Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 26 Phytochemical Investigation of Alkaloids in the Iraqi Echinops heterophyllus (Compositae) Enas J. Khadim *,1 , Alaa A. Abdulrasool ** and Zainab J. Awad * * Department of Pharmacognosy, College of Pharmacy, University of Baghdad,Baghdad,Iraq. * * Chairman of Baghdad University, Baghdad,Iraq. Abstract Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms . They are a large family of compounds synthesized by plants in addition to the bacteria, fungi, and animals, they often have pharmacological effects. The aim of this study is to isolate and identified alkaloids in a newly studied, wild Iraqi plant named Echinops heterophyllus. The medicinal importance of alkaloids, on one hand and the absence of any phytochemical investigation on heterophyllus species of echinops genus on the other hand , acquired this study its importance. Three alkaloids (named E1, E2 and E3) were isolated from seed plant part by two chromatographic methods: Preparative high Performance Liquid Chromatography (PHPLC) and preparative thin layer chromatography (PTLC), one of them identified as(1-Methyl-2,3-dihydro-4(1H)-quinolinone by different chemical analysis like: ultra violet spectrum analysis (UV spectrum), Fourier transforms infrared spectra (FT-IR) , elemental microanalysis (CHN) and Proton 1 H-NMR and carbon 13 C-NMR analysis. Key words: Echinops, heterophyllus, quinoline alkaloids. دراسة كيميائية نهقهويدات في نبات شوك انجمم انعراقي ايناس جواد كاظم *،1 عبد انرسول عبد انحسين ، عالء ** زينب جهيم عواد و * * . انؼزاق ،بغذاد ،جايؼت بغذاد،كهٍت انصٍذنت فزع انؼقاقٍز ًاننباث انطبٍت ، .بغذاد ، انؼزاق ** رئٍش جايؼت بغذاد ، الخالصة انقهٌٌذاث ىً يجًٌػت ين انًزكباث انخً حصنغ ين قبم انكثٍز ين اننباحاث ًحهؼب دًرا ييًا فً يؼانجت ً حنظٍى انكثٍز ين االيزاض .انيذف ين ىذه انذراصت ىٌ انفصم ً انخؼزف ػهى انقهٌٌذاث انًٌجٌدة فً نباث ػزاقً بزي ًاصغ االنخشار اصًو Echinops . نى ٌذرس صابقا االىًٍت انطبٍت نهقهٌٌذاث ً ػذو ًجٌد ينشٌراث ػهًٍت حخناًل انًكٌناث انكًٍٍائٍت نيذه اننبخت اكضبج ىذه انذراصت اىًٍخيا .حى فصم ,M.P, FT.IR, CHN)ثالد قهٌٌذاث ين االجزاء انًخخهفت ين اننباث ً انخؼزف ػهى انخزكٍب انكًٍٍائً نٌاحذ ينيا بٌاصطت قٍاس H 1 NMR and C 13 NMR) : انقهويدات ، نبات شوك انجمم انكهمات انمفتاحية Introduction Echinops, a genus includes many plants which are individually referred to as globe thistle, is made up of more than 120 species of perennials, annuals, and biennials (1,2) . The genus belongs to the daisy family Asteraceae, and its species are found in Eastern and Southern Europe, Tropical and North Africa and Asia (3). In Iraq Ali Al-Rawi mention 11 species of Echinops in his book (4) among them E. heterophyllus P.H. Davis Family (Compositae) which was chosen for this study. This plant was wildly grown and widely distributed in Iraq specially in Erbil and Sulaimani. In Hanara village and surrounding area in Wadi Bastora and Shaklawa in Erbil governorate, the plant is called (Shakroka). The term (Shakroka) is come from that the circle-like part of the plant, before getting harderd in the late spring, is eaten and the taste is sweet, therefore, it is called shakroka: Shakr means sugar , Shakroka sweet like sugar (oral communication) . The Echinopus heterophyllus is a perennial, 40-100 cm high.Stems are simple or branching from the base, sparsely cobwebby- canescent. Leaves are lanceolate or oblong- lanceolate. (5) Echinops plant was reported to possess variety of compounds belonging to various classes like: alkaloids, flavonoids, terpenoids, lipids, steroids and polyacetylenes (6) . 1 Corresponding author E-mail:enassara@yahoo.com Received: 23/1/2013 Accepted: 5/2/2014 http://en.wikipedia.org/wiki/Chemical_compound http://en.wikipedia.org/wiki/Base_(chemistry) http://en.wikipedia.org/wiki/Nitrogen http://en.wikipedia.org/wiki/Bacteria http://en.wikipedia.org/wiki/Fungus http://en.wikipedia.org/wiki/Animal http://en.wikipedia.org/wiki/Pharmacology Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 27 And many literatures survey revealed different pharmacological activities of Echinops plant like, antibacterial activity (7) , Antifungal (8) , Antioxidant activity (9) , Protective effects on testosterone-induced prostatic hyperplasia (10) , Hepatoprotective (11) and antiulcerogenic activity (12) . All the alkaloids isolated from different species of Echinops related to the quinoline type so, the biosynthetic origin of quinoline alkaloids is the aromatic amine anthranilic (2- aminobenzoic) acid involved in the metabolism of the amino acid tryptophan. The skeleton of quinoline alkaloids constitutes a bicyclic system with a fused benzene and pyridine ring (figure 1). The attachment of a furan ring to the pyridine nucleus generates furoquinolines (e.g. furacridone), an important subgroup of quinoline alkaloids. The plant family Rutaceae represents the major source of quinoline alkaloids.Some of these naturally occurring quinolines have profound medicinal properties while others have served as lead structures and provided inspiration for the design of synthetic quinolines as useful drugs (13) . Figure 1: Basic structure of quinoline nucleus This study was emphasized on the isolation and identification of alkaloids found in the Iraqi species of Echinops plant by two methods : Preparative high Performance Liquid Chromatography chromatography (PHPLC) and preparative thin layer chromatography(PTLC) and compare between quantity of these active constituents obtained by both methods. Methods and Materials Plant material The whole plant of Echinops heterophyllus of the Family (Compositae) was collected from Nazali, 71Km north of Erbil . The plant was authenticated by Dr. Abdul-hussien Alkhait specialist in plant taxonomy . The plant seeds were collected during the month of November (2011), while aerial parts (leaves/stem) and roots were collected during the months of May and June (flowering time) and were cleaned, dried at room temperature in the shade then pulverized by mechanical mills and weighed. Thin layer chromatography was done on readymade plates of silica gel GF254nm (20x20cm) of 0.25mm thickness (MERCK), using three different developing solvent systems: S1 = Benzene : methanol: (8 : 2) (14) S2 = Chloroform: acetone: diethylamine (5 : 4 :1) (14) . S3 = Toluene: ethylacetate: diethylamine (70 : 20 : 10) (15) and detected by dragendorffs spraying reagent Preparative HPLC was done using :- acetonitrile : water (65:35) as a mobile phase Column: mediterranea C18 , 5 µm 15 X 2.12 cm, flow rate: 5 ml / min Injection volume: 1 ml. Detection: UV. Detector at λ 205 nm. Experimental work The experimental work is divided into : 1- Preliminary phytochemical screening of various secondary metabolites like alkaloids, flavonoids, steroids, tannins, saponins, anthraquinioin, terpenoids and cardiac glycosides) in the different parts of Echinops plant. 2- Extraction of alkaloids. 3- Isolation and purification of alkaloids. 4- Identification and characterization of the isolated compounds. Preliminary qualitative phytochemical analysis: Chemical tests were carried out using the ethanolic extracts from plants and or the powdered specimens, using standard procedures to identify the active constituents. (16-18) Test for alkaloids : Wagner’s reagents & Mayer’s Test for flavonoids : Lead acetate test & NaOH test Tests for steroids: Liebermann-Burchard test Test for tannins : FeCl3 solution test Test of saponins: foam test Tests for anthraquinones: Borntrager’s test Test for terpenoids NaOH test Test for cardiac glycoside: Keller-kiliani test Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 28 Extraction method Powdered plant material Hexane Hexane extract Defatted plant material (Fat) 80% Ethanol in the soxhlet Exhausted plant material Alcoholic extract (crude extract) 5% HCl / ethylacetate Ethylacetate layer Aqueous layer (Neutral and acids) (Alkaloids and water soluble) 5%NaOH/ CHCl3 Aqueous OH - layer (F-2) CHCl3 Layer (Water soluble) (F-1) Alkaloids Figure:2 General scheme for separation of different plant constituents (17) Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 29 Results and Discussion Preliminary qualitative phytochemical analysis: The results of phytochemical screening are given in table (1).The results of preliminary phytochemical screening of plant extracts showed the presence of alkaloids, flavonoids, steroids, tannins and terpenoids in different parts of Iraqi species in different percentage, and the absence of, saponins, anthraquinoin and cardiac glycosides in all plant parts. These results can be compared with phytochemical screening of other Echinops species for example,: the aerial part of Iraqi heterophyllus species was contained traces amount of alkaloids, unlike Egyptian species E. spinosissimus, its aerial parts was contained about 11.3% alkaloids (19) , also quinoline alkaloids and flavonoids found in the aerial part of Indian E. echinatus with the presence of tannins in the root parts only (20) . Many researchers reported that the concentration of secondary metabolites are varying from plant to plant belong to the same genus and even in the different parts of the same plant (21) , this is due to many factors like environmental heterogeneity, since the effect of environmental heterogeneity is highly scale- dependent. It may create high niche diversity and hence allow species to coexist at a large spatial scale (22) , also the high complexity and heterogeneity of soil, like( soil structure, texture and depth, moisture retention characteristics, aeration) create a big variation in the chemical constituents even in the same country (23) , good example seen in two Iraqi species of Echinops plant : E. tenuisectus and E. heterophyllus, phytochemical analysis of E. tenuisectus revealed the presence of high percentage of silymarine in the seeds (0.878%) and aerial parts (0.095) (24) with the absence of this compound in the heterophyllus species. Table(1) Phytochemical screening of different parts of Echinops heterophyllus Plant part Al- kaloids Flavo- noids Stero- ids Tann- ins Sap- onin Anthra- quinoin Terpen- oids Cardiac glycoside Seeds + + - - - - + - Aerial part Traces + + + - - + - Roots + + + + - - + - +, - represent presence and absence of phytoconstituents respectively. Preliminary identification of different Echinops parts by TLC Thin layer chromatography of fraction 1 (F- 1) obtained from different parts of the Echinops, confirms the following: (a)The presence of three different alkaloids in fraction-1 (named E1, E2 and E3) which is obtained from seeds part and two alkaloids in the same fraction obtained from roots part (E1 and E2) with very traces one compound (E1) in the alkaloidal fraction of aerial plant . figure-3The Rf values of these compounds in the different solvent systems were calculated, table(2). Table (2) Rf values of alkaloids obtained from different plant parts in different developing solvent systems in TLC. Compound Plant part S1 S2 S3 E1 Seed 0.16 .22 0.25 E2 Seed 0.58 0.68 0.66 E3 Seed 0.75 0.8 0.79 E1 Root 0.17 0.25 0.26 E2 Root 0.6 0.7 0.67 E1 Aerial part 0.15 0.21 0.25 R S A Figure 3: TLC of fraction one (F-1) for different Echinops parts. ( roots, seeds, aerial parts) using silica gel GF254nm as adsorbent and S1 as a mobile phase. Detection by dragendorffs spraying reagent R : Roots S : Seeds A : Aerial part Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 30 Isolation and purification of alkaloids: Two chromatographic analysis were carried out to isolate in a pure form three alkaloids (named E1, E2, E3) found in the plant which are: Preparative HPLC and preparative TLC, since seeds contain the largest number and highest quantity of the alkaloids so alkaloids fraction obtained from seeds part was used to separate and isolate these compounds in a pure form. Isolation and purification of alkaloids by preparative HPLC One gram (1 gm) of F-1 obtained from plant seeds dissolved in a minimum quantity of chloroform was injected in to preparative HPLC Figure 4: Preparative HPLC analysis of fraction-1 obtained from seeds plant observing three peaks represent three different compounds, one of them (E2) is a major one. Three samples obtained from preparative HPLC were weighted and subjected to co-TLC .Weight of E1 = 0.07 gm , weight of E2 = 0.5 gm, weight of E3 = 0.16 g Isolation and purification of alkaloids by preparative TLC On a 20cm x 20cm glass plates a slurry of 75 gm of silica gel GF 254 suspended in 150 ml of distilled water was applied in 1mm thickness manually by using Jobling laboratory division plate coater. The freshly coated plates were left until the transparency of the layer disappears. After 10 minutes, the plates stacked in a dry rack and heated in vertical position for 1 hour at 110 o C with occasional opening of the oven door from time to time in order to allow moisture escape. The completely dried and activated plates were kept in a dry and moisture free container containing adsorbent silica gel One gram (1 gm) of F-1 obtained from plant seeds (highest quantity) dissolved in a minimum quantity of chloroform and applied on a number of preparative TLC plates using S1a solvent system. The solvent was allowed to rise to a height of 15cm from the base line. One major and two minor bands were observed after spraying a side of plates with dragendorffs three band had been scrapped off, eluted with chloroform, then filtered. The filtrate evaporated to dryness, in vacuo to give white crystals, upon re-crystallization out of boiling ethylacetate, a fluffy white crystals of E1, E2 and E3 were obtained. Three samples obtained from preparative TLC were weighted and subjected to co-TLC . Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 31 Weight of E1 = 0.037 gm , weight of E2 = 0.327 gm, weight of E3 = 0. 063 gm From the above results, the quantity of compounds obtained in a (pure form) by Preparative HPLC is higher than that obtained by preparative TLC. Classical preparative TLC suffers from several drawbacks, the main disadvantage being the removal of purified substance from the plate and its subsequent extraction from the sorbent, other drawbacks include the length of time required for the separation and degree of purity for the separated compounds (25) , compare with preparative HPLC, which is consider know, the most powerful and versatile method for purification tasks in the pharmaceutical industry (26) . Despite the fact that among the tools used in the large scale purification of pharmaceuticals, Preparative HPLC is one of the more expensive and solvent-consuming approaches, it yields the highest-purity drug substance. The interest in preparative HPLC will continue to grow because of the increasing uncertainty in the market expectations for product purity. Its nearly linear scalability makes preparative HPLC one of the more viable approaches to compound purification (27) . Characterization and Identific -Ation of The Isolated Alkaloids (E2): 1- Melting point:- The isolated compound which is named E2 had a sharp melting point of 160-162 ه C 2- Ultra violate spectra: The isolated alkaloid ( E2 ) show UV absorption near 242nm 3- FT-IR spectra: The identification of the unknown alkaloid (E2 ) was further confirmed by using FT-IR spectroscopy figure (5) . The characteristic IR absorption bands showed by this compound are listed in table(3). Table (3 )Characteristic IR absorption bands( in cm -1 ) of the isolated alkaloids (28) Functional group Group frequency wave number ( in cm -1 ) Assignment N C-H 3306, 3245 2910-2852 N–H stretch (two band for tertiary amine Asymmetric and symmetric stretching of CH3 C=O 1590-1750 C=O stretching vibration (conjugated) C-N 1333,1336 C-N stretching bands of tertiary amine CH3 1430,1480 C-H bending vibration C-H 914, 868, 750 C-H of aromatic group out of plane 4- Elemental micro analysis (CHN): Elemental microanalysis was performed for unknown isolated compounds ( E2) to confirm their chemical structure. The result of this analysis (table 5) showed that the unknown compounds consist of carbon , hydrogen, oxygen and nitrogen in different percentage. Table(4) Elemental microanalysis of the unknown isolated alkaloid Name C% H% O% N% E2 74.07 6.208 10.25 9.463 5- H 1 and C 13 NMR The E2 compounds presented 13 C NMR spectra (DMSO, 75 MHz): with chemical shifts typical of quinoline rings (28) in the ranges of δC 21.12 (C-2), 24.77 (C-3), 170.12(C- 4),126.987 (C-5), 121.825 (C-6), 127.640 (C- 7), 114.951 (C-8), 138.26 (C-9), 123.47 (C- 10), 30.4 (C-11). 1 H NMR (DMSO-d6-, 300 MHz) revealed that E2 compound undergo tautomerism which lead to the appearance of chemical shifts of the hydroxyl group at 10.02 at (C-4), 2.4 (3H, as a singlet of the methyl protons ), 2.6 (2H, d, H- 2),5.09 (1H,s, H-3), 6.84-7.15 (4H, m, H-5 ,H- 6 , H-7 , H-8). Figure (7). Depending on the above results, the expected chemical structure for the isolated E2 compound is: N O CH3 1 2 3 4 5 6 7 8 9 10 11 1-Methyl-2,3-dihydro-4(1H)-quinolinone, It is a new compound isolated (for the first time) from Iraqi Echinops heterophyllus plant, it seen to be the hydrogenated form of echinopsine (1-Methyl-4(1H)-quinolinone), an alkaloid isolated from 14 species of Echinops plant. Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 32 Figure 5: FT-IR spectrum of the isolated alkaloid (E2) Figure 6 : 13 C-NMR analysis of the isolated E2 compound Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 33 Figure 7 : 1 H-NMR analysis of the isolated E2 compound. Conclusion Phytochemical investigation of a new wild Iraqi plant used traditionally for wound healing and snake bit named Echinops heterophyllus was done and the results revealed the presence of alkaloids, flavonoids, terpenoids, tannins and steroids in the different plant parts and in a different percentages, aerial parts contain the highest quantity of flavonoids, while seeds contain the highest amount of alkaloids. General schematic procedure of Jeffirey B. Harborne was used to extract different plant parts using 80% ethanol in soxhlet apparatus.Two chromatographic analysis were carried out to isolate in a pure form three alkaloids from seeds part (which contain highest quantity) : preparative HPLC and preparative TLC, where the quantity of compounds obtained by preparative HPLC was higher than that isolated by preparative TLC. The benefit of preparative HPLC to isolate the maximum amount of desirable products at a desired purity in a minimum of time from different Iraqi medicinal plants to use it as a standard reference or as lead structures for the design of useful drugs in the future studies .Preparative HPLC can be used in pharmaceutical development for troubleshooting purposes or as part of a systematic scale-up process. References 1. Bobrov E.G.: Echinops L. Flora of the USSR . Volume 27, Shishkin BK and Bobrov EG (eds.), Dehra Dun: Bishen Singh, Mahendra Pal Singh and Koelz Scientific Books 1997;p.p 1-70. 2. Susanna A., Garcia-Jacas N.: The tribe Cardueae In: Kadereit J & Kubitzki K (eds.). :Compositae: The Families and Genera of Vascular Plants. Heidelberg: Springer-Verlag 2007; 135-58. 3. Garnatje T., Valles J., Garcia S., Hidalgo O., Sanz M., Canela MA., Siljak-Yakovlev S.: Genome size in Echinops L. and related genera (Asteraceae, Cardueae): karyological, ecological and phylogenetic implications. Biol Cell 2004; 96(2): 117- 24. 4. Al-Rawi A. :Wild plants of Iraq with their distribution. Volume 14,1964, 114p. 5. Rechinger K.H. : Flora Iranica Compositae ІІІ-Cynareae Akademische Druck- u . Verlagsanstalt Graz-Austria No. 139a, 1979. 6. Shukla Y.N.: Chemical, botanical and pharmacological studies on the genus Echinops: Review. J Medic Aromat Plant Sci 2003; 25(3):720-32 . 7. Abdel Rahman S.M., Abd-Ellatif S.A., Deraz S.F., Khalil A.A. Antibacterial activity of some wild medicinal plants collected from wester Mediterranean coast, Egypt: Natural alternatives for infectious disease treatment. Afr J Biotechnol 2011; 10(52):10733-43. 8. Toroğlu S., Keskin D., Vural C., Kertmen M., Çenet M. : Comparison of antimicrobial activity of Echinops viscosus Iraqi J Pharm Sci, Vol.23(1) 2014 Alkaloids of Iraqi Echinops 34 Subsp. Bithynicus and E. microcephalus leaves and flowers extracts from Turkey. Int J Agric Biol 2012; 14(4):637-40. 9. Sharma H., Parihar L., Parihar P.: Review on cancer and anticancerous properties of some medicinal plants. J Med Plant Res 2011; 5(10): 1818-35. 10. Agrawal M., Nahata A., Dixit K.:Protective effects of Echinops echinatus testosterone-induced prostatic hyperplasia in rats. Eu J I M 2012; 4(2):177-85. 11. Abdulrazzaq H.M., Kadeem J.E. , Al- Mohannadi S.S. : Hepatoprotective effect of Echinops tenuisectus (Compositae) on CCl4 induced hepatic damage in rats. Iraqi J Pharm Sci 2008; 17 (1):16-24. 12. Rad A., Najafzadeh H. , Farajzadeh A.: Evaluation of anti-ulcer activity of Echinops persicus on experimental gastric ulcer models in rats. Veterinary Research Forum 2010; 1(3):188 -91. 13. Suarez C., Barrera C., Caballero A. : Quinolone alkaloids and friedelane- ype triterpenes isolated from leaves and wood of Esenbeckia alata kunt Rutaceae. Quim. Nova 2011;34(6): 984-86. 14. Stal E. Thin layer chromatography hand book, 1999. 15. Wagner H. and Bladt S. Plant drug analysis, A thin layer chromatography atlas. (2 nd ed.). Springer-Velag, Berlin, 1996. 16. Evans W. C.:Trease and Evans Pharmacognosy (16 th ed.); Elsevier: Science limited, UK, 2009, pp. 353-56 17. J.B. Harborne, Phytochemical Methods, a guide to modern techniques of plant analysis, chapman and hall, new York, 1973 18. Satyajit D. Sarker, Zahid latif, Alexander I. Gray Natural products isolation second edition 2006 humana Press Inc. 19. Kuete V. , Wiench B., Heqazy M.E., Mohamed T.A., Fankam A.G., Shahat A.A., Efferth T.: Antibacterial activity and cytotoxicity of selected Egyptian medicinal plants. Planta Med 2012; 78(2):193–9. 20. Amish J., Natvarlal M., Amit A., Jitendra P. , Sohan P.: Comparative analgesic activity of root and aerial part methanolic extracts of Echinops echinatus Roxb. IJPI 2011; 1(4): 23-9. 21. Abdul K. K., Palwasha A., Ayeesha M., Safdar Ali K. , Rasool B.T.: Response of plant parts and age on the distribution of secondary Metabolites on plants found in Quetta. Pak J Bot 2009; 41(5): 2129-35. 22. Pausas J. G.1 , Austin M.: Patterns of plant species richness in relation to different environments: An appraisal. Journal of Vegetation Science 2001; 12: 153-166. 23. Karlovsky P.: Secondary Metabolites in Soil Ecology. Volume 14,1 st ed., Springer- Verlag Berlin Heidelberg , 2008, 293p. 24. Al-Mohannadi S.S., Kadeem J.E. ,: Identification of silymarin in Echinops tenuisectus Family Compositae. J Biotech Res Cent 2007; 1 25. Hostettmann K., Marston A., Hostettmann M.: Preparative Chromatography Techniques. Application in Natural Product Isolation. 2 nd ed., Springer, 1997, 255p. 26. Brandt A., Kueppers S., Majors R.: Practical aspects of preparative HPLC in pharmaceutical and development production. LC.GC Europe 2002. 27. Huber U.: Solutions for preparative HPLC – Application Compendium, Agilent Technologies Application 2006. 28. Silversteine R.M and Webster F.X.: Spectrometric identification of organic compounds (6 th ed.). John Wiley and Sons Inc., USA, 1998. http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/S1876382012000066 http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/S1876382012000066 http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/S1876382012000066