Effect of Aqueous extract of Glycyrrhiza on Rabbit Ileum in comparison Acetylcholine, Atropine and Ondansetron

Effect of Aqueous extract of Glycyrrhiza on Rabbit Ileum in comparison Acetylcholine, Atropine and Ondansetron | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Aqueous extract of Glycyrrhiza on Rabbit Ileum in comparison Acetylcholine, Atropine and Ondansetron Tehreem Zulfiqar, Maria Khalid, Tehseen Abaid, Tooba Malik, Ammara Anser This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3521890/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Gastrointestinal disorders like Irritable Bowel Syndrome IBS can decrease life quality and increase risk of anxiety and depression. Drugs like Hyoscyamine, Metoclopramide, 5-HT 3 receptor antagonists like Alosteron are used nowadays for its symptomatic management but their use is associated with adverse effects leading to decreased patient compliance. Nowadays natural plant-based medicine is preferred by people due to its fewer adverse effects therefore, the present study is designed to evaluate the effect of Glycyrrhiza on intestinal smooth muscle of rabbit. The aim of our study is to define the mechanism of action of Glycyrrhiza in promoting or inhibiting gut motility. Methodology and Results: An Experimental in-vitro study was carried out in the Pharmacology Department of Services Institute of Medical Sciences Lahore in January 2023. Rabbit Ileal tissue was used. Ileal smooth muscle activity was recorded using Power lab (AD instruments). After mounting, tissue was given rest for 30 minutes after which baseline contractions were recorded. Then 0.8ml of Acetylcholine (10 -5 ) was added and contractions were recorded for 30 seconds. The tissue was washed with Tyrode’s solution three times and then given rest for 3 minutes. Then ileal tissue was treated with Glycyrrhiza 5%,15% and 20% and their effect was recorded. Acetylcholine served as a positive control and action of Glycyrrhiza 5%, 15%, and 20% solution was compared with it. After that Glycyrrhiza was used in the presence of drugs that inhibit intestinal motility; Atropine and Ondansetron 0.0036µM and 0.036µM. Results showed that Glycyrrhiza 5%, 15% and 20% increased intestinal motility significantly (p-value<0.0001) in comparison with Acetylcholine. However, when Glycyrrhiza was used in the presence of antagonists Atropine and Ondansetron, then it reduced intestinal motility significantly (p-value<0.0001). Conclusion: Aqueous extract of Glycyrrhiza has dual effect on gut motility that is direct muscarinic receptor agonist and indirect modulator of enteric vagus nerve terminal through serotonin 5HT 3 receptors. Glycyrrhiza Acetylcholine Atropine Ondansetron Dopamine Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The enteric nervous system has been described as a "second brain" for several reasons. The enteric nervous system can operate autonomously. It normally communicates with the central nervous system (CNS) through the parasympathetic (e.g., via the vagus nerve) and sympathetic (e.g., via the prevertebral ganglia) nervous systems [ 1 ]. The enteric nervous system also makes use of more than 30 neurotransmitters, most of which are identical to the ones found in CNS, such as acetylcholine, dopamine, and serotonin. More than 90% of the body's serotonin lies in the gut, as well as about 50% of the body's dopamine [ 2 ]. Functional gastrointestinal (GI) disorders are a class of gastrointestinal disorders where there is a malfunction in the normal activities of the gastrointestinal tract mainly due to imbalance of these neurotransmitters but there are no structural abnormalities, Clinical research in neurogastroenterology focuses mainly on the study of common functional gastrointestinal disorders such as irritable bowel syndrome, which is considered the most common functional GI disorder [ 3 ]. Gastrointestinal disorders can lead to decreased life quality and increased risk of anxiety and depression [ 4 ]. These disorders are characterized by recurrent or chronic abdominal pain and in IBS associated with exacerbation or relief by defecation or change in bowel habits [ 5 ]. IBS is also associated with abnormalities of intestinal movement together with symptoms such as diarrhea or constipation and pain. People suffering from diarrhea often demonstrate symptoms such as loose, watery stool [ 6 ]. Currently available drugs for symptomatic management of IBS include anticholinergics like Hyoscyamine, Metoclopramide, 5-HT3 receptor anatgonists like Alosteron. Long term use of these medications is associated with serious adverse effects like Urinary retention, dry mouth and other anticholinergic symptoms with Hyoscyamine, risk of Tardive dyskinesia with prolonged use of Metoclopramide and serious cardiovascular complications with the use of Alosteron [ 7 ]. Due to these adverse effects patient’s compliance with the treatment remains the main hindrance in the management of gastrointestinal disorders like IBS. Nowadays people have started using herbal medicines. According to World Health Organization (WHO) estimates, approximately 4 billion people which is nearly 80% of the world’s population prefer to use herbal medicines for any aspect of their primary health care [ 8 ]. Glycyrrhiza glabra also known as licorice is a herb belonging to the Leguminosae family with numerous species distributed in various geographical regions worldwide. This herb has vast therapeutic applications owing to the presence of numerous Phyto molecules associated with diverse biological effects that are beneficial to health [ 9 ]. Glycyrrhiza glabra is an official plant of the Chinese Pharmacopeia (China Pharmacopoeia Committee, 1999) and forms part of the composition of commercial products indicated in its usage as a flavoring agent in foods, beverages, and tobacco for more than four millennia [ 10 ]. From among different types of licorice, Glycyrrhiza glabra L. is considered as the most valuable type for medical purposes. Glyciritic acid which is an important pharmaceutical composition can be found in the root and stem of this plant, is 50 times sweeter than sugar. The yellow color of licorice rhizome is due to flavonoids the most important of which is liquiritin [ 11 ]. The most important property of licorice rhizome has been reported to be its effect on gastrointestinal tract. This plant is beneficial in treatment of gastritis and many other functional gastrointestinal disorders. [ 12 ]. Based on previous observations and the folk usage of Glycyrrhiza, we decided to study the possible mechanism of action of Glycyrrhiza on rabbit ileal tissue 2. Materials and Methods 2.1 Animals and housing conditions: This was an in vitro experimental study. The study was approved by Institutional Review Board letter no. IRB/2023/1135/SIMS. A healthy rabbit weighing approx. 2–3 kg was purchased from the local market. The animal was kept in animal house of Pharmacology Dept. Services institute of medical sciences Lahore at controlled room temperature (25–27 C) and humidity (45–65%). It was fed with a standard diet consisting of carrots and tap water ad libitum. 2.2 Plant used and extract preparation: Glycyrrhiza in powdered form was purchased from the local market. To prepare its aqueous extract, it was kept soaked in distilled water overnight and then filtered with muslin cloth twice. Following three strengths of Glycyrrhiza were prepared using distilled water: 5% solution 15% solution 20% solution 2.3 Chemicals for study : Acetylcholine Chloride (Sigma Chemical Co. USA), Atropine (Sigma Chemical Co. USA), Dopamine (Howard Pharmaceuticals), Ondansetron Hydrochloride (Werrick Pharmaceuticals Pak) were purchased. All the solutions and dilutions were prepared fresh in distilled water at the time of experiments. Following molar solutions were prepared: Acetylcholine (10 − 5 ) Atropine (10 − 5 ) Ondansetron (0.0036µM and 0.36µM) 2.4 Preparation of tissue A rabbit Ileum strip of 2-3cm in size was dissected out from overnight fasting rabbit. [ 13 ]. The intestinal tissue was thoroughly washed with normal saline, and then transferred to isolated organ bath of 50-milliliter capacity containing Tyrode’s solution (in mM: NaCl, 136.8mM; KCl, 2.7mM; MgCl 2 , 0.5mM; CaCl 2 , 1.3mM; NaH 2 PO 4 , 0.14mM; NaHCO 3 , 12.0mM, Dextrose, 5.5mM) and was aeriated continuously with 100% oxygen. The cumulative dose response curve was made by using power lab (AD instruments). One end of the ileal strip was attached to the bottom of oxygen tube in tissue bath and the other end was connected to a research grade Force Displacement transducer DT-475 (USA) by means of a thread. IWorx / 214 (USA) was connected to the computer. Plug the DIN conector on the cable of the DT-475 Displacement transducer into channel 3 of the iWorx/214 unit. The tissue was allowed a period of equilibration of 15 minutes and physiological solution was changed 2 times. The isotonic ileal smooth muscle activity was recorded through the Displacement Transducer [ 14 ]. 2.5 Preparation of dose response curves: After mounting, tissue was left for equilibration for an interval of 30 minutes. Then 0.8ml of Ach (10 − 5 ) was added to the organ bath. After obtaining maximum contraction of Ach, the tissue was washed with Tyrode’s solution 3 times to washout the agonist. Same procedure was followed for different concentration solutions of Glycyrrhiza i.e 5%, 15%, 20% to record the force of contraction of rabbit ileum. In the next step of experiment Glycyrrhiza 5% was used in the presence of inhibitors of intestinal motility; Atropine (10 − 5 ), Ondansetron 0.0036µM and 0.36µM. this was done to elucidate the underlying mechanism of action of Glycyrrhiza. 2.6 Statistical Analysis: Results were analyzed using Graph Pad Prism version 6. The results were expressed as Means \(\pm\) Standard Deviation (SD). One way analysis of variance (ANOVA) was used to check variation in response among different study groups. Post Hoc Tukey’s test was used to check significant difference in response between different groups. The difference between observations was considered as significant if the p value is less than 0.05. 3. Results 3.1 Effect of acetylcholine and glycyrrhiza (5%, 15%, 20%) on rabbit ileum strip Baseline force of contractions was 0.080mN ± 0.0189. There is an increase in force of contraction in ileum tissue after treatment with acetylcholine that is 1.163mN ± 0.052. 5%, 15% and 20% Aqueous extract of Glycyrrhiza increased the force of contraction 3.910mN ± 0.0562, 1.680mN ± 0.020, 1.022 ± 0.037 respectively. 5% solution has significantly increased force of contraction as compared to normal and Ach induced contraction. *** indicate p \(\le\) 0.0001. Whereas 20% solution was found to have statically significant difference from normal but its response is statistically insignificantly different from Ach. 3.2 Effect of Atropine and Glycyrrhiza 5% on rabbit ileum Baseline force of contraction was 1.045mN ± 0.057. Atropine reduced the baseline force of contraction 0.194mN ± 0.067 significantly p \(\le\) 0.0001. Glycyrrhiza 5% solution when added in the presence of Atropine, forced of contraction further reduced to 0.075mN ± 0.059 which was statistically significant p \(\le\) 0.0001. 3.3 Effect of ondansetron 0.0036µM and glycyrrhiza 5% on rabbit ileum: Low dose of Ondansetron 0.0036 µM reduces force of contraction as compared to normal baseline gut activity significantly (0.1263mN ± 0.05048). It acts as spasmolytic as it has blocked stimulatory effect of serotonin on vagus nerve terminal. When Ileal tissue was treated with 5% Glycyrrhiza in the presence of Ondansetron, it behaves as spasmolytic and the force of contraction is reduces significantly (0.0200mN ± 0.01462) The difference between ondansetron 0.0036µM and glycyrrhiza 5% was statistically non-significant. 3.4 Effect of ondansetron 0.36µM and glycyrrhiza 5% on rabbit ileum strip Baseline force of contraction was 0.1707mN ± 0.02638. The increase in force of contraction in ileum tissue after treatment with 5HT-3 receptor antagonist, ondansetron 0.36µM (1.019 ± 0.05643) in comparison to glycyrrhiza 5% solution (0.3047 ± 0.05144) showed spasmogenic activity. The difference between ondansetron 0.36µM and glycyrrhiza 5% solution was statistically significant, p-value \(\le\) 0.0001. 4. Discussion Motility disorders are used to describe a variety of conditions in which the gut has lost its ability to coordinate muscular activity because of endogenous and exogenous causes [ 15 ]. In the last years, consumers are paying much more attention to natural medicines and principles, mainly due to the general sense that natural compounds are safe. Glycyrrhiza glabra Linn. belongs to the Fabaceae family and has been recognized since ancient times for its ethnopharmacological values. This plant contains different phytocompounds, such as glycyrrhizin, 18β-glycyrrhetinic acid, glabrin A and B, and isoflavones that have demonstrated various pharmacological activities. Pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including anti spasmodic effect on intestinal motility, antibacterial, anti‐inflammatory, antiviral, antioxidant, and antidiabetic activities [ 16 ]. Rabbit ileal tissue was selected for this experiment as it is considered more suitable for studying the response of agonist in the presence of antagonists [ 17 ]. Intestinal motility is mainly controlled by the enteric nervous system through multiple mediators, mainly acetylcholine, histamine, serotonin, bradykinins, dopamine etc. Acetylcholine is a prokinetic by acting on muscarinic receptors while serotonin receptor stimulation leads to nausea, vomiting and abdominal pain. Atropine is an antagonist of muscarinic receptors and Ondansetron is a 5HT 3 receptor antagonist which inhibits colonic motility especially left colon increasing colonic transit time. [ 18 ]. It has been previously demonstrated that isoliquiritigenin, a flavonoid isolated from Glycyrrhiza induces relaxations in the lower part of the intestine by transformation from its glycosides [ 19 ]. In this study, to explore the underlying mechanism of aqueous extract of glycyrrhiza we used isolated rabbit ileal tissue. Acetylcholine served as a gold standard muscarinic agonist that showed a significant (p value < 0.0001) spasmogenic effect which was mediated through muscarinic receptors. Glycyrrhiza 5% solution showed significant spasmogenic effect which was even significantly greater than Acetylcholine (p value < 0.0001). Similar significant spasmogenic effect was seen with Glycyrrhiza 15% and 20% solution. These results are in accordance with a previous study that showed its spasmogenic effect in rat stomach fundus [ 20 ]. Glycyrrhiza is found to be muscarinic agonist as it has increased ileal force of contraction which is even significantly greater than that produced by prototype muscarinic agonist i.e. Acetylcholine. This higher spasmogenic effect as compared to Ach may also be due to its indirect stimulating effect on vagus nerve terminal through serotonergic modulation. This indirect effect is further supported when Glycyrrhiza significantly reduced the gut motility in the presence of low dose of Ondansetron i.e. 5HT 3 receptor antagonist as serotonin receptors were blocked and it was unable to indirectly stimulate gut motility. When Glycyrrhiza is given in the presence of Atropine, the ileal force of contraction is markedly inhibited that may be because Atropine has already blocked muscarinic receptors through which Glycyrrhiza was acting to stimulate the ileal force of contraction. This is also in accordance with a previous study which showed similar results, that is spasmogenic in the presence of Acetylcholine and spasmolytic in the presence of Atropine. The spasmolytic action of Glycyrrhiza can also be attributed to blockade of voltage gated calcium channels [ 21 ]. In our experiment, Ondansetron 5HT 3 antagonist showed dual response; increased intestinal force of contraction at high doses and decreased force of contractions at low dose. This is in accordance with a previous study that showed similar dual response and attributed it to the partial agonistic activity of Ondansetron on peripheral 5HT 3 receptors [ 22 ]. Response of Glycyrrhiza in the presence of low dose 0.0036µM of Ondansetron is explained above. When the ileal tissue treated with 0.36µM Ondansetron was given Glycyrrhiza 5%, it increased intestinal force of contraction as compared to baseline contraction. This elucidates that when 5% Glycyrrhiza is given in the presence of high dose of Ondansetron then its partial agonistic effect is reduced. Hence our study shows that when Glycyrrhiza was used in the presence of drugs which inhibit intestinal motility then its spasmolytic effect unmasks which can be attributed to its calcium channel blocker effect as previously explained in literature. (21) 5. Conclusions From the results of our study, it can be concluded that Aqueous extract of Glycyrrhiza has dual effect on gut motility that is direct muscarinic receptor agonist and indirect modulator of enteric vagus nerve terminal through serotonin 5HT3 receptors. Clinical application of this drugs can be in the management of Irritable Bowel syndrome IBS where altered intestinal motility decreases patients’ quality of life. Further clinical trials are recommended in this domain. Declarations Funding: “The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.” Competing Interest: “The authors have no relevant financial or non-financial interests to disclose.” Authors Contribution: “All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Dr Tehreem Zulfiqar], [Dr. Tooba Malik] [Dr. Maria Khalid] and [Dr. Ammara Ansar]. The first draft of the manuscript was written by [Dr. Tehseen Abaid] [Dr. Tehreem Zulfiqar] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.” Ethical Approval: Ethical Committee and Institutional Review board (IRB) of Services Institute of Medical Sciences Lahore approved the conduct of this study through letter no IRB/2023/1135/SIMS. According to IRB’s directions authors conducted the animal research in accordance with the declaration of World Medical Association [WMA] made at Helsinki regarding the ethical principles for medical Research. All measures were taken to ensure that animal suffering is minimal. Availability of Data and Material: The complete data set supporting this manuscript’s results will be made available by the corresponding author on request of any qualified researcher in the same domain. They can contact through email. References Li,Ying;, Owyang,Chung (2003) Musings on the Wanderer: What's New in Our Understanding of Vago-Vagal Reflexes? V. Remodeling of vagus and enteric neural circuitry after vagal injury". American Journal of Physiology. Gastrointestinal and Liver Physiology. 285 (3): G461-9. 10.1152/ajpgi.00119.2003 ) Martinucci I et al (2015) Genetics and pharmacogenetics of aminergic transmitter pathways in functional gastrointestinal disorders. Pharmacogenomics 16(5):523–539 Kumar A, Rinwa P, Sharma N (2012) Irritable Bowel Syndrome: A Review. J Phys Pharm Adv 2(2):97–108 Anheyer D, Frawley J, Koch AK et al (2017) Herbal medicines for gastrointestinal disorders in children and adolescents: a systematic review, Pediatrics, 139, 6, article e20170062 Holtmann G, Shah A, Morrison M (2017) Pathophysiology of functional gastrointestinal disorders: a holistic overview. 35:5–13Supplement 1 Sadraei H, Ghanadian M, Asghari G, Madadi E, Azali N (2014) Antispasmodic and antidiarrhoeal activities of 6-(4-hydroxy-3-methoxyphenyl)-hexanonic acid from Pycnocycla spinosa Decne. Research in Pharmaceutical Sciences, vol 9. exBoiss, p 279 Black CJ, Burr NE, Camilleri M, Earnest DL, Quigley EM, Moayyedi P, Houghton LA, Ford AC (2020) Efficacy of pharmacological therapies in patients with IBS with diarrhoea or mixed stool pattern: systematic review and network meta-analysis. Gut 69(1):74–82 Ekor M (2014) The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 10:4:177 Wahab S, Annadurai S, Abullais SS, Das G, Ahmad W, Ahmad MF et al (2021) Glycyrrhiza glabra (licorice): a comprehensive review on its phytochemistry, biological activities, clinical evidence and toxicology. Plants 10:2751. 10.3390/plants10122751) Ploeger B, Mensinga T, Sips A, Seinen W, Meulenbelt J, DeJongh J (2001) The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling. Drug Metab Rev 33:125–147 Nassiri M, Hosseinzadeh H (2008) Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother Res 22:709–724 Khayyal MT, el-Ghazaly MA, Kenawy SA, Seif-el-Nasr M, Mahran LG, Kafafi YA et al (2001) Anti ulcerogenic effect of some gastrointestinally acting plant extracts and their combination. Arzneimittelforschung 51(7):545–553 Jabeen Q, Aziz N, Afzal Z, Gilani HA (2007) The spasmogenic and spasmolytic activities of lavandula Stoechas are mediated through muscarinic receptor stimulation and calcium channel blockade. Int J Pharmacol 3(1):61–67 Tanko Y, Alladey O, Ahmad KM, Muhammad A, Musa KY (2012) The effect of methanol leaves extract of Ficus Glumosa on gastrointestinal motility and castor oil induced diarrhoe in laboratory animals. Scholar Res Libr 2(3):360–367 Di Lorenzo C, Youssef NN (2010) Diagnosis and management of intestinal motility disorders. Semin Pediatr Surg 19(1):50–58 Pastorino G, Cornara L, Soares S, Rodrigues F, Oliveira MBPP (2018) Liquorice (Glycyrrhiza glabra): A phytochemical and pharmacological review. Phytother Res 32(12):2323–2339 Iqbal S, Sultana U, Sultana T A et al (2022) Effects of Drugs on Spontaneous Contractions of Rabbit Jejunum. PJMHS 16(2):237–239 Katzung 14th edition page 1096-97, 1022) Sato Y, He JX, Nagai H, Tani T, Akao T (2007) Isoliquiritigenin, one of the antispasmodic principles of Glycyrrhiza ularensis roots, acts in the lower part of intestine. Biol Pharm Bull 30:145–149 Chen G, Zhu L, Liu Y, Zhou Q, Chen H, Yang J (2009) Isoliquiritigenin, a flavonoid from licorice, plays a dual role in regulating gastrointestinal motility in vitro and in vivo. Phytother Res 23(4):498–506 Chen G, Zhu L, Liu Y, Zhou Q, Chen H, Yang J (2009) Isoliquiritigenin, a flavonoid from licorice, plays a dual role in regulating gastrointestinal motility in vitro and in vivo. Phytother Res Apr 23(4):498–506 Afzal A, Khan BT, Bakhtiar S (2016) Ondansetron: A newer aspect of dose response relationship on ileal smooth muscles of rabbit. Pak J Pharm Sci 29(1):119–124 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3521890","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":245242635,"identity":"8c15a567-0814-4266-b782-b27274d57926","order_by":0,"name":"Tehreem 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contraction.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3521890/v1/99705849a4c1aa24544d895a.jpg"},{"id":46024535,"identity":"a4d89fb7-9132-4641-80a3-3cdbcea57b2b","added_by":"auto","created_at":"2023-11-07 17:19:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of ileal contraction in normal, atropine and glycyrrhiza 5% group.*** indicate p \u003c/strong\u003e≤\u003cstrong\u003e 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3521890/v1/21e8c5000da26b71eb8e0e46.jpg"},{"id":46024532,"identity":"e01f9cf6-27bf-4564-b23c-f927debb5c95","added_by":"auto","created_at":"2023-11-07 17:19:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of ileum contraction in normal, ondansetron 0.0036µM and glycyrrhiza 5% group.*** indicate p \u003c/strong\u003e≤\u003cstrong\u003e 0.000\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3521890/v1/95426b4a35ad1c2f0e2c0324.jpg"},{"id":46024533,"identity":"da39643b-e26f-4ced-a1b2-9b756b3c8d9a","added_by":"auto","created_at":"2023-11-07 17:19:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of ileal force of contraction in normal, ondansetron 0.36µM and glycyrrhiza 5% group.*** indicate p \u003c/strong\u003e≤\u003cstrong\u003e 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3521890/v1/756ecd5e9254418360b098c5.jpg"},{"id":46028042,"identity":"2d2a5b22-190f-41c4-a37e-84b2e6c191aa","added_by":"auto","created_at":"2023-11-07 17:43:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":482456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3521890/v1/632682db-eea0-4adf-9db7-9ad417dbcb01.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Aqueous extract of Glycyrrhiza on Rabbit Ileum in comparison Acetylcholine, Atropine and Ondansetron","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe enteric nervous system has been described as a \"second brain\" for several reasons. The enteric nervous system can operate autonomously. It normally communicates with the central nervous system (CNS) through the parasympathetic (e.g., via the vagus nerve) and sympathetic (e.g., via the prevertebral ganglia) nervous systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The enteric nervous system also makes use of more than 30 neurotransmitters, most of which are identical to the ones found in CNS, such as acetylcholine, dopamine, and serotonin. More than 90% of the body's serotonin lies in the gut, as well as about 50% of the body's dopamine [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Functional gastrointestinal (GI) disorders are a class of gastrointestinal disorders where there is a malfunction in the normal activities of the gastrointestinal tract mainly due to imbalance of these neurotransmitters but there are no structural abnormalities, Clinical research in neurogastroenterology focuses mainly on the study of common functional gastrointestinal disorders such as irritable bowel syndrome, which is considered the most common functional GI disorder [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Gastrointestinal disorders can lead to decreased life quality and increased risk of anxiety and depression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These disorders are characterized by recurrent or chronic abdominal pain and in IBS associated with exacerbation or relief by defecation or change in bowel habits [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. IBS is also associated with abnormalities of intestinal movement together with symptoms such as diarrhea or constipation and pain. People suffering from diarrhea often demonstrate symptoms such as loose, watery stool [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Currently available drugs for symptomatic management of IBS include anticholinergics like Hyoscyamine, Metoclopramide, 5-HT3 receptor anatgonists like Alosteron. Long term use of these medications is associated with serious adverse effects like Urinary retention, dry mouth and other anticholinergic symptoms with Hyoscyamine, risk of Tardive dyskinesia with prolonged use of Metoclopramide and serious cardiovascular complications with the use of Alosteron [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Due to these adverse effects patient\u0026rsquo;s compliance with the treatment remains the main hindrance in the management of gastrointestinal disorders like IBS. Nowadays people have started using herbal medicines. According to World Health Organization (WHO) estimates, approximately 4\u0026nbsp;billion people which is nearly 80% of the world\u0026rsquo;s population prefer to use herbal medicines for any aspect of their primary health care [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Glycyrrhiza glabra also known as licorice is a herb belonging to the Leguminosae family with numerous species distributed in various geographical regions worldwide. This herb has vast therapeutic applications owing to the presence of numerous Phyto molecules associated with diverse biological effects that are beneficial to health [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e is an official plant of the Chinese Pharmacopeia (China Pharmacopoeia Committee, 1999) and forms part of the composition of commercial products indicated in its usage as a flavoring agent in foods, beverages, and tobacco for more than four millennia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. From among different types of licorice, \u003cem\u003eGlycyrrhiza glabra L.\u003c/em\u003e is considered as the most valuable type for medical purposes. Glyciritic acid which is an important pharmaceutical composition can be found in the root and stem of this plant, is 50 times sweeter than sugar. The yellow color of licorice rhizome is due to flavonoids the most important of which is liquiritin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The most important property of licorice rhizome has been reported to be its effect on gastrointestinal tract. This plant is beneficial in treatment of gastritis and many other functional gastrointestinal disorders. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Based on previous observations and the folk usage of Glycyrrhiza, we decided to study the possible mechanism of action of Glycyrrhiza on rabbit ileal tissue\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and housing conditions:\u003c/h2\u003e \u003cp\u003eThis was an in vitro experimental study. The study was approved by Institutional Review Board letter no. IRB/2023/1135/SIMS. A healthy rabbit weighing approx. 2\u0026ndash;3 kg was purchased from the local market. The animal was kept in animal house of Pharmacology Dept. Services institute of medical sciences Lahore at controlled room temperature (25\u0026ndash;27 C) and humidity (45\u0026ndash;65%). It was fed with a standard diet consisting of carrots and tap water ad libitum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Plant used and extract preparation:\u003c/h2\u003e \u003cp\u003eGlycyrrhiza in powdered form was purchased from the local market. To prepare its aqueous extract, it was kept soaked in distilled water overnight and then filtered with muslin cloth twice. Following three strengths of Glycyrrhiza were prepared using distilled water:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e5% solution\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e15% solution\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e20% solution\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cb\u003eChemicals for study\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eAcetylcholine Chloride (Sigma Chemical Co. USA), Atropine (Sigma Chemical Co. USA), Dopamine (Howard Pharmaceuticals), Ondansetron Hydrochloride (Werrick Pharmaceuticals Pak) were purchased. All the solutions and dilutions were prepared fresh in distilled water at the time of experiments. Following molar solutions were prepared:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAcetylcholine (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAtropine (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eOndansetron (0.0036\u0026micro;M and 0.36\u0026micro;M)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of tissue\u003c/h2\u003e \u003cp\u003eA rabbit Ileum strip of 2-3cm in size was dissected out from overnight fasting rabbit. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The intestinal tissue was thoroughly washed with normal saline, and then transferred to isolated organ bath of 50-milliliter capacity containing Tyrode\u0026rsquo;s solution (in mM: NaCl, 136.8mM; KCl, 2.7mM; MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5mM; CaCl\u003csub\u003e2\u003c/sub\u003e, 1.3mM; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.14mM; NaHCO\u003csub\u003e3\u003c/sub\u003e, 12.0mM, Dextrose, 5.5mM) and was aeriated continuously with 100% oxygen. The cumulative dose response curve was made by using power lab (AD instruments). One end of the ileal strip was attached to the bottom of oxygen tube in tissue bath and the other end was connected to a research grade Force Displacement transducer DT-475 (USA) by means of a thread. IWorx / 214 (USA) was connected to the computer. Plug the DIN conector on the cable of the DT-475 Displacement transducer into channel 3 of the iWorx/214 unit. The tissue was allowed a period of equilibration of 15 minutes and physiological solution was changed 2 times. The isotonic ileal smooth muscle activity was recorded through the Displacement Transducer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preparation of dose response curves:\u003c/h2\u003e \u003cp\u003eAfter mounting, tissue was left for equilibration for an interval of 30 minutes. Then 0.8ml of Ach (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) was added to the organ bath. After obtaining maximum contraction of Ach, the tissue was washed with Tyrode\u0026rsquo;s solution 3 times to washout the agonist. Same procedure was followed for different concentration solutions of Glycyrrhiza i.e 5%, 15%, 20% to record the force of contraction of rabbit ileum. In the next step of experiment Glycyrrhiza 5% was used in the presence of inhibitors of intestinal motility; Atropine (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), Ondansetron 0.0036\u0026micro;M and 0.36\u0026micro;M. this was done to elucidate the underlying mechanism of action of Glycyrrhiza.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis:\u003c/h2\u003e \u003cp\u003eResults were analyzed using Graph Pad Prism version 6. The results were expressed as Means \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e Standard Deviation (SD). One way analysis of variance (ANOVA) was used to check variation in response among different study groups. Post Hoc Tukey\u0026rsquo;s test was used to check significant difference in response between different groups. The difference between observations was considered as significant if the p value is less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of acetylcholine and glycyrrhiza (5%, 15%, 20%) on rabbit ileum strip\u003c/h2\u003e \u003cp\u003eBaseline force of contractions was 0.080mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0189. There is an increase in force of contraction in ileum tissue after treatment with acetylcholine that is 1.163mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052. 5%, 15% and 20% Aqueous extract of Glycyrrhiza increased the force of contraction 3.910mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0562, 1.680mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020, 1.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037 respectively. 5% solution has significantly increased force of contraction as compared to normal and Ach induced contraction. *** indicate p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e0.0001. Whereas 20% solution was found to have statically significant difference from normal but its response is statistically insignificantly different from Ach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of Atropine and Glycyrrhiza 5% on rabbit ileum\u003c/h2\u003e \u003cp\u003eBaseline force of contraction was 1.045mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057. Atropine reduced the baseline force of contraction 0.194mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067 significantly p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e0.0001. Glycyrrhiza 5% solution when added in the presence of Atropine, forced of contraction further reduced to 0.075mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.059 which was statistically significant p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e0.0001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of ondansetron 0.0036\u0026micro;M and glycyrrhiza 5% on rabbit ileum:\u003c/h2\u003e \u003cp\u003eLow dose of Ondansetron 0.0036 \u0026micro;M reduces force of contraction as compared to normal baseline gut activity significantly (0.1263mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05048). It acts as spasmolytic as it has blocked stimulatory effect of serotonin on vagus nerve terminal. When Ileal tissue was treated with 5% Glycyrrhiza in the presence of Ondansetron, it behaves as spasmolytic and the force of contraction is reduces significantly (0.0200mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01462) The difference between ondansetron 0.0036\u0026micro;M and glycyrrhiza 5% was statistically non-significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of ondansetron 0.36\u0026micro;M and glycyrrhiza 5% on rabbit ileum strip\u003c/h2\u003e \u003cp\u003eBaseline force of contraction was 0.1707mN\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02638. The increase in force of contraction in ileum tissue after treatment with 5HT-3 receptor antagonist, ondansetron 0.36\u0026micro;M (1.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05643) in comparison to glycyrrhiza 5% solution (0.3047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05144) showed spasmogenic activity. The difference between ondansetron 0.36\u0026micro;M and glycyrrhiza 5% solution was statistically significant, p-value \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e0.0001.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMotility disorders are used to describe a variety of conditions in which the gut has lost its ability to coordinate muscular activity because of endogenous and exogenous causes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the last years, consumers are paying much more attention to natural medicines and principles, mainly due to the general sense that natural compounds are safe. Glycyrrhiza glabra Linn. belongs to the Fabaceae family and has been recognized since ancient times for its ethnopharmacological values. This plant contains different phytocompounds, such as glycyrrhizin, 18β-glycyrrhetinic acid, glabrin A and B, and isoflavones that have demonstrated various pharmacological activities. Pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including anti spasmodic effect on intestinal motility, antibacterial, anti‐inflammatory, antiviral, antioxidant, and antidiabetic activities [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Rabbit ileal tissue was selected for this experiment as it is considered more suitable for studying the response of agonist in the presence of antagonists [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Intestinal motility is mainly controlled by the enteric nervous system through multiple mediators, mainly acetylcholine, histamine, serotonin, bradykinins, dopamine etc. Acetylcholine is a prokinetic by acting on muscarinic receptors while serotonin receptor stimulation leads to nausea, vomiting and abdominal pain. Atropine is an antagonist of muscarinic receptors and Ondansetron is a 5HT\u003csub\u003e3\u003c/sub\u003e receptor antagonist which inhibits colonic motility especially left colon increasing colonic transit time. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has been previously demonstrated that isoliquiritigenin, a flavonoid isolated from \u003cem\u003eGlycyrrhiza\u003c/em\u003e induces relaxations in the lower part of the intestine by transformation from its glycosides [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, to explore the underlying mechanism of aqueous extract of glycyrrhiza we used isolated rabbit ileal tissue. Acetylcholine served as a gold standard muscarinic agonist that showed a significant (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) spasmogenic effect which was mediated through muscarinic receptors. Glycyrrhiza 5% solution showed significant spasmogenic effect which was even significantly greater than Acetylcholine (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similar significant spasmogenic effect was seen with Glycyrrhiza 15% and 20% solution. These results are in accordance with a previous study that showed its spasmogenic effect in rat stomach fundus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Glycyrrhiza is found to be muscarinic agonist as it has increased ileal force of contraction which is even significantly greater than that produced by prototype muscarinic agonist i.e. Acetylcholine. This higher spasmogenic effect as compared to Ach may also be due to its indirect stimulating effect on vagus nerve terminal through serotonergic modulation. This indirect effect is further supported when Glycyrrhiza significantly reduced the gut motility in the presence of low dose of Ondansetron i.e. 5HT\u003csub\u003e3\u003c/sub\u003e receptor antagonist as serotonin receptors were blocked and it was unable to indirectly stimulate gut motility. When Glycyrrhiza is given in the presence of Atropine, the ileal force of contraction is markedly inhibited that may be because Atropine has already blocked muscarinic receptors through which Glycyrrhiza was acting to stimulate the ileal force of contraction. This is also in accordance with a previous study which showed similar results, that is spasmogenic in the presence of Acetylcholine and spasmolytic in the presence of Atropine. The spasmolytic action of Glycyrrhiza can also be attributed to blockade of voltage gated calcium channels [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In our experiment, Ondansetron 5HT\u003csub\u003e3\u003c/sub\u003e antagonist showed dual response; increased intestinal force of contraction at high doses and decreased force of contractions at low dose. This is in accordance with a previous study that showed similar dual response and attributed it to the partial agonistic activity of Ondansetron on peripheral 5HT\u003csub\u003e3\u003c/sub\u003e receptors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Response of Glycyrrhiza in the presence of low dose 0.0036\u0026micro;M of Ondansetron is explained above. When the ileal tissue treated with 0.36\u0026micro;M Ondansetron was given Glycyrrhiza 5%, it increased intestinal force of contraction as compared to baseline contraction. This elucidates that when 5% Glycyrrhiza is given in the presence of high dose of Ondansetron then its partial agonistic effect is reduced. Hence our study shows that when Glycyrrhiza was used in the presence of drugs which inhibit intestinal motility then its spasmolytic effect unmasks which can be attributed to its calcium channel blocker effect as previously explained in literature. (21)\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eFrom the results of our study, it can be concluded that Aqueous extract of Glycyrrhiza has dual effect on gut motility that is direct muscarinic receptor agonist and indirect modulator of enteric vagus nerve terminal through serotonin 5HT3 receptors. Clinical application of this drugs can be in the management of Irritable Bowel syndrome IBS where altered intestinal motility decreases patients\u0026rsquo; quality of life. Further clinical trials are recommended in this domain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026ldquo;The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u0026rdquo;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026ldquo;The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Dr Tehreem Zulfiqar], [Dr. Tooba Malik] [Dr. Maria Khalid] and [Dr. Ammara Ansar]. The first draft of the manuscript was written by [Dr. Tehseen Abaid] [Dr. Tehreem Zulfiqar] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical Committee and Institutional Review board (IRB) of Services Institute of Medical Sciences Lahore approved the conduct of this study through letter no IRB/2023/1135/SIMS. According to IRB\u0026rsquo;s directions authors conducted the animal research in accordance with the declaration of World Medical Association [WMA] made at Helsinki regarding the ethical principles for medical Research. All measures were taken to ensure that animal suffering is minimal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete data set supporting this manuscript\u0026rsquo;s results will be made available by the corresponding author on request of any qualified researcher in the same domain. They can contact through email. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi,Ying;, Owyang,Chung (2003) Musings on the Wanderer: What's New in Our Understanding of Vago-Vagal Reflexes? V. Remodeling of vagus and enteric neural circuitry after vagal injury\". American Journal of Physiology. Gastrointestinal and Liver Physiology. 285 (3): G461-9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpgi.00119.2003\u003c/span\u003e\u003cspan address=\"10.1152/ajpgi.00119.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinucci I et al (2015) Genetics and pharmacogenetics of aminergic transmitter pathways in functional gastrointestinal disorders. Pharmacogenomics 16(5):523\u0026ndash;539\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Rinwa P, Sharma N (2012) Irritable Bowel Syndrome: A Review. J Phys Pharm Adv 2(2):97\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnheyer D, Frawley J, Koch AK et al (2017) Herbal medicines for gastrointestinal disorders in children and adolescents: a systematic review, Pediatrics, 139, 6, article e20170062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoltmann G, Shah A, Morrison M (2017) Pathophysiology of functional gastrointestinal disorders: a holistic overview. 35:5\u0026ndash;13Supplement 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadraei H, Ghanadian M, Asghari G, Madadi E, Azali N (2014) Antispasmodic and antidiarrhoeal activities of 6-(4-hydroxy-3-methoxyphenyl)-hexanonic acid from Pycnocycla spinosa Decne. Research in Pharmaceutical Sciences, vol 9. exBoiss, p 279\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlack CJ, Burr NE, Camilleri M, Earnest DL, Quigley EM, Moayyedi P, Houghton LA, Ford AC (2020) Efficacy of pharmacological therapies in patients with IBS with diarrhoea or mixed stool pattern: systematic review and network meta-analysis. Gut 69(1):74\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkor M (2014) The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 10:4:177\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWahab S, Annadurai S, Abullais SS, Das G, Ahmad W, Ahmad MF et al (2021) Glycyrrhiza glabra (licorice): a comprehensive review on its phytochemistry, biological activities, clinical evidence and toxicology. Plants 10:2751. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants10122751)\u003c/span\u003e\u003cspan address=\"10.3390/plants10122751)\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePloeger B, Mensinga T, Sips A, Seinen W, Meulenbelt J, DeJongh J (2001) The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling. Drug Metab Rev 33:125\u0026ndash;147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNassiri M, Hosseinzadeh H (2008) Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother Res 22:709\u0026ndash;724\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhayyal MT, el-Ghazaly MA, Kenawy SA, Seif-el-Nasr M, Mahran LG, Kafafi YA et al (2001) Anti ulcerogenic effect of some gastrointestinally acting plant extracts and their combination. Arzneimittelforschung 51(7):545\u0026ndash;553\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJabeen Q, Aziz N, Afzal Z, Gilani HA (2007) The spasmogenic and spasmolytic activities of lavandula Stoechas are mediated through muscarinic receptor stimulation and calcium channel blockade. Int J Pharmacol 3(1):61\u0026ndash;67\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanko Y, Alladey O, Ahmad KM, Muhammad A, Musa KY (2012) The effect of methanol leaves extract of Ficus Glumosa on gastrointestinal motility and castor oil induced diarrhoe in laboratory animals. Scholar Res Libr 2(3):360\u0026ndash;367\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Lorenzo C, Youssef NN (2010) Diagnosis and management of intestinal motility disorders. Semin Pediatr Surg 19(1):50\u0026ndash;58\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePastorino G, Cornara L, Soares S, Rodrigues F, Oliveira MBPP (2018) Liquorice (Glycyrrhiza glabra): A phytochemical and pharmacological review. Phytother Res 32(12):2323\u0026ndash;2339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIqbal S, Sultana U, Sultana T A et al (2022) Effects of Drugs on Spontaneous Contractions of Rabbit Jejunum. PJMHS 16(2):237\u0026ndash;239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatzung 14th edition page 1096-97, 1022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato Y, He JX, Nagai H, Tani T, Akao T (2007) Isoliquiritigenin, one of the antispasmodic principles of Glycyrrhiza ularensis roots, acts in the lower part of intestine. Biol Pharm Bull 30:145\u0026ndash;149\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G, Zhu L, Liu Y, Zhou Q, Chen H, Yang J (2009) Isoliquiritigenin, a flavonoid from licorice, plays a dual role in regulating gastrointestinal motility in vitro and in vivo. Phytother Res 23(4):498\u0026ndash;506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G, Zhu L, Liu Y, Zhou Q, Chen H, Yang J (2009) Isoliquiritigenin, a flavonoid from licorice, plays a dual role in regulating gastrointestinal motility in vitro and in vivo. Phytother Res Apr 23(4):498\u0026ndash;506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfzal A, Khan BT, Bakhtiar S (2016) Ondansetron: A newer aspect of dose response relationship on ileal smooth muscles of rabbit. Pak J Pharm Sci 29(1):119\u0026ndash;124\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glycyrrhiza, Acetylcholine, Atropine, Ondansetron, Dopamine","lastPublishedDoi":"10.21203/rs.3.rs-3521890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3521890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eGastrointestinal disorders like Irritable Bowel Syndrome IBS can decrease life quality and increase risk of anxiety and depression. Drugs like Hyoscyamine, Metoclopramide, 5-HT\u003csub\u003e3\u003c/sub\u003e receptor antagonists like Alosteron are used nowadays for its symptomatic management but their use is associated with adverse effects leading to decreased patient compliance. Nowadays natural plant-based medicine is preferred by people due to its fewer adverse effects therefore, the present study is designed to evaluate the effect of Glycyrrhiza on intestinal smooth muscle of rabbit. The aim of our study is to define the mechanism of action of Glycyrrhiza in promoting or inhibiting gut motility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology and Results: \u003c/strong\u003eAn Experimental in-vitro study was carried out in the Pharmacology Department of Services Institute of Medical Sciences Lahore in January 2023. Rabbit Ileal tissue was used. Ileal smooth muscle activity was recorded using Power lab (AD instruments). After mounting, tissue was given rest for 30 minutes after which baseline contractions were recorded. Then 0.8ml of Acetylcholine (10\u003csup\u003e-5\u003c/sup\u003e) was added and contractions were recorded for 30 seconds. The tissue was washed with Tyrode’s solution three times and then given rest for 3 minutes. Then ileal tissue was treated with Glycyrrhiza 5%,15% and 20% and their effect was recorded. Acetylcholine served as a positive control and action of Glycyrrhiza 5%, 15%, and 20% solution was compared with it. After that Glycyrrhiza was used in the presence of drugs that inhibit intestinal motility; Atropine and Ondansetron 0.0036µM and 0.036µM. Results showed that Glycyrrhiza 5%, 15% and 20% increased intestinal motility significantly (p-value\u0026lt;0.0001) in comparison with Acetylcholine. However, when Glycyrrhiza was used in the presence of antagonists Atropine and Ondansetron, then it reduced intestinal motility significantly (p-value\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAqueous extract of Glycyrrhiza has dual effect on gut motility that is direct muscarinic receptor agonist and indirect modulator of enteric vagus nerve terminal through serotonin 5HT\u003csub\u003e3\u003c/sub\u003e receptors.\u003c/p\u003e","manuscriptTitle":"Effect of Aqueous extract of Glycyrrhiza on Rabbit Ileum in comparison Acetylcholine, Atropine and Ondansetron","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-07 17:19:40","doi":"10.21203/rs.3.rs-3521890/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"249dd1d4-8fd1-4b37-af99-7fcbf962f9fb","owner":[],"postedDate":"November 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-07T17:19:42+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-07 17:19:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3521890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3521890","identity":"rs-3521890","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}