Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. DOI: https://doi.org/10.31351/vol29iss2pp139-151 139 Separation and Identification of Phenolic Acid from Borago officinalis (F:Boraginaceae) Cultivated in Iraq Ashwaq T. Kareem*,1 and Maha N. Hamad* *Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract The plant Borago officinalis, which belongs to the Boraginaceae family and Celebrated as borage, is one of the useful medicinal plants cultivated in Iraq. It was used in olde medicine in Iraq, Irane, Syria and Europe for management of various diseases. It is commonly used as a tonic, tranquilliser, management of cough,sore throat , pneumonia,urinary tract infection, rheumatoid arthrites antioxidant, and anticancer. This project provides the first comprehensive research done in Iraq to study the phytochemicals and the methods of extraction and isolation of active constituents from Borago officinalis cultivated in Iraq. The plant was harvested in spring from AL-Rifai, Nassiriyah city/ IRAQ in February 2019. The aerial parts were washed carefully, dried under dark for two weeks, and milled in a mechanical grinder to a fine powder.The plant was extracted by cold extraction methods using 85% methanol solvent for three days then Fractionation with petrollium ethere, chloroform, ethyl acetate and n-butanol(n.b) to separate the active constituentse according to the change in polarities. The ethyle acetate fraction and n-butanol fraction were used for identification and isolation of phenolic compounds by TLC,PLC,HPLC and LC/mass. Results of the phytochemical screening exposed the presence of, phenols, tannins, fatty acid, in the plant extract. The phenolic acid(Sinapic acide, Rosmarinic acides, Caffeic acid) were separated and purified by PLC.The isolated compounds were subjected to several chemical ,chromatographic and spectral analytical techniques for their identification such as TLC, HPLC,UV andLC/mass. Keywords: Sinapic acid, Rosmarinic acid, Caffeic acid, HPLC, Lc/Mass. في نبات لسان الثور المستزرع في العراق ةالموجودفصل وتحديد االحماض الفينولية *و مها نوري حمد 1*،اشواق طالب كريم .العراق،بغداد ، جامعة بغداد ،كلية الصيدلة ،*فرع العقاقير والنباتات الطبية الخالصة والتي استزرعت ةالنباتات الطبيه المعروف والمعروف بخبز النحل او الحمحم وهو من (Boraginaceae) نبات لسان الثور من عائلة لمعالجة العديد من االمراض حديثا في العراق ويستعمل في الطب الشعبي في العراق وسوريا وايران وبالد الشام ومناطق البحر المتوسط وكندا واوربا ت المفاصل الروماتيزي وكمضاد وااللتهابات الرئوية والتهابات المسالك البولية والتهابا حيث يستعمل كمقوي ومهدئ للسعال ولعالج التهاب الحلق . تم للتاكسد والسرطانات . يعتبر هذا البحث اول بحث شامل في العراق لدراسة الموادالكيمياويه الموجودة في النبات وطرق استخالصها وفصلها سبوعان وطحنه بالمطحنه وتم غسل و تجفيف النبات لمده ا2019جمع النبات من قضاء الرفاعي في محافظة ذي قار في شهر شباط من سنة ميثانول لمدة ثالثة ايام. تمت عملية التجزئيه باستخدام عدة مذيبات %85الميكانيكيه وتمت عملية االستخالص بالطريقة الباردة طريقة نقع النبات في ادا على االختالف في القطبية بين هذه المكونات هي بالتتابع: االثير البترولي, الكلوروفورم ,خالت االثيل والبيوتانول لفصل المركبات الفعالة اعتم .استخدمت طبقتي خالت االثيل والكحول البيوتانولي في التعرف وعزل االحماض الفينولية بعدة تقنيات مثل كروماتوغرافيا الطبقه الرقيقة ائل والطيف الكتلي وكانت نتيجة الكشوفات الكيميائيه كروماتوغرافيا السائل عالية االداء وكروماتوغرافيا السووكروماتوغرافيا الطبقه التحضيرية حامض الروزمارينك وحامض السينابيك وتم كشف وعزل االحماض الفينولية ) وجود مواد فينوليه واحماض دهنيه ومواد دابغة في المستخلص. ,مطياف االشعه فوق البنفسجيه ,كروماتوغرافيا السائل كروماتوغرافيا السائل عالية االداءوكروماتوغرافيا الطبقه الرقيقة بواسطة وحامض الكافيك( .والطيف الكتلي كروماتوغرافياالسائل والطيف الكتلي.، كروماتوغرافيا السائل عالية االداء،حامض السنابيك,حامض الكافييك، حامض الروزماريك الكلمات المفتاحية: Introduction Borage (Borago officinalis L.) is an annual plant belonging to the family. Boraginaceae. It originates from Western regions of Mediterranean area and grows nearly in whole America, Europe, Canada and Iran (1,2). The plant grows during November to January and reaches a height of 70 to 100 cm (3,4) Its stem is shielded with hairs that secrete a strong smell nearly the aroma of fresh cucumbers while on the tops of the shoots there are the star- shaped inflorescences which initially are pink, later turn blue, seldom white (5,6). Aerial partse have been used in old medicine in Iraq as atonic, tranquillizer, management of cough, pneumonia, sore throat, swelling and inflammatory diseases. The leaves and flower possess biological activities for cancer and heart diseases prevention (7) and have antibiotic properties (8), condense cardiovascular diseases (9) and provide benefits for improving healthe due to their various biological events (10). 1corresponding author E-mail: ashotalib@gmail.com Received: 6 / 2 /2020 Accepted: 20/ 5/2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol29iss2pp139-151 Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 140 Results reported by previous studies shown the presences of phenolics acid, flavonoids (Quercetin, Myricetin, Luteolin and Rutin) and isoflavonoid besides, the dominant individual fatty acids of methanolic extract as Oleic acid which is an unsaturated fatty acid (omega-9), linoleic fatty acids (omega-6i) and Hexadecanoic acid. Thet methanolic extract was more biologically active than ethanolic extract (11). Figure 1. Borago officinal plant cultivated in Iraq.(12) Material and Method Plant collection Borago officinalis plant was harvested from AL-Rifai, Nassiriyah city/ IRAQ in February 2019. The aerial plant was driede in the shadow for two weeks and powdered. The plant was identifiede and authenticated by Prof. Dr Israai Mohammed Department of Biology /College of Sciences/ University of Baghdad. Extraction and fractionation of the different active constituent Two hundred and fifty grams of the powdered plant material was soaked in 1500ml, with 85% methanol and shaking, at roome of temperature. After three days, the methanol soluble materials was filtered off. The filtrate was evaporated until dryness unders vacuum using a rotatory evaporator. A dark greenish residue was obtained. Then suspended in 500ml water and partitioned successively with petroleum ether (B.p. 30-60 ℃), chloroform, ethyl acetate, and n-butanole (3×500ml) for each fraction the first three fractions dried over sodium sulfate anhydrous, filtered, and evaporated to dryness(13,14) . The scheme of fractionation is shown in (Figure 2). Figure 2. Schematic diagram for fractionations aerial part crude extracts of Borago officinalis (14 ) Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 141 Identification of phenolic compounds in Borago officinalis plant extract 1. Preliminary phytochemicals showing of the phenolic compounds using a methanolic extract from the plant using a NaOH test, lead acetate, and ferric chloride test (15,16). 2. Isolation and purification of phenolic compounds from the fractions ethyl acetate and n.butanoleei by preparative layer chromatography. Isolation of phenols was done by using preparative TLC; 2 grame of each fraction dissolve in 10 mlof methanole and applied to the number of PLC plates as a semi concentrated solution in streak using a capillary tube on each plate, then the plate placed inside glass tank which contained the S1 solvent system. The band had been scrapped off, eluted with methanol and then filtered, the filtrate evaporated to dryness, in a vacuum as shown in Figure 3 and 4. 3. Thin-layer chromatography. In this qualitative identification, a ready-made aluminium plates of silica gel of 245 with developing solvent systems were used for detection the plant phenols in fractions ethyl acetate and n.butanoleei with standards, as listed in Table (1). Table 1. Developing solvent systems using in the separation of phenols in Borago Officinalis. No. Composition References S1 chloroform: ethyl acetate : methanol :formic acid (70:14:14:10) (17) S2 Dichloromethan:acetonitrile :formic acid(9.5:0.5:0.1) (17) S3 Chloroform:methanol (90: 10). (17) 4. Qualitative estimate of ethyl acetate and n.butanolee fractions by high-performance liquid chromatography (HPLC) the expected phenols in fractions e were separated by HPLC method and detected in comparison with standard compounds . The mobile phase consisted 1 % aq. Acetic acid solution (A) and acetonitrile (B) solvents, the flow rate was adjusted to 0.7 ml/min, the column was thermostatically controlled at 280C, and the injection volume was kept at 20 μl. Gradient elution was accomplished by varying the proportion of B to A solvents. The gradients elution was changed from 10 % to 40% B in a linear fashione for 28 min, from (40 to 60) % B in 39 min, after that from ( 60 to 90) % B solution in 50 min. The mobile phase mixture back to the initial state of solvents (B: A: 10: 90) in 55 min and permitted to run for another ten min. 5. LC-MS Analytical was done using the Agilent Systems joined to an Appliede Biosystems API 2000 mass spectrometry.Mobile phase solvents acetonitrile and wate A columne of 0.19mm external diameter (75μm I.D.) and 200mm length was packede with Thermo Scientifice Hypersil Gold C18 with 5μm particle size. Samples were run under the following conditionse: m/z range was 250 to 10001, 200K resolution, top 5 configurationse with one MS scane and five MS/MS scans, and dynamic exclusion set to 1 with a limit of 90 seconds. A 2.5 hour LCMS separatione was used for all blanke and standard samples. Results and Discussion Borago officinalis plant active constituents In this studyi, cold extraction method was done by 85% absolute methanol to extract the active constituenti depend on the nature of these active constituents.Each 250 g of plant extract yielded 32 g residue Preliminary qualitative phytochemicale analysis The results of the phytochemical analysis of polyphenol in methanolic crude extract given in ( Table 2). Table 2. Results of the phytochemical analysis of polyphenol in the methanolic crude extract Active group Test Reaction Result Poly phenol Lead acetate test White ppt positive Polyphenol Ferric chloride test Dark green colour positive Poly phenol NaOH test Yellow colour after addition NaOH solution positive The isolation of phenols were done by using preparative TLC, in jar contained the S1 solvent system. As in Figure 3 and 4. Figure 3. Preparative thin layer chromatography plate for ethyl acetate fraction on silica gel GF254 developed in the S1 system, detection by UV light at 254nm. Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 142 Figure4. Preparative thin-layer chromatography plate for n.b fraction on silica gel GF254 developed in the S1 system, detection by UV light at 366nm. Table 3. TLC profile of isolated compound (cpd) number 1 compared with standard rosmarinic acide using the mobile phase solvents ( S1, S2, S3). Solvent system Rf of standard Rosmarinic acid.. Rf of isolated Phenolic acid number 1 S1 0.247 0.246 S2 0.13 0.13 S3 0.2 0.2 Table 4. TLC profile of isolatedi compound number 2 compared with sinapic acide standard using the mobile phase solvents ( S1, S2, S3) Solvent system Rf of standard Sinapic acidee… Rf of isolated Phenolic acid number 2 S1 0.71 0.71 S2 0.47 0.47 S3 0.5 0.51 Table 5. TLC profile of isolatedi compound number 3 compared with caffeic acid standard using the mobile phase solvents ( S1, S2, S3). Solvent system Rf of standard caffeic acide Rf of isolated Phenolic acid number 3 S1 0.517 0.517 S2 0.182 0.182 S3 0.294 0.293 A B Figure 5. TLC chromatography (A) for separated compound number (1) from ethyl acetate fraction and Rosmarinic acid STD using silica gel Gf254 as adsorbant and S1 as mobile phase and (B) separated compound after detected by spraying with five %ferric chlorides. Figure 6. TLC chromatography for separated compound number (1) from ethyl acetate fraction and R.A standard using silica gel Gf254 as adsorbant and mobile phase S2. Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 143 Figure 7. TLC chromatography for isolated compound number (1) from ethyl acetate fraction and Rosmarinic acid standard using silica gel Gf254 as adsorbant and mobile phase S3. A B Figurei8. TLC chromatography(A) for isolated compound number (2) from ethyl acetate fraction and Sinapic acid standard using silica gel Gf254 as adsorbant and mobile phase S1 (B) separated compound after detected by spraying with five %ferric chlorides. Figurei9. TLC chromatography for isolatede compound number (2) from ethyl acetate fraction and standard S.A using silica gel Gf254 as adsorbant and mobile phase S2. Figure 10. TLC chromatography for isolated compound number (2) from ethyl acetate fraction and S.A standard using silica gel Gf254 as adsorbant and S3 as mobile phase . Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 144 A B Figure 11. TLC chromatography (A) for separated compound number (3) from ethyl acetate fraction and Caffeic acid STD using silica gel Gf254 as adsorbant and S1 as mobile phase and (B) separated compound number 3 after detected by spraying with five %ferric chloride. Figure 12. TLC chromatography for isolatede compound number (3) from ethyl acetate fraction and caffeic acidi standard using silica gel Gf254 as adsorbant and mobile phase S2 . Figure 13. TLC chromatography for isolatede compound number (3) from ethyl acetate fraction and caffeic acide standard using silica gel Gf254 as adsorbant and S3 as mobile phase HPLC analysis The result gained from HPLC analysis method. 1. The retention time of Rosmarinic acide standard match with a retention time of isolatedi compound number 1 and UV spectrum of separated compound number 1 match with UVs spectrum of Rosmarinic acide standard as in Figure 14and 15 and Table 6 . 2. The retention time of the isolated compound number 2 match with a retention time of Sinapic standard and UV spectrum of the separated compound number 2 match with standard Sinapic acide as in Figure 16 and 17 and Table 6. 3. The retention time of the isolated compound number 3 match with a retention time of Caffeic acidi standard and UV spectrum of the separated compound number 3 match with Caffeic acid standard as in Figure 18 and 19 and Table 6. Figure 14. HPLC chromatogram of isolated compound number 1 and Rosmarinic acide standard. Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 145 Figure 15. The UV spectrum of isolated compound number1 and Rosmarinic acide standard. Figure 16. HPLC chromatogram of the isolatedi compound number 2 and Sinapic acide standard. Figure 17. The UV spectrum of isolated compound number 2 and Sinapic acide standard. Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 146 Figure 18. HPLC chromatogram of Caffeic acid standard and isolatedi compound number 3. Figure 19. UV spectrum of isolated compound number 3 and caffeic acid standard. Table 6.Retention times in minutes for standards (rosmarinic acide,sinapic acide ,caffeic acid)and isolated cpd 1,2,3. Retention time of Rosmarinicacid standard. Retention time for isolated cpd number 1. 20 20 Retention time of Sinapic acid standard. Retention time for isolated cpd number 2. 15 15 Retentiontime of Caffeic acid standard. Retention time for isolated cpdnumber3. 10 10 LC/mass The result gained from LC/mass. 1. LC/mass chromatograme as shown in( Figure 20) the isolated compound number 1 is Rosmarinic acide with molecular weight 360.31 Gram/mol as shown in Figure (21). 2. LC/mass chromatograme as shown in( Figure 22) the isolated compound number 2 is Sinapic acid with molecular weight 224.21 Gram/mol as shown in Figure(23) . 3. LC/mass chromatogrami as shown in( Figure 24) the isolated compound number 3 is caffeic acidi with molecular weight 180.16 Gram/mol as shown in Figure (25). Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 147 Figure 20. LC/MS chromatogram of isolated compound number 1 Molecular ion peak at m/z 360 that correspond to a molecular formula of C18H16O8(3,4- dihydroxyphenyllactic acid) Rosmarinic acid and Molecular ion peak at m/z359[M-H] of Rosmarinic acid. Figure 21. Chemical structure of Rosmarinic acide with molecular weight 360.31 Gram/mol.(18) Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 148 Figure 22. LC/MS chromatogram of isolated compound number 2. Molecular ion peak at m/z 223 that correspond to the molecular formula of C11H12O5 (3,5-dimethoxy-4- hydroxycinnamic acid) sinapic acid. Figure 23. Chemical structure of Sinapic acide with molecular weight 224 Gram/mol.(19) Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 149 Figure 24. LC/MS chromatogram of isolated compound number 3. Molecular ion peak at m/z 181 that correspond to the isotope of a molecular formula of C9H8O4 (3,4- dihydroxycinnamic) caffeic acid, and molecular ion peak m/z 179.3 [M-H]for caffeic acid. Figure 25. Chemical structure of Caffeic acide with molecular weight 180 Gram/mol.(20) Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 150 Conclusion In the light of the results obtained, the study concluded the following: 1. Phytochemical screening of new Iraqi plant Borago officinalis was done for aerial part of the plant nearly two hundred and fifty grams of the powdered plant material, and the results include the separation and identification of phenolic acids (Rosmarinic acid, caffeic acidi and Sinapic acid). 2. Rosmarinic acid with Mwt 360 gram /mol isolated from ethyle acetate and n.butanol fraction with high quantity nearly 13.3 mg. 3. Sinapic acid with molecular weight 224 Gram/mol isolated from ethyle acetate fraction with small quantity nearly 6 mg. 4. Caffeic acide with molecular weight 180 Gram/mol isolated from ethyle acetate fraction with high quantity nearly 9 mg. 5. All isolated phenolic acids were identified by TLC, preparative TLC, HPLC, UV, LC/Mass. Acknowledgements The authors are grateful to acknowledge the College of Pharmacy -University of Baghdad for providing the necessary facilities to carry out this study. References 1. Ghahreman A. Flora of Iran. Tehran: Research Institute of Forests and Rangelands; 1978. Persian. 2. Pieretti PG, Palmegiano GB, Salamano G. Quality and fatty acid content of borage (Borago officinalis L.) during the growth cycle. Ital J Food Sci 2004; 16: 177-184. 3. Farhadi R, Balashahri MS, Tilebeni HG, Sadeghi M. Pharmacologyof Borage (Borago officinalis L.) medicinal plant. Int J AgronPlant Prod 2012; 3: 73-77. 4. Gudej J, Tomczyk M. Badania chromatograficzne związków polifenolowych w zielu Borago officinalis L.Herba Pol 1996; 42:252-6. 5. Basar SN, Rani S, Farah SA, Zaman R. Review on borage officinalis: a wonder herb. Int J Biol Pharm Res 2013; 4: 582-587. 6. Montaner C, Floris E, Alvarez JM.Geitonogamy :A mechanism responsible for high selfing rates in borage (Borago officinalis L.).Theor Appl Genet .2001;102 9(2):375 – 378 . 7. M. Asadi-Samani, M. Bahmani, M. Rafieian- Kopaei, The chemical composition, botanical characteristic and biological activities of Borago officinalis: a review. Asian Pac. J. Trop. Med. 2014;7:22-28. 8. A. Ratz-Łyko, A. Herman, J. Arct, K. Pytkowska, Evaluation of antioxidant and antimicrobial activities of Oenothera biennis, Borago officinalis, and Nigella sativa seedcake extracts. Food Sci. Biotechnol.2014; 23(4): 1029–1036 . 9. G. Oboh, A.O. Ademosun, Comparative studies on the ability of crude polyphenols from some Nigerian citrus peels to prevent lipid peroxidation in vitro. Asian J. Biochem. 2006; 1(2): 169–177. 10. M.D. Lozano-Baena, I. Tasset, A. Muñoz- Serrano, Á Alonso-Moraga, A. Haro-Bailón, Cancer prevention and health beneficesof traditionally consumed Borago officinalis plants. Nutrients 2016; 48: 8(1). 11. A. Crozier, E. Jensen, M.E.J. Lean, M.S. McDonald, Quantitative analysis of flavonoids by reversed-phase high-performance liquid chromatography. J. Chromatogr. A, 1997; 761: 315–321. 12. Gupta M, Singh S.Borago officinalis Linn. An important medicinal plant of Mediterranean region : Review .Int. J.Pharm Sci Rev Res .2010;5(1):27 – 34 . 13. Cetkovic GS, Dilas SM, Canadanovic-Brunet JM, Tumbas VT. thin-layer chromatography analysis and scavenging activity of marigold (calendula officinalis l.) extracts. Apteff. 2003; 34:93–102. 14. Hamad MN. Detection and isolation of flavonoids from Calendula officinalis (F. Asteraceae) cultivated in Iraq. 2016; 25(2):1–6. 15. Wagner H., Bladt. plant Drug analysis:Thin layer Chromatography Atlas 2nd ed. Springer- velag, Berlin, 1996:384. 16. Muhammed NA, Awad Z J .Phytochemical study of some medicinal compounds present in Cordia myxa L. plant cultivated in Iraq . A Thesis 2017 . p40. 17. Waksmundzka-Hajnos M, Sherma J, Kowalska T, editors. Thin layer chromatography in phytochemistry. CRC Press; 2008:P 348. 18. Ying Zhang, Jonathan P. Smuts, Edra Dodbiba, Rekha Rangarajan, John C. Lang, and Daniel W. Armstrong . Degradation Study of Carnosic Acid, Carnosol, Rosmarinic Acid, and Rosemary Extract (Rosmarinus officinalis L.) Assessed Using HPLC. Journal of Agricultural and Food Chemistry 2012, 60 (36) , 9305-9314. Iraqi J Pharm Sci, Vol.29(2) 2020 Borago officinalis L. 151 19. J. Oszmian´ski ,Kolniak-Ostek J , Wojdyło A .Application of ultra performance liquid chromatographyphotodiode detector- quadrupole/time of flight-mass spectrometry (UPLC-PDA-Q/TOF-MS) method for the characterization of phenolic compounds of Lepidium sativum L. sprouts 2013: ;236,699– 706. 20. M. Nardini, M. D’Aquino, G. Tomassi, V. Gentili, M. Di Felice and C. Scaccini, Inhibition of human low- densitylipoproteinoxidation by caffeic acid and other hydroxy- cinnamic acid derivatives, Free Radical Biol. Med. 1995: ;19, 541. Baghdad Iraqi Journal Pharmaceutical Sciences by bijps is licensed under a Creative Commons Attribution 4.0 International License. Copyrights© 2015 College of Pharmacy - University of Baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/