1. Introduction
Constipation is a chronic disorder of the gastrointestinal tract (GIT) and is defined as bowel movements of fewer than three times per week. Clinically, constipation manifests with heterogeneous symptoms including difficulty in defecation, infrequent bowel movements, hard bowel, and the feeling of incomplete defecation [
1]. Constipation is equally prevalent across the globe and affects all age groups almost equally. According to one estimate, constipation is reported in 2.5–39.6% of the world’s adult population [
2], while in infants to adolescents, the prevalence is in the range of 0.5–26.9% [
3]. Common contributing factors to constipation include insufficient dietary fiber and fluid intake, adverse effects of medications, decreased physical activity, hypothyroidism, and colorectal cancer-induced obstruction, which can be broadly attributed to either genetic predisposition or the socioeconomic status of the patients [
4]. Different drugs are applied to improve complications of constipation, including Correctol, Senna, Exlax, and Gaviscon, but with limited benefits due to the associated adverse effects and treatment costs [
5,
6]. The primary approach to treat constipation involves regulation of GIT motility, for which several drugs have been introduced. For instance, cisapride was among the first developed prokinetic agents but was withdrawn later due it to its effect on increasing cardiac arrhythmias [
7]. Another noteworthy example is a selective 5-hydroxytrptamine-4 (5-HT
4) receptor antagonist, tegaserod, that is currently in practice to diminish constipation despite its role in causing coronary artery diseases and myocardial infarction [
8,
9].
The plant kingdom has served as a remarkable resource for extracts exhibiting prokinetic activity that diminish characteristics of constipation by improving intestinal motility, defecation frequency, and stool weight. Leaf extract from the perineal plant
Ecklonia cava showed a prominent laxative effect on the loperamide-induced constipation model in SD rates in terms of stool recovery and GIT motility. This study confirmed that the underlying principle involved facilitation of GIT hormone secretion and augmenting the downstream signaling pathway of local muscarinic acetylcholine receptors (mAChRs) [
10]. Leaf extract of
Malva sylvestris has been shown to attenuate constipation induced in male Wistar rats by increasing gastric emptying and decreasing intestinal transit time via its stimulatory effect on mAChRs [
11]. In addition to intestinal transit length, a significant increase in stool frequency, weight, and water content was also observed as a result of
Ficus carica treatment [
12]. Reports such as these have encouraged many researchers to investigate medicinal plants to look for therapeutic alternatives that offer no or fewer adverse effects compared to currently available drugs.
Chrozophora tinctoria, commonly known as ‘dyer’s croton’ or ‘turnsole’, belongs to the family Euphorbiaceae. It is an annual plant found in Africa, Europe, and Asia. In Pakistan, it is found in tropical and temperate regions [
13].
Chrozophora species are traditionally used to cure mouth ulcers, skin disorders such as skin burns, fever, abdominal and joint pain, jaundice, menstrual problems, wounds, GIT worms, and migraine. Moreover,
Chrozophora tinctoria is used as an emetic and cathartic and to treat warts [
14]. In Ethiopia and Senegal, the seeds and leaves of the
Chrozophora species are used as a laxative. In Nepal, the juice obtained from its fruit is used as a remedy for cough and cold [
15]. The antibacterial activity of
Chrozophora tinctoria leaves and stem was established by earlier workers [
16]. In addition,
Chrozophora tinctoria is used for producing natural dye [
17].
The presence of arabinose, fructose, glucose, raffinose, sucrose, and ribose in
Chrozophora tinctoria has been confirmed by HPLC [
18]. It has been reported that flavonoids are abundantly prevalent in almost all species of the genus
Chrozophora [
19,
20,
21]. HPLC analysis of the methanolic extract of the aerial parts of
Chrozophora tinctoria reported five flavonoid glycosides. These five flavonoid glycosides are apigenin 7-O-b-d-glucopyranoside, apigenin 7-O-b-d-[(6-pcoumaroyl)]-glucopyranoside, acacetin 7-O-rutinoside, quercetin 3-O-rutinoside, and apigenin 7-O-b-d-[6-(3,4dihydroxybenzoyl)]-glucopyranoside (
Chrozophorine) [
13]. Most of these flavonoids have therapeutic properties and especially antiviral activity [
22,
23]. Other compounds extracted from the ethanolic extract of
Chrozophora tinctoria are kaempferol, kaempferol 3-O-(600-a-rhamnopyranosyl)-b-glucopyranoside, kaempferol 3-O-b-glucopyranoside, two phenolics, namely, methyl gallate and gallic acid, and one steroid, namely, b-sitosterol-3-O-b-glucopyranoside. There are 35 different flavonoids found in various species of the genus
Chrozophora, which have anti-proliferative, antioxidant, antimicrobial, antipyretic, and anti-nociceptive properties [
24]. The leaves of
C. tinctoria possess antidiabetic and hepatoprotective properties [
25]. Rutin present in
Chrozophora has the ability to promote bone cell growth [
26]. Here, we used gas chromatography-mass spectrometry to investigate the phytochemical profile of fractions taken from
Chrozophora tinctoria and evaluate their laxative activity as novel treatment options for constipation.
3. Discussion
Constipation is a worldwide problem that is equally prevalent in all age groups. Different pharmacological and non-pharmacological paradigms are available for relieving the symptoms of constipation. Among the pharmacological approaches, various allopathic and herbal-based therapeutic options are practiced. Even though the allopathic system offers various synthetic and semisynthetic molecules for the treatment of constipation, numerous side effects are attributed to those approaches. Therefore, the search for a safe, effective, and affordable therapeutic agent for constipation is a challenge to current medical science. In this study, the prokinetic effect of Chrozophora tinctoria fractions was investigated to establish pharmacological grounds for the potential use of these fractions in constipation therapy.
AChE is a key enzyme found in the blood and nervous system, mediating various important physiological functions. The principal role of this enzyme is the cessation of nerve impulses at cholinergic synapses by breaking down the neurotransmitter acetylcholine into choline and acetic acid. The inhibition of acetylcholinesterase is a promising strategy against Parkinson disease, myasthenia gravis, ataxia, senile dementia, and Alzheimer disease [
27]. Phytochemicals derived from other plants have shown inhibitory action against acetylcholinesterase [
28]. The EAC fraction of
Chrozophora tinctoria significantly inhibited AchE, with an IC
50 of 10 µg/mL. AChE inhibition implies blockade of ACh degradation, resulting in a higher concentration of ACh. This availability of ACh will result in a prolonged interaction with the muscarinic and nicotinic receptors, maintaining the impulse. Ach is bioavailable in various conformational isomers (gauche and anti-gauche forms), which is the reason behind its interaction with both cholinergic receptors. The cholinergic response will promote diarrhea through its intracellular signaling pathways. The laxative effect of
Chrozophora tinctoria might be related to the cholinergic action of the tested fractions. The laxative activity of both fractions was studied in metronidazole- (7 mg/kg) and loperamide hydrochloride- (4 mg/kg) induced constipated pigeons. The prokinetic potential of the tested fractions was attenuated by loperamide more significantly than metronidazole. The prokinetic effect of the
Chrozophora tinctoria fractions was further confirmed by the charcoal meal propulsion in the intestine of pigeons. Both of the fractions demonstrated significant (
p ≤ 0.05) propulsion of charcoal in the animal intestine.
Contraction of intestinal smooth muscle results from periodic depolarization. Thus, depolarization in intestinal smooth muscle is usually attributed to the release of calcium ions, either from intracellular stores or through the influx of calcium to the inside of the tissue through voltage-gated calcium channels. KCL in high doses is considered a depolarizer, which may trigger cellular calcium influx. Therefore, a relaxing effect in KCL-induced contraction usually indicates that the test substance induces calcium channel blockade [
29]. This experiment was conducted to investigate the direct effects of EAC and DCMC on rabbit jejunum. The antispasmodic effect of EAC on the smooth muscles of the jejunum was detected at a concentration of 1 mg/mL. Similarly, the spasm induced in rabbit’s jejunum by KCL (80 mM) was relaxed by EAC completely at 10 mg/mL. Similarly, at a concentration of 3 mg/kg the DCMC exhibited a relaxing effect on KCL-induced (80 mM) contraction in rabbit’s jejunum with an EC
50 value of 5.04 ± 0.05 mg/kg. It was deduced that the relaxing effect with EC
50 2.11 ± 1.20 mg/mL of EAC and an EC
50 value of 5.04 ± 0.05 mg/kg of DCMC may have been due to the inhibition of voltage-gated calcium channels. Furthermore, it also implies that EAC was more potent than DCMC. The tested fractions were also investigated for acute toxicity on pigeons. The animal model used in this study is considered a paradigm for the induction of emesis [
30], as compared to other emesis models, such as mice and rats, which are usually not as responsive. The acute toxicity study confirmed that the plant fractions used in this study induce emesis, a potential side effect of EAC and DCMC. Strikingly, the tested plant fractions are also anthelmintic as dead worms were observed in the stool passed by animals used in this study. The spasmolytic effect can be a contributing factor to the anthelmintic action of
Chrozophora tinctoria. A further molecular-level study is needed to elaborate on the underlying mechanisms of the EAC and DCMC fractions.
4. Materials and Methods
4.1. Chemicals and Solvents
The chemicals/drugs used in the present study were Maxolon® (Valeant Pharmaceutical International, Inc., Karachi, Pakistan), Gravinate® (The Searle company (PVT) LTD, Karachi, Pakistan), Imodium® (ASPIN Pharma PVT. LTD, Karachi, Pakistan), Flagyl® (Sanofi Aventis (PVT) LTD, Pakistan), Copper Sulphate (Nenza pharmaceuticals (PVT) LTD, Peshawar, KPK, Pakistan), Cisplatin (Pfizer Laboratories LTD, Karachi, Pakistan), Magnesium Sulphate (Nenza Pharmaceuticals (PVT) LTD, Pakistan), castor oil (Karachi Pharmaceuticals Laboratory, Karachi, Pakistan), gum acacia (Shreeji Pharma International), normal saline (Shahzeb Pharmaceutical, Haripur, KPK, Pakistan), methanol (Master Chemical Supplier, Karachi, Pakistan), and ethyl acetate (Master Chemical Supplier, Karachi, Pakistan).
4.2. Instruments
A rotary evaporator (Model RE-111, Bochi, Switzerland), 5-cc, 3-cc, and 1-cc syringes (Shifa disposable syringe), a drip set (Shifa drip set, Pakistan), water bath (Thermostatic controlled-STD/GMP), magnetic stirrer (H3760-S Digital magnetic stirrer), and analytical balance (Shimadzo analytical balance) were used.
4.3. Plant Collection and Identification
Mature Chrozophora tinctoria was collected from the Mohmand Agency, Khyber Pakhtunkhwa, Pakistan, and was authenticated by Dr. Sher Wali, Assistant Professor, Department of Botany, Islamia College Peshawar. The identified plant specimen was given a voucher number (CT-Bot-11082017) and deposited in the Herbarium, Department of Botany, Islamia College Peshawar. The collected plant was washed with clean water to remove dirt, and was dried at room temperature by spreading it in a single layer on blotting paper. After drying, the plant was cut into fine pieces and was powdered using an electric grinder.
4.4. Extraction and Fractionation
Extraction and fractionation of the desired fractions were performed according to the literature with some modifications [
31,
32]. Maceration in methanol was carried out for a total of 10 kg of powdered plant with random shaking for several days followed by filtration through Whatmann filter paper No 1. The filtrate was concentrated by a rotary evaporator to obtain a crude methanolic extract. The dried crude methanolic extract was dissolved in distilled water and then n-hexane was added. After shaking in a separating funnel, the n-hexane fraction was obtained and concentrated by a rotary evaporator. Similarly, dichloromethane, ethyl acetate, and n-butanol fractions were obtained. This method is called solvent–solvent fractionation. Their order of fractionation was from n-hexane then dichloromethane, ethyl acetate, and n-butanol, respectively. DCMC and EAC fractions were used for further activities [
31,
32].
4.5. Gas Chromatography-Mass Spectrometry (GC-MS)
To identify different phytochemicals present in EAC and DCMC fractions, the samples were subjected to GC-MS. The samples were checked using a Thermo Scientific (DSQ-II) GC. The GC device was furnished with a 30-m-long TR-5MS capillary column and a 0.25-µm-thick film and had 0.25 mm of internal diameter. Helium was used as a carrier gas with a flow rate of 1 mL/min. The injection device was run in a split mode at 250 °C. The sample was injected, 1 µL at a time, with an initial oven temperature of 50 °C that was maintained for 2 min followed by gradually elevating the temperature to 150 °C at a rate of 8 °C/min. Finally, the temperature was raised to 300 °C at a rate of 15 °C/min and maintained for 5 min [
33,
34].
4.6. Pharmacological Activities
4.6.1. Animals
Pigeons
Healthy pigeons were defined as those with normal stools (non-diarrheal and non-emetic), that were non-lethargic, and had no weight loss, no nasal dropping, no shedding or feather ruffling, and usual flying movement with regular feeding. Mature pigeons of both sexes were selected with weights in the range of 240–380 g. Animals were provided with standard food (locally available food; millet + wheat grains), fresh water, and a light/dark cycle for 12/12 h for 5–7 days. On the day of the experiment, the pigeons were weighed and health was assessed. Pigeons observed as unhealthy were removed from the experiment. After selection, animals were placed in separate cages to get individual data of each group [
35]. Pigeons were held gently, and fractions were given orally by a feeding tube with the help of an assistant.
Rabbits
Healthy rabbits were defined as being active and alert with no wet nose, runny eyes, scabby ears, or sore spots on their feet, with normal feeding and feces. Mature male rabbits weighing 1.5–3 kg were selected for the experiment. Rabbits were served with standard food and fresh water for several days. On the day of the experiment, the rabbits were examined again and the healthy ones were subjected to cervical dislocation [
36]. The abdomen of the animal was opened after cervical dislocation and about 1.5–2.5-cm slices of jejunum were detached and placed in Tyrode solution in Petri dishes with a continuous supply of carbogen gas (95% O
2 and 5% CO
2). The jejunum was then used for further experiments.
4.6.2. In Vitro Experiments
Acetylcholinesterase Assay
Increasing concentrations of EAC (125, 250, 500, 1000 µg/mL) and DCMC fractions were tested for AChE using a spectrophotometer according to the method of Ellman [
37]. This method for assaying thiols is based on the principle that acetylthiocholine iodide is hydrolyzed by the respective enzyme-producing thiocholine, which reacts with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) (Ellman’s reagent). The final products of the second reaction are 5-thio-2 nitrobenzoate and 2-nitrobenzoate-5-mercaptothiocholine. The absorption of the former product is measured by a spectrophotometer (412 nm). The positive control, galantamine, was prepared in the same concentrations as EAC. The test and control solutions were incubated at 37 °C for 20 min. The enzyme inhibition was calculated from the absorption rate with a change in time [
37,
38].
The percent inhibition was calculated as
where (V
max) is an enzyme activity in the absence of an inhibitor.
4.6.3. In Vivo Experiments
Acute Toxicity
Acute toxicity of EAC and DCMC was established according to an available protocol with slight modifications [
39]. The pigeons were divided into two groups (n = 8). One group received a fraction while the negative control group received distilled water only (6 mL/kg, PO). Each fraction was administered orally with 0.3, 0.5, 1, 2, 3, 4, and 5 g/kg as a single dose using a feeding tube to different groups. All the animals were observed for toxic symptoms, i.e., diarrhea, emesis, lethargy, and motility, for about 72 h [
39].
Laxative Activity
Pigeons were divided into eight groups (n = 8). A white, plastic base was provided in the cages for stool collection and examination. Groups 1, 2, and 3 were administered distilled water and 1-g, 2-g, and 3-g doses of the fraction, respectively. Group 4 was given distilled water (6 mL/kg) and group 5, castor oil (6 mL/kg P.O). Groups 6, 7, and 8 were given 1 g, 2 g, and 3 g of the fractions, respectively. Constipation was induced in all the groups except groups 1, 2, and 3s by administering metronidazole (7 mg/kg) and loperamide hydrochloride (6 mg/kg). After 30 min, the first stool time/latency time (min), number of stools, number of wet stools, and weight of stool (g) were recorded and the percent effect was calculated as follows:
Charcoal Meal Treatment
Pigeons were divided into five groups (n = 8). Constipation was induced in all the groups by loperamide hydrochloride. Group 1 was given distilled water (2 mL) and group 2 was served with castor oil while groups 3, 4, and 5 received fractions with a concentration of 25, 50, and 100 mg/kg, respectively. After 30 min, 2 mL of charcoal meal (a solution of 10% charcoal and 5% gum acacia) was given orally to each pigeon. The animals were then provided food and water, and, after 30 min, they were sacrificed. Then, the whole intestine, starting from the pylorus region up to the ileocecal junction, was removed from the pigeons and was placed on white paper parallel to a ruler. The distance travelled by the charcoal marker was measured and expressed as percent intestinal transit [
40]. The percent effect was calculated as follows:
Spasmolytic Activity
The spasmolytic activity of the fractions was assessed according to earlier reported studies [
36]. Briefly, the abdomen of the rabbit was opened after cervical dislocation and about 1.5–2.5-cm slices of jejunum were detached and placed in Tyrode solution in Petri dishes with a continuous supply of carbogen gas (95% O
2 and 5% CO
2). The mesentery was removed from the separated jejunum tissue and was fixed in the organ bath containing Tyrode solution at a retained temperature of 37 ± 1 °C. After this, the tissue was stabilized in the organ bath for about 20–30 min. The stable tissue with spontaneous response was taken as a baseline control, which is a positive control. The relaxing effect of the tested plant extract was compared.
Then, the effect of the fractions on spontaneous activity of the jejunum preparation was carried out at different concentrations, i.e., 0.01, 0.03, 0.1, 0.3, 3.0, 5.0, and 10 mg/mL, at an interval of 1 to 2 min in a cumulative manner, and the effect was noted. The effect of the fractions was also tested against KCL-induced contraction.
4.7. Ethical Approval
The study was approved by the ethical board of the Department of Pharmacy, Abdul Wali Khan University, Mardan, Pakistan. The ethical approval no. is EC/PhM/AWKUM-871D.
4.8. Statistical Analysis
Data are expressed as the mean. One-way ANOVA followed by Dunnett’s test was applied. The concentration–response curve was plotted using Graph Pad Prism for Windows 6.0 (Graph Pad Software, San Diego, CA, USA).