Gastrointestinal, Selective Airways and Urinary Bladder Relaxant Effects Of

eCommons@AKU

Department of Biological & Biomedical Sciences Medical College, Pakistan

February 2008 Gastrointestinal, selective airways and urinary bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca(2+) channels Anwarul Hassan Gilani Aga Khan University

Arif-Ullah Khan Aga Khan University

Mustafa Raoof Aga Khan University

Muhammad Nabeel Ghayur Aga Khan University

Bina S. Siddiqui

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Recommended Citation Gilani, A., Khan, A., Raoof, M., Ghayur, M., Siddiqui, B., Vohra, W., Begum, S. (2008). Gastrointestinal, selective airways and urinary bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca(2+) channels. Fundamental & Clinical Pharmacology, 22(1), 87-99. Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/76 Authors Anwarul Hassan Gilani, Arif-Ullah Khan, Mustafa Raoof, Muhammad Nabeel Ghayur, Bina S. Siddiqui, Waseem Vohra, and Sabira Begum

This article is available at eCommons@AKU: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/76 doi: 10.1111/j.1472-8206.2007.00561.x

ORIGINAL Gastrointestinal, selective airways and ARTICLE urinary bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca2+ channels

Anwarul Hassan Gilania*, Arif-ullah Khana,b, Mustafa Raoof a, Muhammad Nabeel Ghayura,c, Bina S. Siddiquid, Waseem Vohrad, Sabira Begumd aNatural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi – 74800, Pakistan bDepartment of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan cDepartment of Medicine, McMaster University, St. Joseph’s Hospital, Hamilton, ON, Canada dHEJ Research Institute of Chemistry, University of Karachi, Karachi – 75270, Pakistan

Keywords ABSTRACT airways and bladder selectivity, This study describes the spasmolytic, antidiarrhoeal, antisecretory, bronchodilatory anticholinergic, and urinary bladder relaxant properties of Hyoscyamus niger to rationalize some of Ca2+ antagonist, its medicinal uses. The crude extract of H. niger seeds (Hn.Cr) caused a complete Hyoscyamus niger concentration-dependent relaxation of spontaneous contractions of rabbit jejunum, similar to that caused by verapamil, whereas atropine produced partial inhibition. + Hn.Cr inhibited contractions induced by carbachol (1 lM) and K (80 mM)ina Received 21 June 2007; revised 13 September 2007; pattern similar to that of dicyclomine, but different from verapamil and atropine. 2+ accepted 13 November 2007 Hn.Cr shifted the Ca concentration–response curves to the right, similar to that caused by verapamil and dicyclomine, suggesting a Ca2+ channel-blocking mechanism in addition to an anticholinergic effect. In the guinea-pig ileum, Hn.Cr *Correspondence and reprints: produced a rightward parallel shift of the acetylcholine curves, followed by a non- [email protected] parallel shift with suppression of the maximum response at a higher concentration, similar to that caused by dicyclomine, but different from that of verapamil and atropine. Hn.Cr exhibited antidiarrhoeal and antisecretory effects against castor oil- induced diarrhoea and intestinal ﬂuid accumulation in mice. In guinea-pig trachea and rabbit urinary bladder tissues, Hn.Cr caused relaxation of carbachol (1 lM) and + K (80 mM) induced contractions at around 10 and 25 times lower concentrations than in gut, respectively, and shifted carbachol curves to the right. Only the organic fractions of the extract had a Ca2+ antagonist effect, whereas both organic and aqueous fractions had anticholinergic effect. A constituent, b-sitosterol exhibited Ca2+ channel-blocking action. These results suggest that the antispasmodic effect of H. niger is mediated through a combination of anticholinergic and Ca2+ antagonist mechanisms. The relaxant effects of Hn.Cr occur at much lower concentrations in the trachea and bladder. This study offers explanations for the medicinal use of H. niger in treating gastrointestinal and respiratory disorders and bladder hyperactivity.

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INTRODUCTION MATERIALS AND METHODS Hyoscyamus niger Linn. (Solanaceae), commonly Plant material, preparation of crude extract and known as ‘Henbane’ and ‘Ajwain-i-khurasani’ locally fractions in Pakistan, grows wildly throughout the Himalayan The seeds of H. niger (5 kg) were purchased from the range at altitudes of 8000–11 000 feet and in local market, cleaned and coarsely ground. The pow- many other parts of the world [1]. Its seeds are used dered material was extracted four times with 98% in traditional medicine for treating a variety of methanol at room temperature. The soaked material ailments including asthma, cough, colic, diarrhoea was filtered through a muslin cloth and then through and genito-urinary complaints such as irritable blad- Whatman qualitative grade 1 filter paper [17]. This der [2,3]. procedure was repeated thrice and the combined filtrate Phytochemical studies revealed the presence of was evaporated with a rotary evaporator under reduced hyoscine (scopolamine), hyoscyamine, anisodine, pressure to a thick syrupy mass, the crude extract anisodamine [4,5], aesculetin, coumarin, kaempferol, (Hn.Cr), at a yield of approximately 19.6%. Activity- quercetin, rutin, cuscohygrine, chlorogenic acid, lino- guided fractionation of the parent extract was carried leic acid, myristic acid, oleic acid, stearic acid, pyridine, out by using solvents of increasing polarity. Hn.Cr was trimethylamine [6], b-sitosterol, grossamide, canna- partitioned between ethyl acetate and water. The ethyl bisin D & G, daucosterol, N-trans-feruloyltyramine, acetate phase was dried over Na2SO4 (anhydrous), then 1-O-octadecanoyl glycerol, 1-O-(9Z,12Z-octadecadienoyl) concentrated at reduced pressure to yield an ethyl glycerol, 1-O-(9Z,12Z-octadecadienoyl)-3-O-(9Z-octa- acetate residue. The water layer was dried to obtain decenoyl) glycerol, vanillic acid [7], calystegines [8] the aqueous fraction (Hn.Aq). The ethyl acetate residue and withanolides [9]. The plant is known to possess was treated with petroleum ether to obtain the petro- antispasmodic, analgesic [10], mydriatic [11] and leum ether fraction (Hn.PE) and insoluble fraction. The sedative [12] properties. latter was treated with ethyl acetate to give the ethyl Hyoscine and hyoscyamine, previously identified acetate fraction (Hn.EtAc). The fraction left after as the anticholinergic constituents in H. niger, are extraction with ethyl acetate was dissolved in metha- considered responsible for its antispasmodic effect [13]. nol to obtain the methanolic fraction (Hn.MeOH). However, it is generally believed that the plant Stock solutions of the plant materials were prepared contains multiple compounds acting on different sites. in 10% ethanol, and subsequent dilutions in distilled

The aim of this study was to see if H. niger has an H2O/saline. antispasmodic effect on the gut, airways and urinary bladder through a combination of mechanisms. Plants Chemicals with medicinal use in hyperactive gut and airway Acetylcholine chloride, atropine sulphate, b-sitosterol, disorders usually possess a Ca2+ channel-blocking carbachol, chlorogenic acid, coumarin, dicyclomine, action in combination with other spasmolytic mecha- loperamide hydrochloride, scopolamine, vanillic acid nisms [14–16]. In this investigation, we report the and verapamil hydrochloride were purchased from presence of a combination of anticholinergic and Ca2+ Sigma Chemicals Co (St Louis, MO, USA). Hyoscyamine antagonist effects in H. niger seed extract, studied in sulphate and castor oil were obtained from ChromaDex tissues relevant to gastrointestinal, airway and urinary (Santa Ana, CA, USA) and KCl Pharma (Karachi, systems. Activity-directed fractionation revealed that Pakistan) respectively. Chemicals used for making the anticholinergic component(s) was widely distrib- physiological salt solutions were potassium chloride uted both in the organic and aqueous fractions, while (Sigma Chemicals Co), calcium chloride, glucose, Ca2+ antagonist was concentrated only in the organic magnesium chloride, magnesium sulphate, potassium fraction. Apart from the crude extract and its resultant dihydrogen phosphate, sodium bicarbonate, sodium fractions, the pure compounds of H. niger namely dihydrogen phosphate (Merck, Darmstadt, Germany) b-sitosterol, scopolamine, hyoscyamine, coumarin, and sodium chloride from BDH Laboratory Supplies chlorogenic acid and vanillic acid were also studied. (Poole, UK). All chemicals used were of analytical grade b-sitosterol was found to exhibit spasmolytic effect via and solubilized in distilled H2O except, b-sitosterol which Ca2+ antagonist mechanism. was dissolved in 10% ethanol; subsequent dilutions were

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2+ prepared in distilled H2O. Vehicles used had no effect on To conﬁrm the Ca antagonist action of the test tissue contractility in control experiments. substance, the tissue was allowed to stabilize in normal Tyrode’s solution, which was then replaced with Ca2+- Experimental animals free Tyrode’s solution containing ethylenediaminetetra- Animals used in this study, adult rabbits (1.0–1.2 kg), acetic acid (EDTA; 0.1 mM) for 30 min in order to remove guinea-pigs (500–550 g) and BALB/c mice (20–25 g) Ca2+ from the tissues. This solution was replaced with K+- of either sex and local breed, were housed at the rich and Ca2+-free Tyrode’s solution, having the compo-

Animal House of the Aga Khan University and main- sition (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 tained at 23–25 C. Animals were given tap water ad 11.90, NaH2PO4 0.42, glucose 5.55 and EDTA 0.1. libitum and a standard diet consisting of (g/kg): flour Following an incubation period of 30 min, control 380, chokar 380, molasses 12, sodium chloride 5.8, concentration–response curves of Ca2+ were constructed. nutrivet L 2.5, potassium meta-bisulphate 1.2, vegeta- When the control Ca2+ concentration–response curves ble oil 38, fish meal 170 and powdered milk 150. were super-imposable (usually after two cycles), the Rabbits and guinea-pigs had free access to water, but tissue was pretreated with the plant extract for 60 min food was withdrawn 24 h prior to experiments. They to test for a possible Ca2+ antagonist effect. The concen- were killed by a blow on the back of the head and tration–response curves of Ca2+ were reconstructed in cervical dislocation respectively. Experiments performed the presence of different concentrations of the test complied with the recommendations of the Institute of material. Laboratory Animal Resources, Commission on Life Sciences, National Research Council [18] and approved Guinea-pig ileum by the Ethics Review Committee of the Aga Khan The ileum was dissected and kept in Tyrode’s solution. University. The segments, about 2 cm long, were mounted individually in a 10-mL tissue bath, filled with Tyrode’s Isolated tissue preparations solution, at 37 C and aerated with carbogen. An initial Rabbit jejunum load of 0.7 g was applied to the tissue and isotonic The jejunum was dissected, kept in Tyrode’s solution and contractions were recorded with a Bioscience transducer cleaned of mesentery tissues [19]. Segments about 2 cm coupled to a Harvard oscillograph (Harvard Apparatus, long were suspended individually in a 10-mL tissue bath Holliston, MA, USA). An equilibrium period of 30 min containing Tyrode’s solution maintained at 37 C and was required before administration of drugs. After aerated with a mixture of 95% O2 and 5% CO2 (carbo- equilibration, each tissue preparation was repeatedly gen). The composition of Tyrode’s solution in mM was: treated with sub-maximal concentrations (0.3 lM)of

KCl 2.68, NaCl 136.9, MgCl2 1.05, NaHCO3 11.90, acetylcholine at 3-min intervals until constant responses NaH2PO4 0.42, CaCl2 1.8 and glucose 5.55. Intestinal were recorded. Control acetylcholine concentration– contractions were recorded isotonically using Bioscience response curves were then constructed by addition of transducers and oscillograph (Harvard Apparatus, increasing concentration of agonist and washing be- Holliston, MA, USA). Tissues were allowed to equilibrate tween doses. Acetylcholine curves were then re-deter- for at least 30 min before the addition of any drug, then mined in the presence of increasing concentrations of stabilized with a sub-maximal concentration of acetyl- test and control drugs as described previously [22]. choline (0.3 lM) at 3-min intervals until constant responses were recorded. Under these experimental Guinea-pig trachea conditions, rabbit jejunum exhibits spontaneous rhyth- Trachea was dissected and kept in Kreb’s solution. The mic contractions, allowing the testing of relaxant (spas- tracheal tube was cut into rings, 2–3 mm wide, each molytic) activity directly without the use of an agonist. containing about two cartilages. Each ring was opened For the determination of Ca2+ channel-blocking by a longitudinal cut on the ventral side opposite the + activity, high K concentration (80 mM) was used to smooth muscle, forming a tracheal chain with smooth depolarize the preparations as described by Farre et al. muscle in the centre and cartilaginous portions on the [20]. K+ was added to the tissue bath, which produced a edges. The preparation was mounted in a 20-mL tissue sustained contraction. Test materials were then added in bath containing Kreb’s solution at 37 C, aerated with cumulative fashion to obtain concentration-dependent carbogen. The composition of Kreb’s solution was (mM): inhibitory responses [21]. NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4

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1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). A tension of treatment, each animal received 10 mL/kg of castor oil 1 g was applied to the tracheal strips continuously orally through a feeding needle [25]. Afterwards, the throughout the experiment. The tissue was equilibrated cages were inspected for the presence of diarrhoea for 1 h before the addition of any drug. In some droppings; their absence was noted as a positive result, + preparations, carbachol (1 lM)orK (80 mM) were used indicating protection from diarrhoea at that time. to stabilize the respective preparations until constant responses of each agonist were achieved (usually three to Intestinal ﬂuid accumulation four concentrations). When sustained contractions were Different groups of overnight fasted mice were treated obtained, the relaxant effect of the test substance was with increasing doses of extract intraperitoneally assessed by adding it in a cumulative fashion. The through detachable U-100 insulin syringe with 25G · carbachol cumulative concentration–response curves 1¢¢ (0.50 · 25 mm) needle, 60 min before oral admin- were plotted using increasing concentrations of agonist. istration of castor oil (10 mL/kg). The mice were killed When a threefold increase in concentration produced no 30 min later by cervical dislocation and the entire further increment in response, the tissue was washed to intestine was removed and weighed with care, not re-establish the baseline tension (usually 30–35 min). allowing any intestinal ﬂuid to leak out [25]. The results

The carbachol curves were then replotted in the presence were expressed as (Pi/Pm) · 1000 where Pi is the weight of increasing concentrations of an extract [23]. Isometric (g) of the intestine and Pm is the weight of the animal. responses were recorded on a Grass model 7 Polygraph (Grass Instrument Company, Quincy, MA, USA). Statistical analysis Data are mean ± standard errors of mean (SEM, n = Rabbit urinary bladder number of experiments) for the median effective concen-

The whole urinary bladder was divided into four vertical trations (EC50 values) with 95% conﬁdence intervals. The strips [24]. Each preparation was mounted in a 20-mL statistical parameters applied were Student’s t-test for tissue bath containing Krebs–Henseleit solution (mM): intestinal ﬂuid accumulation, and chi-squared test for

NaCl 119, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 antidiarrhoeal activity. Difference of P < 0.05 was 1.18, MgSO4 1.17, EDTA 0.027, glucose 5.5 and HEPES considered statistically significant. Concentration– 10 aerated with carbogen to yield a pH of 7.4 at 37 C. A response curves were analysed by nonlinear regression tension of 1 g was applied continuously throughout the using GraphPad program (GraphPAD, San Diego, CA, experiment. Tissues were equilibrated for 1 h before the USA). addition of any drug. As was the case with trachea, in + some preparations carbachol (1 lM) and K (80 mM) RESULTS were used to stabilize the respective preparations until constant responses of agonists were achieved. Once Effect on jejunum sustained contractions were obtained, inhibitory effects Figure 1a compares inhibitory effects of Hn.Cr with those of the plant material were assessed by adding it in of dicyclomine, verapamil and atropine on the sponta- a cumulative fashion. The carbachol concentration– neous contractions of isolated rabbit jejunum. The response curves were plotted in the absence and presence respective EC50 values were 5.6 lg/mL (4.1–7.6, 95% of increasing concentrations of the extract. Isometric confidence interval, n = 5), 0.53 lM (95% CI 0.40–0.70, responses were recorded on a Grass model 7 Polygraph. n = 4), 0.23 lM (95% CI 0.17–0.32, n = 5) and 0.001 lM (95% CI 0.0008–0.01, n = 4). The spasmo- In vivo experiments lytic effect was concentration-dependent with the excep- Castor oil-induced diarrhoea tion of atropine, where further increments in the Mice were fasted for 24 h before the experiment. concentration beyond 0.3 lM did not show additional + Animals were housed in individual cages and divided inhibition. When tested against carbachol (1 lM) and K in four groups, each containing five mice. The first group (80 mM)-induced contractions, Hn.Cr inhibited received saline (10 mL/kg p.o.) and served as a negative (Figure 1b) carbachol-induced contractions at a much control. Extract doses were selected on trial basis and lower concentration with EC50 value of 7.2 lg/mL (95% then two increasing doses of the extract were given CI 4.4–12, n = 4) as compared with 300 lg/mL (95% CI orally. A group of mice was treated with loperamide 209.8–428.4, n = 4) for K+. Dicyclomine also showed a (10 mg/kg p.o.), as a positive control. One hour after similar pattern of inhibition (Figure 1c) with respective

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Jejunum (a) 1 min Control (b) 100

Hn.Cr 50 % of control 25 CCh (1 mM)

0.3 1 3 10 30 100 µg/mL K+ (80 mM)

Dicyclomine 0 0.3 3.0 30 300 [Hn.Cr] μg/mL

µM (c) 0.03 0.1 0.3 1 3 100 Verapamil 75

50 µM 0.03 0.1 0.3 1 % of control

Atropine 25 CCh (1 μM)

K+ (80 mM)

µM 0.01 0.1 1 10 0.001 0.003 0.01 0.03 0.1 0.3 1 3 [Dicyclomine] μM

(d) (e) 100 100

75 75

50 50 % of control % of control CCh (1 μM) 25 CCh (1 μM) 25 K+ (80 mM) K+ (80 mM)

0 0 0.003 0.03 0.3 0.0003 0.003 0.03 [Verapamil] μM [Atropine] μM

Figure 1 (a) Tracing showing spasmolytic effect of the crude extract of Hyoscyamus niger (Hn.Cr), dicyclomine, verapamil and atropine on spontaneous contractions and concentration–response curves representing comparison of (b) Hn.Cr; (c) dicyclomine; (d) verapamil and (e) atropine for the inhibitory effect against carbachol (CCh) and K+-induced contractions in isolated rabbit jejunum preparations. Symbols represent mean ± SEM, n = 4–8.

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EC50 values of 0.13 (95% CI 0.11–0.19, n = 6) and rightward shift with suppression of the maximum 3.2 lM (95% CI 1.9–5.3, n = 5). Verapamil on the other response (Figure 3c) and atropine (0.01–0.03 lM) hand, was more potent against K+-induced contractions caused a rightward parallel shift without suppression of with EC50 value of 0.04 lM (95% CI 0.03–0.05, n = 5), the maximum contractile effect (Figure 3d). compared with carbachol [0.16 lM (95% CI 0.13–0.19, n = 5)] as shown in Figure 1d. Atropine relaxed the Effect on castor oil-induced diarrhoea carbachol-induced contractions potently with EC50 value Hn.Cr exhibited a dose-dependent (300–1000 mg/kg) of 0.003 lM (95% CI 0.002–0.004, n = 8), without any inhibitory effect against castor oil-induced diarrhoea in effect on K+ (Figure 1e), as was expected. When tested for mice. The negative control treatment (saline) did not the possible interaction with Ca2+ channels, Hn.Cr protect the animals from diarrhoea. Pretreatment with caused a rightward shift of the Ca2+ concentration– the plant extract led to 40% protection from diarrhoea response curves (Figure 2a), similar to that caused by at 300 mg/kg and 80% protection at 1000 mg/kg verapamil (Figure 2b) and dicyclomine (Figure 2c). (P < 0.05 vs. saline group). Loperamide (10 mg/kg) led to complete protection from diarrhoea in the positive Effect on ileum control group (Table I). Hn.Cr at 1.0 lg/mL caused a rightward parallel shift in the acetylcholine concentration–response curves with- Effect on intestinal fluid accumulation out suppressing maximum contractile response, followed When tested against castor oil-induced intestinal fluid by a non-parallel shift with suppression of the maximum accumulation in mice, Hn.Cr had a dose-dependent response at 3.0 lg/mL (Figure 3a). Dicyclomine (0.03– (300–1000 mg/kg) antisecretory effect (Figure 4). Intes- 0.1 lM) also showed a similar pattern of shift (Figure 3b), tinal fluid accumulation in the saline-treated group was while verapamil (0.01–0.03 lM) produced a non-parallel 114.4 ± 0.77 g, while in castor oil-treated group it was

Jejunum (a) (b) 100 Control 100 Control Verapamil 0.03 μM μ Hn.Cr 100 g/mL Verapmil 0.1 μM Hn.Cr 300 μg/mL 75 75

50 50

25 25 % of control max. % of control max.

0 0

–4.5 –3.5 –2.5 –1.5 –4.5 –3.5 –2.5 –1.5 Log [Ca++] M Log [Ca++] M

50 50 Figure 2 Concentration–response curves of Ca2+ in the absence and 25 25

% of control max. presence of the increasing concentra- % of control max. tions of (a) crude extract of Hyoscyamus 0 0 niger (Hn.Cr); (b) verapamil; (c) dicyclomine and (d) b-sitosterol in isolated –4.5 –3.5 –2.5 –1.5 –4.5 –3.5 –2.5 –1.5 rabbit jejunum preparations. Symbols Log [Ca++] M Log [Ca++] M represent mean ± SEM, n = 4–6.

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Ileum (a) (b) 100 100 Control Control Hn.Cr Dicyclomine (1 μg/mL) (0.03 μM) 75 Hn.Cr 75 Dicyclomine (3 μg/mL) (0.1 μM)

50 50 % of ACh max. % of ACh max. 25 25

0 0 –8 –7 –6 –5 –4 –8 –7 –6 –5 –4 Log [ACh] M Log [ACh] M

50 50 Figure 3 Concentration–response curves of acetylcholine (ACh) in the % of ACh max. absence and presence of (a) crude extract 25 % of ACh max. 25 of Hyoscyamus niger (Hn.Cr); (b) dicyclomine; (c) verapamil and (d) atropine in 0 0 isolated guinea-pig ileum preparations. Symbols represent mean ± SEM, –8 –7 –6 –5 –4 –8 –7 –6 –5 –4 n = 3–6. Log [ACh] M Log [ACh] M

Table I Effect of the crude extract of Hyoscyamus niger (Hn.Cr) on value of 12.3 lg/mL (95% CI 7.9–18.9, n = 5) as shown castor oil (c.oil, 10 mL/kg)-induced diarrhoea. in Figure 5a. Similar to that in ileum, Hn.Cr produced a rightward parallel displacement of the carbachol curves Treatment (p.o.) No. mice/ﬁve with diarrhoea % Protection without suppression of the maximum contractile re- Saline (10 mL/kg) + (c.oil) 5 0 sponse at 1.0 lg/mL, followed by a non-parallel shift Hn.Cr (300 mg/kg) + (c.oil) 3 40 with suppression of the maximum effect at 3.0 lg/mL Hn.Cr (1000 mg/kg) + (c.oil) 1* 80 (Figure 5b). Loperamide (10 mg/kg) + (c.oil) 0** 100

*P < 0.05, **P < 0.01 compared to saline group, chi-squared test. Effect on urinary bladder + Hn.Cr inhibited carbachol (1 lM) and K (80 mM)

122.6 ± 0.70 g (P < 0.001 vs. saline group). Hn.Cr contractions with respective EC50 values of 0.6 (95% at the doses of 300 and 1000 mg/kg reduced the castor CI 0.3–1.0, n = 5) and 6.2 lg/mL (95% CI 4.2–9.1, oil-induced ﬂuid accumulation to 118.6 ± 1.10 g n = 5) as shown in Figure 5c. Hn.Cr caused a rightward (P < 0.05 vs. castor oil group) and 114.4 ± 0.70 g parallel shift of the carbachol curves without suppression (P < 0.001 vs. castor oil group) respectively. of the maximum contractile response at 1.0 lg/mL, followed by a non-parallel shift with the suppression of Effect on trachea maximum effect at 3.0 lg/mL (Figure 5d). In tracheal preparations, Hn.Cr inhibited carbachol

(1 lM) contractions at low concentrations with EC50 Effect of fractions value of 0.8 lg/mL (95% CI 0.5–1.3, n = 4), compared Hn.PE, Hn.EtAc and Hn.MeOH inhibited carbachol + + with that against K (80 mM) contractions with EC50 (1 lM) and K (80 mM) contractions of rabbit jejunum

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Saline DISCUSSION Castor oil Hyoscyamus niger is known to contain tropane alkaloids, 125 # Castor oil + Hn.Cr i.e. hyoscine and hyoscyamine, which are well known for their anticholinergic effect [13]. In view of the wide 120 * distribution of Ca2+ antagonists in medicinal plants, which usually exist in combination with other spasmo- 115 *** lytics [14–16], the present study was undertaken to see whether the antispasmodic action of H. niger is the result 110 of a combination of anticholinergic and Ca2+ antagonist

(Pi/Pm) x 1000 mechanisms. When tested in isolated rabbit jejunum 105 preparations, the plant extract completely inhibited

Intestinal fluid accumulation spontaneous contractions, like that caused by verapamil, 100 a standard Ca2+ channel blocker [26]. Atropine, a 300 1000 muscarinic receptor antagonist [27], caused partial [Hn.Cr] mg/kg relaxation of spontaneous contractions. Gastrointestinal motility is regulated by multiple physiological mediators, Figure 4 Effect of increasing doses of the crude extract of mainly acetylcholine, histamine, 5-hydroxytryptamine, Hyoscyamus niger (Hn.Cr) on castor oil-stimulated fluid accumula- bradykinins, prostaglandins, substance P and cholecysto tion in small intestine of mice. Results shown are mean ± SEM of kinins which achieve their contractile effects through an five animals for each experimental group. Intestinal fluid accumu- ultimate increase in cytosolic Ca2+ [28]. Hence, sub- lation is expressed as Pi/Pm · 1000 (g) where Pi is the weight of the 2+ # stances that evoke non-specific inhibition, like Ca small intestine and Pm is the weight of the mouse. P < 0.001 vs. saline group, *P < 0.05 and ***P < 0.001 vs. castor oil group, antagonists, are considered to more effectively suppress Student’s t-test. gut motility, whereas muscarinic receptor antagonists produce partial inhibition, as cholinergic innervation is

(Figure 6a–c) with respective EC50 values of 9.0 (95% CI one of the several mechanisms responsible for regulating 6.0–13.6, n = 3) and 59.6 (95% CI 30.2–117.8, n = 3), intestinal motility [29,30]. This suggests the presence of 236.8 (95% CI 171.3–327.2, n = 3) and 1117 (95% CI a non-speciﬁc spasmolytic component(s), most likely 738.7–1690, n = 3), 11.7 (95% CI 7.1–19.1, n =3) Ca2+ antagonist in H. niger in addition to the anticho- and 130.3 lg/mL (95% CI 93.3–226.4, n = 5), while linergic constituents (hyoscine and hyoscyamine). This

Hn.Aq relaxed carbachol (1 lM) contractions with EC50 speculation was strengthened when the plant extract value of 8.6 lg/mL (95% CI 6.2–12.0, n = 4) without reversed both carbachol and K+-induced contractions + any effect on K (80 mM), n =5(Figure 6d). (being more potent against carbachol), indicating the presence of at least two different spasmolytic Effect of pure compounds mechanisms, similar to dicyclomine, a dual blocker of Some of the known commercially available pure com- muscarinic receptors and Ca2+ influx [31]. Verapamil, pounds of H. niger were also tested in the jejunum. however, was found to be more potent against the b-sitosterol, like verapamil was found more potent contractions of K+ than carbachol, and atropine relaxed + against K (80 mM)-induced contractions (Figure 7a), carbachol-induced contractions only. + with an EC50 value of 9.6 lg/mL (95% CI 7.1–13.1, High K (>30 mM) is known to cause smooth muscle n = 5), when compared with carbachol [218.6 lg/mL contractions through opening of voltage-dependent (95% CI 129.5–369.0), n = 4] and shifted Ca2+ concen- L-type Ca2+ channels, thus allowing influx of extracel- tration–response curves to the right (Figure 2d). Scopol- lular Ca2+ [32] and a substance that inhibits high K+ 2+ amine and hyoscyamine relaxed only carbachol (1 lM) contractions is considered as a blocker of Ca influx 2+ contractions with respective EC50 values of 0.004 lg/mL [33]. The Ca antagonist effect was further confirmed (95% CI 0.003–0.005, n = 6) and 0.001 lg/mL (95% CI when H. niger extract caused a rightward shift in the 0.0009–0.002, n = 4) without any effect on K+ (Fig- Ca2+ concentration–response curves similar to that ure 7b,c). Coumarin, chlorogenic acid and vanillic acid caused by verapamil and dicyclomine. + failed to relax either carbachol (1 lM)orK (80 mM) The concept of a dual mode of inhibition involving contractions up to 1000 lg/mL. anticholinergic and Ca2+ channel blockade received

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Trachea Urinary bladder (a) (c) 100 100

75 75

50 50 % of control % of control CCh (1 μM) 25 CCh (1 μM) 25 K+ (80 mM) K+ (80 mM)

0 0 0.1 1 10 100 0.1 1 10 100 [Hn.Cr] μg/mL [Hn.Cr] μg/mL

(b) (d) 100 100 Control Control Hn.Cr 1 μg/mL Hn.Cr 1 μg/mL Hn.Cr 3 μg/mL Hn.Cr 3 μg/mL Figure 5 (a, c) represents concentra- 75 75 tion-dependent inhibitory effect of the crude extract of Hyoscyamus niger 50 50 (Hn.Cr) on carbachol (CCh) and K+- induced contractions, while (b) and % of CCh max. (d) show CCh concentration–response 25 % of CCh max. 25 curves in the absence and presence of Hn.Cr in isolated guinea-pig tracheal 0 0 and rabbit urinary bladder preparations. Symbols represent mean ± SEM, –8.5 –7.5 –6.5 –5.5 –4.5 –3.5 –8.5 –7.5 –6.5 –5.5 –4.5 –3.5 n = 4–6. Log [CCh] M Log [CCh] M additional support from acetylcholine concentration– In view of the medicinal use of H. niger in diarrhoea, response data in guinea-pig ileum, as this preparation is its extract was tested for possible protective effect against considered more useful to quantify contractile responses castor oil-induced diarrhoea in mice. Diarrhoea induced of an agonist in the presence of an inhibitor, thus by castor oil is a result of the action of ricinoleic acid allowing exploration of the nature of antispasmodic formed during hydrolysis [37], which affects transport of effect [22]. The plant extract at a low concentration electrolytes and water and generates giant contractions caused a rightward parallel shift in the acetylcholine of the transverse and distal colon [38]. The observed curves without suppressing the maximum response, a antidiarrhoeal effect of H. niger extract, following oral characteristic of a competitive or specific antagonist, like administration, could have resulted from a local or a atropine [27], then at higher concentration caused a combination of local and systemic actions. H. niger also non-parallel shift with suppression of the maximum protected the mice against castor oil-induced intestinal effect, pointing towards an additional non-competitive fluid secretions. This is expected, as both anticholinergic inhibition [34], as expected for Ca2+ antagonists [35,36]. drugs and Ca2+ antagonists possess an antidiarrhoeal Dicyclomine also shifted the acetylcholine curves similar and antisecretory actions [39–41]. to that of extract, while verapamil resulted in a right- As H. niger has uses in hyperactive respiratory and ward but non-parallel shift with suppression of the urogenital ailments, the plant extract was further studied maximum contractile effect at both concentrations used. for possible bronchodilatory and urinary bladder relax- Atropine caused a rightward parallel shift of the acetyl- ant activity. In trachea and bladder preparations, as with choline curves without suppression of the maximum the gut, the extract inhibited both carbachol and K+- response. This confirms the presence of a combination of induced contractions and displaced carbachol curves to muscarinic receptors and Ca2+ channel-blocking activ- the right in a parallel fashion without suppression of the ities in H. niger, similar to dicyclomine. maximum response at low and higher concentrations it

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Jejunum

(a) (b) 100 100

75 75

50 50

% of control μ % of control μ CCh (1 M) 25 CCh (1 M) 25 K+ (80 mM) K+ (80 mM)

0 0 0.3 3 30 1 10 100 1000 [Hn.PE] μg/mL [Hn.EtAc] μg/mL

75 75

50 50 Figure 6 Concentration-response curves showing effect of the Hyoscyamus % of control

CCh (1 μM) % of control niger fractions: (a) petroleum ether 25 25 CCh (1 μM) + (Hn.PE); (b) ethylacetate (Hn.EtAc); K (80 mM) + K (80 mM) (c) methanolic (Hn.MeOH) and (d) 0 0 aqueous (Hn.Aq) on carbachol (CCh) and K+-induced contractions in isolated 0.3 300 3 30 0.1 1 10 rabbit jejunum preparations. Symbols μ μ [Hn.MeOH] g/mL [Hn.Aq] g/mL represent mean ± SEM, n = 3–5. caused a non-parallel shift with suppression of the The presence of a Ca2+ channel-blocking component maximum effect. Interestingly, the plant extract was in H. niger reported here for the first time may enhance found to be a more potent spasmolytic in trachea and the medicinal benefits of the plant, as Ca2+ antagonists bladder than in the gut (Table II). Tissue-selective have the potential to depress cardiac arrhythmias, which behaviour of H. niger could be due to a Ca2+ antagonist usually occur when anticholinergics are used alone [45]. component, as Ca2+ channels are known to be hetero- In smooth muscles, a combination of anticholinergic and geneous and different Ca2+ antagonists have selectivity Ca2+ antagonist components could synergize, making for different organ systems [20,42]. A parallel shift of the the plant more effective for therapy against spasmodic cholinergic agonists’ concentration–response curves conditions [16]. For instance dicyclomine, which constructed in different tissues by similar concentrations possesses dual muscarinic receptors and Ca2+ channel- of the plant extract may rule out any possibility of the blocking action, is considered to be a more efficacious tissue-selective nature of the anticholinergic component, spasmolytic, compared with pure anticholinergics [46]. as expected from the non-selective profile of anticholin- The activity-directed fractionation study revealed that ergic constituents of H. niger, i.e. hyoscine and hyoscy- the Ca2+ antagonist component, as opposed to the amine [43]. Alternatively, a possibility exists that a anticholinergic was concentrated in the petroleum ether difference in physiological modulators among various fraction (around 10 times more potent than the parent tissues and/or the extent of their regulatory influences extract), while the aqueous fraction was devoid of it. This [44] may cause plant constituents to exert a better observation supports our previous findings that the Ca2+ synergistic interaction in trachea and bladder compared channel-blocking component is concentrated in the with the gut. organic fractions [47,48]. The relative potency difference

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Jejunum Table II Comparative inhibitory effects of the crude extract of Hyoscyamus niger (Hn.Cr) on different isolated tissue preparations. (a) 100 Preparation Parameter EC50 values (95% CI; lg/mL)

Jejunum CCh (1 lM) 7.2 (4.4–12), n =4 + 75 K (80 mM) 300 (209.8–428.4), n =4 Trachea CCh (1 lM) 0.8 (0.5–1.3), n =4 + K (80 mM) 12.3 (7.9–18.9), n =5 50 Bladder CCh (1 lM) 0.6 (0.3–1.0), n =5 + K (80 mM) 6.2 (4.2–9.1), n =5 % of control μ 25 CCh (1 M) Values represent geometric means along with 95% conﬁdence intervals in parenthesis. ‘n‘ represents number of observations. K+ (80 mM)

0 + 0.03 0.3 3 30 300 against carbachol and K -induced contractions in the [β-sitosterol] μg/mL organic fractions was less than that in the parent extract, probably because the Ca2+ antagonist compo- (b) 100 nent is entirely concentrated in these fractions, while anticholinergic component is distributed in both organic and aqueous fractions. Among the tested plant constit- 75 uents, b-sitosterol was found to possess Ca2+ antagonist effect, though the presence of additional compounds with 2+ 50 Ca channel-blocking mechanisms cannot be ruled out. In conclusion, H. niger offers an interesting combina-

% of control tion of spasmolytic mechanisms (anticholinergic and 25 CCh (1 μM) Ca2+ antagonist) which might be responsible for its K+ (80 mM) medicinal use in treating disorders of the gut and airways, and urinary bladder hyperactivity. The selec- 0 tivity for the latter two preparations presents an inter- 0.0003 0.003 0.03 0.3 esting scenario, which warrants further studies to ﬁnd μ [Scopolamine] g/mL out whether this tissue-selective behaviour of the plant is due to the selectivity of the Ca2+ antagonist component 100 (c) or the better synergistic interaction of the plant-active constituents in trachea and urinary bladder. 75 ACKNOWLEDGEMENTS 50 This study was supported by funds made available by the Higher Education Commission of Pakistan under the % of control scheme of Distinguished National Professor Research 25 CCh (1 μM) Allowance. We would like to thank Prof. John Connor,

K+ (80 mM) Department of Biological and Biomedical Sciences, Aga 0 Khan University Medical College, Karachi for editorial 0.0003 0.003 0.03 0.3 suggestions. [Hyoscyamine] μg/mL REFERENCES Figure 7 Concentration–response curves showing the effect of Hyoscyamus niger active ingredients: (a) b-sitosterol; (b) scopol- 1 Nadkarni K.M. Indian materia medica, 3rd edn, Popular amine; and (c) hyoscyamine on carbachol (CCh) and K+ (80 mM)- Prakashan, Bombay, 1976. induced contractions in isolated rabbit jejunum preparations. 2 Usmanghani K., Saeed A., Alam M.T. Indusyunic medicine. Symbols represent mean ± SEM, n = 4–5. University of Karachi Press, Karachi, 1997.

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