Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum DOI: https://doi.org/10.31351/vol30iss2pp158-166 158 Extraction and Identification of Phenolic Compounds from the Iraqi Heliotropium europaeum L. plant Walaa H. Jasim *,1 and Maha N. Hamad* **Department of Pharmacognosy and Medicinal Plants, College of pharmacy, University of Baghdad, Baghdad, Iraq. Abstract The Heliotropium europaeum L. (Boraginaceae) are well-known to contain toxic pyrrolizidine alkaloids in addition to other secondary metabolites. Its spread in the Mediterranean area, southern Europe, Central Asia, and Iraq. The present study included extraction of the aerial parts of Iraqi Heliotropium europaeum with methanol (soxhlet apparatus), fractionation, screening the active constituent, and identification by chromatographic techniques. The extract was suspended in distilled water and partitioned with chloroform, ethyl acetate, and n- butanol. The hydrolysis step was done for the two fractions (n-butanol and ethyl acetate). Phytochemical screening and identification of bioactive substances of the plant was done for the two fractions. A qualitative and quantitative analysis of the fractions was carried using a high -performance liquid chromatography technique and twenty–one reference standards. The outcomes of this study were the identifications of new six phenolic compounds (kaempferol (1), Silybin (2), caffeic acid (3), Genistein (4), Apigenin (5), in addition to syringic acid (6)) from Heliotropium europaeum ethyl acetate fraction. Key words: Heliotropium europaeum, soxhelt, Genistein, syringic acid, High -performance liquid chromatography. من النبات العراقي حشيشة العقربالمركبات الفينولية وتشخيص فصل * مها نوري حمد و 1*،والء حامد جاسم .،العراق بغداد ، بغداد جامعة ، الصيدلة ،كلية الطبية والنباتات العقاقير فرع* الخالصة على قلويدات بيروليزيدين السامة باإلضافة إلى نواتج معروف باحتوائه (Boraginaceae) نبات حشيشة العقرب أو التنووم من عائلة الدراسة الحالية على استخالص تتضمن وجنوب أوروبا وآسيا الوسطى والعراق. األيض الثانوية األخرى. ينتشر في منطقة البحر األبيض المتوسط التجزئة ، وفحص المكون النشط ، والتعرف على التقنيات تم اجراء عملية ، و(وكسلتجهاز ساألجزاء الهوائية من الهليوتروبيوم العراقي بالميثانول ) بيوتانول. تم إجراء خطوة التحلل المائي -ن الكروماتوجرافية. تم تعليق المستخلص في ماء مقطر وتم تقسيمه باستخدام الكلوروفورم ، أسيتات اإليثيل ين. تم إجراء تحليل نوعي جزئاء فحص كيميائي نباتي وتحديد المواد النشطة بيولوجًيا في النبات للاإليثيل(. تم إجر اسيتاتبيوتانول و-ن) جزئينلل باستخدام تقنية كروماتوغرافيا سائلة عالية األداء وواحد وعشرون معياًرا مرجعيًا. كانت نتائج هذه الدراسة هي تحديد ستة مركبات الجزاءوكمي ل (( من جزء أسيتات 6، باإلضافة إلى حمض الحقن )أبجنن (5)، جنستين (4)( ، 3، حمض الكافيك ) (2)ليبينس، (1) كامفيرولفينولية جديدة ) .لنبات حشيشة العقرب اإليثيل السائل عالية االداء . كروماتوجرافيا , جينيستين ، حمض الحقن,سوكسيلت ,هيليوتروبيوم يوروبيوم الكلمات المفتاحية: Introduction Large number of plants of Heliotropium genus belong to Boraginaceae family which is biggest family of plant that includes around 250-300 species in the world. These plant species are widespread in temperate and tropical parts of both hemispheres (12) Figure 1. The name “heliotrope” is taken from the fact that these plants move their leaves toward the sun (3,4). The phytochemical studies about Heliotropium europaeum are very limited and majority of these studies are concentrated on pyrrolizidine alkaloids (PAs) (5,6). The toxicity of this type of alkaloids are concentrated usually in the flowering parts of the plants and in the seeds of the Boraginaceae (all genera) (7). Some Pyrrolizidine alkaloids-bearing plants are used as ornamental plants, ground cover, soil improvers, and can contaminate feeds of animal (8). Risks of exposure to Pyrrolizidine alkaloids, in the large countries, originate from so called herbal remedies, folk medicines and herbal teas (7). Pyrrolizidine alkaloids (PAs) are produce by plants and regarded as one of the widespread toxins of natural origin (9, 10). They are esters produced from esterification of various amino alcohol bases called necine and mono or dicarboxylic acids (11). Toxicological studies displayed the reason behind such toxicity of H. europaeum is the alkylation that take place by some biological nucleophiles such as nucleic acids (enzymes, DNA and RNA) and proteins via 1,2-unsaturated PAs metabolites (12,13). The importance of this plant lead to screen the chemical constituents especially the phenolic compounds. 1Corresponding author E-mail: Walaa.Hamid@yahoo.com Received: 30/1/2020 Accepted: 9/5 /2021 Published Online First: 2021-12-11 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol30iss2pp158-166 Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 159 Figure 1. The Iraqi Heliotropium europaeum Materials and Methods Plant materials The aerial parts of Heliotropium europaeum L. were collected from the bank of Tigris river in the periphery of Tikrit city in August 2019. The collected plant purified carefully then identified, authenticated by Dr. Khansaa Rasheed at Iraq natural history research center and museum plant and environment department / University of Baghdad. The aerial parts were cleaned, dried in shade, and pulverized in a mechanical grinder to a fine powder Extraction and fractionation of the different active constituents 100 g of powdered plant was first defatted with hexane (250 mL) for 24 hr. at room temperature (about 25 ℃) then filtered. The residual plant was dried on air and packed in the thimble of soxhlet apparatus. The extraction was carried with 750 mL of methanol till exhaustion. The extract was filtered and the solvent was evaporated by applying a reduced pressure by a rotary evaporator. A dark greenish residue was obtained and then suspended in 150 mL distilled water and partitioned with three solvent chloroform, ethyl acetate, and n-butanol (3x150 mL) for each solvent. The chloroform and ethyl acetate fraction were dried with sodium sulfate (anhydrous) then filtered and evaporated to dryness. Hydrolysis of Ethyl acetate and n-Butanol fractions The hydrolysis step was done for the two fractions by reflux for 10 hr. using 5% HCl (200 mL). The cooled solution was partitioned by extraction with ethyl acetate (3x200 mL). The layers were combined, dried and filtered. The identification of n-butanol and ethyl acetate fractions were carried out by thin layer chromatography (TLC) and HPLC technique. Identification of phenolic compounds in Heliotropium europaeum plant extract 1. Preliminary phytochemical analysis by chemical tests Preliminary qualitative phytochemical analysis for the screening and identification of some important chemical constituents of Heliotropium europaeum plant under study was carried out on crude extract, and fractions using the standard procedures as previous reported (14) . Test for phenolic compound: The plant extract (or fractions) (500 mg) was dissolved in 80% methanol (20mL) and filtered. The filtrate was used for the following tests: (a) NaOH test: The alkaline reagent test for flavonoids was performed by placing 0.5 mg of the crude alcoholic extract in a test tube followed by addition of diluted solution of 10% sodium hydroxide drop wise. The dissolved flavonoids formed an intense yellow color due to formation of sodium phenolate salt as precipitate. This color was changed to faint yellow upon the addition of diluted solution of hydrochloric acid HCl. It was noted that any further addition of diluted solution of HCl to the resulted solution, turns the solution to colorless that indicate the flavonoids presence (15, 16). (b) Ferric chloride test: the plant extract (25 mg) dissolves in distilled water (10mL) and filter. Aqueous ferric chloride (1% FeCl3) solution was added to the filtrate. The appearance of intense green, black or blue color indicates the of phenolic acid presence in the sample due to formation of ferric phenolate salt as precipitate. 2. Thin layer chromatography Using readymade plate aluminum coated TLC sheet G/F254, 0.20 mm stationary phase used was silica gel, using UV light for detection of the spot. Different mobile phases were tried for the detection of plant constituents (phenolic compounds) and it was used for the identifying of phenolic compounds of Heliotropium europaeum L that found in ethyl acetate and n-butanol fractions of the plant. The P1 mobile phase (CHCl3: acetone: formic acid (75:16.5:8.5) (17) proved to be the best for separation of the fractions, and it was adopted for separation. After drying the developed plates were sprayed with 5% ethanolic KOH. 3. The HPLC technique The HPLC conditions for analyzed fractions HPLC system instruments were supplied by Knauer, Germany. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 160 Table 1. HPLC System components. Component Model or version Company and origin 1 Binary high- pressure gradient pump P6.1L Knauer, Germany 2 Diode array detector DAD 2.1L Knuaer, Germany 3 Sample loop (20 µl) and injector D1357 Knuaer, Germany 4 Analyses and system control software Claritychrom, V 7.4.2.107 Dataapex, Czech Republic In this method a C18 column was used for separation with internal diameter of (250 - 4.6 mm). The particle size is 5 µm with a pore size of 80 Å (18) . 1% aqueous acetic acid, solvent A, and acetonitrile, Solvent B, were the mobile phases. Moreover 1 ml/min was the flow rate. The column was thermostatically controlled at 28℃ and the injection volume was fixed at 20 μL. The technique was employed was gradient elution with continuous change of B solvent proportionally to A solvent. Table (2). The instrument is equipped with photo diode UV detector at the following wave lengths (272, 280 and 310 nm) in order to scanned the resulted chromatogram. Table 2.The gradient program used in separation by HPLC system. Time (min) Mobile A (%) Mobile B (%) Flow rate ml/min 0 90 10 1ml/min 28 60 40 1ml/min 39 40 60 1ml/min 60 10 90 1ml/min The detection of each metabolite was performed by matching retention time and absorbance spectrum of the standards. For quantitative analysis the concentration components were calculated by calibration curve in which serial concentrations of reference standards was plotted against their equivalent peak area, the calculation was done by using the straight line equation Y= aX +b, slop = y/x (Y and X: are holding the place of coordinates (x, y) of any point that lies on the line, a: slop, b: y- intercept), where R2: regression factor. Twenty-one standards were used in this study and they were: Quercetin, Apgenin, Luteolin, Chlorogenic acid, Kaempferol, Caffiec acid, Genistein, Ferullic acid, Cinnamic acid, Silybin, Oleuropein, Caffeine, Myricetin, Salicylicacid, Sinapicacids, Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, Daidzein, Syringic acid, Gallic acid. Preparation of stock solutions for standards compounds and the examined samples for HPLC Stock solutions used in HPLC analysis were prepared from 0.04 mg of each standard in 1mL methanol (HPLC grade). The operator data were obtained from the database of the instrument. The examine samples were subjected to ultra- sonication (Baransonsonifier, USA) at 60% duty cycle at 25˚C for 30 minutes. The resulted suspension was centrifuge (at 7500rpm) 20 min. The clear upper layer of sample solution was separated then evaporated under vacuum. The residue was dissolved in 1mL methanol using vortex mixer followed by filtration through disposable filter (2.5 μm). The clear solution temperature adjusted to 4˚C. The used volume for injection of the sample was 20 µL. Results and Discussion In spite of the importance of H. europaeum as a toxic plant, the phytochemical studies concentrated on the alkaloids secondary metabolites especially pyrrolizidine type. Therefore, the chosen of phenolic compounds in this study is the main target because they have different pharmacological activities. In this study, hot extraction method was done by absolute methanol to extract the active constituent depend on the nature of these active constituents. The hydrolysis process was done to remove the glycoside moiety from the compounds by cleavage the ether linkage (glyosidic bond) producing the free a glycon. The screening of these sample revealed that they contain various phytochemical constituents by using p1 solvent system (Chloroform: acetone formic acid) (75:16.5:8.5) which is best mixture of solvents used in the seperation of phenolic compound from fraction and 254 nm and 366 nm UV light. Identification of phenolic compounds in Heliotropium europaeum plant extract 1- Preliminary phytochemical investigation like chemical tests were carried on the H. europaeum aerial parts and showed the following results as shown in Table 3 . Table 3. Qualitative analysis of phytochemical constituents in crude extract of Heliotropium europaeum L. Name of test NaOH test Ferric chloride test Fraction Ethyl acetate +ve +ve n-butanol +ve +ve 2- Preparative Thin Layer Chromatography analysis for the ethyl acetate and n- butanol extract. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 161 Preparative Thin Layer Chromatography technique was used for separation of small amount constituent by using the same separation condition that was followed previously in ordinary TLC technique. The results of preparative TLC were more than one bands in each fraction which represent that the sample contain many of chemical constituents which can be separated easily. Figure (2-3). Figure 2. Preparative Thin layer chromatography on silica gel GF254 for fractions (1) Fraction of ethyl acetate before hydrolysis (2) fraction of n-butanol before hydrolysis. Using solvent system P1 (CHCl3:CH3OCH3: HCOOH) in a ratio (75:16.5:8.5). The detection under UV light 365 and 254 nm, P1 which is best mixture of solvents used in the separation of phenolic compound from fractions. Figure 3. preparative Thin layer chromatography on silica gel GF254 for fractions (1) Fraction of ethyl acetate after hydrolysis (2) fraction of n-butanol after hydrolysis. Using solvent system P1(CHCl3:CH3OCH3: HCOOH) in a ratio (75:16.5:8.5). The detection under UV light 365 and 254 nm. High-performance liquid chromatography (HPLC) HPLC technique can provide a lot of information about the content of the extract. It is also used for qualitative identification of extract constituents by making a comparison of their retention time and the shape of the UV spectrum of the detected compound by the detector of the instrument with that for authenticated reference standards at identical chromatographic conditions. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 162 In this study, the sample was analyzed by this technique, ethyl acetate fraction after hydrolysis (ETA). The resulted chromatogram from fraction was compared with twenty-one standards at the same condition. Figure (4) shows the HPLC chromatogram of ethyl acetate fraction (ETA) matched with six of the used standards. Figure 4. HPLC chromatogram for ethyl acetate fraction (ETA) matched with six detected standards chromatograms. The matching between the spectra of the detected compounds in ethyl acetate fraction with corresponding standards spectra as shown in Figures (5-10) respectively. These figures show an excellent fitness between the detected compounds and the six standards. These results concluded that Heliotropium plant ethyl acetate might contains syringic acid, silybin, kaempferol, apigenin, caffiec acid and genistein. Figure 5. UV spectrum of fraction of ethyl acetate matched with syringic acid standard UV spectrum. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 163 Figure 6. UV spectrum of fraction of ethyl acetate matched with silybin standard UV spectrum. Figure 7. UV spectrum of fraction of ethyl acetate matched with kaempferol standard UV spectrum. Figure 8. UV spectrum of fraction of ethyl acetate matched with genistein standard UV spectrum. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 164 Figure 9. UV spectrum of ethyl acetate fraction matched with caffiec acid standard UV spectrum. Figure 10. UV spectrum of fraction of ethyl acetate matched with apigenin standard UV spectrum. It is important to refer that the ethyl acetate fraction chromatogram has at least three significant compounds did not match with the used standards. This reflects the need to extend study by using additional standard. Because in HPLC technique, the area under the peak of a certain compound is proportional to its concentration therefore calibration curves of the six matched standards were constructed for quantitive analysis and the peak areas of the detected compounds in fraction of ethyl acetate were used to determine the concentration of each one in the fraction, according to the first line equation Y= aX +b, slop = Y (area under the curve)/X (concentration mg/mL). The quantitative concentration of the six phenolic compounds in ethyl acetate fraction revealed that kaempferol has the highest concentration while genistein concentration was the least.as shown in Table 4. Iraqi J Pharm Sci, Vol.30(2) 2021 Heliotropium europaeum 165 Table 4. The HPLC Quantitative analysis for Ethyl acetate fraction. Ethyl acetate fraction peak area (y) µg/ml (x) mg/ml Kaempfero l 5327.83 5 111.39857 0.11039 9 Silybin 3695.17 64.995163 0.06499 5 caffeic acid 1977.47 2 65.970708 9 0.06597 1 Genistein 1209.13 1 6.2299365 2 0.00623 Apigenin 446.711 19.853822 2 0.01985 4 syringic acid 7812.71 2 89.880837 1 0.08988 1 From the above finding, Heliotropium europaeum is a promising plant as it contains different secondary metabolites especially phenolic compounds that was detected novelty in this study Conclusion Little attention was reported about the toxic H. europaeum plant and its secondary metabolites especially its phenolic compounds. Ethyl acetate and n- butanol fractions of this plant were prepared and their phenolic compounds were studied. The technique used was high performance liquid chromatography equipped with UV-vis facility and group of standard phenolic compounds. Six phenolic compounds were isolated and identified for the first time from ethyl acetate extract of this plant. The isolated compounds (syringic acid, silybin, kaempferol, apigenin, caffiec acid and genistein) were evaluated qualitatively and quantitatively. The study revealed that kaempferol has the highest concentration while genistein concentration was the least. The results obtained from H. europaeum study provide a good scientific base to examine the pharmacological effects of this plant in the future. References 1. The Wealth of India. National Institute of Science Communication. Vol. 10. New Delhi: CSIR Publication; 1985. p. 28. 2. Rastogi RP, Mehrotra BN. Compendium of Indian Medicinal Plants: Vol. 2, 1970–1979. Central Drug Research Institute (CDRI), Lucknow, India. 1991:81-4. 3. Al-Turki TA, Omer S, Ghafoor A. Two new species of Heliotropium L.(Boraginaceae) from Saudi Arabia. Botanical Journal of the Linnean Society. 2001; 1,137(2):215-20. 4. von Erhardt-Siebold E. The heliotrope tradition. Osiris. 1937; 1(3):22-46. 5. Goyal N, Sharma SK. 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