A tricyclic labdane type diterpene was isolated for the first time from ethyl acetate soluble part of Phlomis bracteosa. Its structure was confirmed by x-ray which was found to be marrubiin. When studied in isolated rabbit jejunum, marrubiin caused concentration-dependent relaxation of spontaneous and high K+ (80 mM)-induced contractions, like that caused by verapamil, indicating that marrubiin exhibits spasmolytic activity, possibly mediated through Ca++ channel blocking action

Al-Harrasi, Ahmed

Gilani, Anwar

Hussain, Javid

Khan, Arif-ullah

Mabood, Fazal

Shah, Mohammad Raza

Ullah, Riaz

English

eCommons@AKU

eCommons@AKUDepartment of Biological & Biomedical Sciences Medical College, PakistanJanuary 2011Antispasmodic and Ca++ antagonist potential ofmarrubiin, a labdane type diterpene from PhlomisbracteosaJavid HussainKohat University of Science and Technology, Kohat, PakistanRiaz UllahKohat University of Science and Technology, Kohat, PakistanArif-ullah KhanAga Khan UniversityFazal MaboodCollege of Arts and Sciences University of Nizwa, OmanMohammad Raza ShahUniversity of Karachi, Karachi, PakistanSee next page for additional authorsFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_bbsPart of the Alternative and Complementary Medicine Commons, Pharmaceutical PreparationsCommons, and the Pharmaceutics and Drug Design CommonsRecommended CitationHussain, J., Ullah, R., Khan, A., Mabood, F., Shah, M. R., Al-Harrasi, A., Gilani, A. (2011). Antispasmodic and Ca++ antagonistpotential of marrubiin, a labdane type diterpene from Phlomis bracteosa. Journal of Pharmacy Research, 4(1), 178-180.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/227AuthorsJavid Hussain, Riaz Ullah, Arif-ullah Khan, Fazal Mabood, Mohammad Raza Shah, Ahmed Al-Harrasi, andAnwar GilaniThis article is available at eCommons@AKU: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/227Journal of Pharmacy Research Vol.4.Issue 1. January 2011Javid Hussain et al. / Journal of Pharmacy Research 2011,4(1),178-180178-180Research  ArticleISSN: 0974-6943 Available online throughhttp://jprsolutions.info*Corresponding author.Javid HussainDepartment of Chemistry,Kohat University of Science and Technology,Kohat-26000, PakistanE-mail:javidhej@yahoo.com,afridiriaz@yahoo.comINTRODUCTIONThe genus Phlomis belonging to family Labiatea consists of about 100 species inthe world. [1, 2] Some Phlomis species are used as tonics and stimulants in Anatolianfolk medicine. [3]  Different Phlomis species are used in folk medicine as stimu-lant, tonic, analgesic, anti-diarrheal and considered useful for the treatment ofulcers, hemorrhoids and airways disorders. There are a few reports on the phar-macological activities, such as anti-inflammatory, immunosuppressive, anti mu-tagenic, anti-nociceptive, antifibrile, anti-oxidant, anti-malarial and anti-mi-crobial effects. [4] The present work has led to the isolation of a known labdanetype diterpene for the first time from Phlomis bracteosa, identified as marrubiin,which in pharmacological investigation revealed spasmolytic and Ca++ antago-nistic effects.Antispasmodic and Ca++ antagonist potential of marrubiin,a labdane type diterpene from Phlomis bracteosaJavid Hussain1,5, Riaz Ullah1, Arif-ullah Khan2,3, Fazal Mabood5, Mohammad Raza Shah4, Ahmed Al-Harrasi5 and Anwarul Hassan Gilani21Department of Chemistry, Kohat University of Science and Technology,Kohat-26000, Pakistan2Department of Biological and Biomedical Sciences, The Aga Khan University,Karachi-74800, Pakistan3Institute of Pharmaceutical Sciences, Kohat University of Science and Technology,Kohat-26000, Pakistan4ICCBS, HEJ Research Institute of Chemistry, University of Karachi, Karachi, Pakistan5Department of Biological Sciences and Chemistry, College of Arts and Sciences University of Nizwa, OmanReceived on: 15-09-2010; Revised  on: 18-10-2010; Accepted on:13-12-2010ABSTRACTExtraction, Isolation and structure elucidationThe whole plant of Phlomis bracteosa was dried in shade, chopped and ground tocoarse powder. The powdered plant (3 Kg) was initially extracted with methanolfor 25 days at room temp. The combined methanolic extract was evaporatedunder reduced pressure giving residue (165 g). Using separating funnel, the metha-nol extract was partitioned applying different solvents i.e., hexane, chloroform,ethyl acetate, butanol and water (in order of increasing polarity) successively. [5]A portion of the ethyl acetate extract (70 g) was chromatographed over a silicagel column. The elution was carried out with a gradient of increasing polarity ofhexane, ethyl acetate, and methanol. Fraction B was obtained through elutionwith n-hexane-ethyl acetate (1:1) and was subjected to column chromatographywith the solvent system (n-hexane-ethyl acetate).Compound (1) was ob-tained from this fraction after eluting with n-hexane-ethyl acetate (3:7). Com-pound (1) was further purified by recrystallization from methanol. ColumnChromatography (CC) was performed using silica gel, 70–230 mesh. Flash chro-matography was carried out using silica gel 230–400 mesh. Thin layer chroma-tography (TLC) was performed with precoated silica gel G-25-UV254 plates andvisualization was achieved using (UV, 254 nm) and by ceric sulphate in 10 %H2SO4 solution Silica gel (E. Merck, 230–400 mesh) was used for column chro-matography. The IR and UV Spectra were recorded on a Jasco-320-A and Hitachi-UV-240 spectrophotometers, respectively. Optical rotations were measured ona Jasco-DIP-360-digital polarimeter using a 10-cm cell-tube. Mass spectra (EI-and HR-EI-MS) were measured in an electron impact mode on Finnigan MAT 12or MAT 312 spectrophotometers; ions are presented in m/z (%). The 1H-and13C-NMR spectra were recorded on a Bruker AMX-400 spectrometer in CDCl3.The 2D-NMR spectra were recorded on Bruker AMX-400 spectrometer. Chemi-cal shifts in parts per million (ä), relative to tetra methyl silane as an internalstandard, and scalar coupling were reported in Hertz. The pulse conditions wereas follows: for 1H-NMR spectra, spectrometer frequency (SF) 400.032 MHz,acquisition time (AQ) 2.281 s, number of transients (NS) 128, receiver gain(RG) 812.7, temperature (TE) 300K, dwell time (DW) 69.6ms, per scan delay(DE)10ms, dummy scans (DS) 0, F1 2431.15 Hz, F2–1.77 Hz; for the 13C-NMRspectra SF100.613 MHz, AQ 0.6 + 26 s, NS 25000, RG 16384, TE 300 K, DW19.1ms, DE 20ms, DS2, F1 23157.50 Hz; for the COSY 45o spectra SF 400.03MHz, NS32, DS4, pulse (P1) 5.70 ms, P22.70 µs, TE 300 K, RG 267.4, DW145.6ms, DE 10ms; for the NOESY experiments SF 300.133 MHz, NS 64, DE305ms, pulse width (PW) 0.0, F1 2903.69 Hz, F2 2903.69 Hz; for the HMQCspectra, SF 400.032 MHz, AQ 0.1491s, NS 32, DS 16, DE 10 ms, DW 145.6ms,RG 6502, TE 300 K, F1 1688.52 Hz, F2 3071.71 Hz; for the HMBC spectra SF400.032 MHz, AQ 0.1491 s, RG 7296.2, NS 128, DW 145.6ms, DS 16, DE 10ms, TE 300 K, F1 3212.30  Hz, F2 23236.21 Hz.MATERIALS AND METHODSPlant materialWhole parts of Phlomis bracteosa were collected from Swat, NWFP, Pakistanin July 2005 and identified by Muhammad Naveed, Assistant Professor BotanyDepartment University of Peshawar NWFP Pakistan. A Voucher Specimen wasdeposited in the Herbarium of the college.A tricyclic labdane type diterpene was isolated for the first time from ethyl acetate soluble part of Phlomis bracteosa. Its structure was confirmed by  x-ray whichwas found to be  marrubiin.When studied in isolated rabbit jejunum, marrubiin caused concentration-dependent relaxation of spontaneous and high K+ (80 mM)-induced contractions, like that caused by verapamil, indicating that marrubiin exhibits spasmolytic activity, possibly mediated through Ca++ channel blockingaction.Key words: Phlomis bracteosa, labiatae, marrubiin, antispasmodic,  Ca++ antagonist.Figure 1. Structure of compound 1, marrubiin, a labdane type diterpeneisolated from Phlomis bracteosaOOHOO1234 56 7891011121314151618171920Journal of Pharmacy Research Vol.4.Issue 1. January 2011Javid Hussain et al. / Journal of Pharmacy Research 2011,4(1),178-180178-180 02 55 07 51 0 00 . 0 1 0 . 1 1 . 0 1 0A[ M a r r u b i i n ] m M% of Spontaneous Contraction 0 . 0 1 0 . 1 102 55 07 51 0 0B[ V e r a p a m i l ] m M% of Spontaneous Contraction 02 55 07 51 0 00 . 0 1 0 . 1 1 01 . 0A [ M a r r u b i i n ] m M% of K+  (80 mM)-Induced Contraction 0 . 0 1 0 . 1 102 55 07 51 0 0B[ V e r a p a m i l ] m M% of K+  (80 mM)-Induced ContractionFigure 3. Concentration-dependent inhibitory effect of (A) marrubiin,X-rays analysisThe conditions for recording the spectra and the instruments have been de-scribed in (Sigitdinova et al., 1996).[6]Chemicals and animalsAcetylcholine (ACh) and verapamil were obtained from Sigma Chemicals Co.Statistical analysisThe data expressed are mean ± standard error of mean (SEM) and analyzed byusing GraphPad program (GraphPAD, San Diego, CA, USA).When tested in isolated rabbit jejunum, marrubiin inhibited the spontaneouscontractions with EC50 value of 1.3 mM (n=3), thus showing an antispasmodiceffect (Figure 2A). Similarly verapamil, used as a positive control drug, relaxedthe spontaneous contractions with EC50 values of 0.31 mM (n=3) as shown inFigure 2B. The contraction of smooth muscle preparations, including rabbitjejunum is dependent upon an increase in the cytoplasmic free [Ca++], whichactivates the contractile elements. [10] The increase in intracellular Ca++ is due toeither influx via voltage dependant L-type Ca++ channels (VDCs) or to releasefrom intracellular stores in the sarcoplasmic reticulum. Periodic depolarizationregulates the spontaneous movements of intestine and at the height of depolar-ization the action potential appears as a rapid influx of Ca++ via VDCs.[11] Theinhibitory effect of the plant compound on spontaneous movements of je-junum.RESULTS AND DISCUSSIONThe ethyl acetate fraction obtained from methanolic extract of Phlomisbracteosa was subjected to silica gel column chromatography eluted with ethylacetate hexane (8:2), afforded labdane type diterpene, compound 1 (Figure 1).It was isolated in white powder form and its molecular formula established asC20H28O4 by x-rays and spectral techniques. By comparison its data with litera-ture showed, that it is marrubiin.Determination of calcium antagonist activityTo assess whether the antispasmodic effect of the test compound is mediatedthrough calcium channel blockade, high K+ (80 mM) was used to depolarize thepreparations as described by Farre et al.[9] . Addition of high K+ to the tissue bathproduced a sustained contraction. Relaxation of intestinal preparations by thecompound, precontracted with K+, was expressed as percent of the controlresponse mediated by K+.Antispasmodic effectThe spasmolytic activity of the test compound was studied by using isolatedrabbit jejunum as described previously. [8]  Respective segments of 2-cm lengthwere suspended in a 10 ml of Tyrode’s solution and bubbled with carbogen gas at37°C.  The composition of the Tyrode’s solution in mM was KCl 2.68, NaCl136.9, MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, CaCl2 1.8, and glucose 5.55.A resting tension of 1 g was applied to each of the tissues and was kept constantthroughout the experiment. Intestinal responses were recorded isotonicallyusing a Bioscience transducer and Oscillograph. Each tissue was allowed toequilibrate for at least 30 min before the addition of any drug and then stabilizedwith a sub-maximal concentration of acetylcholine (0.3 µM) and the bath fluidwas subsequently replaced with normal Tyrode solution before starting the ex-periment. Under these experimental conditions, rabbit jejunum exhibits sponta-neous rhythmic contractions, allowing testing of the relaxant (spasmolytic)activity directly without the use of any agonist.form and methanol were from Merck (Darmstadt, Germany). Chemicals usedfor making physiological salt solutions were potassium chloride (Sigma Chemi-cals Co, St. Louis, MO, USA), calcium chloride, glucose, magnesium chloride,sodium bicarbonate, sodium dihydorgen phosphate (Merck, Darmstadt, Ger-many) and sodium chloride (BDH Laboratory supplies, Poole, England). Allchemicals used were of the analytical grade. Rabbits (1.2-1.5 kg) of either sex orlocal breed were used in this study, housed at the Animal House of the Aga KhanUniversity, maintained at 23-25°C and given a standard diet and tap water.Rabbits had free access to water, but food was withdrawn 24 hrs prior to experi-ment and killed by a blow on the back of the head. Experiments performedcomplied with the rulings of the Institute of Laboratory Animal Resources,Commission on Life Sciences, National Research Council  [7] and were approvedby the Ethical Committee of the Aga Khan University(St. Louis, MO, USA). The solvents, n-butanol, ethyl acetate, n-hexane, chloroisolated from Phlomis bracteosa and (B) verapamil on K+-induced con-tractions of isolated rabbit jejunum preparations. Values shown aremean ± SEM, n=3.Figure 2. Concentration-dependent inhibitory effect of (A) marrubiin,isolated from Phlomis bracteosa and (B) verapamil on spontaneous con-tractions of isolated rabbit jejunum preparations. Values shown aremean ± SEM, n=3.Journal of Pharmacy Research Vol.4.Issue 1. January 2011Javid Hussain et al. / Journal of Pharmacy Research 2011,4(1),178-180178-180           Source of support: Higher Education Commission, Government of Pakistan, Conflict of interest: None Declaredmay be due to interference either with the Ca++ release or with the Ca++ influxthrough VDCs. In our earlier studies, we have observed that the spasmolyticeffect of the natural compounds is usually mediated through Ca++ channel block-ade.[12-15] To see whether the spasmolytic effect of marrubiin is also mediated viathe same mechanism, it was tested against high K+ (80 mM)-induced contrac-tions, which was relaxed by marrubiin with EC50 value of 1.4 µM (n=3) as shownin Figure 3A. At high concentration (> 30 mM), K+ is known to cause smoothmuscle contractions through opening of VDCs, thus allowing influx of extracel-lular Ca++ causing a contractile effect [16] and a substance causing inhibition of thehigh K+-induced contraction is considered an inhibitor of the Ca++ influx.[17]Verapamil, a standard Ca++ antagonist[18]  inhibited the high K+ (80 mM)-inducedcontractions with EC50 values of 0.13 µM (n=3) as shown in Figure 3B. Thus thespasmolytic effect of the marrubiin may be visualized as the Ca++ channel block-ade mechanism.ACKNOWLEDGEMENTThe authors wish to thank Higher Education Commission, Government ofPakistan for providing financial support for the current study under the Na-tional Research Program for Universities (NRPU).REFERENCES1. Albaladejo RG, Aparicio S. Variation patterns in the Phlomis composite (Lamiaceae) hybridcomplex in Iberian peninsula. Bot. J. Linn. Soc. 2004,145: 97-108.2. Kyriakopoulo I, Magiatis P, Skaltounis AI, Aligiannis N, Harvala C. Samioside. A newphenylethanoid glycoside with free-radical scavenging and antimicrobial activities fromPhlomis samia. J. Nat. Prod. 2001, 64: 1095-1097.3. Calis I, Kýrmýzýbekmez H, Glycoside from Phlomis lunariifolia, Phytochemistry 2004, 65:2619-2625.4.          Sarkhail P, Hamid RME, Gholam RA, Mohammad HSS, Abass S. Phytochemical study ofPhlomis olivieri benth. and Phlomis persica boiss. DARU 2006,14: 115-121.5. Wlliamson EM, Okpako DT, Evans FJ. Selection, Preparation and Pharmacological Evaluationof Plant Material. John Wiley & Sons, Chichester,  1998, 15-23.6. Sigitdinova GB, Makhmudov MK, Tashkhodzhaev B, Mal T. Labdanoid of Marrubium anisodon.Chem. Nat. Comp. 1996, 32: 43-46.7. National Research Council. Guide for the Care and Use of Laboratory Animals. NationalAcademy Press, Washington, pp 1996, 1-7.8. Gilani AH, Bashir S, Khan A. Pharmacological basis for the use of Borago officinalis ingastrointestinal, respiratory and cardiovascular disorders. J. Ethnopharmacol. 2007, 114: 393-399.9. Farre AJ, Columbo M, Fort M, Gutierrez B. Differential effects of various Ca++ antagonists.Gen. Pharmacol. 1991, 22: 177-181.10. Karaki H, Weiss G. Mini review: calcium release in smooth muscles. Life Sci. 1988, 42: 111-122.11. Brading AF. How do drugs initiate contraction in smooth muscles. Trends Pharmacol. Sci.1981, 2: 261-265.12. Khalid A, Choudhary MI, Haq Z, Ghayur MN, Feroz F, Rahman A, Gilani AH. Cholinesteraseinhibitory and spasmolytic potential of steroidal alkaloids. J. Steroidal Biochem. Mol. Biol.2004, 92: 477-484.13. Gilani AH, Ghayur MN, Khalid A, Zaheer-ul-Haq, Choudhary MI, Atta-ur-Rahman. Pres-ence of antispasmodic, antidiarrhoeal, antisecretory, calcium antagonist and acetylcholinest-erase inhibitory steroidal alkaloids in Sarcococca saligna. Planta Med. 2005, 71: 120-125.14. Choudhary MI, Nawaz SA, Zaheer-ul-Haq, Azim MK, Ghayur MN, Lodhi MA, Jalil S, KhalidA, Ahmed A, Rode BM, Atta-ur-Rahman, Gilani AH, Ahmad VU. Juliflorine: a potent naturalperipheral anionic-site- binding inhibitor of acetylcholinesterase with calcium-channel block-ing potential, a leading candidate for Alzheimer’s disease therapy. Biochem. Biophys. Res.Commun. 2005, 332: 1171-1179.15. Atta-ur-Rahman, Khalid A, Sultana N, Ghayur MN, Mesaik MA, Khan MR, Gilani AH,Choudhary MI. New natural cholinesterase inhibiting and calcium channels blocking quinolinealkaloids. J. Enz. Inhib. Med. Chem.  2006, 21: 703-710.16. Bolton TB. Mechanism of action of transmitters and other substances on smooth muscles.Physiol. Rev. 1979, 59: 606-718.17. Godfraind T, Miller R, Wibo M. Calcium antagonism and calcium entry blockade. Pharmacol.Rev. 1986, 38: 321-41618. Fleckenstein A. Specific pharmacology of Ca++ in myocardium, cardiac pacemakers andvascular smooth muscle. Rev. Pharmacol. Toxicol. 1977, 17: 149-166.

Antispasmodic and Ca++ antagonist potential of marrubiin, a labdane type diterpene from Phlomis bracteosa

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Antispasmodic and Ca++ antagonist potential of marrubiin, a labdane type diterpene from Phlomis bracteosa

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