ASIAN JOURNAL OF CHEMISTRY

Transcription

1 Asin Journl of Chemistry; Vol. 7, o. 7 (05), 68-7 ASIA JURAL F CEMISTRY Two ew Biotive Furoquinoline Alkloids from Znthoxylum rmtum ABDUL SAMAD, MUAMMAD ADEEL, DILFARAZ KA,*, SYED BADSA, ZIA MUAMMAD, SAFIULLA KA, nd JAE ARA A Deprtment of Chemistry Goml University, Der Ismil Khn-9050, KPK, Pkistn Deprtment of Chemistry, Koht University of Siene nd Tehnology, Koht-6000, KPK, Pkistn Stte Key Lortory of rgni-inorgni Composite, Beijing University of Chemil Tehnology, Beijing 0009, P.R. Chin Fulty of Phrmy, University of Sydney, SW 006, Austrli *Corresponding uthor: Fx: ; Tel: ; E-mil: dilfrzkhn@gu.edu.pk Reeived: My 0; Aepted: July 0; Pulished online: 0 Mrh 05; AJC-7058 The n-hexne, dihloromethne, ethyl ette nd n-utnol solule frtions of Znthoxylum rmtum were sreened for ntifungl tivity ginst Aspergillus flvus nd Aspergillus niger. The dihloromethne solule frtion of Znthoxylum rmtum inhiited the growth of Aspergillus flvus y.5 % nd Aspergillus niger y. %, while the ethyl ette solule frtion ompound deresed the growth of Aspergillus flvus nd Aspergillus niger y 55.5 nd 6 % respetively. The n-hexne nd n-utnol frtions showed low inhiitions ginst these fungi. Further the hromtogrphi seprtion of the ethyl ette frtion led to the isoltion of two new furoquinoline lkloids; -methoxy-,-dimethylylopent furoquinoline-0-rldehyde nd methyl -methoxy-,- dimethylylopent furoquinoline-0-roxylte. The isolted ompounds were tested for ntioxidnt tivities. The ompound ws found more potent in oth DPP nd ABTS rdil svenging ssy. Keywords: Znthoxylum rmtum, Furoquinoline lkloids, Antifungl, Antioxidnt. ITRDUCTI Znthoxylum is genus of out 50 speies of deiduous nd evergreen trees nd shrus elonging to fmily Rutee, ntive to wrm temperte nd sutropil res worldwide. Vrious plnts of the genus Znthoxylum hve widely een used y trdition helers ginst severl ilments like oli, fltulent, gstro-intestinl disorder nd intestinl prlysis s well s in snke ite remedy. Aniently, the Zulus used the powdered rk of these plnts to relive tooth he nd ginst T.B. Z. gillnet is used in Ghn nd igeri s n ntisepti nd nlgesi. Z. llntoids hve een used in myordium disorder ttenution, one-injury llevition nd old resistne. Some of the speies of this genus re reported to ontin lkloids, espeilly enzophennthriidines nd exoprofini lkloid liriodenine, whih show remrkle nti-tumour tivity nd potent inhiiter of topoisomerse II, respetively 5-7. Z. shinifolium possesses poptogeni tivity ginst humn ute leukemi 8. Z. rmtum is widely distriuted in siren vlley, Mnsehr, Pkistn. Its indi nme is Tejl while lol nme is Timer. In Pkistn, the only one speies of znthoxylem genus is found 9. The essentil oil of fruits of Z. rmtum shows ntiteril, ntifungl, nthelminti tivities nd lso used s leeh repellent nd ure in skin diseses 0, while the seeds nd roots re stomhi nd vermifuge -. The plnt hs lso find uses in the tretment of hest nd rdi pin, for piles nd tmers. A produt nming "Thumuru" from this plnt is used in treting numness nd trophy. In Unni (Greek) mediine, the fruit seeds nd rk re used s rmintive, stomhi nd nthelminti 5. The rk is lso used s remedy for toothhe 6. Phytohemilly, the plnt is reported to ontin is-9-hexdeenoi, monoterpenes, enoi ids nd plmti ids, tmuletin 7,8 long with the sesmin, srinin nd limonene nd znthonitrile 9. Four lkenoi ids were otined from the hexne extrt of the seed of this plnt previously 0. In the present study two new ompounds nd were isolted from the ethyl ette solule frtion of Znthoxylum rmtum (Fig. ). Further the different solvent solule frtion of Znthoxylum rmtum nd the isolted ompounds were sreened for ntifungl nd ntioxidnt tivities. EXPERIMETAL Aluminium TLC pltes (0 0, 0.5 mm thik) pre-oted with sili gel 60 F 5 (0 0 m, 0. mm thik; E. Merk, Drmstdt, Germny) were used for TLC to hek the purity of the ompounds. Column hromtogrphy (CC) ws rried out using sili gel of 0-00 mesh (E. Merk, Drmstdt, Germny). Preprtive TLC glss pltes (0 0, mm thik)

2 Vol. 7, o. 7 (05) Two ew Biotive Furoquinoline Alkloids from Znthoxylum rmtum methoxy-, -dimethylylopentfuroquinoline-0-rldehyde () Methyl -methoxy-,-dimethylylopentfuroquinoline-0-roxylte () Fig.. 8 Strutures of ompounds nd isolted from Znthoxylum rmtum pre-oted with sili gel 60 F 5 (0.5 mm lyer thikness; E. Merk, Germny) were used for the purifition of semi pure ompounds. Ceri sulphte nd potssium permngnte solutions were used s visuliztion regents. The UV spetr were reorded on Shimdzu UV-700 spetrophotometer (Shimdzu, Jpn) in Me. Mss Spetr ws reorded on Bruker TF Mss spetrometers (Billeri, USA) using eletrospry ionistion (ESI). The MR nd C MR spetr were reorded on Bruker DPX-00 MR spetrometer (Billeri, USA). Further ssignments were mde y DEPT, CSY, MQC nd MBC experiments. The whole plnt of Znthoxylum rmtum ws olleted from Sirn vlley, zr division, Distrit Mnsehr, in 008 nd ws identified y Professor Dr. Mnzoor Ahmd, Plnt Txonomist, Deprtment of Botny, Government Degree College Aotd, Pkistn, (Aession o. C-008). Extrtion nd isoltion: The shde dried whole plnt of Znthoxylum rmtum ws ground nd extrted with methnol t room temperture. The omined methnoli extrt ws evported under redued pressure to otin thik greenish gummy mteril (rude). It ws suspended in wter nd suessively prtitioned with suitle solvents to yield n- hexne, dihloromethne, ethyl ette nd n-utnol solule frtions, respetively. The ethyl ette solule frtion ws sujeted to olumn hromtogrphy over sili gel (70-0 mesh) eluting with n-hexne: EtA (:9-9:), EtA (00 %), EtA: Me (:9-5:), in inresing order of polrity to otin ompound 8 frtions E0-E08. The sufrtion Et07 gve two spots on TLC with good R f vlue nd hene ws rehromtogrphed over sili gel olumn hromtogrphy eluted with n-hexne: EtA (5:-:) to get the two spots seprtely whih were pled in fume hood to get fine rystls of ompound. Sufrtion E08 gve severl spots on TLC nd ws loded over sili gel olumn hromtogrphy eluted rndomly with n-hexne: EtA (0:-:) to fford ompound s white needles Methoxy-,-dimethylylopent furoquinoline- 0-rldehyde (): White rystl; m.p. 0- C; U.V (Me) λ mx (log ε): (.), 9 (.), 6 (.), 55 (.7), 66 (.6), 78 (.6), 0 (.9), 8 (.95), 85 (.5); IR (KBr, ν mx, m - ): 960, 90, 850, 80, 60, 65, 60, 585, 50, 80, 5, 5, 09, 0; MR nd C MR (CDCl, 00 nd 00 Mz) Tle-; R- EI-MS m/z 9.5 [M + ] + Cld. for C 8 5, 9.). Methyl -methoxy-,-dimethylylopent furoquinoline-0-roxylte (): White needles; m.p: - C; U.V (Me) λ mx (log ε): 0 (.7), 9 (.), 7 (.8), (.0), (.90) nd (.5) nm; IR (KBr, ν mx, m - ): 9, 86, 75, 60, 65, 600, 595, 50, 65, 6, 5, 09, 05; MR nd C MR (CDCl, 00 nd 00 Mz) Tle-; R-EI-MS m/z.6 [M + ] + Cld. for C 9 7,.). TABLE- AD C MR DATA F CMPUDS AD (δ I ppm, BTAIED I CDCl ) Compound Compound o. δ δ (J in δ C δ (J in z) C z). 7.6 d (.9) d (.) d (.9) d (.) d (8.) d (8.5) d (8.) d (8.5) s s C s s C 6..6 s 6.9. s C C s -C= s A Antifungl ssy: The ntifungl tivity of different solvent solule frtions of Znthoxylum rmtum ws determined y using the the protool. mg/ml stok solution of rude hexne, dihlormethne, ethyl ette frtion nd n- utnol frtions were prepred in DMS. Furthermore, ml solution of 00 µg/ml ws prepred from the stok solution using DMS s diluting solvent. In the sme wy, terinofine stok solution of mg/ml (positive ontrol/ntifungl gent) ws prepred in DMS. Moreover, ml solution of the required onentrtion (00 µg/ml) ws prepred from the stok solution in DMS. Correspondingly, ml DMS ws tken, used s negtive ontrol from the ottle. In order to grow fungus for inoulums preprtion, Sourud dextrose gr (MERCK) ws used, omposed of peptone omplex 0 g/l, gluose 0 g/l nd gr 5 g/l. 6.5 g SDA medi ws dissolved in 00 ml distilled/utolved wter in flsk for fungus growth nd utolved t C for 5 min. ml of this medi ws poured in the ll the utolved test tues nd mrked up to

3 70 Smd et l. Asin J. Chem. 0 m in the Lminr flow inet for two fungl strins. From the required onentrtion (00 µg/ml) of the solution, 67 µl of extrt solution ws put in ll the test tues, whih were speified nd duplite for the two fungl strins. In the sme wy, the terinofine solution of 67 µl (for positive ontrol) of the required onentrtion (00 µg/ml) ws put in ll the two test tues (one for eh) of the two fungl strins. Similrly, 67 µl DMS (negtive ontrol) ws poured in nother set of two test tues (one for eh) of the two fungl strins. After the ompletion of this whole proess, ll the test tues were ple in the lminr flow in slnting position for solidifying the medi in the test tues t room temperture. After the solidifition proess, 5 spores from 7 dys old ulture were pled of eh fungus strin in ll the test tues (extrt + ontrol) nd were speified very refully for eh strin. All the test tues were pked ir tightly nd were pled in inutor t 6 C for 7 dys. Their growth ws mesured fter 7 dys nd lulted their % inhiition. DPP rdil svenging ssy: The ntioxidnt DPP rdil svenging ssy ws done ording to the stndrd proedure with some modifitions. In the ssy 900 µl methnoli solution of DPP ( mm), 00 µl from eh of the isolted ompound previously solulized in methnol ws dded t vrious onentrtions, from 50 to 500 µg/ml in finl volume of ml. An equl mount of methnol ws dded to the ontrol. After 0 min, sorne ws reorded t 57 nm. Experiment ws done in duplite nd performed in triplite. ABTS rdil svenging ssy: The,'-zino-is-(- ethylenzthizoline-6-sulphoni) (ABTS) hve high ility to t s n eletron donor for the redution of oxo speies. ABTS ssy ws mde ording to the stndrd method with some modifition. Assy proedure equl volumes of 7 mm ABTS nd 5 mm K S 8 were mixed nd inute for h. 00 µl of the smple, from eh onentrtion i.e., (50, 00, 50, 00, 50, 500 µg/ml eh of these onentrtion ws tken in duplite) ws mixed with 800 µl of ABTS solution ws mixed with it for eh onentrtion. Similr proess ws repeted for sori id onentrtion. All test tues (ompounds - nd sori id) leled seprtely were shken well nd inuted in the drk t 5 C for 0.5 h. The kineti sorne ws reorded t 7 nm y spetrophotometer fter nd 6 min for eh onentrtion nd men ws tken for eh reding. The potentil to svenge the ABTS rdil ws lulted using the following eqution: (A A ) ABTS rdil svenging effet (%) = 00 A where A is the sorne of the ontrol (ABTS solution without test smple) nd A is the sorne in the presene of the test smple. RESULTS AD DISCUSSI The n-hexne, dihloromethne, ethyl ette nd n- utnol frtions were sreened for ntifungl tivities ginst Aspergillus flvus nd Aspergillus niger. The ethyl ette solule frtion showed mximum inhiition nd deresed the fungl growth y 55.5 nd 6 % ginst Aspergillus flvus nd Aspergillus niger, respetively, dihloromethne frtion showed moderte inhiition while n-hexne nd n-utnol frtions showed minimum inhiitions ginst the orresponding fungi (Tle-). TABLE- ATIFUGAL ACTIVITY n-exae, DICLRMETAE, ETYL ACETATE, AD n-butal FRACTIS F Xnthoxylum rmtum AGAIST Aspergillus flvus AD Aspergillus niger Frtions, solvent nd ontrol Aspergillus flvus Fungl growth (m) Inhiition (%) Aspergillus niger Fungl growth (m) Inhiition (%) n-exne Dihloromethne Ethyl ette n-utnol DMS TER Furthermore, the ethyl ette solule frtion ws sujeted to olumn hromtogrphy over sili gel (70-0 mesh) nd preprtive TLC to fford ompounds nd (Fig. ). Compound ws isolted s white rystls. The EI-MS fforded the moleulr ion t m/z 9 implying the moleulr formul C 8 5 whih ws onfirmed y R-EI-MS ([M] + ; found: 9.5 Cld: 9.). The presene of -quinoline system in the moleule ws indited y the UV sorption t, 6, 66 sh, 8 nd 85 nm. The IR spetrum lso showed nd t 67 m - suggesting the presene of α, β-unsturted ldehyde whih ws onfirmed y the presene of formyl signl t δ 0.09 in MR. ther MR signls were oserved t δ.6 (C ),.8 (C ). A ouple of doulet found t δ 7.9 (, J = 8. z), 7.69 (, J = 8. z) were ssigned to -5 nd -6 proton, respetively. Another ouple of doulet displyed t δ 7.6 (, J =.9 z) nd 7.0 (, d, J =.9 z) were hrteristis of - nd - of the furoquinoline moiety. The proton t β-position of formyl group resonted t 7. (, s). C MR displyed 7 signls. Signls for romti rons were found t δ 65.9 (9), (C-), 8. (C-8), 5.7 (C-8),.96 (C-7),. (C-), 9.6 (C-6),.8 (C-), 9.89 (C-5), 09. (C-) nd (C-). The α nd β ron of D-ring resonted t δ 6.56 (C-0) nd 6.5 (C-), respetively. The methoxy nd methyl rons were found t δ 6. (C ), 7.8 (C ) while the pek t δ 5.08 ws ssigned to C-. The nd C MR dt ws onfirmed y MBC experiments whih showed J orreltions etween J orreltions ws found etween - nd C- nd C-9, - nd C-9, -5 nd C-7 nd C-8, -6 nd C- nd C-8, - nd C-7, C-8 nd C=. Formyl proton showed J orreltion with C-0 while methoxy proton showed J orreltion with C-. Similrly J orreltion ws found etween the two methyl protons nd C- nd C-8. Aording to the ove dt nd MBC experiments (Fig. ) the struture of ompound ws eluidted s -methoxy-,-dimethylylopent furoquinoline-0-rldehyde. Compound ws isolted s white needles. Its moleulr formul ws suggested s C 9 7 from the moleulr ion pek t m/z in EI-MS spetrum whih ws lso onfirmed y R-EI-MS nlysis. The UV sorption t 9,,

4 Vol. 7, o. 7 (05) Two ew Biotive Furoquinoline Alkloids from Znthoxylum rmtum 7 Fig.. Importnt MBC orreltions of ompound Svenging DPP (%) A. Aid Compound Compound nd were similr to tht of ompound nd were hrteristis of furoquinoline nuleus. The presene of α,β-unsturted ester in moleule ws reveled y IR nds t 75 m - nd ws onfirmed y MR pek t.7 ppm. The MR spetrum of ompound ws similr to tht of ompound exept tht the ldehydi pek of formyl group ws repled y methoxy pek of ester group. The presene of furoquinoline nuleus ws lerly demonstrted from two pirs of doulet of doulet resonted t δ 8.0 (, d, J = 8.5 z, -5), 7.9 (, d, J = 8.5 z, -6), 7.68 (, d, J =. z, -) nd 6.89 (, d, J =. z, -). In ddition the presene of methoxy group t C- nd the methyl grops t C- were onfirmed y signls t δ.5 (, s) nd.0 (6, s), respetively. C MR dt ws similr to ompound exept nd dditionl pek t δ 55. for C group of ester moiety. Aording to the ove dt the struture of ompound ws ssignes s methyl -methoxy-,-dimethylylopent furoquinoline-0-roxylte whih ws further onfirmed y MBC experiment (Fig. ). 0 0 Fig Svenging ABTS (%) Conentrtion (µg/ml) DPP svenging tivities of vrious onentrtions of sori id nd pure ompounds nd. The dt represent the men of three different experiments done in duplite. The different letters represent sttistilly different from eh other A. Aid Compound Compound 0 Fig.. Importnt MBC orreltions of ompound The isolted ompounds were studied s ntioxidnt. The vrious rnge onentrtion of ompounds nd (50, 00, 50 00, µg/ml) showed signifint DPP rdil svenging tivity ompred with sori id using s stndrd of sme onentrtion. Furthermore, the ompound hs less nti-oxidnt tivity thn ompound (Fig. ). Similr experiments for ABTS (,'-zino-is(-ethylenzthizoline-6-sulphoni) rdil svenging ssy were designed to verify the ntioxidnt tivity of the isolted ompounds nd from Znthoxylum rmtum. The ABTS (,'-zino-is(-ethylenzthizoline-6-sulphoni) rdil svenging ssy lso support the dt tht indited tht ompound hs higher svenging thn ompound (Fig. 5). 0 Fig. 5. Conlusion Conentrtion (µg/ml) ABTS svenging tivities of vrious onentrtions of sori id nd isolted ompounds -6. The dt represent the men of three different experiments done in duplite. Eh vlue represents men ± SEM In onlusion, it is interesting to note tht Znthoxylum rmtum tht hs een used for long time for vrious purposes; in ft the new sientifi report my suggest new pplitions. ACKWLEDGEMETS The uthor is thnkful to igher Edution Commission, Pkistn for finnil support under IRSIP Fellowship Progrm t University of Sydney, Austrli. The uthor lso knowledges Dr. Jne nrhn, Fulty of Phrmy, University of Sydney for her guidne in the spetrl nlysis of the ompounds. Dr. Zi Muhmmd, Koht University of Siene nd Tehnology Koht, KPK, Pkistn is lso herey knowledged for his ssistne in rrying out the iotivities of the onstituents.

5 7 Smd et l. Asin J. Chem. REFERECES. V. Thoms, G. Rin, J.V. Gogh nd J. Adendorff, Sppi Tree Spotting, ighlnds, ighveld, Drkenserg, Estern Cpe Mountins, edn, p. 60 (00).. J.M. Wtt nd M.G.B. Brndwijk, The Mediinl nd Poisonous Plnts of Southern nd Estern Afri, E&S Livingstone Ltd., Edinurgh, pp (96).. D.K. Aiw, Useful Plnts of Ghn: Intermedite Tehnology Pulitions nd the Royl Botni Grdens, Kew, UK, pp. 98- (990).. Y.C. Chung, C.T. Chien, K.Y. Teng nd S.T. Chou, Food Chem., 97, 8 (006). 5. K. Deyun, A.I. Gry, T.G. rtley nd P.G. Wtermn, Biohem. Syst.,, 87 (996). 6. M. Del Poet, S.-F. Chen, D. Von off, C.C. Dykstr, M.C. Wni, G. Mnikumr, J. eitmn, M.E. Wll nd J.R. Perfet, Antimiro. Agents Chemother.,, 86 (999). 7. S.. Woo, M.C. Reynolds,.J. Sun, J.M. Cssdy nd R.M. Snpk, Biohem. Phrmol., 5, 67 (997). 8. D.Y. Jun, J.S. Kim,.S. Prk, C.R. n, Z. Fng, M.. Woo, I.K. Rhee nd Y.. Kim, Crinogenesis, 8, 0 (007). 9. "Znthoxylum", In Flor of Pkistn, Pulished y Siene Press (Beijing) nd Missouri Botnil, Grden Press, p M.B. Meht, M.D. Khry, R. Srivstv nd K.C. Verm, Indin Perfumer., 5, 9 (98).. D.R. th,.g. Ds nd S.C. Ds, Ind. J. Med. Res. Setion A Infet. Dis., 97, 8 (99).. J.C. Uphof, The Ditionry of Eonomi Plnts, Weinheim (959).. G. Usher, A Ditionry of Plnts Used y Mn, Constle (97).. A Brefoot Dotors Mnul, Running Press, Phildelphi (977); ISB X. 5. C.P. Khre, Indin erl Remedies, Springer, pp (00). 6. R.. Chopr, S.L. yr nd I.C. Chopr, Glossry of Indin Mediinl Plnts Counil of Sientifi nd Industril Reserh, ew Delhi (986). 7. F. Ahmd, I. Ahmd nd S.M. smn, J. Am. il Chem. So., 57, (980). 8. A.G.R. ir, G.A. ir nd C.P. Joshu, Phytohemistry,, 8 (98). 9. T.P. Singh nd.m. Singh, Ind. J. t. Prod. Resour.,, 75 (0). 0. A. Ahmd, L.. Misr nd M.M. Gupt, J. t. Prod., 56, 56 (99).. V. Duripndiyn nd S. Ignimuthu, J. Ethnophrmol.,, 9 (009).. M. Sreejyn, Arzneim.- Forsh. Drug Res., 6, 69 (996).. M.A. Gymfi, M. Yonmine nd Y. Aniy, Gen. Phrmol.,, 66 (999).