 inhibition did not follow dose dependent pattern. Based on in vitro observations, the molecular docking has been performed on the basis of interaction with TNF-α. In in silico studies, it was observed that IC showed hydrogen bonding with GLN 47 amino acid residue of TNF-α protein. Conclusions IC possibly produces anti-inflammatory activity through inhibition of TNF-α and NO production. Electronic supplementary material The online version of this article (doi:10.1186/s12906-015-0942-7) contains supplementary material, which is available to authorized users.


Background
Inflammation is the complex biological response to pathophysiological events mediated by various signalling molecules produced by leukocytes, macrophages and mast cells [1]. During inflammation, enhanced vascular permeability coupled with migration of blood corpuscles into the inflammatory site/s causes oedema, erythema and pain. Various inflammatory mediators, namely nitric oxide (NO), prostaglandin-2 (PGE-2), interleukins (ILs) and tumor necrosis factor (TNF-α) play an imperative role during the progress of inflammation [2]. Inflammation is major cause of morbidity throughout the world [3]. If untreated, it may lead to various associated diseases like arthritis, atherosclerosis, and even cancer [4][5][6]. Serious adverse effects of most of commercially available antiinflammatory drugs largely encourage the development of new, target specific and less toxic anti-inflammatory agents from plants [7]. Many Indian herbs have been claimed to exert notable anti-inflammatory activity without producing considerable untoward effects [8,9]. Recently computer-aided drug design by in silico computer aided drug design is being employed in rational drug discovery to understand the inhibitor-receptor interactions and predict the inhibitory activity of new compounds. Therefore, the combination of ethnopharmacological literature and modern scientific tools including molecular docking is now believed to offer a holistic approach of novel drug discovery.
Zizyphus nummularia Aubrev. (Rhamnaceae), a thorny small bush or shrub, grows in abundance in the grazing lands of the arid and semi-arid regions of India. The plant is used in traditional medicine as analgesic, antiinflammatory, antitussive, anthelmintic, and anti-cancer drug [10][11][12]. Leaves and root bark of the plant are used as the remedy of inflammation by the local communities of eastern India. While, much work exists on extracts of leaves in animal model proving the antiinflammatory action [13][14][15][16]. On other hand, there is no literature on rationalization of anti-inflammatory activity of root bark of Z. nummularia by experimental models. However, the root barks of different species under the same genus, Zizyphus, have been reported to possess significant anti-inflammatory activity [17][18][19]. Considering the ethnopharmacological relevance and the existing literatures in support of anti-inflammatory activities of the root bark of Zizyphus species, the present study was designed to rationalize the anti-inflammatory activity of the crude extract and of the isolated compound, octadecahydro-picene-2,3,14,15-tetranone, from the root bark of Z. nummularia employing in vivo animal models. The earlier reports regarding anti-inflammatory activity of some small molecules bearing similar pentacyclic structure [20][21][22] encouraged us to pursue the study of antiinflammatory activity of this isolated compound. Based on the observation of the in vivo bioassay, the mechanism of action of the isolated compound was studied with respect to in vitro assays in murine monocytic macrophage cell line (RAW 264.7). Therefore, IC was further subjected to in silico study to predict its possible orientation at receptor level.

Carrageenan-induced paw oedema
Mice were divided into six groups (n = 6) and the acute inflammation was induced by carrageenan [23]. Pedal inflammation was induced by a single subcutaneous injection of carrageenan (0.1 ml, 1 %, w/v, in normal saline) into the right paw of each mouse under the subplantar aponeurosis. Thirty minutes prior to carrageenan injection, the first group of mice received orally tween-80 (1 %) and served as inflammation control. The second and third group were treated orally with EE at the doses of 100 and 200 mg/kg, respectively, while, fourth and fifth groups were treated with IC at the doses of 400 and 600 μg/kg, respectively. A group of mice was treated with the standard anti-inflammatory drug, aspirin (positive control). The paw volume was measured by dipping the foot in the mercury bath of a plethysmometer up to the anatomical hairline on lateral malleolus and compared with control animals. The paw volumes were recorded at 0, 1, 2, 3, 4 and 5 h following carrageenan injection.

Arachidonic acid and xylene-induced right ear oedema
Inflammation was induced in mice (n = 6/group) by applying on the inner and outer surfaces of the right ear 30 μl of following irritants: arachidonic acid 0.1 mg/μl in acetone [24] and xylene [25]. Thirty minutes prior to irritants' treatment, two groups of mice received orally tween-80 (1 %) and served as control group. The other groups were treated orally with EE (100 and 200 mg/kg) and IC (400 and 600 μg/kg), respectively. Two groups of mice were treated with the standard anti-inflammatory, aspirin (10 mg/kg) (positive Table 1 The Qikprop® properties and descriptors Lipinski's rules of five are: mol_MW < 500, QPlogPo/w < 5, donorHB ≤ 5, accptHB ≤ 10. Compounds that satisfy these rules are considered drug like. (The "five" refers to the limits, which are multiples of 5).
Maximum is 4 control). The left ear served as normal control. Thirty minutes after the arachidonic acid injection, the mice under different groups were subjected to CO 2 euthanasia and sacrificed by cervical dislocation. Both ears were removed and weighed. The data were represented as percent of oedema weight.

Cotton pellet-induced granuloma
This study was carried out following the protocol of Ismail and co-authors [26] with little modification. Sterile cotton pellets (10 ± 0.5 mg) were implanted subcutaneously on the backs of mice. The six groups (n = 6/group) of mice were treated with aqueous tween 80 (1 %), EE (100 and 200 mg/kg), IC (400 and 600 μg/kg) and aspirin (10 mg/kg) orally, once daily over 7 consecutive days. On day 8, the mice were subjected to CO 2 euthanasia and sacrificed by cervical dislocation. The cotton pellet were removed, dried overnight at 60°C and weighed.
In vitro anti-inflammatory activity of IC Cell culture The RAW 264.7 cells were maintained in DMEM supplemented with FBS (10 %), glucose (4.5 g/l), sodium pyruvate (1 mM), L-glutamine (2 mM), streptomycin (50 μg/ml) and penicillin (50 U/ml) at 37°C and 5 % CO 2 . The medium was routinely changed on alternate days. The cells were passaged by trypsinization (Trypsin-EDTA) to disrupt cell monolayer at confluence while splitting RAW264.7 cells for the routine culture and plating the cells for the in vitro assays.

Cytotoxicity assay
The cytotoxic effect of IC was determined by cell viability assay. Briefly, Cell suspension was seeded into a 96-well plate (~4 × 10 5 cells/well) and incubated for 12 h (37°C; 5 % CO 2 ) to allow cell attachment. The cells were then incubated with IC (1-1000 μM) at 37°C and 5 % CO 2 tension. The cell viability was assessed at 2 h and 4 h by MTT assay [27]. IC did not cause any loss of cell viability up to 100 μM as compared with untreated RAW264.7 cells (data were shown in Additional file 1: Figure S1).

Estimation of NO, PGE-2 and TNF-α inhibitory activity
Cell suspension was seeded into a 96-well plate (~4 × 10 5 cells/well) and incubated for 12 h (37°C; 5 % CO 2 ) to allow cell attachment. The cells were then stimulated with lipopolysaccharide (1 μg/ml) and different concentrations of IC. Nitrite accumulation, an indicator of NO synthesis, was measured in culture media based on a diazotization reaction using the Griess reagent [28]. The nitrite concentration was measured using sodium nitrite as a standard. PGE-2 and TNF-α in the supernatant were measured using ELISA kits (eBioscience, USA) according to manufacturer's instructions.
In silico ADME prediction and molecular docking studies of IC The pharmacokinetic profile of IC was assessed using absorption, distribution, metabolism, elimination (ADME) prediction methods.

Preparation of the protein and prediction of active site
A docking study was carried out at the receptor site of TNF-α protein to find out the putative binding mode of the isolated compounds. The crystal structure of recombinant human TNF-α with a resolution of 2.30 Å was retrieved from the protein data bank (PDB ID: 1A8M) [29]. The structure was prepared by the protein preparation wizard within the Maestro Schrödinger®9.0 module, which was further utilized to predict the possible active site. As the selected TNF-α protein was devoid of associated co-crystallized ligand, therefore the location of the primary binding site on a receptor was unknown.   [30] and the highest scored ( Table 2) binding site (Figs. 2 and 3) was selected for the molecular docking.

Statistical analysis
The experimental data were statistically analyzed by the One-way Analysis of Variance (ANOVA) and expressed as mean ± S.E.M. followed by Dunnett's t-test using computerised GraphPad InStat version 3.05, Graph pad software, USA. The differences are considered significant when p < 0.05.

Results and discussion
In vivo studies to validate the effect of EE and IC Effects on carrageenan-induced right paw edema Carrageenan-induced inflammation model is a wellestablished method used for acute inflammation. The edema expansion follows biphasic responses. The early phase (within 1 h) is mediated by the discharge of autacoids, viz. histamine, kinins and serotonin, while the later phase (after 1 h) involves prostaglandins; the link between the two phases is provided by kinins [31]. Oral administration of EE (100 and 200 mg/kg) exhibited significant inhibition of carrageenan-induced inflammation (Fig. 4a)

Effect on arachidonic acid and xylene-induced right ear oedema
The arachidonic acid and xylene can cause an acute inflammatory response and lead to severe vasodilation and oedematous changes [32]. The results showed that EE (200 mg/kg) exhibited significant (p < 0.05) suppression of arachidonic acid and xylene-induced ear oedema in mice, while the effect of IC (400 and 600 μg/kg) is more pronounced (p < 0.01) (Fig. 4b). The activity of IC (600 μg/kg) was found nearly comparable to that of positive control aspirin (10 mg/kg). Arachidonic acid is a precursor of PGE-2. Besides, arachidonic acid also can act as a second messenger to regulate many cellular processes including nitric oxide formation [33]. Therefore, the observed effect may be due to inhibition of PGE-2 and/or NO production. On other hand, xylene causes the release of pro-inflammatory mediators from sensory neurons that act on peripheral target cells such as mast cells and other immune cells producing neurogenic inflammation [34].

Effect on granuloma tissue formation
The inflammatory granuloma tissue formation is a feature of chronic inflammation. It evaluates the effects on macrophage dysfunction and granuloma formation [3]. Macrophage activation during the process of chronic inflammation causes release of pro-inflammatory mediators, including TNF-α, and participates in the subsequent process of inflammation [35]. Figure 4c depicted that the effect of EE and IC on granuloma tissue formation. EE exhibited significant (p < 0.05) inhibition of dry weight of respectively. The effect of IC (600 μg/kg) was found nearly comparable to that of positive control, the standard anti-inflammatory agent, aspirin (10 mg/kg), which exhibited an inhibitory value of~43.3 %. The inhibitory effect of test materials may be due to inhibition of macrophage activation. Based on the results of in vivo studies, IC was further subjected to in vitro experiment to elucidate its possible mechanism.
In vitro studies to predict the probable mechanism Effect of IC on NO , PGE-2 and TNF-α production The effects of IC on LPS stimulated NO , PGE-2 and TNF-α production in RAW 264.7 cells were depicted in Fig. 5. LPS-induced macrophage activation increased the production of pro-inflammatory cytokines and inflammatory mediators, including NO, PGE-2 and TNF-α [36]. IC caused a concentration-dependent inhibition of LPS-stimulated NO production up to~67.4 % at the highest used dose of 50 μM. NO plays an important role in various inflammatory conditions and in tissues is susceptible to manipulation by pro-inflammatory cytokines [37]. The inhibition of TNF-α production also follows a concentration-dependent manner. IC caused a maximum inhibition of LPS stimulated TNF-α production of~84.5 % at the dose of 50 μM and the steady state inhibition (>80 %) attained between 1 and 50 μM  The key amino acids were shown within the active site of TNF-α after docking concentration range. However, the PGE-2 inhibition did not follow a dose-dependent pattern. The maximum inhibition of~61.4 % was observed between the 1 and 5 μM. Based on in vitro observations, it would be hypothesized that IC possibly acts through inhibition of TNF-α production. Therefore, molecular docking has been performed on the basis of interaction with TNF-α.
In silico observations ADME The predicted value of Lipinski's rule of five for IC is within the range of stipulated values. The predicted values of each individual parameters necessary for Lipinski's rule of five (mol_MW, QPlogPo/w, donorHB and accptHB) are also well within the recommended range ( Table 3) which indicate that IC has property of drug-likeness [38].
The calculated values of total solvent accessible surface area (SASA) along with its hydrophobic component (FOSA), hydrophilic component (FISA), the π component (PISA) and volume of IC are within the stipulated ranges which favours the fixation of IC with hydrophilichydrophobic contour of TNF-α receptor. The predicted qualitative human oral absorption of IC is high (3 in our case) and the percent of human oral absorption value is 77.61 %, very close to the recommended values. The number of reactive functional groups that can produce reactivity and toxicity problems in vivo is zero. Finally, the predictive central nervous activity of IC is −2, which indicates that IC is CNS inactive.

Active site prediction and molecular docking
In the present assessment of ligand-receptor interactions using Glide, IC showed hydrogen bonding with GLN 47 amino acid residue (Fig. 6). The G score of −3.286 obtained by the dock pose of IC complemented by the hydrophilic-hydrophobic contour of TNF-α protein (Fig. 7). The hydrogen bond interaction occurred with oxygen atom of the isolated compounds and GLN 47. The oxygen atom served as hydrogen bond acceptor with the -NH 2 group of glutamine. This also correlates with the calculated values of accptHB of IC (5.5 in our case). Molecular docking study also reveals the best possible environment necessary for drug receptor interaction. The hydrophilic-hydrophobic domain of the predicted active site of TNF-α comprises of ARG 131, ASP 45, LYS 90, GLN 47, ASN 46, GLU 135, GLN 27, GLN 25, LEU 26, GLU 23 and GLY 24 amino acid residues (Fig. 8). The similar types of molecular docking were reported earlier to understand the probable interactions between TNF-α protein and IC [39,40].

Conclusion
In the present study, we found that the root bark of Z. nummularia exhibited significant anti-inflammatory activity. We also observed that, octadecahydro-picene-2,3,14,15-tetranone isolated from the root bark of Z. nummularia exhibited significant anti-inflammatory activity. To predict the mechanism, in vitro assays were performed to see the effect of IC on LPS stimulated NO , PGE-2 and TNF-α production in RAW 264.7 cells. Among the tested mediators, IC significantly inhibited LPS stimulated TNF-α and NO production. However, the inhibitory effect on TNF-α production has been found more pronounced. Based on quantitative value/ inhibition characteristics, molecular docking studies were performed on TNF-α protein. Molecular docking study further helped in supporting the observed TNF-α selectivity. Based on these observation, IC can be regarded as an anti-inflammatory agent with possible inhibitory effect on NO and TNF-α production. Therefore, the compound may have clinical potential for the treatment of inflammation in future.