Gut and Airways Relaxant Effects of Carum Roxburghianum Munasib Khan Aga Khan University

eCommons@AKU

Department of Biological & Biomedical Sciences Medical College, Pakistan

June 2012 Gut and airways relaxant effects of Carum roxburghianum Munasib Khan Aga Khan University

Arif-ullah Khan

Najeeb-ur-Rehman 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 Pharmacology Commons

Recommended Citation Khan, M., Khan, A., Najeeb-ur-Rehman, ., Gilani, A. (2012). Gut and airways relaxant effects of Carum roxburghianum. Journal of Ethnopharmacology, 141(3), 938-946. Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/81

Journal of Ethnopharmacology 141 (2012) 938–946

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

jo urnal homepage: www.elsevier.com/locate/jethpharm

Gut and airways relaxant effects of Carum roxburghianum

a,b c a a,∗

Munasib Khan , Arif-ullah Khan , Najeeb-ur-Rehman , Anwarul-Hassan Gilani

Natural Products Research Unit, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi-74800, Pakistan

Department of Pharmacology, Faculty of Pharmacy, University of Karachi, 75270, Pakistan

Institute of Pharmaceutical Sciences, Kohat University of Science and Technology, Kohat-26000, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Carum roxburghianum is traditionally used in hyperactive gastrointesti-

Received 3 November 2011

nal and respiratory disorders. The present study was carried out to investigate the possible gut and

Received in revised form 5 March 2012

airways relaxant potential of Carum roxburghianum to rationalize its folk uses.

Accepted 18 March 2012

Materials and methods: Crude extract of Carum roxburghianum (Cr.Cr) was studied in in vivo and in vitro Available online 4 April 2012

techniques.

Results: Cr.Cr exhibited protective effect against castor oil-induced diarrhea in mice at 100–1000 mg/kg. In

Keywords: +

rabbit jejunum preparations, Cr.Cr (0.03–3.0 mg/mL) caused relaxation of spontaneous and K (80 mM)-

Carum roxburghianum

Antidiarrheal induced contractions at similar concentrations, like papaverine. Pretreatment of tissues with Cr.Cr

Antispasmodic (0.1–1.0 mg/mL) shifted Ca concentration–response curves (CRCs) to right, like verapamil. Cr.Cr (0.03

Bronchodilator and 0.1 mg/mL) caused leftward shift of isoprenaline-induced inhibitory CRCs, similar to papaverine. In

Gastrointestinal and respiratory diseases isolated guinea-pig ileum, Cr.Cr (0.01 and 0.03 mg/mL) produced rightward parallel shift of acetylcholine-

curves, like atropine. Cr.Cr (1.0–30 mg/kg) caused suppression of carbachol (CCh, 100 ␮g/kg)-induced

increase in inspiratory pressure of anaesthetized rats. In guinea-pig trachea, Cr.Cr (0.03–1.0 mg/mL)

relaxed CCh and high K -induced contractions, shifted isoprenaline-induced inhibitory CRCs to left at 0.1

and 0.3 mg/mL and CCh-curves parallel to right (0.01 and 0.03 mg/mL). Cr.Cr did not cause any mortality

of mice up to 10 g/kg dose.

Conclusion: These results indicate that Carum roxburghianum possess combination of antidiarrheal, anti-

spasmodic and bronchodilatory effects, which provides pharmacological basis to its traditional use in the

disorders of gut and airways hyperactivity, like diarrhea, colic and asthma.

1. Introduction bronchitis cough, common cold, dyspepsia, lethargy, loss of con-

sciousness, palpitation, vomiting, pain in bladder and kidneys

Carum roxburghianum (DC) Craib & Benth. (family: Api- (Lis-Balchin and Deans, 1997; Saroja et al., 1998; Babu and Madhavi,

aceae/Umbelliferae), commonly known as “Wild celery/Ajmud” 2003; Wyk and Wink, 2004; Lokhande et al., 2006) as well as con-

and locally as “Ajmud” is native to tropical Asia, Africa and is sidered useful as anthelmintic, antigout, antimicrobial, cardiotonic,

cultivated as spice and medicinal plant in Bangladesh, India, carminative, condiment, digestive, diuretic, emmenagogue, stimu-

China, Pakistan and Indonesia (Nadkarni, 1976; Papini et al., 2007; lant and stomachic (Hanelt et al., 1986; Khare, 2004; Deshpande

Mishra, 2009). Carum roxburghianum is used in traditional system et al., 2008).

of medicine to treat diarrhea, abdominal spasm (colic), asthma, Phytochemical studies on the plant revealed pres-

ence of limonene, sabinene, terpinen-4-ol, (Z)-ligustilide,

␥

-terpinene, menthol, citronellal, 3,6-dimethyl-2,3,3a,4,5,7a-

hexahydrobenzefyran citronellol, geraniol, benzene,

Abbreviations: ACh, acetylcholine; ANOVA, analysis of variance; CaCl2, cal-

1-methoxy-4-(1-propenyl) phenol, 2-methoxy-4-(1-propenyl)-

cium chloride; CCh, carbachol; CI, conﬁdence interval; C.oil, castor oil; CRCs,

Neryl propionate, ␤-elemene, apiol, ␥-eudesmol, camphor,

concentration–response curves; EC , median effective concentration; EDTA,

50 ␣

patchuli alcohol, -pinene, ␤-pinene, ␤-myrcene, ␣-hellandrene,

ethylenediaminetetra acetic acid; Cr.Cr, Carum roxburghianum; KCl, potassium chlo-

␣

ride; KH2PO4, potassium dihydrogen phosphate; KPK, Khyber PakhtunKhwa; MgCl2, ocimene, -4-dimethylstyrene, decane, 3-methyl-cis-p-mentha-

magnesium chloride; MgSO , magnesium sulfate; mmHg, millimeter of mercury;

4 2,8-dien-1-ol, dihydrocarvone, trans-carveol, eugenol, myristicin

n, number of experiments; NaCl, sodium chloride; NaH2PO4, sodium dihydrogen

and elemicin (Chowdhury et al., 2009). Total lipids (seed) include

phosphate; NaHCO3, sodium bicarbonate; p.o., Persa ora (oral); SEM, standard error

mean. hydrocarbons, wax esters, sterol esters, triacylglycerols, free

∗ fatty acids, diacylglycerols, lysophosphatidylethanolamines and

Corresponding author. Tel.: +92 21 34864571; fax: +92 21 493 4294/494 2095.

E-mail address: [email protected] (A.-H. Gilani). phosphatidylinositols. Oleic acid was found as major component

M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946 939

in all lipid classes, whereas palmitic, linoleic and linolenic acids Council (1996) and approved by the Ethical Committee of the Aga

were present in lesser quantities. Arachidic acid was identiﬁed Khan University.

as minor component in only seven out of twelve lipid classes.

Major constituents of the essential oil from seed are: d-limolene, 2.4. In vivo experiments

␣

-terpinene, d-linalool and d-piperitone (Waheed et al., 2003;

Deshpande et al., 2008). Other constituents are bergaptene, 2.4.1. Castor oil-induced diarrhea

␤-sitosterol, cuminol, 7-methoxy-6-methylcoumarin, thymol, Mice were fasted for 24 h before the experiment. Animals were

thymoquinol and umbelliferone (Khare, 2004). housed in individual cages and divided in ﬁve groups, each con-

Despite the fact that extensive phytochemical research has taining 10 mice. The ﬁrst group received saline (10 mL/kg, p.o.)

been carried out on Carum roxburghianum, reports related to phar- and served as negative control. The doses of the test extract were

macological investigation are limited, only citing its antitumor selected on trial basis and three increasing doses of extract, 100,

activity (Alluri et al., 2005). In this study, we provide evidence 300 and 1000 mg/kg were given to three different groups. A group

that Carum roxburghianum exhibits antidiarrheal, antispasmodic of mice was treated with loperamide (10 mg/kg, p.o.), as positive

and bronchodilatory activities, which justify Carum roxburghianum control. One hour after treatment, each animal received 10 mL/kg

usefulness in overactive gut and airways disorders. of castor oil (Gilani et al., 2008a). The aforementioned test mate-

rial was administered orally to mice through feeding needle, ﬁtted

to U-100 insulin syringe. Afterward, the cages were inspected for

2. Materials and methods

the presence of diarrhea droppings, their absence was noted as a

positive result, indicating protection from diarrhea at that time.

2.1. Plant material and extraction

2.4.2. Bronchodilatory activity

Seeds were purchased from grocery shop in Peshawar, Khyber

Rats were anaesthetized with sodium thiopental (Pentothal,

PakhtunKhwa (KPK), Pakistan. The plant material was identi-

80–100 mg/kg, i.p.), than incubated with 5 cm long tracheal tube

ﬁed with help of a taxonomist, Dr. Ilyas Iqbal, Department of

(Intramedic Polyethylene Tubing, ID 1.77 mm. OD 2.80 mm, Clay

Botany, University of Malakand, KPK, Pakistan. A voucher speci-

Adams, Division of Becton Dickinson and Company, Parsippany, NJ,

men (UOM/BGH/148) has been submitted to the herbarium of same

USA) and ventilated with volume ventilator (Miniature ideal pump,

university. The seed were clean up adulterants and approximately

Bioscience, UK) adjusted at rate of 70–80 strokes/min to deliver

909.09 gm of the seeds were soaked in 4.0 L aqueous-methanol

7–10 mL/kg of room air. A polyethylene catheter was inserted into

(70%) at room temperature for three days with occasional shak-

jugular vein for drugs administration. Changes in the inspiratory

ing. The soaked material was ﬁltered through a muslin cloth and

pressure (mmHg) were measured by pressure transducer (MLT-

then through Whatman qualitative grade 1 ﬁlter paper (Williamson

1199) connected to side arm of tracheal cannula and recorded

et al., 1998). This procedure of soaking and ﬁltration was repeated

by PowerLab 4/25 with running chart software via Quad bridge

twice more. All the ﬁltrates were combined and evaporated to dry-

− ampliﬁer (AD Instruments, Bella Vista, NSW, Australia). Bron-

ness on rotary evaporator under reduced pressure ( 760 mmHg)

◦ choconstriction was induced with CCh (100 ␮g/kg), which was

at 35–40 C to obtain crude extract of Carum roxburghianum (Cr.Cr)

reversed within 7–10 min (Khan et al., 2012). Test drug was given to

having 23.25% yield. Cr.Cr was completely soluble in normal saline

animals 5–8 min prior to administration of CCh. The responses were

(0.9%, w/v) and distilled water for in vivo and in vitro experiments.

expressed as percent reduction of CCh-induced bronchospasm.

2.2. Chemicals

2.4.3. Acute toxicity test

Mice were divided in groups of ﬁve mice each. The test was per-

Acetylcholine chloride (ACh), atropine, carbachol (CCh), dicy-

formed using increasing doses of the plant extract, given orally in

clomine, isopreanaline, loperamide, papaverine and verapamil

10 mL/kg volume to different groups serving as test groups. Another

were purchased from Sigma Chemicals Co., St. Louis, MO, USA.

group of mice was administered saline (10 mL/kg, p.o.) as negative

Aminophylline, pentothal sodium (thiopental) and castor oil were

control. The mice were allowed food ad libitum and kept under

respectively obtained from GlaxoSmithKline, Abbot Laboratories

regular observation for lethality recorded after 24 h (Gilani et al.,

and KCL Pharma, Karachi, Pakistan. Chemicals used for mak- 2005a).

ing physiological salt solutions were: potassium chloride (KCl,

Sigma Chemicals Co., St. Louis, MO, USA), calcium chloride (CaCl2),

2.5. Isolated tissue preparations

Ethylenediaminetetra acetic acid (EDTA), glucose, magnesium

chloride (MgCl2), magnesium sulfate (MgSO4), potassium dihydro-

2.5.1. Rabbit jejunum

gen phosphate (KH2PO4), sodium bicarbonate (NaHCO3), sodium

The rabbit abdomen was opened and jejunum was dissected

dihydrogen phosphate (NaH2PO4, Merck, Darmstadt, Germany)

out, kept in normal Tyrode’s solution and cleaned of mesenter-

and sodium chloride (NaCl) from BDH Laboratory supplies, Poole,

ies. Each segment of about 2 cm length was suspended in 10 mL

England. All chemicals used were of analytical grade and solubilized

tissue bath containing Tyrode’s solution (pH 7.4), maintained at

in distilled H2O/saline. ◦

37 C and aerated with a mixture of 95% O2 and 5% CO2 (carbogen).

The composition of Tyrode’s solution was (mM): NaCl: 136.9, KCl:

· ·

2.3. Animals 2.7, MgCl2 6H2O: 0.5, NaHCO3: 11.9, NaH2PO4 2H2O: 0.32, CaCl2:

1.8 and glucose: 5.05. One end of the segment was attached to

Rabbits (1–1.2 kg), guinea-pigs (500–550 g), Sprague-Dawley metal tissue hook and other was attached by cotton thread to an

rats (200–250 g) and Balb-C mice (20–25 g) of local breed and either isotonic Bioscience transducer, connected to Student Oscillograph

sex were used for this study housed at the Animal House of the Aga (Harvard Apparatus, Holliston, MA, USA). An initial load of 1 g was

◦

Khan University, maintained at 23–25 C and were given a standard applied to each tissue and was allowed to equilibrate for 30 min

diet and tap water. Rabbits and guinea-pigs starved for 24 h were before the addition of any drug. Following equilibration period,

sacriﬁced by blow on back of head and cervical dislocation. Exper- each preparation was then stabilized with sub-maximal concen-

␮

iments performed complied with rulings of Institute of Laboratory tration of ACh (0.3 M) at 3 min interval until constant responses

Animal Resources, Commission on Life Sciences, National Research were recorded (Gilani et al., 2005a). The inhibitory effects of test

940 M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946

Table 1

substances were measured as percent change in jejunum spon-

+ Effect of the crude extract of Carum roxburghianum (Cr.Cr) on castor oil (C.oil,

taneous contractions. High K (80 mM) was used to depolarize

10 mL/kg)-induced diarrhoea in mice.

the preparations. Once plateau of the induced contraction was

Treatment (p.o.) No. of mice/10 with % protection

achieved (usually within 7–10 min), the test material was then

diarrhea

added in cumulative fashion to obtain concentration-dependent

inhibitory responses. To construct Ca concentration–response Saline (10 mL/kg) + C.oil 10 0

Cr.Cr (100 mg/kg) + C.oil 9 10

curves (CRCs), the tissue was allowed to stabilize in normal Tyrode’s

++ Cr.Cr (300 mg/kg) + C.oil 8 20

solution, which was then replaced with Ca -free Tyrode’s solution *

Cr.Cr (1000 mg/kg) + C.oil 5 50

containing EDTA (0.1 mM) for 30 min in order to remove Ca from *

Dicyclomine (50 mg/kg) + C.oil 7 30

+ ++ **

tissue. This solution was further replaced with K -rich and Ca -free Dicyclomine (100 mg/kg) + C.oil 1 90

Loperamide (10 mg/kg) + C.oil 0 100

Tyrode’s solution, having composition (mM): NaCl: 91.03, KCl: 50,

· ·

MgCl2 6H2O: 0.50, NaHCO3: 11.9, NaH2PO4 2H2O: 0.32, glucose: p < 0.05 compared to saline group, Chi-square test.

5.05 and EDTA-Na2·2H2O: 0.1. Following an incubation period of p < 0.01 compared to saline group, Chi-square test.

++ ++

30 min, control CRCs of Ca were obtained. When control Ca -

CRCs were found super-imposable (usually after two cycles), the

++ 2.6. Statistical analysis

tissue was pretreated with test drug for 1 h. The CRCs of Ca were

reconstructed in the presence of different concentrations of test

The data expressed are mean ± standard error of mean (SEM,

material. The isoprenaline-induced inhibitory CRCs against CCh-

n = number of experiment) and median effective concentrations

induced contractions were constructed in absence and presence of

(EC50) with 95% conﬁdence intervals (CI). The statistical param-

test substance (Gilani et al., 2005b).

eters applied were Chi-square-test for antidiarrheal assay, one

way analysis of variance (ANOVA) followed by Dunnett’s test

for bronchodilatory activity. Difference of p < 0.05 was consid-

2.5.2. Guinea-pig ileum ered statistically signiﬁcant. The CRCs were analyzed by non-linear

The ileum was dissected out and kept in Tyrode’s solution. The regression and two-way ANOVA followed by Bonferroni’s post-test

segments, each of about 2 cm length, were mounted individually in correction was used for multiple comparisons of CRCs with respec-

◦

a 10 mL tissue bath, ﬁlled with Tyrode’s solution, at 37 C and aer- tive control. All the graphing, calculations and statistical analysis

ated with carbogen. An initial load of 0.7 g was applied to the tissue were performed using GraphPAD program (GraphPAD, San Diego,

and isotonic contractions were recorded with a Bioscience trans- CA, USA).

ducer coupled to Harvard Oscillograph. After equilibration period

of 30 min, each tissue preparation was repeatedly treated with sub-

3. Results

␮

maximal concentration (0.3 M) of ACh (Gilani et al., 2008a) until

constant responses were recorded. Control bolus curves to ACh

3.1. Effect on castor oil-induced diarrhea

were then constructed by the addition of increasing concentrations

of agonist. The ACh-CRCs were then re-determined in presence of

Cr.Cr exhibited dose-dependent (100, 300 and 1000 mg/kg)

increasing concentrations of crude extract (0.01 and 0.03 mg/mL)

protective effect against castor oil-induced diarrhea in mice. The

and atropine (0.003 and 0.01 ␮M), as described earlier (Choo and

negative control group (saline treated) did not show any protec-

Mitchelson, 1978).

tion against castor oil-induced diarrhea. Pretreatment of animals

with Cr.Cr, showed 10% protection from diarrhea at 100 mg/kg,

20% at 300 mg/kg and 50% protection at 1000 mg/kg (p < 0.05).

2.5.3. Guinea-pig trachea Dicyclomine caused protection of 30% (p < 0.05) and 90% (p < 0.01)

Trachea was dissected from guinea-pig and kept in normal at respective doses of 50 and 100 mg/kg. Loperamide (10 mg/kg)

Kreb’s solution. The tracheal tube was cut into rings, 2–3 mm wide, showed 100% protection from diarrhea (p < 0.01) in the positive

each containing about two cartilages. Each ring was opened by lon- control group (Table 1).

gitudinal cut on ventral side opposite the smooth muscle, forming

a strip with smooth muscle in center and cartilaginous portions

3.2. Effect on jejunum

on edges. The preparation was mounted in 20 mL tissue bath,

◦

containing Kreb’s solution (pH 7.4), at 37 C and aerated with car-

Cr.Cr caused concentration-dependent (0.03–1.0 mg/mL) relax-

bogen. The composition of Kreb’s solution was (mM): NaCl: 118.2,

ation of jejunum spontaneous contractions (Fig. 1). Fig. 2 shows

NaHCO : 25.0, CaCl : 2.5, KCl: 4.7, KH PO : 1.2, MgSO ·7H O:

3 2 2 4 4 2 the comparative inhibitory effect of Cr.Cr, papaverine and vera-

1.2 and glucose: 11.7. A tension of 1 g was applied to tracheal +

pamil against spontaneous and K (80 mM)-induced contractions.

strips continuously throughout experiment. Each tissue was equi- +

Cr.Cr was found to be equally effective against spontaneous and K

librated for 1 h before addition of any drug. In some preparations,

(80 mM)-induced contractions with EC50 values of 0.37 (0.32–0.48,

carbachol (1 ␮M) or K (80 mM) were used to stabilize respec-

95% CI, n = 4) and 0.34 mg/mL (0.28–0.42, n = 3) respectively as

tive preparations (Gilani et al., 2008b), until constant responses

shown in Fig. 2A. Papaverine also showed similar pattern of

of each agonist were achieved. When sustained contractions were

non-speciﬁc inhibitory response (Fig. 2B) with respective EC50 val-

obtained, relaxant effect of test substance was assessed by adding

ues of 3.4 (2.9–4.8, n = 4) and 3.6 ␮M (3.1–4.7, n = 4), whereas,

in cumulative fashion. Like in jejunum, isoprenaline-CRCs were +

verapamil was found more potent against K (80 mM)-induced

constructed in trachea, as described previously (Shah and Gilani,

contractions with EC50 value of 0.045 ␮M (0.04–0.06, n = 3), as com-

2010). Cumulative curves to CCh were constructed using increasing

pared to spontaneous contractions [0.30 ␮M (0.2–0.4, n = 3)] as

concentration of agonist. When a 3-fold increase in concentration

shown in Fig. 2C. Pretreatment of tissue with Cr.Cr (0.1–1.0 mg/mL)

produced no further increment in response, the tissue was washed ++

caused rightward shift in the Ca CRCs accompanied by sup-

to re-establish base-line tension. The CCh-CRCs were repeated

pression (p < 0.05 and p < 0.001) of the maximum contractile

in presence of increasing concentrations of test drug. Isometric

effect (Fig. 3A), similar to that caused by verapamil (0.03–0.3 ␮M,

responses were recorded on Grass model 7 Polygraph (Grass instru-

p < 0.01 and p < 0.001) as shown in Fig. 3B. When tested for pos-

ment company, Quincy, MA, USA).

sible interaction with isoprenaline, pretreatment of tissues with

M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946 941

Fig. 1. Tracings showing concentration-dependent relaxant effect of the crude

extract of Carum roxburghianum (Cr.Cr) on spontaneously contracting isolated rabbit

jejunum preparations. Test material was added to tissue bath in cumulative fashion

and the concentrations shown are the ﬁnal bath concentration.

Cr.Cr (0.03 and 0.1 mg/mL) shifted isoprenaline-induced inhibitory

CRCs to left (p < 0.05 and p < 0.01), showing potentiating effect

(Fig. 4A). Papaverine caused similar concentration-dependent (0.3

␮

and 1.0 M) leftward shift (p < 0.05 and p < 0.01) in the CRCs of

isoprenaline (Fig. 4B).

3.3. Effect on ileum

Cr.Cr at 0.01 and 0.03 mg/mL caused rightward parallel shift

of ACh-curves without suppressing (p > 0.05) maximum response

(Fig. 5A). Similarly, atropine (0.003 and 0.01 ␮M) caused rightward

parallel shift, without suppression (p > 0.05) of maximum stimulant

effect (Fig. 5B).

3.4. Effect on carbachol-induced bronchoconstriction

Cr.Cr at the doses of 1, 3, 10 and 30 mg/kg (n = 6) caused

± ± ± ±

16.8 4.6, 27.3 5.54 (p < 0.01), 36.8 7.7 (p < 0.01) and 43.3 7.1%

(p < 0.01), respective suppression of the control (CCh, 100 ␮g/kg)-

induced increase in the inspiratory pressure of anaesthetized rats

(Fig. 6A). Aminophylline was used as positive control, inhibited

␮

the CCh (100 g/kg)-mediated bronchoconstriction at 1, 3, 10 and

30 mg/kg (n = 4) by 10 ± 7.4, 13.2 ± 4.15, 28.0 ± 7.7 (p < 0.05) and

37.0 ± 9.3% (p < 0.01) respectively (Fig. 6B).

3.5. Effect on trachea

Cr.Cr was found devoid of any stimulant action, when

screened on tracheal resting baseline. When tested against

␮ +

CCh (1 M) and K (80 mM)-induced contractions, Cr.Cr caused

concentration-dependent inhibition with respective EC50 values of

0.09 (0.04–0.12, n = 4) and 0.50 mg/mL (0.26–0.96, n = 3) as shown in

Fig. 7A. Papaverine relaxed the CCh (1 ␮M) and K (80 mM)-induced

contractions at similar concentration (Fig. 7B) with respective EC50

values of 2.9 (2.6–3.6, n = 5) and 3.1 ␮M (2.9–3.9, n = 4), whereas

verapamil was found more potent in its inhibitory effect against

K (80 mM)-induced contractions with EC50 value of 0.027 ␮M

(0.02–0.03, n = 4), when compared with CCh-induced contractions

[0.57 ␮M (0.38–0.85, n = 3)] as shown in Fig. 7C. Pretreatment of

tracheal preparations with Cr.Cr shifted the isoprenaline-induced

inhibitory CRCs to left (Fig. 8A) in concentration-dependent manner

(0.1 and 0.3 mg/mL, p < 0.01 and p < 0.001), similar to that caused

by papaverine (1.0 and 3.0 ␮M, p < 0.05, p < 0.01 and p < 0.001),

showing potentiating effect (Fig. 8B). Cr.Cr at 0.01 and 0.03 mg/mL

caused rightward parallel shift of CCh-curves, without suppress-

ing (p > 0.05) maximum contractile response (Fig. 9A). Similarly,

␮

atropine (0.01 and 0.03 M) caused rightward parallel shift without

Fig. 2.

suppression (p > 0.05) of maximum contractile effect (Fig. 9B).

942 M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946

Fig. 3. Concentration–response curves of Ca in the absence and presence of the

increasing concentrations of (A) crude extract of Carum roxburghianum (Cr.Cr)

and (B) verapamil in isolated rabbit jejunum preparations. Values shown are

* ** ***

mean ± SEM, n = 3–6. p < 0.05, p < 0.01 and p < 0.001 compared to respective con-

centrations values in the Ca control curves, two-way analysis of variance followed

by Bonferroni’s post-test.

Fig. 4. Inhibitory concentration–response curves of isoprenaline against carbachol

(CCh)-induced contractions in the absence and presence of different concentrations

3.6. Acute toxicity

of (A) crude extract of Carum roxburghianum (Cr.Cr) and (B) papaverine in isolated

rabbit jejunum preparations. Values shown are mean ± SEM, n = 3. p < 0.05 and

The three different groups of mice were given Cr.Cr in graded p < 0.01 compared to respective concentrations values in the isoprenaline control

curves, two-way analysis of variance followed by Bonferroni’s post-test.

doses of 1, 5 and 10 g/kg respectively and animals were observed

for mortality after 24 h of drug administration. The extract did not

cause any mortality up to the dose of 10 g/kg.

4. Discussion

In view of traditional use of Carum roxburghianum in hyper-

Fig. 2. Concentration-dependent inhibitory effect on spontaneous and K (80 mM)- active gut disorder, diarrhea, its extract was evaluated for the

induced contractions of (A) crude extract of Carum roxburghianum (Cr.Cr), (B)

possible antidiarrheal action in mice. In castor oil-induced diarrhea

papaverine, and (C) verapamil in isolated rabbit jejunum preparations. Values shown

** model, Carum roxburghianum extract in dose-dependent fashion

are mean ± SEM, n = 3–4. p < 0.01 compared to respective concentrations values in

+ showed protective effect, like that caused by dicyclomine and lop-

high K -curve (Cr.Cr) and spontaneous contractions (verapamil), two-way analysis

of variance followed by Bonferroni’s post-test. eramide, standard antidiarrheal drug (Reynolds et al., 1984). Castor

M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946 943

Fig. 5. Concentration–response curves of acetylcholine (ACh) in the absence and

presence of (A) crude extract of Carum roxburghianum (Cr.Cr) and (B) atropine in

isolated guinea-pig ileum preparations. Values shown are mean ± SEM, n = 5–7. The

“ns” shows non-signiﬁcant difference (p > 0.05) at the maximum response level of

each ACh curve, two-way analysis of variance followed by Bonferroni’s post-test.

Fig. 6. Dose-dependent suppressant effect of (A) crude extract of Carum

roxburghianum (Cr.Cr) and (B) aminophylline on carbachol (CCh)-mediated bron-

oil induces diarrhea as a results of the action of ricinoleic acid

choconstriction in anaesthetized rats. Values shown are mean ± SEM, n = 4–6.

formed during the hydrolysis of oil, which produces changes in * **

p < 0.05 and p < 0.01 vs. control CCh group, one way analysis of variance followed

transport of electrolytes and water, leading to generation of giant by Dunnett’s test.

contractions of intestine (Iwao and Terada, 1962; Croci et al., 1997).

Thus, a potential antidiarrheal agent may exhibit its antidiarrheal

effect by inhibiting bowel contractions. The antidiarrheal activ- K -induced contractions. Pretreatment of tissue with plant extract

ity of Cr.Cr following oral administration appears to be virtue shifted the Ca -CRCs to right, with suppression of maximum

of gastrointestinal relaxant component(s) presence in Carum rox- contractile effect, similar to that caused by verapamil. The

burghianum. extract potentiated isoprenaline-induced relaxant effect, simi-

For investigation of possible spasmolytic effect, isolated rab- lar to that caused by papaverine. The ACh-CRCs in presence of

bit jejunum preparation was used, which is known to exhibit different concentrations of extract were constructed in guinea-

spontaneous rhythmic contractions, thus allowing the testing pig ileum, as this preparation is considered more useful to

of relaxant activity. In jejunum, Cr.Cr inhibited both sponta- quantify contractile responses of an agonist in presence of an

neous and high K -induced contractions with similar potency. inhibitor (Daniel et al., 2001). The plant extract caused right-

Papaverine, a phosphodiesterase and Ca inﬂux inhibitor (Rang ward parallel shift in ACh-curves, without suppressing maximum

et al., 1999) caused similar pattern of inhibitory effect with stimulant response, a characteristic of competitive or speciﬁc

comparable potency against spontaneous and K -induced contrac- antagonist (Arunlakhshana and Schild, 1959). Atropine, a stan-

tions, while verapamil, a standard Ca antagonist (Fleckenstein, dard antimuscarinic agent (Eglen and Harris, 1993) also caused

1977) was relatively selective in its inhibitory effect on the similar rightward parallel shift of ACh-curves. The observed

944 M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946

Fig. 8. Inhibitory concentration–response curves of isoprenaline against carbachol

(CCh)-induced contractions in the absence and presence of different concentrations

of (A) crude extract of Carum roxburghianum (Cr.Cr) and (B) papaverine in isolated

guinea-pig tracheal preparations. Values shown are mean ± SEM, n = 4–5. p < 0.05,

** ***

p < 0.01 and p < 0.001 compared to respective concentrations values in the iso-

prenaline control curves, two-way analysis of variance followed by Bonferroni’s

post-test.

antidiarrheal and antispasmodic effects of Carum roxburghi-

anum justify its traditional use in diarrhea and abdominal

colic.

Based on the folkloric reputation of Carum roxburghianum

application in asthma, it was tested for possible bronchodilatory

effect in anaesthetized rats. Cr.Cr dose-dependently suppressed

the CCh-evoked bronchospasm, like that caused by aminophylline

Fig. 7. Concentration–response curves showing inhibitory effect of (A) crude extract

of Carum roxburghianum (Cr.Cr), (B) papaverine, and (C) verapamil on carbachol

(CCh) and K (80 mM)-induced contractions in isolated guinea-pig tracheal prepa-

± * ** ***

rations. Values shown are mean SEM, n = 3–5. p < 0.05, p < 0.01 and p < 0.001

compared to respective concentrations values in high K -curve (Cr.Cr) and CCh-

contractions (verapamil), two-way analysis of variance followed by Bonferroni’s

Fig. 7. post-test.

M. Khan et al. / Journal of Ethnopharmacology 141 (2012) 938–946 945

Acknowledgments

This study was supported by Higher Education Commission of

Pakistan. The author, Munasib Khan is on leave from University of

Malakand for the PhD study.

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