Journal of Pharmaceutical Research

INTRODUCTION

The gastrointestinal (GI) system consists of the digestive tract and a series of accessory organs. The tract extends from the oral cavity through the pharynx, oesophagus, stomach, small intestine, large intestine, and terminates at the anal canal. The accessory organs include the teeth and the tongue, as well as glandular organs such as the salivary glands, liver, gallbladder, and pancreas [1].

Together, these components perform essential physiological functions: ingestion, digestion, absorption of vital nutrients, secretion of necessary enzymes and fluids, and ultimately Excretion of undigested food remnants from the body [2]. The gastrointestinal tract (GIT) relies on a precisely regulated sequence of muscular movements - segmental and peristaltic contractions - to perform its functions effectively [3]. 

The motor functions of the gastrointestinal tract are facilitated by two layers of smooth muscle within its wall: an inner circular layer and an outer longitudinal layer. These layers work together to generate coordinated contractions that enable the movement of contents through the digestive system [4]. This motility pattern in the small intestine is predominantly controlled by the enteric nervous system, which coordinates local mixing and propulsive movements essential for digestion and nutrient absorption., with neurotransmitters like acetylcholine playing a key excitatory role in stimulating smooth muscle contraction [5]. Disruptions in these processes underlie a variety of gastrointestinal disorders [6].

The pharmacological management of gastrointestinal disorders is often accompanied by a range of adverse effects. In response to these concerns, there has been growing patient interest in herbal remedies [7].

Across civilizations, herbal remedies have served as the cornerstone of therapeutic practice, evolving through empirical knowledge inherited from ancestors. These traditional systems, rooted in the medicinal flora of each region, continue to inform modern approaches to natural and integrative healthcare [8].

Many plants have been studied and reported to have the potential to treat gastrointestinal disorders. For example, Radix aucklandiae, Zygophyllum gaetulum, Prangos ferulacea, Allium elburzense, Plectranthus barbatus, Thymus vulgaris, Artemisia vulgaris, Matricaria recutita, Galphimia glauca, Allium cepa, Eucalyptus camaldulensis, Viburnum prunifolium, Phyllanthus emblica and Acorus calamus were found to have antispasmodic property [9-23].

It has been observed that these plants and herbs contain bioactive compounds, including flavonoids and tannins, which exert therapeutic impact on the human body.

Urtica dioica, commonly known as stinging nettle, is a perennial herb renowned for its wide range of medicinal and nutritional properties. It is from the Urticaceae family, long valued for both its nutritional and medicinal properties. Traditionally, the roots have been used as diuretics, astringents, anthelmintics and in the treatment of ailments such as cough, cold, jaundice, and asthma. Additionally, leaf paste is traditionally used to treat diarrhoea and dysentery [24].

Phytochemicals, including flavonoids, phenolic acids, amino acids, carotenoids, organic acids, and fatty acids are reported from the different plant parts of Urtica dioica [25]. The different flavonoids present in stinging nettle such as apigenin, luteolin and quercetin have been proven to have antispasmodic property [24, 26].

Hence, the aim of this study is to determine antispasmodic effects of Urtica dioica on spontaneous motility and contractility of the smooth muscle preparation of chicken in vitro. 

MATERIALS AND METHODS

Chemicals: 

All chemicals used were of analytical grade and purchased from standard companies. All the ingredients of solution were prepared freshly before the experiment.

Plant collection:

Urtica dioica leaves were collected from Lingmoo, South Sikkim. After the collection, they were washed and shade dried for 2 weeks. The leaves were then cut into small pieces and was grinded by using grinder. Then, they were prepared for extraction process ([Fig. 1]).

Preparation of extract: 

The leaves were air-dried in a shaded area to preserve their bioactive compounds for about 30 days and processed by grinding into fine powder in a blender. The powder was extracted using maceration with ethanol. For this purpose, 100 g of powder was macerated in 1 l of ethanol (70% the volume of the fraction) for three days with occasional stirring and the solution was then filtered using filter paper (Whatman No. 1) and then concentrated in a rotary evaporator at 40°C and stored at –20°C in the refrigerator for subsequent evaluation of anticholinergic activity [27].

Phytochemical screening:

Preliminary phytochemical screening was performed for alkaloids, flavonoids, glycosides, terpenoids, proteins, amino acids, saponins, volatile oils, carbohydrates and tannins  by standard procedures [28, 29].

Pharmacological test: 

Segments of healthy chicken ileum were freshly collected from a local slaughterhouse. The ileum is the small intestinal segment extending from Meckel’s diverticulum to the ileo–caecal junction where the paired ceca begin. Terminal portions measuring approximately 1–1.5 cm in length were dissected and transferred into 30 mL organ baths containing Tyrode’s solution (NaCl, 40; KCl, 1; MgCl2, 5; NaH2PO4, 0.25; CaCl2, 1; NaHCO3, 5; glucose, 10).

The chicken ileum was maintained in Tyrode solution to preserve tissue viability during the experiment at 37 ± 2 °C and continuously oxygenated using an aerator. The tissue was then allowed to equilibrate in the organ bath for 30 minutes. Graded concentrations of acetylcholine (1, 2, and 4 µg/mL) were successively introduced into the bath to induce contractile responses, and control cumulative concentration–response curves were plotted.

Subsequently, a low dose (2 mg/mL) and a high dose (4 mg/mL) of the ethanolic extract of Urtica dioica, as well as atropine (2 µg/mL), were added separately to the organ bath 10 minutes prior to recording the corresponding concentration–response curves. Acetylcholine was then tested along with the plant extract and the standard antagonist (atropine). The anticholinergic activity of the extracts and atropine was assessed against a standardized effective dose of acetylcholine (1–4 µg/mL). The inhibitory effects on acetylcholine-induced contractions were subsequently represented graphically [30, 31].

RESULT

Preliminary qualitative phytochemical analysis:

Results of the preliminary phytochemical investigation of aqueous extract of Urtica dioica exhibited presence of flavonoids and tannins ([Table. 1]).

Yield:

The percentage yield of the extract was calculated as follows: 

Weight of dried extract/ weight of dried leaf powder x 100 (w/w) 

The percentage yield of Urtica dioica= (2.5/23.25) x100 = 10.75%

Specifications:

The environmental conditions maintained during the experiment were as follows: the bath volume was 30ml, speed of recording was 0.25mm/sec, equilibrium time was 30 minutes, the interval between dose is 1minute, and the dosing method was cumulative ([Table. 2]).

Response for different doses of acetylcholine alone and in the presence of HDUD, LDUD, Atropine:

Concentration-response curve in [Fig. 4] showed that both 4 and 2mg/mL of Urtica dioica as well as 2µg/mL of atropine caused concentration dependent, decrease in contractile response as compared to concentration-response curve of Ach alone. 

Half Maximal Response (EC50) values for acetylcholine administered alone and along with high and low dose of aqueous extract of Urtica dioica and atropine:

According to the determination of EC50, EC50 of ACh alone was 1.99µg/mL. After the addition of 4 and 2mg/mL concentrations of Urtica dioica extract, the EC50 of ACh increased, indicating a reduction in acetylcholine potency in the presence of the extract. In the presence of HDUD and LDUD, EC50 were 5.49 and 3.6µg/mL respectively. EC50 of ACh in the presence of 2µg/mL atropine was 29.5µg/mL.

Force of contraction in gram generated by chicken ileum in response to addition of ACh, high and low dose of aqueous extract of Urtica dioica and atropine:

The peaks presented in [Fig. 5] showed the force of contractions in chicken ileum generated by graded doses of ACh from 1, 2 and 4 µg/mL. After addition of 4 and 2mg/mL of Urtica dioica as well as 2 µg/mL atropine, there were significant reductions in the peaks.

DISCUSSION

Herbal remedies have been integral to disease management and quality-of-life improvement since the earliest human civilizations [32].

The main objective of this study was to assess the antispasmodic effects of the ethanolic extract derived from the leaves of Urtica dioica, using isolated chicken ileum tissue as the experimental model. Acetylcholine (Ach) was observed to produce concentration-dependent contractions, with the maximum response recorded at 4 µg/mL. Prior to the administration of varying doses of the plant extract or the standard antagonist, the ileum tissue was rinsed for 3–5 minutes to restore contractility to baseline levels.

The concentration–response analysis revealed that Urtica dioica extract at both 2 mg/mL and 4 mg/mL, as well as atropine (2 µg/mL), produced a concentration- dependent inhibition of acetylcholine-induced contractions. The higher dose of the extract exhibited a more pronounced inhibitory effect compared to the lower dose.

It is well established that acetylcholine-induced intestinal contractions are predominantly through muscarinic M2 and M3 receptors present in intestinal smooth muscle. Binding of acetylcholine to M2 receptors reduces cyclic adenosine monophosphate (cAMP)-mediated relaxation, while its interaction with M3 receptors stimulates phosphoinositide hydrolysis, resulting in muscle contraction [33].

The outcomes of this study suggest that Urtica dioica extract, similar to atropine, likely exerts its antispasmodic activity by inhibiting signaling through M2 and M3 muscarinic receptor pathways. Both concentrations of Urtica dioica extract and atropine, significantly reduced acetylcholine-induced contractile responses compared to acetylcholine alone. These findings support the idea that suppression of muscarinic receptor activity by the extract promotes intestinal smooth muscle relaxation and may contribute to the alleviation of gastrointestinal spasms. It is well-established that blocking the  muscarinic receptors in the gastrointestinal tract leads to the relaxation of smooth muscles [34].

The EC50 (effective concentration required to produce half maximal response for acetylcholine (Ach) alone was determined to be 1.99 µg/mL. When given along with Urtica dioica extract, the EC50 value of Ach increased to 3.6 µg/mL with 2 mg/mL of extract and 5.49 µg/mL with 4 mg/mL of extract. By comparison, the addition of atropine (2 µg/mL) caused a marked shift in the EC50 of Ach to 29.5 µg/mL. The observed increase in EC50 implies that a greater concentration of Ach is required to elicit half of the maximal contraction, indicating that Urtica dioica extract attenuates Ach-induced intestinal contractions and demonstrates significant antispasmodic potential.

[Fig. 5] illustrates the contractile force of chicken ileum in response to increasing concentrations of acetylcholine (1, 2, and 4 µg/mL). A significant reduction in contraction amplitude was observed following the administration of Urtica dioica extract at 2 mg/mL and 4 mg/mL, as well as atropine at 2 µg/mL. This inhibition confirms that both the extract and atropine effectively antagonize acetylcholine-induced intestinal contractions.

Previous studies have identified that Urtica dioica leaves are rich in phenolic and polyphenolic compounds, which are products of secondary plant metabolism. These compounds include simple phenolics, phenolic acids, stilbenes, flavonoids, coumarins, tannins, lignans, and others, known for their potent biological activities [35]. Among these phytochemicals, flavonoids have a well-established anti-spasmodic property [36].

From an earlier study it is also established that the antidiarrheal activity is directly linked with high content of flavonoids and tannins [37]. Specifically, flavonoids like quercetin, kaempferol, Luteolin, apigenin, isorhamnetin and rutin are found in the leaves of Urtica dioica [24]. Among the flavonoids identified, apigenin, luteolin, and quercetin have been detected in Achillea millefolium s.l., a plant demonstrated to possess notable antispasmodic activity [26]. Similarly, Phyllanthus emblica has been studied for its antispasmodic activity, which is attributed partly to kaempferol—a flavonoid also present in Urtica dioica exhibiting spasmolytic effects through interactions with muscarinic receptors and calcium channel blockade [22]. 

In addition, Matricaria chamomilla methanolic extract contains quercetin and isorhamnetin, both of which are also present in Urtica dioica, further supporting the role of these shared phytochemicals in contributing to the plant’s antispasmodic effects [38].

Hence, from our experiment it was proved that leaves of Urtica dioica possess anti spasmodic effect probably because of the presence of phytochemicals like flavonoids. 

CONCLUSION

The result indicates that both 2 mg/mL and 4 mg/mL concentrations of ethanolic leaf extract of Urtica dioica produced a dose-dependent reduction in acetylcholine-induced contractions of the chicken ileum, demonstrating clear antispasmodic activity.

The contractile force generated by acetylcholine was significantly reduced with both low and high doses of Urtica dioica extract and atropine, compared to acetylcholine alone. These results suggest that the extract, may inhibit muscarinic receptors in a manner similar to atropine, thereby reducing intestinal contractions. It is well-established that muscarinic receptor inhibition in the gastrointestinal tract promotes the smooth muscle relaxation.

Overall, the data support the potential of Urtica dioica as a therapeutic agent for managing gastrointestinal conditions such as diarrhoea and IBS. However, additional clinical investigations are warranted to validate these effects in human subjects.

ACKNOWLEDGEMENT

We sincerely thank Dr. H.P. Chhetri, Director of Himalayan Pharmacy Institute, Sikkim, and Dr. N.R. Bhuyan, Principal of Himalayan Pharmacy Institute, Sikkim for extending the essential facilities that enabled us to conduct this research work.