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 Chromatography)، ثم كروماتوغرافیا الطبقة الرقیقة(Thin layer 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 Department of pharmacognosy, College of pharmacy, University of Baghdad Abstract The aerial part of Iraqi Anabasis aphylla (Chenopodiaceae) had been investigated for its alkaloidal contents.The alkaloid anabasine [2-(3-pyridyl)-piperidine] [1] & aphyllidine were isolated from an ethanolic extract of the plant. Isolation of the alkaloid was done by column chromatography followed by preperative thin layer chromatography. Identification of the isolated alkaloid was done by different spectroscopic methods (UV,IR), refractive index & TLC using authentic sample & preparation of a salt. Introduction The Chinopodiaceae contains 102 genera & 1400 species most grow naturally in soils containing much salts (halophytes). Genera include Beta (6spp), Chenopodium (100-150), Salicornia, Atriplex &Anabasis.(2) Investigation of certain Anabasis species revealed that they contain triterpenoid sapogenins &/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 aphylline[lll], aphylline N-oxide, aphyllidine[IV] & oxaphylline[V]. Lupinine as well as other alkaloids were also detected in A. aphylla.[3, 4, 5] The presence of the alkaloids above was confirmed by paper chromatography[4] Although nicotine is the 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 studied since anabasine was at one time widely used as insecticide.[6] Anabasine, like lobeline, has antismoking & respiratory muscle stimulatory action, & like nicotine it exhibits insecticidal properties . Anabasine also was used as a mental anticorrosive [7, 8]. Studies revealed that anabasine is teratogenic ,where by it can induce arthogrypotic congenital defect in pigs.[9] Anabasine as well as other minor tobacco alkaloids , nor nicotine & anatabine , are known to posses nicotinic receptor agonist activity, although they are relatively less potent than S-(-)-nicotine , the principal tobacco alkaloid.[10] Biosyntheticaly the pyridine ring of (-)- anabasine is derived from nicotinic acid, but the piperidine ring is not. This was demonstrated by oxidation of the anabasine to nicotinic acid & decarboxylation of the latter to pyridine .[11] Anabasine is recommended in the form of its hydrochloride salt for extensive medical use for the treatment of chronic nicotination & technology for its preparation has been developed. No phytochemical studies had been done in Iraq on this species before, therefore we are reporting here the first phytochemical study in Iraq. Experimental Plant material: The plant material was collected from Al Therthar district, west of Iraq in April & was identified by the Iraqi National Herbarium. Apparatus UV spectra were recorded using Shimadzu UV-300 spectrophotometer. lR were measured by using Beckman Acculab -8 spectrophotometer . nD was measured by using Abb'8 refractometer, TLC was performed on a pre coated silica gel plates & PLC was carried out on silica gel GF254 plates 20X20 cm, 0.5 mm thickness. Extraction & isolation The aerial parts of the plant were air dried & ground into a fine powder. About (700 gm) of the powder was extracted exhaustively with 80% aqueous ethanol in a mixer. The extract was filtered & evaporated to dryness, to yield 83 gm oily residue. The residue was dissolved using 2% citric acid (pH 3-4), filtered & extracted with chloroform (3x250ml) , the chloroform layers were combined , filtered, dried over un hydrous sodium sulfate & evaporated to dryness (Fraction A). IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1) 2010 The acidic fraction was basified to pH 5-5.5 with 10% ammonia solution (pH meter) , then extracted with chloroform , the chloroform layers were combined , filtered, dried over unhydrous sodium sulfate & evaporated to dryness (Fraction B). The aqueous layer was further basified to PH 8-9 with 10% ammonia solution & extracted with CHCl3 , the chloroform layers were combined , filtered, dried over unhydrous sodium sulfate & evaporated to dryness (Fraction C). The overall method of extraction is shown in scheme 2. Scheme (2): Method of extraction and fractionation of Anabasis aphylla aerial part Powdered aerial part Extract with 80% aqueous ethanol Ethanolic filterate Evaporate to dryness 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 Isolation of aphyllidine Fraction A gave a negative test for alkaloids (M ayer 's reagent), fraction B gave a positive Dragendorff's & Mayer’s reagent, it revealed the presence of two minor spots showed positive reaction. About 0.4gm of this fraction was further fractionated by column chromatography using a column of alumina (Grade II ,50gm), eluted with benzene, then with benzene-MeOH 1/2,3&5% [12]. The benzene fractions were further purified by PLC on silica gel GF254 plates using ( acetone –water 100-8 ) as a mobile phase ,to reveal 19 mg crystals (m.p.110-113 °C )which is identical with that reported for aphyllidine. [12] Aphyllidine was further identified by UV λmax 238nm; IR Vmax at 1630 cm -1 (C=0), 2920 and 2845 cm -1 (methylene CH) and by TLC using Acetone-water 100:8 on silica gel; ether- CHCl3 100:70 on alumina to give identical Rf values with the reported one. [13, 14] Isolation of anabasine: Fraction C showed positive tests with both Mayer & Dragendorff's reagent . About 1 gm of this fraction was fractionated by passing it through a column of silica gel (60-120 mesh) using about 70 gm of silica gel mixed with hexane & packed in a column (2 cm diameter x 80 cm hight) . The column was eluted with hexane, then with CHCL3 then with CHCL3-MeOH 1,2,5,10%. About 7-10 ml fractions were collected. Similar fractions (TLC) were combined . Fractions containing anabasine (authentic) were further fractionated by PLC on silica gel using (CHC13-MeOH-NH4OH 60:10:1) as a mobile phase. [15, 16] (Mode of separation by column chromatography is shown in figure 1). Anabasine (C10H14N2) was isolated as an oil (73 mg), B. P. 105-107 0C n20/D 1.500, UV λmax 210, 260 nm, IR Vmax 3450, 3100-2900, 1580, 1490-1410, 1300-1100, 800, 715 cm-1. Anabasine HC1 m.p. 213-216oC. Results and Discussion The differences in basicity of alkaloids of Anabasis aphylla gave opportunity for fractionation of the mixture of alkaloids by step wise basification. Column and TLC of fraction B revealed the presence of two alkaloids, the one eluted from the benzene fraction was confirmed to be aphyllidine which showed UV absorption at 238 nm which is characteristic for the chromophore C=C-N-C=O, in addition the IR spectrum showed absorption band at 1650 cm -1 due to the lactam carbonyl. Column & thin layer chromatography of fraction C revealed the presence of not less than three alkaloids the major band of them was isolated & identified. The identification started by comparing the isolated alkaloid with standard anabasine by TLC using five different solvent systems / using silica gel GF254 as a stationary phase , the solvent systems are: [16, 17] 1- CCl4 : Me2CO : MeOH 3: 7 :0.5 II- CHCI3: MeOH: NH4OH 60:10:1 III -CHCL3 : MeOH: Acetic acid 60:10:1 IV-Toluene : Methanol: chloroform 90: 30 : 10 ( on basic SG 0. IN KOH) V-CHCl3-EtOH 9:l The isolated alkaloid gave identical Rf values with the standard alkaloid, using single & mixed IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1) 2010 spots (HRf values are shown in table I). The UV spectrum showed absorption maxima at 210 & 260 nm (figure 1) which is identical for compounds containing the pyridine-piperidine moieties. IR showed bands at 3450cm -1 (N-H stretching vibration), 1300-1100 cm -1 (C-N stretching), (figure 2), [18, 19, 20, 21]. Further identification of anabasine was confirmed by preparation of a salt which is anabasine HC1 which showed m.p at 213 -216 °C which is identical with the reported m.p . As a conclusion ethanolic extract of the aerial part of Anabasis aphylla revealed the presence of about five compounds showed a positive reactions for alkaloids, the major one was isolated from fraction C & was proved to be anabasine . A minor one was isolated from fraction B which was confirmed to be aphyllidine. These alkaloids are reported here in the Iraqi species for the first time. References 1.Pouchert , C.J. (1978) The Aldrich Library of infrared spectra ; Aldrich chemical company , Inc. p.1156 F. 2. Trease , G.E. and Evans , W.C. (2002) Pharmacognosy ; 15th edition ;WB saunders company Ltd London ; p. 36, 340 , 493. 3. Rizh , A.M. (1986) The phytochemistry of the flora of Qatar ; king print 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 Massagetov, p .s.(1964) (chem. & pharm. Res. Inst. Moscow) Med prom. SSSR; 18(12):34-5. 6. Mansk’e, R.H.F.(1953) The Alkaloids: Chemistry & physiology; Academic press, Inc. Newyork. 7. Cordell , G.A. (1981) Introduction to alkaloids John Wiley & sons , Inc. Newyork p. 143-199. 8. Sadykov , A.S. ( 1957) Abhandle . deut. Akad . Wiss. Belin Kl. Chem..Geol . u. Biol. 9. Keeler , R.F. ; Crowe , M.W. and Lambert , E.A.( 2005)Teratology; 30 :61 – 69. 10- Ting,I.P.( 1982) Plant physiology ; Addison-Wesley Publishing company, P 314. 11- Ayers,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 , Marcel Dekker , Inc. P. 155 :17- 172. 13. Späth, E.; Galinovsky, F.and Mayer, M.( 1942)Ber 75 B, 805-13, . C.A. 37: 34368. 14. CHO, Y. D. and Martin, R. O.(1971) Canadian Journal of chemistry, 49:265-270. 15. Tsuda, Y. and Marion, L.(1964) Canadian Journal of chemistry 42:768. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1) 2010 16. Nurimov, E.and Lovkova, M. Ya.(1973) Prikl. Biokim. Mikrobiol. 9(5): 789-96. C. A. 80: 30635 d 17. Forostyan , yu.N. and Novikov , V.I .(1968) Zh . obshch . Khim . ; 38(6) :1222- 3. 18. Egon stahl (1969) Thin layer chromatography springer – 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.Silverstein, R.M. and Webster , F.X .(1996) Spectrometric identification of organic compounds ; John Wiley & sons , Inc. p. 103 – 109. Table (1): HRf value of standard and isolated anabasine Solvent system HRf standard HRf sample I 35 33 II 50 52 III 06 04 IV 83 80 V 34 37 Fig.(1): Mode of separation of fraction C by column chromatography Adsorbent : Silica gel GF254 Anabasine Fraction number 0 0. 2 0. 4 0. 6 0. 8 1 1. 2 Solvent system : CHC3-MeOH-NH4OH 60:10:1 IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL. 23 (1) 2010 Figure 1: UV spectrum of anabasine Fig(3): IR spectrum of anabasine Fig(2):