In vitro Antimotility and Antispasmodic effects of Nephelium lappaceum on isolated chicken ileum

 

Sasikala Chinnappan,  Mogana R., Tan Xue Qin

UCSI University, Taman Connaught, Kuala Lumpur, Malaysia 56000.

*Corresponding Author E-mail: hasipharm@gmail.com

 

ABSTRACT:

Objectives of this study were to determine the antimotility and antispasmodic effects of both methanolic and aqueous extracts of Nephelium lappaceum on contractile response induced by acetylcholine and histamine and on isolated chicken ileum. Methanolic and aqueous extracts of N. lappaceum were prepared by using maceration technique. Graded doses of acetylcholine and histamine were used to trigger the contractile response. Atropine, mepyramine and N. lappaceum extracts were used as the antagonists to reduce the intestinal contractions. Concentration response curves in the absence and in the presence of both extracts were plotted separately. Half maximal effective concentration (EC50) in the presence of extracts were higher than EC50 in the absence of extracts. EC50 of acetylcholine in the presence of 4mg/mL and 8 mg/mL methanolic extracts were 1.59µg/mL and 1.71µg/mL respectively whereas EC50 of acetylcholine alone is 1.38µg/mL. In the presence of 2mg/mL and 4mg/mL of aqueous extracts, EC50 of acetylcholine also increased to 1.98 and 2.58µg/mL respectively. EC50 of acetylcholine in the presence of 2µg/mL atropine was the lowest which was 9.84µg/mL.  EC50 of histamine alone is 0.93µg/mL. After addition of 1 mg/mL and 2mg/mL methanolic extracts as well as 2mg/mL and 4 mg/mL aqueous extracts, EC50 increased to 1.08, 1.24, 1.03 and 1.09µg/mL respectively. In the presence of 2 µg/mL mepyramine, EC50 of histamine is 1.55µg/mL. In conclusion, both methanolic and aqueous extracts showed antimotility and antispasmodic properties by blocking acetylcholine and histamine.

 

KEYWORDS: Acetylcholine, Atropine, Histamine, Mepyramine, N. lappaceum.

 

 


INTRODUCTION:

According to Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015), diarrhea caused more than 1.3 million worldwide and became the 4th leading cause of death among 5-year-old children in 2015. As observed from statistics done by GBD in 2013 and 2015, mortality rate of diarrhea among children younger than 5 years old decreased from 1.87 million to 1.3 million globally. Despite the substantia reduction in mortality rate, diarrhea remains a preventable health disorder which with considerable high mortality rate. GBD 2015 concluded that mortality rate of diarrhea is due to poor control of hygiene, malnutrition and poor access to appropriate therapy agent.1

 

Nephelium lappaceum L. (Family- Sapindaceae), popularly known as ‘Rambutan’, is an evergreen tree about 10-12m tall with grayish brown branches.2 Because of presence of numerous hairy protuberances on the fruit, the word rambutan has been derived from the Malay word 'rambut' which means “hair”. The tree is native to Malaysia but grown in other parts of the world.3 The leaves are glossy green and compound. Inflorescences many branched, flowers are yellowish green to white, small sized and occur in large bunches, no petal, with mild sweet scent, dioecious (male and female flowers on separate trees) or bisexual, rich in nectar, attract bees.4 Each flower holds six to eight stamens. The superior ovary possesses one to two lobes with a single style. The tree flowers twice a year.3 Fruits are edible, oval to spherical drupe, leathery skin with flexible hairy spines, mature from green to red. Aril white, fleshy, edible and sweet and surrounds single large seed.4 N. lappaceum has been used as traditional medicine for centuries especially as a remedy for diabetes and high blood pressure.5 The fruit is believed to be stomachic, astringent, anthelmintic and believed to be a good remedy to treat diarrhea and dysentery. The leaves are used in poultices for treating headache.6 Dried fruit rind is sometimes used as an ingredient in manufacture of soap. The roots, leaves and bark are used in the manufacturing of dyes. In Malaysia, the decoction of the root is used in traditional treatment while the bark is used as an astringent for tongue diseases.7 Aim of this study is to determine the antimotility and anti-spasmodic effects of Nephelium lappaceum extracts on contractions of isolated chicken ileum. This study focus on antagonistic action of N. lappaceum extracts on intestinal contraction.

 

MATERIAL METHODS:

Chemical and Drugs:

Drugs that were used in current study included acetylcholine, histamine, atropine, mepyramine from Cayman Chemical. All the ingredient of solutions of analytical grade and prepared freshly before the experiment.

 

Collection of Plant Material and Preparation of Extracts:

Nephelium lappaceum fruits were purchased from local markets in Cheras. Pericarps of the fruits were peeled off and left to dry at shade and followed by under room temperature. After the pericarps were completely dried, they were grinded into fine powders. Dried pericarps in powder form were then extracted within methanol and distilled water separately by using maceration technique at a ratio of 1:20.8 1 gram of the sample powders were mixed with 20mL of methanol and purified water separately. Throughout 7 days of maceration process, the solutions which contained extraction solvent and N. lappaceum were monitored daily and shaked well. After 7 days of maceration, methanolic and aqueous extracts of N. lappaceum were filtered and then concentrated by vacuum under reduced pressure in rotary evaporator.

 

Pharmacological test:

Fresh ileum of healthy chickens was obtained from a slaughter house. Terminal segments of ileum about 1–1.5cm in length was prepared and placed in 30mL baths filled with Tyrode solution (NaCl, l36.7; KCl, 2.68; MgCl2.2H2O, 1.05; NaH2PO4, 0.42; CaCl2.2H2O, 1.80; NaHCO3, 11.90; glucose, 5.55mM). Tyrode solution containing fresh chicken ileum was kept at 37 to 40°C and oxygenated continuously. Initial tension was 2 grams. Then, fresh chicken ileum was allowed to stabilize for around 45–60 min inside the Tyrode solution. Isometric contractions were recorded in power lab by using transducer. Graded doses of acetylcholine and histamine from 1, 2, 4, 8 16µg/mL were added into the organ bath to trigger contractile response. Control cumulative concentration–response curves for each acetylcholine and histamine were plotted.

 

Both methanolic and aqueous extracts, 2µg/mL of atropine and mepyramine were then added to the bath 10 min before the corresponding concentration–response curve was recorded.9 Each agonist was tested in the presence of methanolic extracts, aqueous extracts and standard antagonist. Anticholinergic effect of extracts and atropine were evaluated against a fixed minimally effective dose of acetylcholine from 1 to 16µg/mL. Antihistamine effect of extracts and mepyramine were evaluated against a fixed minimally effective dose of histamine from 1 to 16µg/mL. Inhibitory effects on contractile response caused by extracts and standard antagonists were plotted in graphs.

 

Statistical Analysis:

All the values were recorded as mean ± standard error of mean (S.E.M.) with n=5. The data were analyzed by using Student’s t-test with differences of the means considered significant at p<0.05.10


 

Figure 1: Concentration response curve that shows effect of N. lappaceum extracts and atropine on contractile response of acetylcholine on chicken ileum significantly (p< 0.05)

 

Figure 2: Concentration response curve that shows effects of N lappaceum extracts and mepyramine on contractile response of histamine on chicken ileum significantly (p< 0.05)

 


RESULTS:

Concentration-response curve in Figure 1 showed that both 4 and 8mg/mL MER, 2 and 4mg/mL AER as well as 2µg/mL atropine caused significant (p<0.05) concentration dependent decrease in contractile response as compared to concentration-response curve of ACh alone. Force of contraction induced by ACh in the presence of 4mg/mL and 8mg/mL MER, 2mg/mL and 4 mg/mL AER and 2µg/mL atropine is less than that induced by ACh alone. These findings suggested that N. lappaceum extracts and atropine may inhibit muscarinic receptors and decrease intestinal contractions. A study of Henry et al stated that inhibition of muscarinic receptors in GIT can lead to relaxation of intestinal smooth muscles.11,12  Concentration-response curve in Figure 2 showed that both 1 and 2mg/mL MER, 2 and 4 mg/mL AER as well as 2µg/mL mepyramine caused significant (p< 0.05) concentration dependent decrease in contractile response as compared to concentration-response curve of histamine alone. Force of contraction induced by histamine in the presence of 1mg/mL and 2 mg/mL MER, 2mg/mL and 4mg/mL AER and 2µg/mL mepyramine is less than that induced by histamine alone. Study by Leurs et al showed that histamine receptor antagonist such as mepyramine can inhibit electrically-induced twitches in guinea pig ileum and therefore lead to relaxation of smooth muscle.13 According to table 1, EC50 of ACh alone is 1.38µg/mL. After addition of increasing concentration of MER and AER, EC50 of ACh increased. In the presence of 4 mg/mL and 8mg/mL of MER, EC50 were 1.59 and 1.71 µg/mL respectively. Increase in EC50 indicates that higher concentration of ACh was required to elicit 50% of the maximum contractile response. In other words, MER can antagonise ACh-induced contraction in GIT. In the presence of 2mg/mL and 4mg/mL of AER, EC50 of ACh also increased to 1.98 and 2.58µg/mL respectively. EC50 of ACh in the presence of 2µg/mL atropine was the lowest which was 9.84µg/mL.

 

Table 1: Half Maximal Response (EC50) for acetylcholine alone and in the presence of MER, AER and atropine

Drugs/Extracts

EC50 (µg/mL)

ACh 1-16µg/mL

1.38

ACh + MER 4mg/mL

1.59

ACh + MER 8mg/mL

1.71

ACh + AER 2mg/mL

1.98

ACh + AER 4mg/mL

2.58

ACh + Atropine 2µg/mL

9.84

 

Table 2: Half Maximal Response (EC50) for histamine alone and in the presence of MER, AER and mepyramine

Drugs/Extracts

EC50 (µg/mL)

Histamine 1-16µg/mL

0.93

Histamine + MER 1mg/mL

1.08

Histamine + MER 2mg/mL

1.24

Histamine + AER 2mg/mL

1.03

Histamine + AER 4mg/mL

1.09

Histamine + Mepyramine 2µg/mL

1.55

 

According to table 2, EC50 of histamine alone is 0.93 µg/mL. After addition of increasing concentration of MER and AER, EC50 of serotonin increased. In the presence of 1mg/mL and 2mg/mL of MER, EC50 were 1.08 and 1.24µg/mL respectively. In the presence of 2 mg/mL and 4mg/mL of AER, EC50 of histamine also increased to 1.03 and 1.09µg/mL respectively. EC50 of histamine in the presence of 2µg/mL mepyramine was the lowest which was 1.55µg/mL. Increase in EC50 indicates that higher concentration of histamine was required to elicit 50% of the maximum contractile response. In other words, MER and AER can antagonizes histamine-induced contraction in GIT. The peaks presented in Figure 3 showed the force of contractions in chicken ileum generated by graded doses of ACh from 1, 2, 4, 8 to 16µg/mL. After addition of 4 and 8 mg/mL MER, 2 and 4mg/mL AER as well as 2 µg/mL atropine, there were significant reductions in the peaks. These reductions indicate that MER, AER and atropine antagonized ACh-induced intestinal contractions. The peaks presented in Figure 4 showed the force of contractions in chicken ileum generated by graded doses of histamine from 1, 2, 4, 8 to 16 µg/mL. After addition of 1 and 2 mg/mL MER, 2 and 4 mg/mL AER as well as 2 µg/mL mepyramine, there were significant reductions in the peaks. These reductions indicate that MER, AER and mepyramine antagonized histamine-induced intestinal contractions.


 

1-16µg/mL ACh    4 and 8mg/mL MER    2 and 4mg/mL AER    2µg/mL Atropine

 

Figure 3: Force of contraction in gram generated by chicken ileum in response to addition of ACh, MER, AER and atropine

 

1-16µg/mL Histamine   1 and 2mg/mL MER    2 and 4mg/mL AER   2µg/mL Mepyramine

 

Figure 4: Force of contraction in gram generated by chicken ileum in response to addition of histamine, MER, AER and mepyramine

 


DISCUSSIONS:

As shown by the concentration-response curves in Figure 1 and 2, acetylcholine and histamine exhibited concentration dependent increase in contractile response of chicken ileum. Contractile response of chicken ileum increased when higher concentration of agonists was added. Maximum contractile response was obtained after concentration of agonists reached 16 µg/mL. After 16 µg/mL, contractile response reached plateau due to saturation of the receptors. Before addition of different concentrations of each control, extract and antagonist, a period of washing out for about 3 to 5 minutes is required to ensure recovery of contractile response to the initial tension. Increasing concentration of acetylcholine and histamine even in the presence of atropine and mepyramine at concentration of 2 µg/mL showed increase in contractile response. However, sensitivity of chicken ileum can diminish gradually with repeated large doses of agonists. This phenomenon is known as tolerance.14

 

Frederick et al studied different muscarinic receptors on laboratory animals such as guinea pig, rat and cow. His findings found out that M2 and M3 receptors are mainly involved in intestinal contractions.15 Both M2 and M3 receptors are present in isolated preparation of intestinal smooth muscles with M2 receptors outnumber the M3 receptors by a factor of 4. Therefore, it may be safe to assume that in this study, acetylcholine binds to M2 receptors to inhibit relaxant effect caused by cyclic adenosine monophosphate (cAMP) as well as binds to M3 receptors to facilitate phosphoinositide hydrolysis and causes intestinal contractions. Both methanolic and aqueous extracts of N. lappaceum were then blocked the M2 and M3 receptors to inhibit intestinal contractions.

 

Leurs et al’s study on effects of H1, H2 and H3 receptors on guinea pig ileum intestines showed that histamine-induced intestinal contractions are mainly regulated by H1 receptors whereas inhibition of intestinal contractions are mediated by H3 receptors.16 Study by Carlos et al demonstrated that mepyramine is the H1 receptors antagonists thus inhibiting H1 receptors-induced intestinal contractions.17 In this study, extracts of N. lappaceum may mimic mepyramine by inhibiting H1 receptors-induced intestinal contractions or may act on H3 receptors to cause intestinal relaxation.

 

N. lappaceum extracts have inhibitory actions on intestinal contractions. The results are compatible with previous study on inhibitory effect of N. lappaceum extracts by Dash et al.18 This study investigated the methanolic extracts of N. lappaceum seeds on rat models which were supplied with castor oil to trigger diarrhea. Results of that study showed that methanolic extracts of N. lappaceum extracts have significant reduction in fecal dropping as compared to loperamide. Thus, Dash et al.concluded that N. lappaceum possess antidiarrheal activity.18

 

There is also another report of antispasmodic activity of other plants of Sapindaceae family.19 This study by A. Rojas et al compared the spasmolytic activity of various plants with D. lanosa. Methanolic extracts of D. lanosa was used as the positive control in that study due to its high content of atropine and scopolamine. Youmans et al reported that both atropine and scopolamine have inhibitory action on intestinal motility.20 Results by A. Rojas et al demonstrated that methanolic extract of D. viscosa exhibited same antagonistic potency as D. lanosa.19  

 

Therefore, a few studies were found to prove the presence of flavonoids in N. lappaceum and eventually to support roles of flavonoids on intestinal contractions. Umadevi et al screened 19 plants of Sapindaceae family for the presence of flavonoids.21 N. lappaceum was one of the plants included in Umadevi et al study. Results of the screening tests showed that flavonoids are present in all plants of Sapindacea family except Glennia.21 Therefore, study by Umadevi et al proved the presence of flavonoids in plants of Sapindaceae family such N. lappaceum. It also further supported the antispasmolytic activity of flavonoids as mentioned by A. Rojas et al study.

 

Capasso et al investigate the actions of flavonoids on contractions of guinea pig ileum. Results presented by Capasso et al indicated that flavonoids such as apigenin, quercetin, kaempferol, crysin and flavone can inhibit contractions on guinea pig ileum.22 Inhibitory action of the flavonoids was concentration and time-dependent. These findings are similar to the results by my study in which both methanolic extracts and aqueous extracts of N. lappaceum showed concentration dependent inhibition on contractile response of chicken ileum.

 

CONCLUSIONS:

Results of the present work demonstrated that both methanolic and aqueous extracts of N. lappaceum show antimotility and anti-spasmodic activity. N. lappaceum extracts showed concentration dependent reduction in contractile response of chicken ileum induced by acetylcholine and histamine. These findings support that N. lappaceum may become a potential treatment for gastrointestinal disorders such as diarrhoea.

 

ACKNOWLEDGEMENT:

We are grateful to Faculty of Pharmaceutical Sciences, UCSI University for providing the facilities during this study.

 

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Received on 17.10.2019            Modified on 21.11.2019

Accepted on 28.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4346-4350.

DOI: 10.5958/0974-360X.2020.00768.4