Curculigo recurvata W.T.Aiton exhibits anti‐nociceptive and anti‐diarrheal effects in Albino mice and an in silico model

Abstract Background Curculigo recurvata (C. recurvata) is an enthnomedicinally important herb reported to have significant medicinal values. The present study aimed to explore the in vivo and in silico anti‐nociceptive and anti‐diarrheal effects of a C. recurvate rhizome methanol extract (Me‐RCR). Methods The analgesic effects of Me‐RCR were assessed using acetic acid‐induced writhing and the formalin‐induced flicking test. The drugs were administered intraperitoneally (IP) at doses of 200 and 400 mg/kg body weight (bw). Anti‐diarrheal activity was evaluated by assessing intestinal motility, hypersecretion, and fecal score in mice at oral doses of 200 and 400 mg/kg·bw. Computer facilitated analyses for anti‐nociceptive and anti‐diarrheal activities of three isolated compounds from C. recurvata were undertaken to identify the best‐fit phytoconstituents. Results The Me‐RCR showed significant (P < .05) peripheral anti‐nociception at the highest dose. The extract inhibited both early and late phases of nociception in the formalin‐induced writhing test. In the castor oil‐induced diarrhoea model, the extract significantly (P < .05) prolonged the onset time of diarrhoea, inhibited percentage of diarrhoea, and decreased both the volume and weight of intestinal contents. Rates of intestinal fluid accumulation inhibition were (33.61 ± 1.00)% and (46.44 ± 0.89)% at Me‐RCR doses of 200 and 400 mg/kg·bw, respectively. Moreover, a significant (P < .05) reduction in gastrointestinal motility was observed. An absorption, distribution, metabolism, excretion and/or toxicity (ADME/T) test showed that the selected compounds yielded promising results, satisfying Lipinski's rule of five for predicting drug‐like potential. Notably, of the three phytoconstituents curculigine and isocurculigine possessed the highest affinity for the COX‐1 and COX‐2. Isocurculigine was also identified as the most effective anti‐diarrheal compound in the computer‐facilitated model. Conclusion An extract of the plant C. recurvata showed potential analgesic and anti‐diarrheal activity due to the presence of one or more active secondary metabolite(s).


| INTRODUC TI ON
The use of herbal formulations as therapeutics in traditional medicine is widespread and historically ancient. Folk practitioners have long used such formulations based on tradition and experience with no proper scientific validation. 1,2 Therefore there is increasing need to bridge the gap between traditional uses of herbal preparations and scientifically generated natural products.
Pain is an unpleasant sensory and emotional response accompanying actual or potential tissue damage. The presence of pathologic conditions such as tumor, muscle spasm, inflammation, and nerve damage or exposure to noxious chemicals, or mechanical or thermal stimuli have been established as contributing factors of this damage. 3 The injured tissue and migrating cells release various pro-and anti-inflammatogenic agents including cytokines, interleukins, serotonin, histamine, prostaglandins, and nitric oxides that are responsible for regulation of the inflammation. 4,5 To relieve pain, opioid analgesics (morphine and codeine), non-opioid analgesics (aspirin and diclofenac), and adjuvant analgesics are used to suppress the pain signal. However, their usage is limited by adverse effects on particular conditions such as peptic ulcers, heart failure, hypertension, and renal dysfunction, the allergic reactions caused by NSAIDs, and the state of tolerance and dependence induced by opioids. 1,6,7 Diarrhea is associated with increased frequency of loose stools often accompanied by abdominal cramp. In many parts of the world, diarrhea has been shown to be a chronic disease or the result of infectious etiology. 8 It is a common symptom of gastrointestinal infection due to ingestion of bacteria, viruses, or parasites transmitted by water, food, utensils, hands, and flies. 9 The major causative agents of diarrhoea in humans include the Shigella species (Shigella flexneri, Shigella sonnei, Shigella boydii, Shigella shiga, and Shigellla dysentery), Staphylococcus aureus, Escherichia coli and Salmonella typhi. 10 The World Health Organization (WHO) has encouraged the conduct of studies of the treatment and prevention of diarrhoeal diseases using traditional medical practices. 11 Such efforts to find new medicinal agents with fewer toxic effects may lead to new drugs with improved pharmacological properties, thus substantially helping to extend the range of effective and safe therapies. such as snake bites, consumptive cough, impotence, asthma, jaundice, diarrhea, colic, gonorrhea, and arthropod stings, 12,13 as well as for the treatment of menoxenia, bleeding disorders, nephritis, arthritis, and leucorrhea. [13][14][15] The traditional uses of RCR have been validated by animal and clinical studies, including hypoglycemic, antibacterial, anthelmintic, 16 antithrombotic, and cytotoxic studies. 14 Mustakim et al, 17 for example, confirmed that a crude methanol extract of C. recurvata rhizomes possesses potential antioxidative and cytotoxic properties. Two novel diastereoisomer glucosides, curculigine and isocurculigine, have been identified from this species, 18 and glucosides of curculigo rhizome have been shown to significantly improve premenopausal syndrome, 19 characterized by hysteria, depression, and melancholia. 20 Accumulating evidence suggests that other species of the Curculigo genus possess various therapeutic effects. For instance, the C. orchioides has shown neuroprotective, 21,22 anticancer, 22 antiosteoporotic, 24 immunostimulatory, 25 and estrogenic activities. 26 Traditional usage in the treatment of several diseases and accumulating pharmacologic evidence suggest that C. recurvata is a plant with rich potential for therapeutic studies. The folkloric value of C. recurvata encouraged us to investigate the pharmacological activity of this plant in this study. The objective of the study was to assess the anti-nociceptive and anti-diarrheal activities of a methanol extract of C. recurvata (Me-RCR), in order to determine its relevance for the treatment of pain and diarrhea.

| Chemicals and reagents
Formalin and acetic acid were obtained from Merck (India). DMSO and methanol were from Merck (Germany). Loperamide was from Square Pharmaceuticals Ltd, Bangladesh. Castor oil was from WELL's Heath Care, Spain. Diclofenac-Na was from Sigma-Aldrich, USA.
Analytical reagent grade chemicals were used for all experiments.

| Preparation of crude extract
The RCR was air dried (23 ± 0.5)°C followed by mechanical drying (Ecocell, MMM Group, Germany) at 55-60°C. The dried rhizomes were ground into a coarse powder with a mechanical grinder analgesic, anti-diarrheal, anti-nociceptive, curculigine, Curculigo recurvata, isocurculigine (NOWAKE, Japan). The powdered rhizome (500 gm) was soaked in methanol (800 mL) for 7 days with occasional shaking and then filtered using Whatman #1 filter paper (Whatman International, Maidstone, Kent, UK). The filtrate was then evaporated under reduced pressure at 50°C using a rotary evaporator (RE200, Bibby Sterling Ltd, UK) to yield a brown mass of 35.5 gm of crude methanolic extract of RCR (Me-RCR). The extract was stored in a freezer until used for the experiments.

| Acetic acid-induced writhing test
Twenty four mice were divided into 4 groups (n = 6). The first group, the normal control (NC), received normal saline (10 mL/ kg·bw), the second group, the reference control (DS), received the standard drug diclofenac sodium (10 mg/kg·bw), and the remaining two groups received Me-RCR at doses of 200 and 400 mg/kg·bw. Thirty minutes after saline, diclofenac sodium or plant extract injection, the animals were treated IP with 10 mL/kg·bw of acetic acid (1% (v/v)). After 5 min of acetic acid injection, abdominal constrictions were counted for 10 min and the responses were compared with control group. 28,29 Inhibition of writhing was calculated using the following formula:

| Formalin-induced licking test
The formalin-induced biphasic method used in mice models has been described previously. 29,30 A volume of formalin solution (2.5%, 20 µL) was prepared by 0.9% saline solution and injected into the sub-plantar region of the right hind paw of mice to induce pain. Animals were pretreated by IP injection of vehicle (saline water), diclofenac sodium (10 mg/kg·bw), and different doses (200 and 400 mg/kg·bw) of Me-RCR 60 minutes before formalin injection. Responses such as licking and biting of the right hind paw were considered as nociception.
Responses measured during the first 5 minutes after formalin injection were considered as the first phase and responses 15-30 minutes after injection were considered as the second phase. First and second phase responses were judged to describe neurogenic and inflammatory pain, respectively. Anti-nociceptive activity was calculated as the percentage inhibition of licking time.

| Castor oil-induced diarrhea
The anti-diarrheal effects of the Me-RCR were determined according to the method described by Taufiq and Bellah. 6,31 Swiss albino mice were fasted for 24 hours and divided into 4 groups (n = 6).

| Castor oil-induced enteropooling
The castor oil-induced enteropooling test was performed using the method described previously by Robert. 32 Swiss albino mice were were collected into a graduated tube and the volume of the content was measured. The intestines were reweighed and the difference between full and empty intestines was calculated.

| Gastrointestinal motility test
This test was carried out by the method described by Mascolo. 33 Swiss albino mice were fasted for 24 hours with free access to water and divided into 4 groups (n = 6). The first group received 10 mL/kg·bw of Tween 80 (1% Tween 80 in water) orally as the normal control (NC), the second group received loperamide (5 mg/kg·bw as oral suspension) as the reference control (RC) and the remaining two groups received Me-RCR orally at doses of 200 and 400 mg/kg·bw. One hour after treatment, the animals received 1 mL of charcoal meal (10% charcoal suspension in 5% gum acacia) orally. After 1 hour, the animals were sacrificed using 70% (v/v) ethanol in 0.9% sterile saline as an anesthetic and the distance traveled by the charcoal meal from pylorus to caecum was measured and expressed as a percentage of the total distance of the intestine. Percentage inhibition was calculated using the following formula:

| Selection of compounds for PASS prediction
The three compounds curculigine, isocurculigine, and nyasicoside were selected for PASS prediction analysis on the basis of availability, as the main compounds isolated from RCR identified by a literature review. 18 The chemical structures of the selected compounds were obtained from the PubChem data base.

| In silico experiment to predict the activity spectra for substances (PASS)
The suitability of the selected compounds (curculigine, isocurculigine, and nyasicoside) 18 for studying anti-nociceptive activity was confirmed using the PASS prediction program. This program predicts the theoretical biological or pharmacological activity of a compound as probable activity (P a ) and probable inactivity (P i ) 29 based on the results of analysis of the structure-activity relationship of 205 000 candidate compounds, predicting more than 3750 kinds of biological

| Ligand preparation
The structures of the selected compounds (curculigine, isocurculigine, and nyasicoside) were obtained from Pubchem databases.

| Receptor grid generation
Receptor grids were measured for prepared proteins so that different ligand poses could bind within the predicted active site during docking. In glide, grids were generated in so as to keep the default parameters of Van der Waals scaling factor and the charge cutoff value as 1.00 0.25, respectively, subject to OPLS 2005 force field.
A cubic box of specific dimensions centered on the centroid of the active site residues was generated as a receptor. The bounding box was set to 14 Å × 14 Å × 14 Å for docking experiments.

| Absorption, Distribution, metabolism, excretion and/or toxicity (ADME/T) property analysis
The

| Statistical analysis
Data are expressed as means ± SEM. One-way ANOVA followed by a post-hoc test (Dunnet's t test) was used to find significant differences between test and control groups. GraphPad Prism Version 6.0 (GraphPad software Inc, San Diego, CA) was used for the statistical analysis. P values (<.05 and <.001) were considered as statistically significant.

| Formalin-induced licking test
The biphasic nociceptive potential of Me-RCR against the formalininduced pain test is shown in Figure 2. Me-RCR showed significant

| Castor oil-induced enteropooling in mice
Castor oil (also known as recinolic acid) has a tendency to produce

| Gastrointestinal motility test
Me-RCR significantly (P < .001) reduced the gastrointestinal transit of marker diet in castor oil-induced mice at all doses (Table 3).

| In silico PASS prediction
The compounds curculigine, isocurculigine, and nyasicoside were evaluated by the PASS program for their anti-nociceptive effects.
All the compounds demonstrated a higher P a than P i (Table 4).
Curculigine and isocurculigine showed the highest P a value for antinociceptive activity (P a = 0.480), followed by nyasicoside (P a = 0.454).

| In silico molecular docking analysis for antidiarrheal activity
Computer aided anti-diarrheal activity was performed to assess the binding pattern of molecules with the amino acids present in the active pocket of the protein. In this study, two major receptors (M3 muscarinic acetylcholine receptor, PDB: 4U14 and PDB: 5AIN) involved in intestinal motility were used to explore the possible anti-

| ADME/T property analysis
The absorption, distribution, metabolism and excretion (ADME) properties of curculigine, isocurculigine, and nyasicoside were clarified with the QikProp module of Schrodinger ( Table 8). The selected properties are known to be cell permeability, metabolism, and bioavailability. The predicted properties of curculigine, isocurculigine, and nyasicoside showed promising results, satisfying Lipinski's rule of five for identification of drug like potential. The colors indicate the residue (or species) type: red: acidic (Asp, Glu); green: hydrophobic (Ala, Val, Ile, Leu, Tyr, Phe, Trp, Met, Cys, Pro); purple: basic (Hip, Lys, Arg); blue: polar (Ser, Thr, Gln, Asn, His, Hie, Hid); light gray: other (Gly, water); darker gray: metal atoms. Interactions with the protein are marked with lines between ligand atoms and protein residues: solid pink: H-bonds to the protein backbone; dotted pink: H-bonds to protein side chains; green: π-π stacking interactions; orange: π-cation interactions. Ligand atoms that are exposed to solvent are marked with gray spheres. The protein "pocket" is displayed with a line around the ligand, colored with the color of the nearest protein residue. The gap in the line shows the opening of the pocket analgesic and anti-inflammatory drugs may be found by screening medicinal plants that have traditional analgesic and anti-inflammatory uses, 40,41 and efforts should be made to enrich drug arsenals with the products of medicinal plants used against inflammation and pain. 42,43 In the present study, Me-RCR was first tested for anti-nociceptive properties using the acetic acid-induced writhing test and the formalin-induced pain test. Pain is induced by acetic acid administration and writhing is initiated when free arachidonic acid is released from tissue phospholipids through biosynthesis of prostaglandins (PGs) resulting in a local inflammatory response. 44 The The formalin-induced paw licking test is characterized by two distinct pathways -early and late phase pathways. In the early phase, pain, termed neurogenic pain, is induced just after formalin administration. In this phase, pain is associated with the direct stimulation of nociceptive neurons through the release of bradykinin and substance P. 6,28,46 The late phase, on the other hand, is initiated within 15 minutes of formalin injection, and is associated with the actions of PG, bradykinin, serotonin, and histamine in peripheral tissues. 47  H-bonds to protein side chains; green: π-π stacking interactions; orange: π-cation interactions. Ligand atoms that are exposed to solvent are marked with gray spheres. The protein "pocket" is displayed with a line around the ligand, colored with the color of the nearest protein residue. The gap in the line shows the opening of the pock motility and hypersecretion of water as well as electrolytes. 32 The ricinoleic acid forms ricinoleate salts with Na + and K + in the lumen of the intestine. These ricinoleate salts inhibit Na + -K + ATPase, and thus increase the permeability of intestinal epithelium and increase the secretion of water and electrolytes. 6,32 In this study, the significant anti-diarrhoeal action exerted by the ME-RCR extract may be F I G U R E 6 Docking analysis for anti-diarrheal activity of curculigine (A), isocurculigine (B), nyasicoside (C) and loperamide (D) docking with M3 muscarinic receptor (PDB: 5AIN). The colors indicate the residue (or species) type: red: acidic (Asp, Glu); green: hydrophobic (Ala, Val, Ile, Leu, Tyr, Phe, Trp, Met, Cys, Pro); purple: basic (Hip, Lys, Arg); blue: polar (Ser, Thr, Gln, Asn, His, Hie, Hid); light gray: other (Gly, water); darker gray: metal atoms. Interactions with the protein are marked with lines between ligand atoms and protein residues: solid pink: H-bonds to the protein backbone; dotted pink: H-bonds to protein side chains; green: π-π stacking interactions; orange: π-cation interactions. Ligand atoms that are exposed to solvent are marked with gray spheres. The protein "pocket" is displayed with a line around the ligand, colored with the color of the nearest protein residue. The gap in the line shows the opening of the pocket F I G U R E 7 Docking analysis for anti-diarrheal activity of nyasicoside (C) and loperamide (D) docking with M3 muscarinic receptor (PDB: 4U14). The colors indicate the residue (or species) type: red: acidic (Asp, Glu); green: hydrophobic (Ala, Val, Ile, Leu, Tyr, Phe, Trp, Met, Cys, Pro); purple: basic (Hip, Lys, Arg); blue: polar (Ser, Thr, Gln, Asn, His, Hie, Hid); light gray: other (Gly, water); darker gray: metal atoms.

| D ISCUSS I ON
Interactions with the protein are marked with lines between ligand atoms and protein residues: solid pink: H-bonds to the protein backbone; dotted pink: H-bonds to protein side chains; green: π-π stacking interactions; orange: π-cation interactions. Ligand atoms that are exposed to solvent are marked with gray spheres. The protein "pocket" is displayed with a line around the ligand, colored with the color of the nearest protein residue. Natural product researchers should aim to carry out virtual screening for previously isolated potentially active molecules that have not yet been assayed against specified drug targets to check for the possibility of good ADME/T profiles.Y 51 It is clear that among the compounds tested, curculigine satisfied Lipinski's rule of five, with lower toxicity and better pharmacokinetic profiles, and can therefore be considered a potential drug candidate.

| CON CLUS ION
The present study showed that a methanol extract of C. recurvata rhizome (Me-RCR) may contribute to anti-diarrheal and anti-nociceptive activities in vivo due to the presence of secondary pharmacologically active metabolites. In addition, PASS program analysis of curculigine, isocurculigine, and nyasicoside indicated the anti-nociceptive potential of the compounds, molecular docking analysis predicted a higher affinity for binding with COX-1 and COX-2, and lastly ADME/T analysis revealed better pharmacokinetic and toxicity profiles. Further analysis of the herb is required, which should include HPLC, NMR, and MS to confirm the exact molecule(s) responsible for the aforesaid activities. Indepth mechanistic investigations should also be performed using animal models, including a dose-response study to explain the anti-nociceptive and anti-diarrheal activities of selected potential compounds.

ACK N OWLED G EM ENTS
The authors are grateful to the International Islamic University

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
SA, NC, AH and MSHK carried out the study. MSN, ASMAR, SI, SMT, AHMKA, MAH and MSIA contributed in the analysis and interpretation of data, and drafting the manuscript. MAH and ASMAR coordinated the research, revised the manuscript and approved the final version to be submitted for publication. All authors read and approved the final manuscript.

E TH I C A L A PPROVA L
The protocol used in this study using mice as the animal model for