Phytochemistry Letters 7 (2014) 57–61

New diterpene polyester and phenolic compounds from Pycnocyclaspinosa Decne. Ex Boiss with relaxant effects on KCl-inducedcontraction in rat ileum

Mustafa Ghanadian a,*, Hassan Sadraei b, Samar Yousuf c, Gholamreza Asghari a,b,M. Iqbal Choudhary c, Masood Jahed a,b

a Department of Pharmacognosy, Isfahan Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Islamic Republic

of Iranb Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iranc H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

A R T I C L E I N F O

Article history:

Received 19 June 2013

Received in revised form 30 August 2013

Accepted 26 September 2013

Available online 16 October 2013

Keywords:

Pycnocycla spinosa Decne

Polycyclic diterpene

Spasmolytic effect

Activity guided isolation

A B S T R A C T

Using a bioassay-directed fractionation of Pycnocycla spinosa Decne. Ex Boiss. var. spinosa, a new

polycyclic diterpenoid, 3,7,10,14,15-pentaacetyl-5-butanoyl-13,17-epoxy-8-myrsinene (1), two known

compounds; vanillin (2) and isoacetovanilon (3), and a new phenolic compound, 6-(4-hydroxy-3-

methoxyphenyl)-hexanoic acid (4) with inhibitory effects on KCl-induced smooth muscle contractions

on the rat isolated ileum were obtained. Compound 1, the most active of the series, which exerted potent

antispasmodic activity with IC50 value of IC50 = 0.062 � 0.011 mM, is likely the main active ingredient of

the extract. The structures of the isolated compounds were established based on 13C and 1H NMR as well as

2D NMR, IR, HR-MS, and X-ray crystallographic methods.

� 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

1. Introduction

The umbelliferae is a large family of the aromatic floweringplants, including over 300 genera and 2500–3000 species as herbsor shrubs with hollow inter nodes (Heywood, 1998). Within thisfamily, the genus Pycnocycla is distributed in the dry arid regionsfrom North Africa to West of Pakistan. In Iran, about 8 species arereported, all of which are endemic (Mozaffarian, 1996). Amongthem, Pycnocycla spinosa Decne. Ex Boiss. va. spinosa, known as‘‘Sagdandan-e-khardar’’ in Persian is a naturally growing plant thatis grassed by sheep and spread widely in Tehran, and central partsof Iran (Mozaffarian, 1996). The aerial parts oil of Pycnocycla

spinosa was reported to have thirty three components. Geranylisovalerate, caryophyllene oxide, a-eudesmol,b-citronellol, ele-micin, r-cymene, citronellyl acetate, and a-cadinol were found asmajor components (Asghari et al., 2001). Seasonal variation on theessential oil composition of plant was revealed (Asghari et al.,2002). Hydro alcoholic extract from the plant aerial part and seedswas previously shown to have spasmolytic action in vitro (Sadraei

* Corresponding author. Tel.: +98 913 316 7326; fax: +98 311 668 0011.

E-mail addresses: ghanadian@pharm.mui.ac.ir, Ghannadian@gmail.com

(M. Ghanadian).

1874-3900/$ – see front matter � 2013 Phytochemical Society of Europe. Published by

http://dx.doi.org/10.1016/j.phytol.2013.09.016

et al., 2003a,b). Using a bioassay-directed fractionation, newditerpenoid polyester, and three phenolic compounds includingone new 4-hydroxy-3-methyl-phenyl derivative responsible forthe observed spasmolytic activities in vitro on isolated rat ileumhave been isolated, and we report here their structure elucidation.However, it is the first time that a myrsinane type diterpene isreported to have significant relaxant effects on KCl-inducedcontractions on rat isolated ileum. Myrsinanes are polycyclicditerpenoids biosynthesized probably from 6,20-epoxy-lathyrolderivatives (Ahmad et al., 1998). They were previously reportedfrom different species of spurge family (Euphorbiaceae) andtherefore, P. spinosa (Fam. Umbelliferae) could be a new source forthis chemo-type useful in the treatment of diseases related to thesmooth muscle contraction (Fig. 1).

2. Results and discussion

Compound 1 exhibited a positive HRESI-MS pseudo-molecularion [M+NH4]+ at m/z 682.3445 (calcd. 682.34386) suggesting itsquasi-molecular formula as C34H48O13 + NH4

+. The IR absorptionsindicated the presence of carbonyls (1739 cm�1), C–O (1039–1232 cm�1), and 55C–H (3010 cm�1), with no absorption forhydroxyl group. Signals of five acetyl methyl singlets at dH 2.15,2.05, 2.05, 2.04, and 1.98 together with protons of butanoyl ester at

Elsevier B.V. All rights reserved.

Fig. 1. Macrocyclic diterpene (1) and aromatics (2–4) from Pycnocycla spinosa.

M. Ghanadian et al. / Phytochemistry Letters 7 (2014) 57–6158

dH 2.21 (m, H-200), 1.56 (m, H-300), and 0.94 (t, J = 7.6 Hz, H-400)(Ahmad et al., 2005) were observed in the 1H NMR spectrum. TheESIMS confirmed the presence of acetates at m/z 605 [M�Ac], 545[M�2 Ac], 485 [M�3 Ac], 443 [M�4 Ac], 385 [M�5 Ac], and the lossof butanoate at m/z 457 [M�2 Ac�88] in the molecule. Takentogether, the 13C NMR spectra (BB and DEPT) and the elevendegrees of unsaturation, supported six ester carbonyls, one pairof double bond and therefore four rings in the main skeleton.Without esters, the remaining twenty carbons of the main coreskeleton represented four methyls, ten methines, two methy-lenes, and four quaternary carbons of which 8 were oxygenated.The 1H NMR spectrum showed signals for one pair of double bondat dH 6.17 (dd, J = 10.4, 6.8 Hz, H-9), 5.95 (dd, J = 10.4, 6.0 Hz, H-8),and also six deshielded signals due to the proton geminal tooxygen-bearing groups; four were from oxymethine groups dH

[5.90 (d, J = 11.2 Hz, H-5), 5.20 (t, J = 3.6 Hz, H-3), 5.00 (s, H-14),and 4.60 (d, J = 6.0 Hz, H-7)] and two from oxymethylene groupsdH 4.01 (d, J = 8.8 Hz, H-17a), and 3.45 (d, J = 8.8 Hz, H-17b)] withrelatively small geminal J values (J = 8.8 Hz) suggesting atetrahydrofuran ring on a Myrsinane-type skeleton (Ghanadianet al., 2012; Ahmad and Jassbi, 1999). Other downfield-shiftedprotons were observed at dH 3.34 (m, H-11), 2.70 (dd, J = 16.0,11.2 Hz, H-1a), and 2.53 (dd, J = 16.0, 9.2 Hz, H-1b) which seemsto be located in anisotropic fields characteristic of myrsinane-type skeleton (Ahmad et al., 2005). The anisotropic effect causedthe down filed shift of H-12 (4.07) for decipinone (Ahmad et al.,2005) but for this compound the carbonyl group is no longer existand therefore, the signal appeared at 3.12 ppm. 1H–1H COSYconnectivities of H-1 to H-5, H-7 to H-12, and HMBC correlationsas is shown in Fig. 2a enabled estimated proposal of the structure

Fig. 2. (a) COSY (in bold), and key HMBC correlations (!); (b

similar to decipinone which was first described from Euphorbai

tehranica (Ahmad et al., 1998), and Euphorbia decipiens (Ahmadand Jassbi, 1999). HMBC correlations of H-5 with carbonyl carbonresonated at dC 171.6 indicated the position of butanoyl ester atC-5, and therefore, other acetate groups on C-3, C-7, C-10, C-14,and C-15 (Fig. 2a). The stereochemistry was determined by theNOESY spectrum and 1H NMR coupling constants. The NOEcorrelations were detected between H-4/H-2, H-14; H-14/H-20;H-20/H11; H-11/H-7; H-7/H-17a, and H-17b, in addition to thesmall coupling constants of J = 3.6 Hz between H-4 and H-3indicated that these protons lie on the same face of the molecule.Relatively large J-value between H-4 and H-5 (J = 11.2 Hz) and theNOE of H-5 with H-12 supported H-5 and H-12 oppositeorientations (Fig. 2b). In order to firmly establish the absoluteconfiguration of the molecule, single-crystal X-ray diffractionanalysis was carried out to establish the structure of compound 1(Fig. 3). The ORTEP diagrams of 1 (Fig. 3) showed four trans fusedrings A, B, C, and D. Two five membered rings A (C8–C12) and C(O1/C6/C14–C15) found in envelop conformations. Seven mem-ber ring B found to attain distorted conformation with pseudoaxially oriented acetyl (O4–O5/C24–C25) and pseudo equato-rially oriented (O10–O11/C31–C34) groups. The olefinic bondcontaining six membered ring D exist in half chair conformationwith pseudo axially oriented acetyl (O12–O13/C16–C17)) and(O2–O3/C18–C22) substituents.

Comparison of the 1H and 13C NMR as well as mass spectral datawith those published before, and co-TLC with authenticatedstandards allowed us to establish the structures of the compounds2 and 3 as vanillin and isoacetovanilon (Huang et al., 2012; Fieldinget al., 2011).

) key NOESY cross-peaks ($) detected for compound 1.

Fig. 3. Structure of compound 1 established by single X-ray crystallography.

Hydrogens are omitted for clarity except those on stereocenters.

Fig. 4. Log concentration-inhibition response curve of compounds 1–4 from

Pycnocycla spinosa on KCl-induced smooth muscle contractions in rat isolated

ileum. Results are given as mean and vertical bars indicate SEM (n = 6).

M. Ghanadian et al. / Phytochemistry Letters 7 (2014) 57–61 59

Compound 4 was obtained as white solid with positive reactionto methanolic ferric chloride. It exhibited a positive HRESI-MSpseudo-molecular ion [M+NH4]+ at m/z 256.1565 (calcd.256.1543) suggesting its quasi-molecular formula asC13H18O4 + NH4

+. The IR absorptions indicated the peaks ofcarbonyl (1684 cm�1), C–O (1030–1113 cm�1), aromatic bonds(3099, 1597, and 1435), and free hydroxyl group (3485). The 13CNMR spectrum supports the existence of the aromatic ringshowing six carbon peaks dC 122.1 (s, C-10), 112.4 (d, C-20),147.2 (s, C-30), 151.1 (s, C-40), 114.4 (d, C-50), 123.8 (d, C-60) at thearomatic region. The carbon resonances of C-20, C-50 and C-60 wereassigned by the use of HSQC spectrum. The 1H NMR spectrumshowed ABX spin pattern for H-50, H-60, and H-20 at dH 6.86 (d,J = 8.4 Hz, A of ABX), 7.56 (dd, J = 8.4, 1.5 Hz, B of ABX), and 7.58 (d,J = 1.5 Hz, X of ABX), respectively in addition to deshielded singletsignal due to the aryl methoxy group at dH 3.91 (s) similar to thoseof vanillin (Huang et al., 2012). Five other methylene protons wereobserved at dH 3.19 (t, J = 7.6 Hz, H-6), 1.53 (pent, J = 7.6 Hz, H-5),1.35 (pent, J = 7.6 Hz, H-4), 1.63 (pent, J = 7.6 Hz, H-3), and 2.20 (t,J = 7.6 Hz, H-2). 1H–1H COSY, as well as HMBC correlations,confirmed the coupling between these protons and characteristicconnectivities of alkyl C5-H (dH 3.19) with C-10 (dC 122.1), and C2-H(dH 2.20) with carboxylic carbon at dC 174.6 (C-1) which ascribedthe chain as hexanoic acid attached to the phenolic ring. Thisstructure was more confirmed through EI mass ion fragments at m/z 238 [M], 193 [238-COOH], 123 [238-alkylchain] and allowed us toestablish the structure of compound 4 as 6-(40-hydroxy-30-methoxyphenyl)-hexanoic acid.

KCl-induced smooth muscle contraction attributed to the K (+)-depolarization of membrane potential leading to an increase in thecytosolic free Ca (2+) through activation of voltage-dependentcalcium channels. The contraction is inhibited in the presence of L-type voltage-operated Ca (2+) channels blocker or transientreceptor potential channel blockers (Ratz et al., 2005). As clearedfrom Fig. 4, after KCl stimulus, compound 1–4 dose dependentlyinhibited contraction with IC50 values of 0.062 � 0.011, 0.84 � 0.11,1.05 � 0.09, and 0.46 � 0.15 mM, respectively. Nifedipine as astandard voltage-dependent calcium channel blocker drug showedspasmolytic effect with IC50 values of 0.0025 mM. So, as presented inFig. 4 the compound 1 is the major component in the series, stronglyinhibited the contraction at least 10 times more potent thancompound 2–4.

In conclusion, it is the first report of a myrsinane type diterpenepossessing potent KCl-induced contraction inhibitory effect in

vitro. To have a better judge about its relaxant effect, it is

recommended to check the spasmolytic properties of thiscompound against other spasmogens like electrical field stimula-tion, acetylcholine and histamine.

3. Experimental

3.1. General experimental methods

Isolation of pure compounds were done with CC usingLichroprep1 Si 60, RP-18 (40–63 mm, Merck, Germany), TLC (Silicagel 60 F254, Merck, Germany) and recycling preparative HPLCequipped with UV and RI detectors (LC-908, Hitachi, Japan) using aYMC-Pack-Sil column (250 � 20 mm i.d., YMC, Japan). Thestructures of the compounds were elucidated using spectralmethods 1H NMR, 13C NMR (BB, and DEPT), COSY, HMBC, FT-IR, UVand HRESI-MS. The NMR spectra were acquired with Bruker AV-300 (1H) and AV-600 (13C). The Infra red spectra were acquired byJASCO 302-A spectrophotometer, with KBr discs. The HRESI-MSspectra were obtained with waters Q-TOF Micro YA019 massspectrometer in m/z and EI-MS spectra with Varian MAT 112 orMAT 312 spectrometers. Single-crystal X-ray diffraction data wascollected on Bruker Smart APEX II, CCD area detector diffractome-ter. Data reduction was performed using SAINT program. Thestructure was solved by direct methods (Altomare et al., 1993), andrefined by full-matrix least squares on F2 by using the SHELXTL-PCpackage (Sheldrick, 1997).

3.2. Plant material

The aerial parts of plant were collected at autumn from Isfahanprovince at the central part of Iran at 1590 m altitude in 2011. Theplant was identified and the voucher specimen A24 was depositedat the herbarium of the Faculty of Pharmacy of Isfahan Universityof Medical Sciences, Iran.

3.3. Extraction and isolation

The air dried plant material powder (4 kg) was macerated with28 L methanol (72 h � 3 times) at room temperature, concentratedunder reduced pressure (325 g) and applied to VLC over RP-18 silica-gel using MeOH:H2O (6:4). Evaporation of the defatted fraction gavea gummy residue (42.5 g) which chromatographed on normalcolumn using gradient mixtures of hexane: ethyl-acetate (5 ! 100)to six fractions. Relaxant effect of the resulted fractions (Fr.1–Fr.6)was compared in vitro on KCl-induced contraction in rat ileumsmooth muscles (Sadraei et al., 2003a,b). Fractions Fr.1–Fr.6 showedIC50 values of 3.5 � 0.7, 11 � 3.1, 15 � 3.9, 8 � 2.9, 14 � 2.4, and58 � 14.5 mg/ml, respectively. The most active fractions, Fr.1 (2.1 g),Fr.2 (1.5 g), and Fr.4 (2.9 g), were subjected separately to silica gel

1 CCDC contains the supplementary crystallographic data for this paper. These

data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/

retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:

+44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).

M. Ghanadian et al. / Phytochemistry Letters 7 (2014) 57–6160

columns, using hexane/ethyl acetate (5 ! 100%). Guiding by 1H NMRspectrum, Fr.1c eluted with hexane/EtOAc 80:20, was subjected tomore purification on HPLC using YMC-Pak-Sil column (250 � 20 mm)and hexane/EtOAc (75:25) as mobile phase to yield compound 1(47 mg). Fr.3c eluted with hexane/EtOAc 85:15, submitted on columnchromatography with hexane/acetone (5 ! 30%). Fr.3c2 eluted withhexane/acetone 90:10, was purified on HPLC using hexane/EtOAc(15 ! 20%) to yield compounds 2 (18 mg) and 3 (15 mg). Finally, Fr.4 geluted with hexane/EtOAc 60:40, submitted on polyamide column(DCM/MeOH, 4 ! 15). Fr.4g3 eluted with DCM/MeOH (91:9) furtherpurified through thin layer chromatography with DCM/MeOH (9:1) togive compound 4 (43 mg).

3.4. 3,7,10,14,15-Pentaacetyl-5-butanoyl-13,17-epoxy-8-myrsinene

(1)

Colorless crystal; [a]D �29.6 (c 0.73, CHCl3); UV (CHCl3) lmax:291 nm; IR (KBr) nmax: 3010, 2960, 2933, 1739, 1458, 1371, 1232,1149, 1086, 1039, 968 cm�1; 1H NMR (400 MHz, CDCl3): 2.70 (dd,J = 16.0, 11.2 Hz, 1H, H-1a), 2.53 (dd, J = 16.0, 9.2 Hz, 1H, H-1b), 2.15(m, 1H, H-2), 5.20 (t, J = 3.6 Hz, 1H, H-3), 3.01 (d, J = 11.2, 3.6 Hz, 1H,H-4), 5.90 (d, J = 11.2 Hz, 1H, H-5), 4.60 (d, J = 6.0 Hz, 1H, H-7), 5.95(dd, J = 10.4, 6.0 Hz, 1H, H-8), 6.17 (dd, J = 10.4, 6.8 Hz, 1H, H-9), 3.34(m, 1H, H-11), 3.12 (d, J = 2.8 Hz, 1H, H-12), 5.00 (s, H-14), 0.80 (d,J = 6.8 Hz, 3H, H-16), 4.01 (d, J = 8.8 Hz, 1H, H-17a), 3.45 (d, J = 8.8 Hz,1H, H-17b), 1.63 (s, 3H, H-18), 1.49 (s, 3H, H-19), 1.26 (s, 3H, H-20),2.04, and 2.05 (s, 3 � 3H, 3-OAc, 7-OAc and 10-OAc), 1.98 (s, 3H, 14-OAc), 2.15 (s, 3H, 15-OAc), 2.21 (m, 2H, Bu-H-20), 1.56 (m, 2H, Bu-H-30), 0.94 (t, J = 7.6 Hz, 3H, Bu-H-40); 13C NMR (100 MHz, CDCl3): 43.3(t, C-1), 36.6 (d, C-2), 77.2 (d, C-3), 51.8 (d, C-4), 68.5 (d, C-5), 54.1 (d,C-6), 63.2 (d, C-7), 125.0 (d, C-8), 131.1 (d, C-9), 85.9 (s, C-10), 41.7 (d,C-11), 37.1 (d, C-12), 90.1 (s, C-13), 81.7 (d, C-14), 90.2 (s, C-15), 14.2(q, C-16), 69.5 (t, C-17), 23.2 (q, C-18), 24.1 (q, C-19), 24.7 (q, C-20),170.9 (s, 3-OAc), 21.1 (q, 3-OAc), 171.6 (s, 5-Bu-C-10), 36.1 (t, 5-Bu-C-20), 17.9 (t, 5-Bu-C-30), 13.8 (q, 5-Bu-C-40), 170.4 (s, 7-OAc), 21.1 (q, 7-OAc), 170.5 (s, 10-OAc), 22.7 (q, 10-OAc), 170.4 (s, 14-OAc), 21.0 (q,14-OAc), 168.2 (s, 15-OAc), 22.8 (q, 15-OAc); HRESI-MS positivemode m/z 682.3445 (calc. for C34H48O13 + NH4

+, 682.3439, D0.9 ppm); positive ESI-MS m/z: 605[M�Ac]+, 545 [M�2Ac]+, 531,485 [M�3Ac]+, 457, 443[M�4Ac]+, 397, 337, 297,277.

3.5. 4-Hydroxy-3-methoxybenzaldehyde (vanillin) (2)

White solid; IR (KBr) nmax: 3384, 3188, 2918, 2848, 2733, 1670,1589, 1508, 1464, 1433, 1400, 1290, 1267, 1207, 1153, 1122, 1030,860, 731 cm�1; 1H NMR (400 MHz, CDCl3): 9.83 (s, 1H, CHO), 7.44–7.41 (overlapped, 2H, H-2,6), 7.05 (d, J = 8.8 Hz, 1H, H-5), 6.25 (s,1H, OH), 3.97 (s, 3H, 3-OMe); 13C NMR (100 MHz, CDCl3): 191.0,151.7, 147.2, 129.9, 127.7, 114.4, 108.8, 56.2; EI-MS (m/z):152[M]+, 151, 123, 109, 97, 95, 83, 81, 69, 57.

3.6. 1-(3-Hydroxy-4-methoxyphenyl)-1-ethanone (isoacetovanillon)

(3)

White solid; IR (KBr) nmax: 3178, 1664, 1587, 1508, 1464, 1429,1298, 1265, 1200, 1171, 1155, 860, 733 cm�1; 1H NMR (400 MHz,CDCl3): 7.55 (dd, J = 8.8, 2.0 Hz, 1H, H-6), 7.54 (d, J = 2.0 Hz, 1H, H-2), 6.95 (d, J = 8.8 Hz, 1H, H-5), 6.09 (s, 1H, OH), 3.96 (s, 3H, 4-OMe),2.57 (s, 3H, COMe); 13C NMR (100 MHz, CDCl3): 196.9, 150.5, 146.7,130.3, 124.1, 113.8, 109.7, 56.2, 26.3; EI-MS (m/z): 166 [M]+, 165,151,137, 125, 111, 109, 97, 95, 83, 81, 69, 57.

3.7. 6-(4-Hydroxy-3-methoxyphenyl)-hexanoic acid (4)

White solid; [a]D �10.6 (c 0.71, MeOH); UV (MeOH) lmax:217.7, 260.1, 290.9 nm. IR (KBr) nmax: 3485, 3099, 2954, 1869,

1684, 1597, 1523, 1473, 1435, 1377, 1298, 1238, 1203, 1113, 1030,918, 881, 806,758 cm�1; 1H NMR (400 MHz, CDCl3): 2.20 (t,J = 7.6 Hz, 2H, H-2), 1.63 (pent, J = 7.6 Hz, 2H, H-3), 1.35 (pent,J = 7.6 Hz, 2H, H-4), 1.53 (pent, J = 7.6 Hz, 2H, H-5), 3.19 (t,J = 7.6 Hz, 2H, H-6), 7.58 (d, J = 1.5 Hz, 1H, H-20), 6.86 (d, J = 8.4 Hz,1H, H-50), 7.56 (dd, J = 8.4, 1.5 Hz, 1H, H-60), 3.91 (s, 3H, 30-OMe);13C NMR (100 MHz, CDCl3): 174.6 (s, C-1), 35.6 (t, C-2), 25.3 (t, C-3),26.1 (t, C-4), 28.7 (t, C-5), 38.7 (t, C-6), 122.1 (s, C-10), 112.4 (d, C-20),147.2 (s, C-30), 151.1 (s, C-40), 123.8 (d, C-60), 114.4 (d, C-50), 55.0 (s,30-OMe); HRESI-MS positive mode m/z 256.1565 (calc. forC13H18O + NH4

+, 256.1543, D 8.6 ppm); EI-MS m/z: 238[M]+, 237[M�1]+, 221 [M�OH]+, 207 [M�MeO]+, 193 [M�COOH]+, 179, 165,151, 137, 123 [M�alkyl chain]+, 111, 109, 97, 95, 71, 69,57.

3.8. Crystal data of compound 1

C34H48O13, Mr = 664.72, Monoclinic, space group P211,a = 11.4850(9) A, b = 12.9877(10) A, c = 12.8614(10)A, b =109.297(2)8, V = 1810.7(2) A3, Z = 2, rcalc = 1.219 mg/m3, F(0 0 0) =712, m(Mo Ka = 0.71073 A, max/min transmission 0.9780/0.9655,crystal size 0.38 � 0.37 � 0.24, 1.688 < u < 25.58, 10741 reflectionswere collected, of which 3646 reflections were judged observed(Rint = 0.0230). The R values were: R1 = 0.0447, wR2 = 0.1208 forI > 2s(I), and R1 = 0.0504, wR2 = 0.1271 for all data; max/minresidual electron density: 0.271/�0.256 e A�3. The structure wassolved by the direct methods and expanded by using Fouriertransformation techniques (Beurskens et al., 1994) and refined by afull-matrix least-square calculation on F2 with the aid of programSHELXL97 (Sheldrick, 1997). Crystallographic data for compound 1has been deposited in the Cambridge Crystallographic Data Center.The crystallographic information can directly be obtained free ofcharge from CCDC data center (CCDC 924704).1

3.9. Effects on KCl-induced smooth muscle contraction in rat ileum

Male Wistar rats (200–250 g) were killed by a blow to the baseof the skull in accordance with the internationally acceptedprinciples for laboratory animal use and care (Committee for theUpdate of the Guide for the Care and Use of Laboratory Animals,2010), and the Ethics Committee of Isfahan University of MedicalSciences.

Bioactivity of compounds on KCl-induced contraction wasmeasured on six different tissues as described previously (Sadraeiet al., 2003a,b). In brief, after cervical dislocation, the ileum was cutand dissected into 2–3 cm pieces. The tissue was then suspendedvia a thread in the organ bath containing Tyrode solution at 37 8C.From a resting tension of 1 g by maximally effective dose of KCl(80 mM), isotonic contractions were recorded using a Harvardtransducer and displayed on a Harvard Universal Oscillograph penrecorder device. Relaxant effect on test samples (10–320 mg/ml)and nifedipine as standard antispasmodic drug were recorded bycumulative addition to the bath until a full concentration responsecurve was achieved. Each concentration of test samples remainedin contact with the tissue for at least 15 min before its effect wasevaluated. Experiments were conducted in parallel with appropri-ate vehicle treated time matched control tissues.

3.10. Statistical analysis

Contractions were measured as maximum amplitude ofcontraction records from pre-contraction baseline 5 min before

M. Ghanadian et al. / Phytochemistry Letters 7 (2014) 57–61 61

addition of the next drug concentration and expressed aspercentage of initial values in absence of testing drug. Resultsare expressed as mean � S.E.M. of 6 independent experiments.Student’s t-test was used for statistical analyses; P values > 0.05 wereconsidered to be significant.

Acknowledgements

This paper is part of a thesis by M. Jahed, submitted in partialfulfilment of the requirements for the degree of Pharm D. Wewould like to thank Isfahan University of Medical Sciences, Isfahan,Iran for providing a Grant (no:189093), and the H.E.J. ResearchInstitute of Chemistry, International Center for Chemical andBiological Sciences, University of Karachi, Pakistan for theirsupport.

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