1. Introduction
Due to ongoing reports of antibiotic resistance by various pathogens, researchers have refocused their interest in the use of natural antimicrobial agents to treat infections instead of established antibiotics [
1]. Although some conventional antibiotics may be bactericidal, they remain unable to inhibit the release of bacterial toxins which complicates the clinical picture [
2]. Analogous to bacterial infections, helminths can persistently infect both humans and animals throughout their lifespan. Helminths exhibit greater complexity than other pathogens and are capable of producing chronic disease, yet these diseases are often neglected in developing regions [
3]. The association of bacteria and parasites with gastrointestinal disorders is a common situation in developed and developing countries. Various etiological (
Salmonella,
Campylobacter,
Escherichia,
Shigella,
Yersinia enterocolitica, parasites, and viruses) agents responsible for enteric infections may lead to dysentery-like chronic diarrhea [
4]. Such infectious diseases cannot be cured easily at present, due to rapid resistance to available drugs; therefore screening of new therapeutic avenues such as plants may provide an alternate and effective approach for the development of novel agents.
Throughout the ages, plants have served humans for innumerable therapeutic interventions, ranging from the common cold to life-threatening conditions. The value of phytomedicinal approaches still resonates with the R&D departments of modern pharmaceutical giants [
5]. The development of modern medicines has, in many instances, stemmed from ethnic medicinal uses, and meticulous investigation of naturally occurring bioactive compounds derived from plant screening programs assists the development of new synthetic drugs [
6]. As plant-derived drugs contain a pool of metabolites with potential complementary pharmacological actions, their use in mitigating chronic diseases through synergism is an area of intense interest [
7]. In this light, indigenous knowledge can help to contribute to the rational drug discovery and development of new drugs from medicinal plants [
8]. While indigenous communities typically have a rich knowledge of ethnic medicines, these uses are based on empirical evidence, and proper mechanistic knowledge of biological or pharmacological properties necessitates a scientifically sound investigation, followed by the documentation and characterization of bioactive components of the studied species [
9,
10]. Hence, proper research on medicinal plants is invaluable in the search for novel bioactive agents for the management of the disease. Cognizant of these principles, we selected the ethnomedicinal plant
Ophiorrhiza rugosa var.
prostrata for the present study.
Ophiorrhiza rugosa var.
prostrata (D.Don) Deb & Mondal (syn:
Ophiorrhiza harrisiana B.Heyne ex Hook.f, Ophiorrhiza prostrata D.Don) is an annual herb belonging to the Rubiaceae family, which naturally grows in Chittagong and both the Chittagong Hill Tract and Sylhet regions of Bangladesh, where it is variously known as ‘Jari’ or ‘kalashona’ (Chakma), ‘Jariphul’ (Tanchangya) or ‘Pahari mehedi’ (Marma).
O. rugosa var.
prostrata is used by the Tanchangya, Marma, and Chakma indigenous communities for the treatment of different diseases. For example, a paste of the leaves is used for the treatment of skin infections (boils) by the Tanchangya people. The Marma community prepares a tea from the leaves, which is drunk daily for the treatment of body aches and chest pain, while the Chakma community applies sun-dried crushed leaves to the ears for the treatment of earache (personal communication). In addition, the crushed roots of the plant are used for the treatment of dysentery [
11,
12]. Juice from the leaves is drunk in the treatment of diarrhea within the Marma community (personal communication). However, despite such widespread use, there has been no scientific investigation to date on either pharmacological or phytochemical aspects of the plant to validate its traditional uses. Therefore, we aimed to investigate the bioactive components of
O. rugosa var.
prostrata leaves using gas chromatography-mass spectrometry (GC-MS). As plants contain a mixture of phytochemicals, robust separation and identification methods are important to elucidate potential bioactive and toxic constituents [
13]. GC-MS, coupled with appropriate detection systems is an invaluable tool for the separation and identification of the components of complex, volatile mixtures [
14]. Many plant secondary metabolites are sufficiently small, adequately volatile, and thermostable in the GC environment to be easily analyzed by GC-MS [
15]. In addition to the phytochemical investigation, we aimed to investigate the known therapeutic applications of the plant through a combination of in vivo (antidiarrheal and anti-inflammatory), in vitro (anthelmintic and antibacterial) and in silico (molecular docking, ADME/T and PASS) analyses.
3. Discussion
Infectious and parasitic diseases continue to represent intimidating issues for developing countries, due to the lack of useful and safe drugs and the increasing resistance of pathogens to available antibiotics or anti-parasitic agents. A common manifestation of these issues is infectious diarrhea, attributable to both enteric bacterial pathogens and parasites [
16]. Such infectious agents may evoke not only adverse effects on intestinal functions but also increase systemic risk via compromising host immunity, leading to increased morbidity and mortality [
17]. To treat such infectious diseases, different plant parts, plant extracts, and plant-derived products have been used in traditional medicine. However, many of these traditional medicines have not been formally reported in the literature to date. Recent comprehensive reports on plants used for the treatment of infectious diseases, including diarrhea and dysentery have indicated their possible applications as alternative therapies [
18]. In Ethiopia for example, a range of medicinal plants including
Calpurnia aurea,
Croton marcostachyus, and
Echinops kebercho have been scientifically validated as anti-infective agents [
19]. In addition, combined screening of anti-diarrheal and anti-infective properties of medicinal plants could prove a valid strategy to identify novel therapeutics. A study conducted by
Taylor et al. 2013 suggested that plants demonstrating significant anti-bacterial activity against entero-pathogens could be considered as potential diarrheal treatments [
20]. In vitro and in vivo investigation of
Rhus plants including
Rhus semialata,
Rhus javanica, and
Rhus tripartitum produced significant anti-bacterial and antidiarrheal effects and the authors concluded that the presence of antibacterial agents might mediate the diarrhea prevention [
21,
22]. However, to recognize the intrinsic value of plant extracts, the involvement of both in vitro and in vivo approaches is important in the clinically search for effective anti-infective agents. Studies of plants with established ethnomedicinal uses must consider ethnomedicinal preparation practices when evaluating materials scientifically in the laboratory environment. Thorough extraction protocols are important to completely evaluate both therapeutic and toxicological potential of medicinal plants. Typically, plant phytochemicals possess diverse chemical functionalities, yet most are readily soluble in methanol or ethanol, due to their high extractability and high polarity. Many nonpolar compounds are also soluble in this solvent [
23,
24]. Therefore methanol and ethanol are frequently used for extraction of medicinal plants prior to evaluation of their therapeutic potential, and we selected ethanol for our extraction of
O. rugosa leaves, the most commonly used part of the plant. Our study identified potential novel active components from the ethanol extract of
Ophiorrhiza rugosa leaves (EEOR), having antidiarrheal, anti-inflammatory, anthelmintic and antibacterial properties.
To verify the ethnomedicinal uses of
Ophiorrhiza rugosa, we examined its antidiarrheal activity, as well as its possible mechanism(s) of action in different animal diarrheal models. In all diarrheal experiments, a high dose of the natural laxative castor oil (0.5 mL) was administered to each mouse. The active metabolite of the oil (ricinoleic acid) is liberated via the action of small intestinal lipases, thus altering the motility of gastrointestinal smooth muscle [
25,
26]. Upon binding of the metabolite with EP3 prostanoid receptors on smooth muscle cells, it inhibits water and electrolyte absorption from the intestine, resulting in accumulation of fluid and interruption of secretory functions, which in turn generates a deleterious effect in the intestine [
27,
28]. Apart from its laxative effect, ricinoleic acid causes intestinal dysfunction via local inflammation and stimulation of prostaglandin biosynthesis, which also inhibits reabsorption of ions and water [
29]. In all antidiarrheal assays, loperamide was used as a standard drug, which enhances the rate of absorption by reducing the volume and movement of intestinal contents [
30].
In castor oil-induced diarrhea, the ethanol extract of
O. rugosa produced a remarkable inhibitory effect, in terms of both defecation rate and diarrhea. The extract, at all doses (100, 200, 400 mg/kg) decreased the total number of feces at 1h intervals over 4h, while diarrheal feces were reduced, indicating an alteration of defecation frequency and consistency. Among all three doses of EEOR, 200 and 400 mg/kg significantly (
P < 0.001) reduced defecation numbers by 52.05% and 60.27% respectively, which indicates a dose-dependent antidiarrheal action. A dose of the extract with 400 mg/kg EEOR exhibited inhibition (62.50%) of diarrhea that was comparable to the standard drug loperamide (65.62%). This demonstrates that a relatively high dose of EEOR is required to evoke the desired response, and a similar phenomenon has been observed by similar studies on different plant species [
31].
The anti-enteropooling potential of EEOR was investigated to explore its antidiarrheal efficacy further and to aid mechanistic interpretation. Our results show that the extract markedly inhibited castor oil-induced enteropooling into the small intestine, likely through suppressing castor oil stimulated prostaglandin biosynthesis. All tested doses significantly decreased intraluminal fluid compared to the control, with the highest dose of 400 mg/kg decreasing both the volume by 32.29% (
P < 0.05) and weight of intestinal contents by 49.57% (
P < 0.001). These results confirm the antidiarrheal efficiency of our extract and are comparable with an analogous study conducted by Agbon et al. [
32].
To further characterize the effect of EEOR in reducing intestinal hypermotility, we investigated gastrointestinal motility using a charcoal meal tracer. We observed that the administration of the extract delayed the transit of the charcoal marker through the entire intestine. This inhibitory effect was seen with all doses employed and implies that an anti-motility action underlies the mechanism of action of the extract. Maximal inhibition of the peristaltic index was exhibited following a dose of 400 mg/kg (41.66%,
P < 0.001), and was equipotent with the standard drug loperamide (42.26%,
P < 0.001). Our findings suggest that the extract both decreases hypermotility and increases the transit time through the suppression of intestinal muscle spasm, thus extending the time for absorptive processes [
33].
As aforementioned, castor oil promotes prostaglandin biosynthesis, which leads to the release of various pro-inflammatory mediators, leading to inflammation and irritation. Non-steroidal anti-inflammatory drugs (NSAIDs) may prevent diarrhea through inhibition of castor oil stimulated prostaglandin synthesis [
34]. In this study, we assessed the anti-inflammatory activity of EEOR following histamine challenge. Histamine causes contraction of the smooth muscle of small intestine, uterus, bronchi, and bronchioles through activation of H1-receptors [
35]. The mechanism of the local inflammatory response induced by histamine is through the activation of vasodilation, edema formation, vascular permeability, and cytokine release. [
36]. Our results showed that EEOR significantly (
P < 0.001) suppressed histamine-induced paw edema, which provides evidence of a potential anti-inflammatory effect. EEOR may thus ameliorate an acute inflammatory response via inhibition of prostaglandins or other inflammatory mediators.
In the anthelmintic study, we utilized the aquatic worm
Tubifex tubifex, a species of aquatic oligochaete that is a suitable host for the
Myxobolus cerebralis parasite, responsible for whirling disease in salmonid fish [
37]. Our data revealed that exposure to EEOR dose-dependently reduced (
P < 0.001) both paralysis and death times of the worm, indicating the presence of a potential anthelmintic compound(s). The reference drug levamisole (a nicotinic receptor agonist) activates excitatory nicotinic acetylcholine (nACh) receptors on the muscle of the worm, causing paralysis and death [
38], and a similar mechanism may account for the anthelmintic action of EEOR.
We investigated the antimicrobial activity of EEOR through the disc diffusion method, and the extract induced a significant zone of inhibition against both
Bacillus subtilis (a model Gram-positive microorganism) and
Escherichia coli (Gram-negative microorganism) at concentrations of 500, 800 and 1000 μg/disc. The lowest concentration (500 μg/disc) failed to show activity against
Salmonella typhi (Gram-negative microorganism), but the other two concentrations exhibited significant antibacterial activity. These results indicate the existence of a broad-spectrum antibiotic effect of the plant extract and represent the first such data on the extract. On the other hand, we did not find any noticeable effect of our extracts on the Gram-positive
Staphylococcus aureus or
Bacillus cereus, or on the Gram-negative organisms
Salmonella paratyphi or
Pseudomonas aeruginosa, even at 1000 μg/disc. Broadly, our results suggest that EEOR constituents may interrupt general cellular functions or disrupt bacterial membrane potential [
39,
40].
Generally, plants are rich in secondary metabolites with diverse biological actions, acting as natural defense mechanisms against bacteria, insects, viruses, and fungi. Our preliminary phytochemical evaluation suggested a distinct phytoconstituent profile in EEOR. Among these, alkaloids, flavonoids, phenols, tannins, terpenoids, and saponins are commonly reported to possess both antibacterial and anthelmintic activities [
41,
42]. Reports on various plant extracts suggest that antidiarrheal effects may also be mediated through the action of saponins, tannins, steroids flavonoids and alkaloids [
43], whereas tannins and flavonoids are well known to aid reabsorption of intestinal fluids and electrolytes [
44]. Additionally, tannins reduce intestinal motility by inhibiting bowel irritation, thereby exhibiting an antidiarrheal effect [
45]. Various phytochemicals including flavonoids, steroids, and phenols have been ascribed anti-inflammatory actions [
46]. As EEOR showed significant anthelmintic and antibacterial activity, especially on certain entero-pathogenic (
Bacillus subtilis,
Salmonella typhi and
Escherichia coli) organisms, coupled with its observed effects on gut motility, this supports its possible utility in infectious diarrhea.
GC-MS analysis of EEOR identified a total of thirty different compounds. Based on the literature, thirteen of these have already been documented to be bioactive. Loliolide [
47], ethyl linoleate [
48], 2-palmitoylglycerol, and erucamide [
49] have been shown to possess antibacterial activity, while γ-sitosterol, stigmasterol, vitamin E, and squalene [
47] have both antibacterial and anti-inflammatory activities. Phytol and methyl palmitate have nematicidal, pesticidal, antibacterial, and anti-inflammatory activities. Notably, phytol is very active against
Salmonella typhi [
49]. Finally, neophytadiene [
50] and methyl linoleate [
47] have demonstrated anti-inflammatory activity.
Molecular docking studies have been widely used for the prediction of ligand-target interactions and to obtain better insights into the biological activity of natural products. It also gives additional clues about possible mechanisms of action and binding modes inside the binding pocket of various enzymes [
51]. In order to obtain better insight into the observed biological activity (antidiarrheal, anthelmintic, and antibacterial) of EEOR constituents, thirteen representative compounds within EEOR were selected for docking analyses. These compounds were then docked against four targets, namely the M3 muscarinic acetylcholine receptor (PDB ID: 4U14), the 5-HT3 receptor (PDB ID: 5AIN), tubulin (PDB ID: 1SA0) and GlcN-6-P synthase (PDB: 1XFF).
Molecular docking studies with the M3 muscarinic acetylcholine receptor (PDB ID: 4U14) revealed that, among the thirteen compounds, seven interacted with several amino acid residues through hydrogen bonds and π-π stacking interactions (Tyr529, Tyr533, Ile222, Leu225, Asn152, Ser151, Tyr148), with docking scores ranging between −2.00 and −8.80 kcal/mol. On the other hand, five compounds interacted with a number of amino acid residues (Ile116, Glu191, Arg57, Arg57, and Thr34) within the 5-HT3 receptor (PDB ID: 5AIN) with docking scores ranging from −0.69 to −5.47 kcal/mol. From these results, we can conclude that the studied phytoconstituents may in part be responsible for the antidiarrheal activity of EEOR through interaction with these target proteins.
In the anthelmintic docking study, the thirteen compounds were docked with tubulin (PDB ID: 1SA0) and showed docking scores ranging from −0.59 to −7.13 kcal/mol. From the results, it is clear that the phytoconstituent stigmasterol displayed the highest score against tubulin, followed by γ-sitosterol, vitamin E, ethyl linolenate, loliolide, erucamide, phytol, methyl linoleate, methyl palmitate, and neophytadiene. It has been previously reported that phytol and methyl palmitate possess nematicidal and pesticidal activities [
49], and the anthelmintic activity of EEOR may be related to these phytoconstituents. In the case of the antibacterial docking study, loliolide had the highest binding affinity towards the GlcN-6-P synthase enzyme (PDB: 1XFF), followed by ethyl linolenate, erucamide, 2-palmitoylglycerol, phytol, methyl linoleate, neophytadiene, methyl palmitate, and methyl stearate. The antibacterial activity of the EEOR may thus be explained by the presence of loliolide, ethyl linolenate, erucamide, 2-palmitoylglycerol, and phytol, which have good docking scores and for which bioactivity has previously been reported [
47,
48].
All bioactive compounds were further characterized using the online-based prediction program ADME analysis to explore their drug-likeness, pharmacokinetics and physiochemical characteristics. Almost all compounds, except for γ -sitosterol, squalene, stigmasterol and vitamin E exhibited orally active drug-likeness properties, according to Lipinski’s rule. It is reported that compounds with lower molecular weight, lipophilicity, and hydrogen bond capacity have high permeability [
52], good absorption and bioavailability [
53,
54]. However, this analysis does not assess if a compound has any particular pharmacological effect.
To predict a likely pharmacological profile of the compounds, we utilized the structure-based biological activity prediction program Prediction of Activity Spectra for Substances (PASS). The results suggested several activities, among these, we established probable activity values (Pa range 0.235–0.826) for all 13 compounds for anthelmintic, antibacterial, anti-inflammatory, spasmolytic and antiprotozoal actions, supporting our laboratory investigations of EEOR. Moreover, other activities were predicted, suggesting the broader potential of this species. In summary, our comprehensive analyses, utilizing complementary tools, support the traditional uses of EEOR. The observed effects may be due to the combined actions of several phytoconstituents, both those documented herein and potentially other as yet uncharacterized compounds.
4. Materials and Methods
4.1. Drugs and Chemicals
All drugs and chemicals used in this research were of analytical grade. Loperamide was obtained from Square Pharmaceuticals Ltd. (Dhaka, Bangladesh), levamisole from ACI Limited (Dhaka, Bangladesh), and castor oil from WELL’s Health Care (Madrid, Spain). Ethanol (Merck, Darmstadt, Germany), Kanamycin (Sigma Chemical Co., St. Louis, MO, USA) and histamine (BDH Chemicals Ltd. Poole, UK) were procured from the mentioned sources.
4.2. Chemical Compounds Studied in This Article
Loliolide (PubChem CID: 100332); Ethyl linolenate (PubChem CID: 6371716); Methyl linoleate (PubChem CID: 5284421); Erucamide (PubChem CID: 5365371); γ-Sitosterol (PubChem CID: 457801); 2-Palmitoylglycerol (PubChem CID: 123409); Methyl Palmitate (PubChem CID: 8181); Methyl stearate (PubChem CID: 8201); Neophytadiene (PubChem CID: 10446); Phytol (PubChem CID: 5280435); Squalene (PubChem CID: 638072); Stigmasterol (PubChem CID: 5280794); Vitamin E (PubChem CID: 14985).
4.3. Plant Collection, Identification, and Extraction
The leaves of Ophiorrhiza rugosa var. prostrata (D.Don) Deb & Mondal were collected in September 2017 from Kaptai National Park (22°30′08″N 92°12′04″E), Rangamati District, Chittagong Division, Bangladesh. The plant was certified and authenticated by Dr. Shaikh Bokhtear Uddin, Professor, Ethno-botany and Pharmacognosy Lab, Department of Botany, University of Chittagong, Bangladesh, with a voucher specimen (accession no: 7609 CTGUH) deposited in the Herbarium of the University of Chittagong (CTGUH). After subsequent washing with normal and distilled water, the collected leaves were cut and oven-dried for a week at constant temperature (50 °C), before milling into a coarse powder using an automatic grinder. Then, the fine powder (350 g) was soaked in 850 mL of ethanol for seven days at room temperature, with regular shaking and stirring on a shaker machine (model VTRS-1, Nunes Instruments, Tamil Nadu, India). After 7 days, the macerate was filtered through a sterilized cotton plug followed by Whatman filter paper No. 1, and the eluting solvent evaporated on a rotary evaporator (RE 200, Sterling, Norman Way Industrial Estate, Cambridge, UK) at room temperature to afford a semisolid extract (EEOR: 10 g), which was kept in a refrigerator (−4 °C) until further use.
4.4. Animals and Ethical Statements
Adult Swiss albino mice (20–25 g) of both sexes were obtained from Jahangir Nagar University, Savar, Dhaka, Bangladesh. The animals were housed in polypropylene cages for adaptation, under standard laboratory conditions (room temperature 25 ± 2 °C; relative humidity 55–60%, 12 h light/dark cycle), with food pellets and water ad libitum. All animals were acclimatized for 2 weeks and fasted overnight before starting all experiments. This experiment was designed based on the Ethical Principles and Guidelines guided by The Swiss Academy of Medical Sciences and the Swiss Academy of Sciences. All tests were run in a remote and noiseless ambiance, between 9.00 a.m. and 5.00 p.m. The study protocol was approved by both the Ethical review committee and the P&D committee of the Department of Pharmacy, International Islamic University Chittagong, Bangladesh under the code Pharm-P&D-61/08′16-122.
4.5. GC-MS (Gas Chromatography-Mass Spectroscopy) Analysis of EEOR
GC-MS analysis of EEOR was evaluated using a model 7890A capillary gas chromatograph along with a mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The column was a fused silica capillary column of 95% dimethyl-poly-siloxane and 5% phenyl (HP-5MSI; length: 90 m, diameter: 0.250 mm and film: 0.25 µm). Parameters for GC-MS detection were an injector temperature of 250 °C, an initial oven temperature of 90 °C gradually raised to 200 °C at a speed of 3 °C/min for 2 min and with a final increase to 280 °C at 15 °C/min for 2 min. The total GC-MS run time was 36 min, using 99.999% helium as a carrier gas, at a column flow rate of 1 mL/min. The GC to MS interface temperature was fixed at 280 °C, and an electron ionization system was set on the MS in scan mode. The mass range evaluated was 50–550 m/z, where MS quad and source temperatures were maintained at 150 °C and 230 °C respectively. The NIST-MS Library 2009 was used to search and identify each component, and to measure the relative percentage of each compound, relative peak areas of the TIC (total ionic chromatogram) were used, with calculations performed automatically.
4.6. Acute Toxicity Testing of EEOR
Acute toxicity testing was assessed under standard laboratory conditions following OECD guidelines [
55]. Animals (
n = 6) of the control and test groups were each administered 1% Tween-80 or a single oral dose (5, 50, 100, 200, 400, 1000 and 2000 mg/kg body weight) of the test extract (EEOR). Before administration of extract, mice were kept fasting overnight, and food was also delayed for 3 to 4 h after receiving the extract. All experimental animals were observed individually, paying particular attention to any unexpected responses, including behavioral changes, allergic syndromes (itching, skin rash), and mortality over the next 72 h.
4.7. Qualitative Phytochemical Screening of EEOR
Qualitative phytochemical analysis of EEOR was carried out following standard procedures, as previously reported by Tiwari et al. [
56].
4.8. Antidiarrheal Activity Evaluation of EEOR (In Vivo)
4.8.1. Castor Oil-Induced Diarrhea
The conditions of Awouters et al. [
34] were followed with slight modifications. Mice were fasted overnight prior to the experiment. Experimental animals were separated randomly into control and test groups consisting of 6 mice in each category. Group-I served as a negative control, and received 1% Tween-80 in distilled water; group-II (positive control) received loperamide (5 mg/kg BW; p.o), while test groups III-V were treated with EEOR (100, 200 and 400mg/kg BW; p.o) respectively. After 1 h, each mouse was put into an individual cage and diarrhea induced (0.5 mL castor oil, p.o). Blotting paper on the floor of each cage was monitored to observe both the number and consistency of fecal droppings. Blotting papers were replaced every 60 min during the 4h observation period. The total numbers of both dry and wet feces excreted by the animals were counted. The following equation was used to calculate percent inhibition of diarrhea:
4.8.2. Castor Oil-Induced Enteropooling
Intraluminal fluid accumulation was evaluated by the method described by Robert et al. [
57]. Dosing treatments were as for castor oil-induced diarrheal testing, again with six animals per group. One hour post administrations of each test dose, animals were treated with castor oil (0.5 mL) to induce diarrhea. Two hours later, the mice were sacrificed, and the small intestine was isolated from pyloric sphincter to caecum. The small intestine was weighed (g) and the volume of intestinal contents (ml) was measured by milking into a graduated tube. The intestine was reweighed, and the differences between full and empty intestines were calculated. To calculate, the percentage volume and weight of intestinal contents were determined using the following formula:
4.8.3. Gastrointestinal Motility
This experiment was performed based on the method of Mascolo et al. [
58], with the treatment of animals of each group (
n = 6) as described in the castor oil-induced diarrhea test. In brief, 0.5 mL of castor oil was administered to each animal to induce diarrhea. One hour after administration of each test dose, animals were treated orally with 1 mL of a charcoal meal (10% charcoal suspension in 5% gum acacia). After 1 h, animals were sacrificed and the distance traveled by the charcoal meal from the pylorus to caecum was measured (cm) and expressed as a percentage of the total distance of the intestine. The following formulae were used to express the percentage of inhibition and Peristalsis index:
4.9. Histamine-Induced Paw Edema
The anti-inflammatory activity of EEOR was evaluated following injection of histamine into the plantar surface of the mouse hind paw [
59]. Animals were divided into four groups (
n = 6); Group I (negative control) received 1% Tween-80 (2 mL/kg); Group II (Positive control) received diclofenac sodium (10 mg/kg BW; p.o); and Groups III and IV received EEOR (200 and 400 mg/kg BW; p.o) respectively. 30 min following treatment, 0.05 mL histamine (1 mg/kg, in 1% Tween-80 with D.W) was injected in the sub-plantar area of the right paw of each mouse to induce acute inflammation, and micrometer slide calipers were used to measure the paw volume at 1, 2, 3 and 4 h. The percentage inhibition of the inflammatory effect of the extract was calculated using the following expression:
4.10. Anthelmintic Activity of EEOR (In Vitro)
Anthelmintic activity was assessed following the method of Ajaiyeoba et al. with slight modifications [
60,
61]. In this experiment, the sludge worm, or sewage worm (
Tubifex tubifex, size: 2 to 2.5 cm in length), was used for its physiological and anatomical relevance to intestinal worms, e.g., Annelida. Testing was performed in triplicate. In brief, 5 to 10 worms were randomly placed in each Petri dish, divided into four groups (I, II, III and IV). To each, 3 mL of either EEOR at a specified concentration (5, 8 or 10 mg/mL) or the standard drug levamisole (1 mg/mL) added. Anthelmintic activity was calculated at two different stages, namely ‘time of paralysis’ and ‘time of death’ of the worms. Time to paralysis was counted as the time when worms lost their natural movement. The time of death was recorded after confirming that the worms moved neither when vigorously shaken nor when dipped in slightly warm water.
4.11. Antibacterial Activity of EEOR (In Vitro)
The antibacterial effect of EEOR was evaluated by the disc diffusion technique [
62]. Prepared Nutrient agar was placed into Petri dishes under laminar airflow for solidification. Overnight cultures of Gram-positive
Bacillus subtilis (ATCC 6633),
Staphylococcus aureus (ATCC 6538) and
Bacillus cereus (ATCC 14579) and Gram-negative
Salmonella typhi (ATCC 29629),
Salmonella paratyphi (ATCC 9150),
Escherichia coli (ATCC 8739) and
Pseudomonas aeruginosa (ATCC 9027) organisms were each prepared with 100 µL bacteria (bacterial inocula were adjusted to 10
8 CFU/mL), spread smoothly on the agar surface. Dry sterile discs (6mm diameter) were laid upon the seeded agar plate using a sterile forceps. Each desired concentration of EEOR (500, 800 or 1000 μg) was loaded on these discs and then incubated (at 37 °C for 24 h). The diameter of each zone of inhibition was recorded and measured in mm. As a positive control, kanamycin (30 μg/disc) was used.
4.12. In silico Molecular Docking
The major bioactive compounds of EEOR, as detected by GC-MS, were selected for molecular docking studies, to understand better possible molecular interactions based on their affinity to interact with different target proteins. Docking studies were performed using the Schrödinger suite-Maestro v10.1, LLC, New York, NY, USA, and Accelrys Discovery Studio 4.0 software (BIOVIA, San Diego, CA, USA) was used for visualization of 3D structures.
4.12.1. Ligand Preparation
The structures of thirteen major compounds were obtained from the PubChem compound repository, and the ligands prepared using the LigPrep tool embedded in Maestro v 10.1 (Schrödinger suite, LLC New York, NY, USA), neutralized at pH 7.0 ± 2.0 using Epik 2.2, and minimized by force field OPLS_2005.
4.12.2. Receptor Preparation
3D crystal structures of the proteins used for the test were downloaded from the Protein Data Bank; RCSB PDB [
63], GlcN-6-P synthase (PDB ID: 1XFF) [
64], tubulin (PDB ID: 1SA0) [
65] 5-HT3 receptor (PDB ID: 5AIN) [
66] and M3 muscarinic acetylcholine receptor (PDB ID: 4U14) [
67]. The Protein Preparation Wizard of the Schrödinger suite-Maestro version 10.1 was used to prepare and refine the crystal structures. Charges and bond orders were assigned, hydrogens added to heavy atoms and selenomethionines and selenocysteines converted into methionines and cysteines respectively, followed by removing all water molecules. Using force field OPLS_2005, minimization was performed to set a maximum heavy atom RMSD to 0.30 Å.
4.12.3. Grid Generation and Molecular Docking
Receptor grid generation and molecular docking experiments were performed using Glide (Schrödinger suite-Maestro version 10.1) [
68,
69] For each protein, a grid was produced using the following default parameters: van der Waals scaling factor 1.00 and charge cut-off value 0.25, subjected to the OPLS_2005 force field. A cubic box of definite dimensions centered on the centroid of the active site residues was generated for the receptor, and the box size was set to 14 Å × 14 Å × 14 Å for docking. Docking experiments were carried out using the Standard Precision (SP) scoring function of Glide, and only the best scoring fit with docking score was noted for each ligand.
4.13. In Silico ADME Analysis
The pharmacokinetic properties of all major identified compounds were evaluated using Lipinski’s rule of five [
70]. Lipinski stated that a compound could show drug-like behavior if it does not fail more than one of the following criteria: (i) molecular weight not more than 500; (ii) H-bond donors ≤5; (iii) H-bond acceptors ≤10; (iv) Lipophilicity <5; and (v) molar refractivity between 40 and 130. The web tool Swiss ADME [
71] was used to assess the ADME parameters of all compounds. Compounds which obey Lipinski rule are considered as ideal drug candidates.
4.14. In Silico PASS Prediction
Possible biological activities of identified major compounds were evaluated using the online computer program PASS (Prediction of Activity Spectra for Substances) [
72]. This tool predicts up to 3750 biological properties of a compound, associated with an analysis of its chemical structure. The outcomes of this analysis were denoted as Pa (probable activity) and Pi (probable inactivity), where the values of both Pa and Pi may differ from 0.000 to 1.000. We considered values of P
a > P
i and Pa > 0.700 to indicate biological activity for a compound [
73].