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Pharmacological Explanation for the Medicinal Use of Juniperus Excelsa in Hyperactive Gastrointestinal and Respiratory Disorders

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Pharmacological Explanation for the Medicinal Use of Juniperus Excelsa in Hyperactive Gastrointestinal and Respiratory Disorders eCommons@AKU Department of Biological & Biomedical Sciences Medical College, Pakistan April 2012 Pharmacological explanation for the medicinal use of Juniperus excelsa in hyperactive gastrointestinal and respiratory disorders. 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 Natural Products Chemistry and Pharmacognosy Commons Recommended Citation Khan, M., Khan, A., Najeeb-ur-Rehman, ., Gilani, A. (2012). Pharmacological explanation for the medicinal use of Juniperus excelsa in hyperactive gastrointestinal and respiratory disorders.. Journal of Natural Medicines, 66(2), 292-301. Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/111 J Nat Med (2012) 66:292–301 DOI 10.1007/s11418-011-0605-z ORIGINAL PAPER Pharmacological explanation for the medicinal use of Juniperus excelsa in hyperactive gastrointestinal and respiratory disorders Munasib Khan • Arif-ullah Khan • Najeeb-ur-Rehman • Anwarul-Hassan Gilani Received: 8 July 2011 / Accepted: 2 September 2011 / Published online: 3 December 2011 Ó The Japanese Society of Pharmacognosy and Springer 2011 Abstract Crude extract of Juniperus excelsa (JeExt), antagonist and phosphodiesterase inhibitory effects, which which tested positive for the presence of anthraquinone, provides a pharmacological basis for its traditional use in flavonoids, saponins, sterols, terpenes and tannin, exhibited disorders of gut and airways hyperactivity, such as diar- a protective effect against castor oil-induced diarrhoea in rhoea, colic and asthma. mice at 100–1000 mg/kg. In rabbit jejunum preparations, JeExt (0.01–1.0 mg/mL) caused relaxation of spontaneous Keywords Juniperus excelsa Á Ca2? channel blocker Á and K? (80 mM)-induced contractions at similar concen- PDE inhibitor Á Gut and airways disorders trations to papaverine, whereas verapamil was relatively more potent against K?. JeExt (0.03–0.3 mg/mL) shifted Ca2? concentration–response curves to the right, like Introduction papaverine or verapamil. JeExt (0.003–0.01 mg/mL) caused a leftward shift of isoprenaline-induced inhibitory Juniperus excelsa Bieb. (Cupressaceae/Coniferae), com- concentration–response curves, similar to papaverine. monly known as ‘‘pencil cedar/Juniper’’ and locally as JeExt (1.0–30 mg/kg) caused suppression of carbachol ‘‘Dhup Guggal’’ is found throughout the eastern Mediter- (CCh, 100 lg/kg)-induced increase in inspiratory pressure ranean from northeastern Greece and southern Bulgaria of anaesthetized rats. In guinea-pig trachea, JeExt across Turkey to Syria and the Caucasus mountains at an (0.001–3.0 mg/mL) relaxed CCh (1 lM)- and high K?- altitude of 2000–4000 m. It also occurs in Alborz and other induced contractions and shifted isoprenaline-induced mountains of Iran, east to northwestern Pakistan and Oman inhibitory curves to the left. This study suggests that Ju- [1, 2]. Juniperus excelsa is used in folk medicine to treat niperus excelsa possibly exhibits a combination of Ca2? diarrhoea, abdominal spasm, asthma [3], fever, gonorrhoea, headache and leucorrhoea [4, 5] as well as being consid- ered useful as an antihypertensive, diuretic, appetizer, M. Khan was on leave from University of Malakand for the PhD carminative, stimulant, anticonvulsant and flavouring agent study. [6]. Phytochemical studies on the plant revealed the pres- ence of (?)-cedrol, (?)-sabinene, (?)-limonene, menthene, M. Khan Á Najeeb-ur-Rehman Á A.-H. Gilani (&) Natural Products Research Unit, Department of Biological terpinene-4-ol, a-cedrene, b-cedrene, p-cymene, b-phel- and Biomedical Sciences, Aga Khan University Medical landrene, a-copaene, muurolene, b-guaiene, guaiazulene College, Karachi 74800, Pakistan [7], a-thujene, a-fenchene, camphene, a-phellandrene, c-3- e-mail: [email protected] carene, a-terpinene, trans-ocimene, c-terpinene, terpino- M. Khan lene, endo-fenchol, cis-pinene hydrate, a-campholenal, Department of Pharmacology, Faculty of Pharmacy, trans-pinocarveol, camphor, borneol, c-terpineol, naph- University of Karachi, Karachi 75270, Pakistan thalene, a-terpineaol, myrtenol, verbenone, trans-carveol, endo-fenchyl acetate, piperitone, bornyl acetate, carvacrol, A. Khan Institute of Pharmaceutical Sciences, Kohat University b-cubebene, thujopsene a-cadinene, a-humulene, b-aco- of Science and Technology, Kohat 26000, KPK, Pakistan radiene, b-cadinene, c-muurolene [8], toluene, tricyclene, 123 J Nat Med (2012) 66:292–301 293 thujene, pinene, camphene, triene cycloheptane 1,3,5- phosphate (Merck, Darmstadt, Germany) and sodium trismethylene, b-myrcene, o-allyl toluene, m-cymene, d,l- chloride (BDH Laboratory Supplies, Poole, UK). The limonene, a-pinene oxide, a-terpinolene, 3-thujanone and chemicals used in phytochemical analysis include: acetic a-campholene aldehyde [9]. anhydride, aluminum chloride, ammonium hydroxide, Despite the fact that extensive phytochemical research ferric chloride (Sigma), benzene, chloroform, hydrochloric has been carried out on Juniperus excelsa, reports related acid and petroleum ether (BDH). All chemicals used were to pharmacological investigation are limited, only citing its of analytical grade and dissolved in distilled H2O/saline. antibacterial [10] and antifungal [11] activities. In the present research, we provide evidence that Juniperus Phytochemical screening excelsa exhibits antidiarrheal, antispasmodic and bron- chodilatory activities, occurring via a combination of Ca2? Preliminary investigation of the plant extracts for the channel blockade and phosphodiesterase (PDE) inhibitory presence of various phytochemical classes, such as sapo- pathways, which explains the medicinal use of Juniperus nins, coumarins, sterols, terpenes, flavonoids, anthraqui- excelsa in hyperactive gut and airways disorders such as nones and tannins was done according to reported methods diarrhea, colic and asthma. [13]. The presence of saponins was detected based on the appearance of froth upon vigorous shaking of diluted samples. The observation of yellow fluorescence under Materials and methods ultraviolet light on examination of filter paper previously exposed to the vapours from boiling plant material indi- Plant material and extraction cated coumarins. For the detection of sterols and terpenes, plant material was treated with petroleum ether and sub- The aerial parts (stem ? leaves) of Juniperus excelsa were sequently extracted with chloroform. The appearance of collected from northern areas of Pakistan (Chitral) in green to pink (for sterols) and pink to purple colours (for September 2007. The plant was identified with the help of a terpenes) was then noted after treatment of the chloroform taxonomist, Dr. Ilyas Iqbal, Department of Botany, Uni- layer with acetic anhydride and concentrated HCl in suc- versity of Malakand, KPK, Pakistan. A voucher specimen cession. Plant material was noted as positive for flavonoids (UOM/BGH/150) has been submitted to the herbarium of when it gave a yellow colour with aluminum chloride the same university. Plant material was cleaned, shade- reagent, and for tannins when green or black colour was dried and coarsely ground. The powdered material produced with aqueous ferric chloride. Lastly, for detecting (580.27 g) was soaked in aqueous methanol (70%) at room anthraquinones, the extract was dissolved in 1% HCl, then temperature (25 ± 2.0°C) for 3 days with occasional in benzene, and observed if the extract showed a pink, shaking. It was filtered through muslin cloth and then violet or red colour with ammonium hydroxide. through Whatman qualitative grade 1 filter paper [12]. The procedure of maceration and filtration was repeated twice Experimental animals more. All the filtrates were combined and evaporated to dryness in a rotary evaporator under reduced pressure Rabbits (1–1.2 kg), guinea pigs (500–550 g), Sprague– (-760 mmHg) at 35–40°C to obtain crude extract of Dawley rats (200–250 g) and BALB/c mice (20–25 g) of Juniperus excelsa (JeExt), yielding approx. 24%. JeExt local breed and either sex were used for this study and were was dissolved in normal saline/distilled water for use in housed at the Animal House of the Aga Khan University, in-vivo and in-vitro experiments. maintained at 23–25°C and given a standard diet and tap water. Rabbits starved for 24 h were killed by a blow to the Chemicals back of the head, and guinea pigs by cervical dislocation. The experiments complied with the rules of the Institute of Acetylcholine chloride (ACh), carbachol (CCh), isoprena- Laboratory Animal Resources, Commission on Life Sci- line, loperamide, papaverine and verapamil were purchased ences, National Research Council [14] and were approved from Sigma Chemical Co., St Louis, MO, USA. Ami- by the Ethical Committee of the Aga Khan University. nophylline, pentothal sodium (thiopental) and castor oil were obtained from GlaxoSmithKline, Abbott Laboratories Castor oil-induced diarrhea and KCL Pharma, Karachi, Pakistan, respectively. Chem- icals used for making physiological salt solutions were: Mice were fasted for 24 h before the experiment. Animals potassium chloride (Sigma), calcium chloride, glucose, were housed in individual cages and divided in five groups, magnesium chloride, magnesium sulfate, potassium dihy- each containing 10 mice. The first group received saline drogen phosphate, sodium bicarbonate, sodium dihydrogen (10 mL/kg, orally) and served as a negative control. The 123 294 J Nat Med (2012) 66:292–301 doses of
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