2010) 1(32المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة ( Anabasis aphylla)عزل لقلویدات نبات االناباسیا مها نوري حمد جامعة بغداد ،كلیة الصیدلة ، العقاقیر واالعشاب الطبیةقسم الخالصة من وقلوید أفلدین فصل قلوید االناباسین .قي من القلویدات محتویات الجزء الهوائى لنبات االناباسیا العرا تدرس تم الفصل باستخدام طریقة الكروموتوغرافیا العمودیة . المستخلص الكحولي للجزء الهوائي من نبات االناباسیا العراقي (Column Chromato graphy Thin lay)ثم كروماتوغرافیا الطبقة الرقیقة ،( er Chromatography تو شخص ( واالشعة تحت الحمراء، وقیاس معامل ،مثل طیف االشعة فوق البنفسجیة ،ق التحلیل المختلفةائباستخدام طر ةالمعزول اتالقلوید .االنكسار ومقارنة القلوید بالقلوید القیاسي باستعمال كروماتوغرافیا الطبقة الرقیقة وتحضیر ملح IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 Investigation of alkaloids of Anabasis aphylla (Chenopodiaceae) M. N. HAMAD Departme nt of pharmacognosy, College of pharmacy, Unive rsity of Baghdad Abstract The aerial p art of Iraqi Anabasis aphylla (Chenopodiaceae) h ad been investigated for its alkalo idal contents.T he alkaloid anabasine [2-(3-p y ridy l)-p iperidine] [1] & aphy llidine were isolated from an ethanolic extract of the p lant. Isolation of the alkaloid was done by column chromatogr aphy followed by p rep erative thin layer chromatogr aphy . Identification of the isolated alkalo id was done by different sp ectroscop ic methods (UV,IR), refractive index & TLC using authentic sample & preparation of a salt. Introduction The Chinopodiaceae contains 102 genera & 1400 sp ecies most grow naturally in soils containin g much salts (halop hytes). Genera include Beta (6sp p ), Chenopodium (100-150), Salicornia, Atriplex &Anabasis.(2) Investigation of certain Anabasis sp ecies revealed that they contain triterpenoid sap ogenins &/or alkaloids. In addition to anabasine (neonicotine)[l], N N H N N N CH 3 (I) (II) N N O N N O (III) (IV) N N O OH (VI) IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 anabasamine[ll], A. aphylla contains several quinolizidine alkaloids which were identified as aphy lline[lll], aphy lline N-oxide, aphy llidine[IV] & o xaphy lline[V]. Lupinine as well as other alkalo ids were also detected in A. aphylla.[3, 4, 5] The presence of the alkaloids above was conf irmed by p aper chromatography [4] Although nicotine is t he best known alkaloid of tobacco, anabasine is the major alkaloide, as it is in Nicotiana glauca & Anabasis aphylla & it’s large scale isolation from Nioctiana & other genera was extensively st udied since anabasine was at one time widely used as insecticide.[6] Anabasine, like lobeline, has antismoking & resp iratory muscle st imulatory action, & like nicotine it exhibits insecticidal p rop erties . Anabasine also was used as a mental anticorrosive [7, 8]. Studies revealed that anabasine is teratogenic ,wher e by it can induce artho gry p otic congenital defect in p igs.[9] Anabasine as well as other minor tobacco alkaloids , nor nicotine & anatabin e , are known to p osses nicotinic receptor agon ist activity , although they are relatively less p otent than S-(-)-nicotine , the principal tobacco alkaloid.[10] Biosy ntheticaly the py ridine ring of (-)- anab asine is derived from nicotinic acid, but the p ip eridine ring is not. This was demonstrated by oxidation of the anabasine to nicotinic acid & decarboxy lation of the latter to py ridine .[11] Anabasine is reco mmended in the form of its hy drochloride salt for e xtensive med ical use for the treatment of chronic nicotination & technolo gy for its p rep aration has been develop ed. No p hytochemical studies had been done in Iraq on this sp ecies before, therefore we are r eporting here the first p hytochemical study in Iraq. Experime ntal Plant material: The p lant material was collected from Al Therthar district, west of Iraq in Ap ril & was identified by the Iraqi National Herbariu m. Apparatus UV sp ectra were recorded using Shimadzu UV-300 sp ectrop hotometer. lR were measured by usin g Beck man Ac culab -8 sp ectrop hotometer . nD was measured by using Abb'8 refractometer, TLC was p erformed on a p re coated si lica gel p lates & PLC was carried out on silica gel GF254 p lates 20X20 cm, 0.5 mm thickness. Extraction & isolation The aerial p arts of the plant were air dried & ground into a fine p owder. About (700 gm) of the p owder was extracted exhaustively with 80% aqueous ethanol in a mixer. The extract was filtered & evaporated to dry ness, t o y ield 83 gm oily residue. The residue was dissolved using 2% citric acid (p H 3-4), filtered & extracted with chloroform (3x250ml) , the chloroform lay ers were combined , filtered, dried over un hydrous sodium sulfate & evaporated to dry ness (Fraction A). IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 The acidic fraction was basified to p H 5-5.5 with 10% ammonia solution (p H meter) , then extracted with chloroform , the chloroform lay ers were combin ed , filtered, dried over unhy drous sodium sulfate & evap orated to dry ness (Fraction B). The aqueous lay er was further basified to PH 8-9 with 10% ammonia solution & extracted with CHCl3 , the chloroform lay ers were combined , f iltered, dried ov er unhy drous sodium sulfate & evap orated to dry ness (Fraction C). The overall method of extraction is shown in scheme 2. S cheme (2): Method of extraction and fractionati on of Anabasi s aphylla aerial part Powdered aerial part Extract with 80% aqueous ethanol Ethanolic filterate Evaporate to dr yness Dissolve in 2 % citric acid Extract with chloroform Aqueous layer organic layer Evaporate Fraction A Extract with chloroform Basify with ammonia pH 5-5.5 Extract with chloroform Basify to pH 8-9 Organic layer Evaporate Fraction B Fraction C Oily residue (83 gm) IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 Isol ation of aphyllidine Fraction A gave a negative test for alkaloids (M ayer 's reagent), fraction B gave a p ositive Dragendorff's & M ayer’s reagent, it revealed the p resence of two minor sp ots showed positive reaction. About 0.4gm of this fraction was further fractionated by column chromatography using a column of alumina (Grade II ,50 gm), eluted with benzene, then with benzene-M eOH 1/2,3&5% [12]. The benzene fr actions were further p urified by PLC on silica gel GF254 p lates using ( acetone –water 100-8 ) as a mobile p hase ,to reveal 19 mg cryst als (m.p .110-113 °C )which is identical with that reported for aphy llidine. [12] Ap hy llidine was further identified by UV λm ax 238nm; IR Vm ax at 1630 cm -1 (C=0), 2920 and 2845 cm -1 (methy lene CH) and by TLC using Acetone-water 100:8 on silica gel; ether- CHCl3 100:70 on alumin a to give identical Rf values with the rep orted one. [13, 14] Isol ation of anabasine: Fraction C showed p ositive tests with both M ay er & Dragendorff's reagent . About 1 gm of this fraction was fractionated by p assing it through a column of silica gel (60-120 mesh) using about 70 gm of silica gel mixed with hexane & p acked in a column (2 cm diameter x 80 cm hight) . The colu mn was eluted with hexane, then with CHCL3 then with CHCL3-M eOH 1,2,5,10%. About 7-10 ml fractions were collected. Similar fractions (TLC) were combined . Fractions containin g anab asine ( authentic) were further fractionated by PLC on silica gel usin g (CHC13-M eOH-NH4OH 60:10:1) as a mobile p hase. [15, 16] (M ode of sep aration by column chromatogr aphy is shown in figure 1). Anabasine (C10H14N2) was isolated as an oil (73 mg), B. P. 105-107 0 C n20/D 1.500, UV λm ax 210, 260 nm, IR Vm ax 3450, 3100-2900, 1580, 1490-1410, 1300-1100, 800, 715 cm -1 . Anabasine HC1 m.p . 213-216 o C. Results and Discussion The differences in b asicity of alkaloids of Anabasis aphylla gave op p ortunity for fractionation of the mixture of alkaloids by st ep wise basification. Column and TLC of fraction B revealed the p resence of two alkalo ids, the one eluted from the benzene fraction was conf irmed to be ap hy llidine which showed UV absorp tion at 238 nm which is characterist ic for the chromop hore C=C-N-C=O, in addition the IR sp ectrum showed absorption band at 1650 cm -1 due to t he lactam carbony l. Column & thin lay er chromatography of fraction C revealed the p resence of not less than three alkalo ids the major band of them was isolated & identified. The identification st arted by comp aring the isolated alkaloid with st andard anabasine by TLC using five differ ent solvent sy stems / using silica gel GF254 as a stationary p hase , the solvent systems are: [16, 17] 1- CCl4 : M e2CO : M eOH 3: 7 :0.5 II- CHCI3: M eOH: NH4OH 60:10:1 III -CHCL3 : M eOH: Acetic acid 60:10:1 IV-Toluene : M ethanol: chlorofor m 90: 30 : 10 ( on basic SG 0. IN KOH) V-CHCl3-EtOH 9:l The isolated alkalo id gave identical Rf values with the standard alkaloid, using sin gle & mixed IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 sp ots (HRf values are shown in table I). The UV sp ectrum showed absorption maxima at 210 & 260 nm (figure 1) which is identical for comp ounds containing the py ridine-pip eridine moieties. IR showed bands at 3450cm -1 (N-H st retching vibration), 1300-1100 cm -1 (C-N stretching), (figur e 2), [18, 19, 20, 21]. Furt her identification of anabasine was confirmed by p rep aration of a salt which is anabasine HC1 which showed m.p at 213 -216 °C which is identical with the rep orted m.p . As a conclusion ethanolic extract of the aerial p art of Anabasis aphylla revealed the p resence of about five co mpounds showed a positive reactions for alkaloids, the major one was isolated from fraction C & was p roved to be anabasine . A minor one was isolated from fraction B which was confirmed to be aphy llidine. These alkaloids are reported here in the Iraqi sp ecies for the first time. Re ferences 1.Pouchert , C.J. (1978) The Aldrich Library of infrared sp ectra ; Aldrich chemical co mpany , Inc. p.1156 F. 2. Trease , G.E. and Evans , W.C. (2002) Pharmacognosy ; 15 th edition ;WB saunders co mpany Ltd London ; p. 36, 340 , 493. 3. Rizh , A.M . (1986) The phytochemist ry of the flora of Qatar ; king p rint of Richmond ; England ; p . 27-28. 4. Sadykov, A.S. and Tumur , B . ( 1960) Dakaldy Akad . Nauk usbek.S.S. R . ; 1 :27-29. 5. Brutko, L.I.and M assagetov, p .s.(1964) (chem. & pharm. Res. Inst . M oscow) M ed p rom. SSSR; 18(12) :34-5. 6. M ansk’e, R.H.F.(1953) T he Alkaloids: Chemistry & p hy siology ; Academic press, Inc. Newy ork. 7. Cordell , G.A. (1981) Int roduction to alkaloids John Wiley & sons , Inc. Newy ork p . 143-199. 8. Sadykov , A.S. ( 1957) Abhandle . deut. Akad . Wiss. Belin Kl. Chem..Geo l . u. Biol. 9. Keeler , R.F. ; Crowe , M .W. and Lambert , E.A.( 2005)T eratology ; 30 :61 – 69. 10- Ting,I.P.( 1982) Plant p hy siology ; Addison-Wesley Publishing comp any, P 314. 11- Ay ers,J.T . ; Xu,R.; Dwoskin,L.P. and Crooks, P.A.(2005)The aaps Journal. 27(3): 752-758. 12. Dalton , D. R . (1979)The alkaloids : the fundamental chemistry , M arcel Dekker , Inc. P. 155 :17- 172. 13. Sp äth, E.; Galinovsky , F.and M ay er, M .( 1942)Ber 75 B, 805-13, . C.A. 37: 3436 8 . 14. CHO, Y. D. and M artin, R. O.(1971) Canadian Journal of ch emistry , 49:265-270. 15. Tsuda, Y. and M arion, L.(1964) Canadian Journal of chemist ry 42:768. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 16. Nurimov, E.and Lovkov a, M . Ya.(1973) Prikl. Biokim. M ikrobiol. 9(5): 789-96. C. A. 80: 30635 d 17. Forostyan , y u.N. and Novikov , V.I .(1968) Zh . obshch . Khim . ; 38(6) :1222- 3. 18. Egon st ahl (1969) Thin layer chromatography sp ringer – verlag Berlin . Heidelberg . New York . 19. Amat , M . ; Canto , M . ; Lior , N . and Bosch , J . (2002)Chem comm. ; 5:526 – 527. 20. Yang , C.M . ; Tanner , D.D. (1997) can . J. chem.; 75: 616 – 620. 21.Silv erstein, R.M . and Webst er , F.X .(1996) Sp ectrometric identification of organic comp ounds ; John Wiley & sons , Inc. p . 103 – 109. Table (1): HRf value of standard and isolated anabasine S olvent syste m HRf standard HRf sample I 35 33 II 50 52 III 06 04 IV 83 80 V 34 37 Fig.(1): Mode of se paration of fraction C by column chromatography Adsor bent : Sili ca gel GF254 Anaba sine Fraction number 0 0.2 0.4 0.6 0.8 1 1.2 S olvent syste m : CHC3-MeOH-NH4OH 60:10:1 IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1 ) 2010 Figure 1: UV sp ectrum of anabasine Fig(3): IR spectrum of anabasine Fig(2):