Antidiarrhoeal and Spasmolytic Activities of the Methanolic Crude Extract of Alstonia Scholaris L. Are Mediated Through Calcium Channel Blockade

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Department of Biological & Biomedical Sciences Medical College, Pakistan

January 2010 Antidiarrhoeal and spasmolytic activities of the methanolic crude extract of Alstonia scholaris L. are mediated through calcium channel blockade. Abdul Jabbar Shah Aga Khan University

Saqib Ali Gowani Aga Khan University

Akber Jalil Zuberi Aga Khan University

Muhammad Nabeel Ghayur Aga Khan University

Anwar Gilani Aga Khan University

Follow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs Part of the Natural Products Chemistry and Pharmacognosy Commons, and the Physiology Commons

Recommended Citation Shah, A., Gowani, S., Zuberi, A., Ghayur, M., Gilani, A. (2010). Antidiarrhoeal and spasmolytic activities of the methanolic crude extract of Alstonia scholaris L. are mediated through calcium channel blockade.. Phytotherapy Research, 24(1), 28-32. Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/25 PHYTOTHERAPY RESEARCH Phytother. Res. 24: 28–32 (2010) 28 Published online 16 June 2009 in Wiley InterScienceA. J. SHAH ET AL. (www.interscience.wiley.com) DOI: 10.1002/ptr.2859

Antidiarrhoeal and Spasmolytic Activities of the Methanolic Crude Extract of Alstonia scholaris L. are mediated through Calcium Channel Blockade

Abdul Jabbar Shah1,2, Saqib Ali Gowani1, Akber Jalil Zuberi1, Muhammad Nabeel Ghayur1,3 and Anwarul Hassan Gilani1* 1Drug Discovery and Natural Products Research Unit, Department of Biological and Biomedical Sciences, Aga Khan University Medical College, Karachi-74800, Pakistan 2Department of Pharmaceutical Sciences, COMSATS, Institute of Information Technology, University Road, Abbottabad 22060, Pakistan 3Department of Medicine, McMaster University, St Joseph’s Hospital, Hamilton, Canada

This study was aimed to provide a pharmacological basis to the medicinal use of Alstonia scholaris as an antidiarrhoeal and antispasmodic by using in vivo and in vitro techniques. In the in vivo study the crude extract of Alstonia scholaris (As.Cr), which tested positive for the presence of alkaloids, provided 31–84% protection against castor oil-induced diarrhoea in mice at 100–1000 mg/kg doses, similar to loperamide. In + isolated rabbit jejunum preparation, the As.Cr caused inhibition of spontaneous and high K (80 mM)-induced

contractions, with respective EC50 values of 1.04 (0.73–1.48) and 1.02 mg/mL (0.56–1.84; 95% CI), thus showing spasmolytic activity mediated possibly through calcium channel blockade (CCB). The CCB activity was further conﬁrmed when pretreatment of the tissue with the As.Cr (0.3–1 mg/mL) caused a rightward shift in the Ca++ concentration-response curves similar to verapamil, a standard calcium channel blocker. Loperamide also inhibited spontaneous and high K+ precontractions as well as shifted the Ca++ CRCs to the right. These results indicate that the crude extract of Alstonia scholaris possesses antidiarrhoeal and spasmolytic effects, mediated possibly through the presence of CCB-like constituent(s) and this study provides a mechanistic base for its medicinal use in diarrhoea and colic. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: Alstonia scholaris; antidiarrhoeal; rabbit jejunum; antispasmodic; calcium channel antagonist.

antibacterial (Khan et al., 2003) and hepatoprotective INTRODUCTION (Lin et al., 1996) activities. This investigation was aimed at providing pharmacological basis for some of the Alstonia scholaris (L.) also known as Echites scholaris medicinal use of Alstonia scholaris in gastrointestinal (devil’s tree) belongs to the family Apocynaceae and disorders, such as diarrhoea and gut spasms. is found throughout tropical Eastern Asia and the Malayan Archipelago. The plant has been used in the traditional system of medicine for various gastrointestinal disorders such as diarrhoea, colic, dysentery, ulcers, MATERIALS AND METHODS dyspepsia and liver disorders (Wiart, 2002). Phytochemical studies of Alstonia scholaris shows that it contains Plant materials and extraction. Aerial parts of the mainly alkaloids (Chatterjee et al., 1965) isolated Alstonia scholaris (2.5 kg) were collected from the Aga from its leaves such as scholarisine A (Cai et al., Khan University campus Karachi, and authenticated 2008), monoterpenoid indole alkaloids (19,20) E- by Dr Rubina Abid, Department of Botany, University alstoscholarine (1) and (19,20) Z-alstoscholarine (2) of Karachi, Pakistan. A specimen voucher (AS-AP-05- (Cai et al., 2007), manilamine (1) (Macabeo et al., 2005), 02-51) was deposited at the herbarium located at the akuammiginone (1) (Salim et al., 2005), ditamine and Department of Biological and Biomedical Sciences, echitamine (echitenine) along with oxalates of calcium, Aga Khan University, Karachi. The plant material crystallizable acid and several fatty resinous substances was cleaned, shade dried and soaked in 70% aqueous- (Nadkarni, 1976). The plant is reported to possess methanol at room temperature (23–25 °C) for 3 days neuropharmacological (Bhattacharya et al., 1979), thrice. The extract was filtered after each 3 days through anticancer (Jagetia and Baliga, 2006), bronchodilatory a muslin cloth and finally through filter paper (Whatman (Channa et al., 2005), antifertility (Gupta et al., 2005), qualitative grade 1). The combined filtrate was then concentrated in a rotary evaporator (35–40 °C), under * Correspondence to: Professor Anwarul Hassan Gilani, Department reduced pressure (−760 mmHg) to a thick, dark brown of Biological and Biomedical Sciences, Aga Khan University Medical coloured crude extract (As.Cr) with a yield of 2% (40 g) College, Karachi-74800, Pakistan. − E-mail: [email protected] and stored at 4 °C for future use. The crude extract Contract/grant sponsor: Pakistan Science Foundation. was dissolved in normal saline and distilled water for

Copyright © 2009 John Wiley & Sons, Ltd. Phytother.Received Res. 24: 2528–32 May (2010) 2008 RevisedDOI: 11 March 10.1002/ptr 2009 Copyright © 2009 John Wiley & Sons, Ltd. Accepted 11 March 2009 GASTROINTESTINAL EFFECTS OF ALSTONIA SCHOLARIS L. 29 in vivo and in vitro experiments, respectively, and dilu- and aerated with a mixture of 5% carbon dioxide tions were made fresh on the day of experiment. in oxygen (carbogen). The composition of Tyrode’s

solution in mM, was: NaCl 136.9, KCl 2.7, MgCl2 1.1, Preliminary phytochemical analysis. Alstonia scholaris NaHCO3 11.9, NaH2PO4 0.4, CaCl2 1.8 and glucose 5.6 crude extract was screened for the presence of saponins, (pH 7.4). A preload of 1 g was applied and the tissues flavonoids, flavanols, flavones, tannins, phenols, coumarins, kept undisturbed for an equilibrium period of 30 min sterols, terpenes, alkaloids and anthraquinones by using after which control responses to a sub-maximal dose methods described by Wall et al. (1952). of acetylcholine (0.3 µM) were obtained and the tissue presumed stable only after the reproducibility of the Drugs and standards. The following reference chemicals said responses. were obtained from the sources specified: loperamide Under these experimental conditions, the rabbit jejunum hydrochloride, acetylcholine chloride, verapamil hydro- exhibits spontaneous rhythmic contractions, allowing the chloride, potassium chloride (Sigma Chemical Company, relaxant (spasmolytic) activity to be tested directly with- St Louis, MO, USA) and castor oil (Karachi Chemical out the use of an agonist (Gilani et al., 1994). Industries, Karachi, Pakistan). All chemicals used were of the highest purity grade. Stock solutions of all the Determination of calcium antagonist activity. To assess chemicals were made in distilled water and the dilutions whether the spasmolytic activity of the test substances, were made fresh in normal saline on the day of the was through calcium channel blockade, K+, as KCl, was experiment. used to depolarize the preparations (Farre et al., 1991). + K (80 mM) was added to the tissue bath, which produced Animals. The experiments performed complied with the sustained contraction. Test materials were then added rulings of the Institute of Laboratory Animal Resources, in a cumulative fashion to obtain concentration-dependent Commission on Life Sciences, National Research inhibitory responses (Van-Rossum, 1963). The relaxa- Council (NRC, 1996) and approved by the Ethical Com- tion of intestinal preparations, precontracted with K+ c mittee of Aga Khan University Karachi. Balb mice (80 mM), was expressed as the percent of the control (20–25 g) and local rabbits (1.5–2 kg) of either sex used response mediated by K+. in the study were bred and housed in the animal house To confirm the calcium antagonist activity of test sub- of the Aga Khan University under controlled environ- stances, the tissue was allowed to stabilize in normal ment (23–25 °C). Animals were given tap water ad Tyrode’s solution, which was then replaced with Ca++- libitum and a standard diet consisting of (g/kg): flour free Tyrode’s solution containing EDTA (0.1 mM) for 380, chokar 380, molasses 12, salt 5.8, nutrivet L 2.5, 30 min in order to remove calcium from the tissues. potassium meta bisulphate 1.2, vegetable oil 38, fish This solution was further replaced with K+-rich and Ca++- meal 170 and powdered milk 150. free Tyrode’s solution, having the following composi-

tion: NaCl 91.04, KCl 50, MgCl2 1.05, NaHCO3 11.90, Castor oil-induced diarrhoea. The in vivo antidiarrhoeal NaH2PO4 0.42, glucose 5.55 and EDTA 0.1 mM. activity of the extract was conducted following the Following an incubation period of 30 min, control methods described previously (Awouters et al., 1978). concentration-response curves (CRCs) of Ca++ were In the present study Balbc albino mice were fasted for obtained. When the control CRCs of Ca++ were found 18 h. The animals were divided in five groups, housed to be superimposable (usually after two cycles), the in five steel cages, five in each, and the bottom of each tissue was pretreated with the plant extract for 60 min cage was covered with a blotting sheet. The first group to test the possible calcium channel blocking effect. The received saline as the vehicle control (10 mL/kg, p.o.) CRCs of Ca++ were reconstructed in the presence of and so acted as the negative control. The doses of the different concentrations of the test material. crude extract of Alstonia scholaris used were selected on a trial basis and were administered orally (100, 300 Statistics. The data expressed are mean ± standard and 1000 mg/kg) by an intragastric feeding needle as a error of the mean (SEM), and the median effective suspension to three groups of animals. The fifth group concentrations (EC50 values) are given with 95% con- received loperamide (10 mg/kg) as a suspension, for fidence intervals (CI). The statistical parameter applied comparison and served as a positive control. One hour is the Student’s t-test with p < 0.05 noted as signifi- after treatment each animal received 10 mL/kg of castor cantly different (GraphPad Prism). oil orally and was then observed for defaecation. Up to 6 h after the castor oil challenge, the presence of characteristic diarrhoeal droppings was noted on the blotting sheets in the individual mouse cages. The per- RESULTS AND DISCUSSION cent protection against the castor oil-induced diarrhoea was calculated based on the number of dry faeces in Based on the medicinal use of Alstonia scholaris in each cage in comparison with the wet. hyperactive gut disorders such as diarrhoea and spasm (Wiart, 2002), its crude extract was tested for a possible Isolated rabbit jejunum tissue preparations. The isolated antidiarrhoeal effect in mice. When tested against the tissue experiments were carried out as previously described castor oil-induced diarrhoea, As.Cr like the standard (Gilani et al., 2005). The animals had free access to antidiarrhoeal agent, loperamide, inhibited significantly water but were fasted for 24 h before the experiment. (p < 0.05) the frequency of defaecation when compared The animals were killed by cervical dislocation, the with untreated mice (i.e. mice receiving neither As.Cr, abdomen was cut open and the jejunal portion isolated. nor loperamide, but castor oil only). Both substances Preparations 2 cm long were mounted in 10 mL tissue also reduced greatly the wetness of the faecal droppings baths containing Tyrode’s solution maintained at 37 °C and provided around 31–84% and 94% protection,

Table 1. Effect of the crude extract of Alstonia scholaris on castor oil-induced diarrhoea in mice

Total number Total number of Group Dose of faeces in 4 h wet faeces in 4 h % protection

Control (saline) 10 mL/kg 14.80 ± 1.10 0.20 ± 0.31 98.46 ± 0.88 Castor oil 10 mL/kg 11.80 ± 0.80 11.6 ± 0.77 1.33 ± 0.82 + As.Cr 100 mg/kg 19.80 ± 1.03 14.20 ± 0.96 31.73 ± 1.02a + As.Cr 300 mg/kg 20.80 ± 1.14 9.20 ± 0.58 52.85 ± 1.88b + As.Cr 1000 mg/kg 13.80 ± 1.15 2.40 ± 0.87 84.38 ± 1.93c + Loperamide 10 mg/kg 8.80 ± 1.18 0.4 ± 0.34 94.82 ± 1.39

p values were calculated vs control (saline treated). a p < 0.05, b p < 0.01, c p < 0.001. As.Cr, crude extract of Alstonia scholaris. respectively (Table 1). The induction of diarrhoea by preparations, including rabbit jejunum, is dependent castor oil results from the action of ricinoleic acid upon an increase in the cytoplasmic free [Ca++], which formed in the hydrolysis of the oil (Iwao and Terada, activates the contractile elements (Karaki and Wiess, 1962), which produces changes in the transport of 1983). The increase in intracellular Ca++ occurs either water and electrolytes resulting in a hypersecretory via an influx through voltage-dependent Ca++ channels response and generation of giant contraction of the (VDCs) or its release from intracellular stores in the transverse and distal colon (Croci et al., 1997). It was sarcoplasmic reticulum. Periodic depolarization and studied further in the in vitro model to see if the plant repolarization regulates the spontaneous movements extract inhibited gut motility. of the intestine and at the height of depolarization, the Spontaneously beating isolated rabbit jejunum prepa- action potential appears as a rapid influx of Ca++ via ration is used routinely to test the possible inhibitory VDCs (Brading, 1981). Thus the inhibitory effect of (spasmolytic) effect of test substances without use of the As.Cr on spontaneous movements of rabbit jejunum spasmogen (Gilani et al., 1994). When tested in isolated may appear be due to interference either with the Ca++ rabbit jejunum preparations, cumulative addition of the release or with the Ca++ influx through VDCs. As.Cr, loperamide and verapamil caused concentration- It was observed previously that the spasmolytic con- dependent inhibition of the spontaneous contractions stituents present in different medicinal plants mediate (Fig. 1), with a respective EC50 value of 1.04 mg/mL (0.734– their effect usually through a CCB effect (Gilani et al., 1.484), 37.16 µM (190.09–72.36) and 0.10 µM (0.06–0.15) 1999, 2005, 2006). To see whether the spasmolytic (Fig. 2), thus showing intestinal smooth muscle relaxant effect of the plant extract observed in this study is also (spasmolytic) activity. The contraction of smooth muscle mediated through CCB, a high concentration of K+ (80 mM) was introduced to depolarize the tissue. The crude extract was then added in a cumulative fashion, where it caused a dose-dependent relaxation of the in-

duced contractions with an EC50 value of 1.02 mg/mL (0.56–1.84) as shown in Fig. 2A, suggesting that the spasmolytic effect is possibly mediated through CCB. Similarly, verapamil and loperamide also caused a dose- related inhibitory effect against high K-induced contractions. When the inhibitory effect against K+ was compared with that on spontaneous contractions, the crude extract showed a similar potency, as was shown by loperamide, while verapamil was more potent against K+-induced contractions, a typical characteristic of CCB (Triggle, 1992). Loperamide is also known to exhibit the CCB effect (Reynolds et al., 1984), in addition to its spasmolytic effect through opioid receptors (Croci et al., 1997). It is possible that the plant extract also possesses some relaxant component in addition to its CCB effect. + The contractions induced by high K (>30 mM) are dependent on the entry of Ca++ into the cells through voltage-dependent calcium channels (VDCs) (Bolton, 1979) and a substance which can inhibit high K+- induced contractions is therefore considered to be a CCB (Godfraind et al., 1986). Thus, the inhibition of + high K (80 mM)-induced contractions of rabbit jejunum by As.Cr may reﬂect the restricted Ca++ entry via VDCs. Figure 1. Spasmolytic effect of the crude extract of Alstonia This hypothesis was further strengthened when pretreat- scholaris (As.Cr), loperamide and verapamil on spontaneous ment of the tissues with As.Cr (0.3–1 mg/mL) caused ++ contractions in isolated rabbit jejunum preparation. a rightward shift in the Ca CRCs (Fig. 2C), similar

Figure 2. Concentration-response curves of (A) the crude extract of Alstonia scholaris (As.Cr), (B) loperamide and (C) verapamil on + spontaneous and K (80 mM)-induced contractions. Figures (D), (E) and (F) show the inhibitory effect of As.Cr, loperamide and verapamil, respectively, on Ca++ concentration response curves in isolated rabbit jejunum preparations. Values shown are mean ± SEM (n = 3–5). to verapamil (Fig. 2F). Loperamide pretreatment also might be the active candidate(s) responsible for its caused a rightward shift in the Ca++ CRCs (Fig. 2E), medicinal use in diarrhoea and colic, although addi- which is in accordance with its known CCB effect at tional mechanisms cannot be ruled out. antidiarrhoeal concentrations (Reynolds et al., 1984). This study thus showed that the crude extract of These data indicate that As.Cr possesses a CCB-like Alstonia scholaris possesses a spasmolytic effect medi- effect similar to verapamil, which provides a pharma- ated through a CCB-like effect, which may provide a cological basis to its antispasmodic and antidiarrhoeal pharmacological base to its medicinal use in diarrhoea effects, as the CCBs are considered useful as and spasms. antidiarrhoeal and antispasmodic (Brunton, 1996). The presence of alkaloids in the crude extract revealed by preliminary phytochemical analysis, support the CCB Acknowledgements effect of the plant because plant derived alkaloids have been found to be CCB (Takemura et al., 1995), which This study was supported in part by Pakistan Science Foundation.

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