Pharmacological and computational evaluation of fig for therapeutic potential in hyperactive gastrointestinal disorders

Background Ficus palmata (Fig), are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including inflammation, tumor, epilepsy, jaundice, influenza and bacillary dysentery. The present study aimed to evaluate the antidiarrheal, antisecretary, antispasmodic, antiulcer and anti motility properties of Ficus palmata. Methods In-vivo, in-vitro and in-silico techniques were used to investigate various gastrointestinal effects of Ficus palmata. Antidiarrheal, antisecretary, antispasmodic, antiulcer, anti motility and molecular docking were performed using castor oil induced diarrhea and fluid accumulation, isolated tissue preparations, ethanol-HCl induced ulcer assay, charcoal meal transit time and Auto Doc Vina. Results Ficus palmata crude extract (Fp.Cr) exhibited protection against castor oil-induced diarrhea in mice and dose-dependently inhibited intestinal fluid secretions. Fp.Cr caused relaxation of spontaneous and K+ (80 Mm)-induced contractions in isolated rabbit jejunum preparations. It showed protective effect against gastric ulcers induced by ethanol-hydrochloric acid in rats. Fp.Cr reduced distance travelled by charcoal meal in the gastrointestinal transit model in mice. The plant constituents: psoralenoside and bergapten showed high binding affinities (E-value ≥ − 6.5 Kcal/mol) against histaminergic H1, calmodulin and voltage gated L-type calcium channels, while showed moderate affinities (E-value ≥7 Kcal/mol) against dopaminergic D2, adrenergic α1, muscranic M3, mu-opioid, whereas revealed lower affinities (E-value ≥9.5 Kcal/mol) vs. muscranic M1, histaminergic H2 and H+/K+ ATPase pump. Germanicol acetate and psoralene exhibited weak affinities against aforementioned targets. Conclusion This study reveals that Ficus palmata possesses anti-diarrheal, anti-secretory, anti-spasmodic, anti-motility and anti-ulcer activities. The various constituents reveal different binding affinities against target proteins, which mediate the gastrointestinal functions.


Background
High prevalence of gastrointestinal disorders among Asian population has caused significant suffering and it is generally considered by the health care professionals to be a leading cause for the incidence of a number of other concomitant disease conditions. Evidence based medicine has not yet succeeded in introducing any sufficiently safe and efficacious drug for the cure of gastrointestinal ailments. Most of the available remedies provide only short-term relief accompanied by various undesirable effects. Nevertheless, conventional phytotherapies have proved to be more cost-effective and long-lasting in the treatment of gastric problems [1]. These herbal products have emerged as a remarkable source to contribute to the discovery of a number of lead compounds and contemporary marketed medicines. Around 61% of the total drugs launched globally have been extracted from naturally originated herbal products [2].
In-vivo evaluation of crude plant extracts aids to screen out newer bioactive lead molecules and their further processing for structural exploration can lead to the development of novel therapies. For obtaining desirable therapeutic effects, pure form of bioactive constituents can be formulated into appropriate dosage forms as well as doses and dosing frequency can be decided [3]. Many chronic disorders have been successfully cured by phytotherapies particularly by edible fruits being consumed as functional foods as well as their active constituents. Several previous studies have demonstrated the potential of crude extracts of a number of edible fruits to treat the gastrointestinal tract diseases [4].
Ficus palmata commonly known as ' Fig' and locally "Injeer" belongs to the family Moraceae that consists of about 800 species [5]. It is found in the Himalayan region, so also named as Wild Himalayan Fig and is mainly the native of Northern areas of Pakistan. Majority of the members of the family are very tall trees, shrubs and sporadically herbs often with milky juice [6]. Variety of Ficus species are used in folk medicine as anti-inflammatory, anti-tumor and tonic medicament [7]. Diseases such as epilepsy, jaundice, influenza, whooping cough, tonsillitis, bronchitis, enteritis, bacillary dysentery, toothache and bruises are also reported to be cured by Ficus extracts. Antioxidant activity was exhibited by Ficus palmata [8]. Various pharmacological activities such as nephroprotective, hepatoprotective and anticoagulant activities are also possessed by this plant [9].
In the present study, we report anti-diarrheal, antisecretary, anti-spasmodic, anti-motility and anti-ulcer effects. Aforementioned ethnomedicinal uses of the plant were validated by using baseline data from traditional uses and previous studies. Molecular docking of its constituents with known structure is done to find out the potential lead molecule responsible for pharmacological effects.

Plant material and extraction
Superior quality of Ficus palmata fruit weighing 2 kg were purchased from local market in Feb 2017. Plant was authenticated by a taxonmist Dr. Mushtaq Ahmad, at Department of Plant Sciences, Quaid-i-Azam University, Islamabad. Voucher specimen no. (ISL-B-24) was collected after submitting sample of specimen of these species to the herbarium at same department. The fruit (2 kg) was air-dried, crushed into powdered form and extracted at room temperature with aqueous-methanol (70:30) three times to obtain Ficus plmata crude extract (Fp.Cr).

Animals
Sprague-Dawley rats (180-220 g), Balb/C mice (25-30 g) and rabbits (1.0-1.2 kg), of either sex were obtained from animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad. The animals were kept in 595 × 380 × 200 mm plastic cages at standard temperature (23-25°C) and a 12:12 light: dark cycle with lights on at 08:00 and off at 20:00. They were fed with standard animal feed and tap water ad libitum. Animals were fasted before each experiment for 24 h. During housing, animals were monitored twice daily for health status. No adverse events were observed. All the animal experimental protocols were approved by Research and Ethics Committee of RIPS (Ref. no. REC/RIPS/2017/008) which were performed in accordance with the guidelines of "Principles of Laboratory Animal care" [12]. All sections of this report adhere to the Animal Research:Reported of In-vivo Experiments (ARRIVE) Guidelines for reporting animal research. A completed ARRIVE guidelines checklist is included in Checklist S1.

Castor oil-induced diarrhea
This method was previously reported by Umer et al. [13]. All the test animals were fasted for 24 h prior to commencement of experimentation and were divided in five groups (n = 5). The floor of cage was lined with blotting paper in which animals were placed. First group was assigned as negative control group and received normal saline (10 mL/kg) orally, while second group was given with a dose of loperamide hydrochloride (10 mg/kg, p.o.), assigned as positive control. Third, fourth and fifth groups received 50, 100 and 300 mg/kg body weight of the extract orally respectively. After one hour of administration of the respective doses and treatments, all animals received castor oil (10 mL/kg, p.o.). Post treatment evaluation was carried out after waiting 4 h in order to analyze the diarrheal droppings presence, absence of diarrheal droppings was documented as a positive result.

Assessment of intestinal fluid accumulation
This method was previously described by Teke et al. [14]. To study the intestinal fluid accumulation, enteropooling assay was used. Overnight fasted mice were taken and put into five assigned cages with five mice in each. Group I and II were administered with normal saline (10 mL/kg) and castor oil (10 mL/kg, p.o.) respectively. Extract doses of 50, 100 and 300 mg/kg intraperitoneally were given to Group III, IV and V respectively.
Standard drug atropine at dose 10 mg/kg was given to last group, 1 h prior induction with castor oil (10 mL/kg, p.o.). Mice were sacrificed by cervical dislocation after 30 min, then intestine was removed and weighed. The results were calculated as: (Pi/Pm) × 1000 where Pi is the weight (g) of the intestine and Pm is the weight of the animal. Later the dead animals were disposed off by burial method.

Isolated tissue preparation
Before experiment rabbits were fasted for 24 h. Jejunal portion was isolated after cervical dislocation of rabbit and washed with Tyrode's solution. In tissue organ bath containing Tyrode's solution 2 cm of jejunal segment was suspended. Temperature of bath was kept at (37°C) and proper aeration of 95% O 2 and 5% CO 2 (carbogen) is ensured. 1 g initial load was applied to tissue and allowed to equilibrate for 30 min before the addition of any drug. Following equilibration period, each preparation was then stabilized with sub-maximal concentration of ACh (0.3 μM) at 3 min interval until constant responses were recorded via a force displacement transducer (model FT-03) coupled with bridge 7 amplifier and power Lab 4/25 data acquisition system connected to computer running Lab-Chart 6 software (AD Instrument, Sydney Australia). The % change in the voluntary contractions of jejunum were recorded for Fp.Cr (0.01-3 mg/mL) [15]. Later the dead animals were disposed off by burial method.

Ethanol-HCl induced ulcer assay
Rats weighing 250-280 g of either sex were distributed in 5 groups (n = 5). Group 1 served as a negative control received normal saline 10 mL/kg body weight, group 2 received 20 mg/kg, (p.o.) omeprazole as standard drug, group 3, 4 and 5 received 50, 100 and 300 mg/kg, (p.o.) of Fp.Cr respectively. All the animals were treated with 1 mL/100 g of ethanol-HCl mixture (p.o.) i.e. (0.3 M Hydrochloric acid and ethanol 60%) after 1 h to induce gastric ulcer. Animals were sacrificed via cervical dislocation one hour after administration of ethanol-HCl mixture. The stomach were removed and lesion index was estimated by measuring each lesion in mm along its greater curvature. Each lesion surface area was measured and scoring was done as described previously by [16]. For each stomach lesion, ulcer index was taken as mean ulcer score (US). For each stomach injury sum of the lengths (mm) of all sores was utilized as the ulcer index (UI). The gastro protective assessment was displayed as an inhibition percentage (I %) calculated by the following formula: I (%) = (USc -USt) 100/USc. Where USc = ulcer surface area of control and USt = ulcer surface area of test drug group.
Later the dead animals were disposed off by burial method.

Charcoal meal transit time
Gastrointestinal transit time was estimated utilizing the charcoal meal test [17]. Rats were fasted for 24 h, the test groups received the extract at 50, 100 and 300 mg/ kg body weight doses, where as positive control group received atropine sulfate (0.1 mg/kg, i.p.), while the negative control group received normal saline (10 mL/kg, p.o.). 30 mins after all treatments, all the animals were sacrificed by cervical dislocation. The small intestine was excised after which the distance travelled by charcoal meal through the organ was expressed as a percentage of the length of the small intestine according to the following expression.
Later the dead animals were disposed off by burial method.

Computational studies
3-D structures of the test compounds bergapten, psoralene, psoralenosid and germanicol acetate were constructed by using the software of Gauss View 5.0. 3 dimensional structures of reference drugs were prepared through Discovery Studio Visualizer (2016) as shown in Fig. 1. Through same software, polar hydrogen atoms (H-atoms) were added in it and then saved as PDB file. Reference drugs included phenoxy benzamine, verapamil, calmozoilum, domperidone, ranitidine, piranzapine, atropine, loperamide, omeprazole and pyrilimiine. 3-D structures of selected targets possibly involved in the gut physiology, were retrieved from the website of RCSB protein data bank as represented in Water molecules and ligands were removed along with addition of polar H-atoms by using same software and then saved in PDB format. Autodock Vina which is a geometry based automatic docking tool is used through which molecular docking was performed. A definite

Statistical analysis
Data was expressed as Mean ± SEM (n = 5) and median effective concentrations (EC50) having 95% confidence intervals. Statistical analysis of the results was analyzed using one-way ANOVA followed by post-hoc Tukey's test. Chi square test was used in the case of the antidiarrheal data, where p < 0.05 was regarded as significant. Non-linear regression using Graph 10 Pad program (GraphPAD, SanDiego, CA-USA) was used to analyze the concentration-response curves.

Docking evaluation
Assessment of E-value is an important contributor which helps in docking evaluation. Table 4 shows the values of atomic energy in Kcal/mol for complexes formed between ligand and target receptor. Binding residues involved in the formation of polar hydrogen bonds and total number of hydrogen bonds is expressed in Table 5. Total number of pi-pi bonds and binding residues forming these bonds are represented in Table 6. Other hydrophobic bonding of best models for ligand-target complexes are represented in Table 7. Formation of bonding and interaction by bergapten, psoralene, psoralenoside, germanicol acetate and standard drugs against adrenergic α 1 receptor, muscarinic M 1 , muscarinic M 3 , dopaminergic D 2 , calmodulin, mu-opioid, voltage gated L-Type calcium channel, histaminergic H 1 , histaminergic H 2 and H + / K + ATPase pump are shown in Figs. 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 respectively.

Discussion
Based on ethnopharmacological use of Ficus palmata in hyperactive gut diseases, such as colic and diarrhea, its extract was evaluated for the possible anti-diarrheal, anti-secretory, charcoal meal gastrointestinal motility and anti-ulcer effects in rodents. Isolated intestinal tissue was used for the elucidation of possible underlying mechanism(s) to rationalize aforementioned ethnomedicinal uses of the plant and it was further supported by virtual screening tools.
Fp.Cr demonstrated an excellent antidiarrheal activity against castor oil induced diarrhea similar to the effect produced by loperamide, a standard drug [13]. Castor oil cause increases in the peristaltic activity and induce permeability changes to electrolytes and water in the mucosal membrane of the small intestine similar to an effect which is associated with prostaglandin release. Ricinoleic acid which is an active metabolite of castor oil is another major factor in causing diarrhea through a series of actions including activation of small intestinal peristaltic activity with reduction of Na + /K + ATPase activity. These changes eventually results in disturbance in the intestinal mucosa, electrolyte permeability, hypersecretion of intestinal contents and a slogging of the transport time in the intestine [19]. Thus, a potential  Omeprazole (20 mg/kg) + Ethanol-HCl 1 ± 0.11 *** 88.8 *** p < 0.001 compared to control saline group, one-way analysis of variance, followed by Post-hoc Tukey's test, n = 5 agent may exhibit its anti-diarrheal activity by these mechanisms. Intracellular Ca 2+ levels had a huge impact on secretary functions of the gastrointestinal organs which lead towards consequences such as discharge of gastric acids and intestinal fluid release. This effect might be affected by some drugs that hinder calcium influx [20]. Fp.Cr shows protection against castor oil induced intestinal fluid secretions in mice. The anti-diarrheal and anti-secretory activities of Fp.Cr might be because of gastrointestinal relaxant component(s) present in the Fp.Cr [10].
Dose-dependent inhibition of spontaneous contractions in isolated rabbit jejunum preparations were exhibited by Fp.Cr, thus showing antispasmodic action. High K + (80 Mm), as KCl, was used to depolarize the tissue to analyze whether the spasmolytic activity of this extract was mediated through calcium channel blockade or some other mechanism. High K + (80 Mm) is known to cause smooth muscle contractions with opening of voltage gated L-type calcium channels, thus permitting the inflow of extracellular calcium causing contractility and the substance causing      this inhibition of high K + -induced contractions is considered as calcium channel blocker [15]. Verapamil, a specific calcium antagonist have inhibitory effect against K + -induced contractions. Against spontaneous and K + -induced contractions Fp.Cr produces inhibitory pattern just like verapamil. Gastric ulcer is the result of an imbalance between aggressive and defensive factors of the gastric mucosa which results in rupturing of mucosal protection and expose gastric lining to gastric acids. To explore the anti-ulcer effect of Fp.Cr, ethanol-HCl induced gastric model was used which through variety of mechanisms stimulates ulcer including mucus exhaustion, mucosal damage, release of superoxide anion, hydro-peroxy free radicals, all these mechanisms prolonged the tissue oxidative stress and release of inflammatory mediators. Fp.Cr significantly decreased the surface ulceration as compared to that of control animals which received saline. In view of these results, methanol extract showed significant cytoprotection. The potential of Fp.Cr to produce anti-ulcer effect might be due to its CCB effect, as Ca 2+ antagonist are well known to demonstrate such effects [21]. In pathophysiology of gastric ulcers, oxidative stresss plays a vital role. Antioxidant and nitric oxide free scaveneging activity has been reported by Ficus palmata [10] which may be responsible for its effectiveness as anti-ulcer agent.
Intestinal transit travelling, an important measurement factor that regulates the bioavailability of orally administered drugs/foods and determines the absorption intensity of luminal contents. More oftenly, timely oral administration of active charcoal, as marker, to experimental animals are useful to measure the intestinal transit rate. This experimental model is sensitive to agents that inhibit/stimulate intestinal peristalsis regulated by autonomic nervous system. This is the rationale for using this assay to  investigate the influence of natural products on intestinal peristalsis [22]. In the small intestinal transit test, Fp.Cr produces suppression of the propulsion of charcoal marker at all test doses just like atropine sulphate a standard drug, that has been reported to have anticholinergic effect on intestinal transit [21]. A decrease in the motility of gut muscles increases the stay of substances in the intestine, thus allows better water absorption. This finding suggests that Fp.Cr has the ability to influence the peristaltic movement of intestine thereby indicating the presence of an anti-motility activity. It is therefore presumed that the reduction in the intestinal propulsive movement in the charcoal meal model may be due to antispasmodic properties of the Fp.Cr [22]. The observed therapeutic effects of Ficus palmata may be due to the presence of phytochemicals, tannins and flavonoids, as these phytocontinuents are well known for gastrointestinal effects. Anti-diarrheal, anti-secretory, anti-ulcer and anti-spasmodic activities may be due to flavonoids [23].
Molecular docking is an effective tool for evaluating the affinity of various protein targets that may possibly be associated with the pathophysiology of gastric disorders. The traditionally acclaimed use of Ficus palmata in the management of gastric related diseases has been supported with scientific evidence using virtual screening tool and different chemicallyinduced gastrointestinal models like castor oil induced diarrhea, castor oil induced enteropooling, and charcoal meal gastrointestinal motility. The use of computational methodologies has end up being fundamental component of drug discovery and development process therefore computational screening becomes broadly employed tool in identifying the potential of structural chemical compounds before initiation of wet lab research. Since early 90's, docking has been observed to play a primary role for virtual screening of different chemical structures and it is still an extremely dynamic region to look forward [24]. Lead compounds are virtually screened through molecular docking that turns out to be time saving as well as economically affordable on premise of structural conformations [25].
In this study, Auto Dock Vina program was used through PyRx [26]. Through gradient optimization   [27] which are contributing factors in increasing affinity with protein and stabilization of ligand-receptor complex. It has been found that psoralenoside showed excellent score of binding against α 1 receptor with lowest E-value. This binding efficacy is greater than majority of the target proteins with better affinity as compared to other test compounds and standard drugs. Order of affinity of test compounds for α 1 and M 1 receptor was; psoralenoside > bergapten > psoralene > germanicol acetate. Compounds with higher affinity all together formed stronger pi-pi bonds, high number of hydrophobic interactions and polar hydrogen bonding against muscarinic M 1 and α 1 receptors. The order of affinity for ligands against muscranic M 3 receptor was found as; bergapten > psoralene > psoralenoside > germanicol acetate.   Order of affinity of test compounds for dopaminergic D 2 receptor was found as; psoralenoside > bergapten > psoralene > germanicol acetate. Alongside hydrogen and hydrophobic interactions, different types of interactions, for example alky, pi-alky and vander waal interactions are appeared with high proclivity by test compounds. The affinity order of ligands against calmodulin was found as; bergapten > psoralene > psoralenoside > germanicol acetate. In addition, hydrogen bond is considered to be vital for Fig. 12 a,   restricting sites. Order of affinity of test compounds for H + /K + ATPase receptor was found as; bergapten > psoralenoside > psoralene > germanicol acetate. Hydrogen and hydrophobic associations are observed to be essential but no such interactions of test compounds with stated restricting site were seen [28]. In this regard, SER 477 is considered as important and vital amino acid. The affinity order of ligands against histaminergic H 2 receptor was found as; psoralenoside > psoralene > bergapten > germanicol acetate. Order of affinity of test compounds for mu-opioid receptor was found as: bergapten > psoralene > psoralenoside > Fig. 14 a,