Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L DOI: https://doi.org/10.31351/vol30iss2pp78-85 78 Isolation of Alkaloids from Papaver rhoeas (Papaveraceae) Wildly Grown in Iraq Amenah Ayad Lafta*,1 and Maha N. Hamad* *Department of Pharmacognosy and Medicinal Plant, College of Pharmacy, University of Baghdad, Iraq Abstract The plant Papaver rhoeas ,which belongs to family Papaveraceae and known as common poppy is wildly grown in Iraq .It was used in traditional medicine in wide range of diseases including inflammation, diarrhea, sleep disorders, treatment of cough, analgesia, and also to reduce the withdrawal signs of opioid addiction. The project provide the first comprehensive research done in Iraq to study the phytochemical and the methods of extraction and separation of alkaloids from Papaver rhoeas wildly grown in Iraq .The plant was harvested in April 2019 from Zurbatiya is an Iraqi town located at the northeast of Waist province in Iraq.The collected plant was washed thoroughly, dries under shade, and grounding in a mechanical grinder to fine powder. The plant was extracted by hot extraction method using Methanol then fractionation was done to separate alkaloids from chloroform Fraction by TLC and PTLC .The alkaloids were isolated and purified by PTLC then subjected to various analytical techniques for alkaloids identification such as UV, LC mass and IR .The result was indicated of three alkaloids (dihydrocodien, chelidonine and papaverrubine C) in Papaver rhoeas plant. Keywords: Papaver rhoeas, Dihydrocodien, chelidonine, papaverrubine C. نبات الخشخاش المنثور البري في العراقمن القلويداتعزل *و مها نوري حمد 1*,آمنه اياد لفته العراق . ،بغداد ،جامعه بغداد ،كلية الصيدله ،*فرع العقاقير والنباتات الطبيه الخالصه نبات الخشخاش والمعروف بالخشخاش المنثورالذي ينمو بريا في العراق ويستخدم في الطب الشعبي على مدى واسع لمعالجة االلتهابات ت الموجوده ,االسهال ,مشاكل النوم ,معالجة السعال,مسكن اآلم وتقليل اعراض االدمان .يعتبر هذا البحث اول بحث شامل في العراق لدراسة االلكلويدا وتم 2019ي نبات الخشخاش البري في العراق وطرق استخالصها وفصلها . تم جمع النبات من قضاء زرباطيه في محافضة واسط في شهر شباط ف ويدات غسل وتجفيف النبات في الظل ثم طحنه بالمطحنه الميكانيكيه.تمت عملية االستخالص ب الطريقه الحاره ثم تمت عملية التجزئه لفصل االلكل الكتلي واالشعه والطيف السائل وكروماتوغرافيا التحضيرية الطبقه وكروماتوغرافيا الرقيقة الطبقه كروماتوغرافيابقه الكلوروفورم بواسطه من ط ( في نبات Cالكيميائيه وجود القلويدات )الدايهايدروكواديين والجليدونين وبابافروبين الكشوفات نتيجة وكانت تحت الحمراء واالشعه فوق البنفسجيه .الخشخاش .cبابافروبين ،الجليدونين ،الدايهيدروكودايين ،المفتاحية : الخشخاش المنثور الكلمات Introduction Poppy (Papaver rhoeas L.) figure 1 is a temperate native with a very wide distribution area, from Africa to temperate and tropical Asia andEurope(1). It grows in fields, beside roads, and on Grassland .Papaver rhoeas is a variable, erect annual, forming a long-lived soil seed bank that can germinate when the soil is disturbed. In the northern hemisphere it generally flowers in late spring (between May and October but if the weather is warm enough other flowers frequently appear at the beginning of autumn. It grows up to about 70 cm (28 in) in height 2).The stems hold single which are large and showy, 5–10 cm (2–4 in) across with four petals that are vivid red, most commonly with a black spot at their base(3).The plant has been used for medicinal proposes a long time ago for treatment of a wide range of diseases including inflammation, diarrhea, sleep disorders, treatment of cough, analgesia, and also to reduce the withdrawal signs of opioid addiction (4)Furthermore, it is known to claim intestinal and urinary irritation and to be useful in various conditions such as bronchitis, pneumonia, and rash(5). Pharmacological studies have shown that the plant extract may have some radical scavenging properties(6).Investigations also indicated that the Papaver rhoeas extract also possess properties of anti-ulcer genic (7), Anti- nociception (8), anti in flammatory effect (9) and Stress amelioration effect (10). This study was conducted for identification of alkaloids that extracted from Papaver rhoeas. 1Corresponding author E-mail: nnuna714@gmail.com Received: 1/11/2020 Accepted: 1/ 3/2021 Published Online First: 2021-12-09 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol30iss2pp78-85 Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 79 Figure 1. Iraqi Papaver rhoeas. Materials and Methods Collection of plant material The arial parts of Papaver rhoeas L. (papaveracea) are collected from Zurbatiya which is an Iraqi township in the north east of Wasit province in Iraq , a border crossing with Iran in April (2019) The plant authenticated by Dr.sukiana Abbas Alewi in College of Sciences, University of Baghdad. The Plant parts cleaned and dried in shade for two weeks then the dried plant material was coarsely powdered using electrical grinder and weighed. Extraction of alkaloids About 200gm of the dried plant material was defatted by maceration in hexane for 24 hours then filtered and the dried defatted plant was placed in a soxhlet, and a sufficient amount of 85% methanol (1 L) was added to the apparatus for 14 hours until complete exhaustion was achieved. The alcoholic extract was filtered by filter paper and the filtrate was evaporated to dryness using rotary evaporator to obtain 50gm dark-greenish residue. The residue was suspended in about 70ml of 6% HCl (pH 4), and partitioned with chloroform (70ml x 3). The upper aqueous acidic layer was separated and basified by ammonium hydroxide (23-25%) added gradually by a dropper in room temperature with stirring until getting pH 10 then the basified aqueous layer partioned three times with equal volume of chloroform in separatory funnel. The lower chloroform layer was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The steps of alkaloids extraction were shown in the following scheme. Figure 2. Steps of alkaloids extraction. Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 80 Identification, isolation and purification of alkaloids from Papaver rhoeas plant 1. Preliminary phytochemical screening of alkaloid compound for the methanolic plant extract using dragendroff and Mayer reagent 2. Thin –layer chromatography (TLC):- few milligrams from the extracted alkaloids was re suspended with 1 ml absolute methanol then applied on an analytical TLC plate pre coated with silica gel 0.25mm using the mobile phase: Cyclohexane: Chloroform: Diethyl amine (70:20:10)(12) . 3-Isolation and purification of alkaloid compounds by preperative thin layer chromotagraphy :- A readymade pre coated silica gel glass plate with 0.5mm thickness was placed in oven for 5 minute for activation At 100C then the plate was placed into glass jar contained 100 ml mobile phase then closed tightly, and left for saturation for about one hour far from sunlight and air current, After development, the plates were taken out of the jar then left at room temperature to dry then the bands were determined and scrubbed by needle under UV light using a wavelength of 254. 5- LC mass (Liquid chromatography–mass spectrometry):- The analytical LC-MS was performed using Agilent System Joined to an Applied Bio systems API 2000 mass spectrometer. 6- FT-IR (Fourier-transform infrared spectroscopy):-Fourier transform infrared spectroscopy is a technique for material analysis it offers an qualitative analysis of the sample. FTIR identified chemical bands in molecules, the range of scanning 4000-400 cm-1. 7- UV (Ultraviolet–visible spectroscopy):- Identification of isolated compound was done by measuring the absorbance by measuring their UV absorption at 240nm Results Preliminary Identification of Alkaloid n Papaver Rhoeas Plant by preperative thin layer chromatography Isolation and purification of alkaloids was carried out by using preparative TLC, in jar contains: Cyclohexane: Chloroform: Diethyl amine (70:20:10) as mobile phase, Figure 3. Preparative TLC chromatogram for Papaver rhoeas alkaloids on silica G F254 plate using mobile phase Cyclohexane: Chloroform: Diethyamine (70:20:10) under U.V light Identification of the isolated compounds by LC- Mass Technique Identification of compound A LC mass (Liquid chromatography–mass spectrometry) for compound A are shown in figure 4 below Figure 4. LC mass for compound A. https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 81 The molecular ion peak at m/z 304 [M]+ was nearly correspond to a molecular formula of dihydrocodeine (C18H23NO)which is 301 , also the abundance of peak 304 is (80538) which is the second highest one between other ions as as shown in figure 4 above. Depending on the analysis above, the expected chemical structure for The isolated compound A is demonstrated in figure 5 below Figure 5.Chemical structure of compound a (dihydrocodeine). The ʎ max spectrum and FTIR chart for compound A were shown in figure 6, 7 and table 1 Figure 6. The ʎ max spectrum for dihydrocodeine. The ʎ max spectrum for the extracted alkaloid was 211 nm which is a typical spectrum for dihydrocodeine alkaloid 211nm(13). Figure 7. IR spectrum for compound A Table 1 .The FTIR spectrum regions indicated the major functional groups in compound A IR band of compound A Interpretation 3308 ,2971 O-H starching of phenol and carboxylic. 2877 Asymmetric and Symmetric stretching of CH2 1378 O-H bending of phenol 1273 C-O-C stretching of ether 1066,1043 C-H bending of aromatic (in plane) 887,802,654 C-H and C=C bending of aromatic in and out and in-plane Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 82 Finally, the data obtained from IR, UV, LC/MS of the isolated compound A were identical with the Data of dihydrocodeine, which indicate that compound A could be Dihydrocodeine alkaloids(14). Identification of compound B The LC mass (Liquid chromatography– mass spectrometry) for compound A are shown in figure 8 Figure 8. LC mass for compound B. The molecular ion peak at m/z 370 [M]+ and 371 which represent M and M+H Respectively that correspond to a molecular formula of Papaverrubine C (Epiporphyroxine) 370 , also the abundance of peak 370 is (75756) which is the second highest peak between other ions . Depending on the analysis above, the expected chemical structure for The isolated compound is demonstrated in figure 11. Figure 9. chemical structure of compound B indicated Papaverrubine C (Epiporphyroxine). The ʎmax spectrum and FTIR chart for compound B were shown in figure 10, 11 and table 2 . Figure 10. The ʎmax spectrum for Papaverrubine C compound. The ʎmax spectrum for the extracted alkaloid was 232 and 285nm which is a typical spectrum for Papaverrubine alkaloid 285 nm(15). Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 83 . Figure 11. IR spectrum for compound B Table2 .The FTIR spectrum regions indicated the major functional groups in compound B IR band of compound B Interpretation 3280, 2971 O-H starching of phenol and carboxylic 2877 Asymmetric and Symmetric stretching of CH2 1648 C=C starching of alkene 1379 O-H bending of phenol 1068.1043 C-H bending of aromatic (in plane) 879,803,681 C-H and C=C bending of aromatic in and out and in- plane Finally, the data obtained from IR, UV, LC/MS of the isolated compound B were identical with the Data of papaverrubine C (Epiporphyroxine), which indicate that compound B could be papaverrubine C (Epiporphyroxine) which is Rhoeadines/ papaverrubines type of alkaloids(16,17). Identification of compound C The LC mass (Liquid chromatography– mass spectrometry) for compound C are shown in figure 12. Figure 12. Lc mass diagram for compound C. The molecular ion peak at m/z 353 [M] + and 354 which represent M and M+H Respectively that that correspond to a molecular formula of chelidonine (C20H19NO5, also the abundance of peak 353 is (65582) which is the highest peak between other ions .Depending on this analysis, the expected chemical structure for The isolated compound is demonstrated in figure 13 Figure 13. chemical structure of chelidonine. The ʎmax spectrum and FTIR chart for compound C were shown in figure 14, 15 and table 3. Iraqi J Pharm Sci, Vol.30(2) 2021 Papaver rhoeas L 84 Figure 14. The ʎ max spectrum for compound C The major absorption maxima are 201 which is the same that also observed for chelidonine at 204. Figure 15. IR spectrum of chelidonine. Table 3 .The FTIR spectrum regions indicated the major functional groups in compound C. IR band of compound C Interpretation 3318,2971 O-H starching of phenol and carboxylic 2878 Asymmetric and Symmetric stretching of CH2 1652 C=C starching of alkene 1378,1325 O-H bending of phenol 1273 C-O stretching of alkyl aryl ether 1086,1043 C-H bending of aromatic (in plane) 878,802,662 C-H and C=C bending of aromatic in and out and in- plane Finally, the data obtained from IR, UV, LC/MS of the isolated compound C were identical with the Data of chelidonine, which indicate that compound C could be chelidonine which is isoquinoline type of alkaloid. Conclusion Phytochemical investigation of wild Iraqi plant Papaver rhoeas was done to the whole plant and the results include the presence of different type of alkaloids[ Dihydrocodeine ( Morphinans ),papaverrubine C (Rhoeadines/ papaverrubines ), chelidonine (isoquinoline)] and these types detected by LC mass,UV,IR . 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