Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 18 Phytochemical Investigation for the Main Active Constituents in Arctium lappa L. Cultivated in Iraq Dhuha A.Al-Shammaa *,1 , Kawkab Y. Saour ** and Zaid M. Abdul-Khalik *** * Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad, Iraq. ** Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. *** Baghdad College of Pharmacy, Baghdad, Iraq. Abstract Burdock ( Arctium lappa), is among the most popular plants in traditional medicine and it is associated with several biological effects. Literature survey revealed the presence of phenylpropanoid compounds .The most widespread are hydroxycinnamic acids ( mainly caffeic acid and chlorogenic acid) and lignans (mainly arctiin and arctigenin). This work will confirm the presence of these compounds in Arctium lappa, cultivated in Iraq, in both root and leaf samples. The dried plant samples were extracted by soxhlet with 80% methanol then separated the main constituents by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Identification of the isolated compounds was carried out by UV, IR, and compared with reference standards using TLC, HPLC and HPTLC. Keywords: Actium lappa, hydroxycinnamic acid, Lignan, Phytochemical. دراسة كيميائية للمواد الفّعالة في نبات االرلطيون المستسرع في العراق ضحى عبذ الصاحة الشماع *،1 ، كوكة يعموب ساعور ** و زيذ محمذ عبذ الخالك *** * .قسى انعقاقٍش وانُباحاث انطبٍت ، كهٍت انصٍذنت ،جايعت بغذاد ، بغذاد، انعشاق ** .، كهٍت انصٍذنت ،جايعت بغذاد ، بغذاد، انعشاققسى انكًٍٍاء انصٍذالٍَت *** .بغذاد، انعشاق كهٍت بغذاد نهصٍذنت ، الخالصة . يٍ انُباحاث االكثش شعبٍت فً انطب انخقهٍذي وٌشحبط بانعذٌذ يٍ انقعانٍاث انبٍىنىجٍت, ٌعذ َباث االسقطٍىٌ وانًعشوف باسى انبهسكاء خصىصا حايض انكافٍك (االكثش اَخشاساهً حىايض هٍذسوكسً سٍُايكو . انسابقت وجىد يشكباث انفُم بشوباَىٌذ أظهشث انذساساث وهزا انعًم سىف ٌثبج وجىد هزِ انًشكباث فً َباث االسقطٍىٌ (.بصىسة سئٍسٍت األسكخٍٍ و األسكخٍجٍٍُ)وانهكُاٌ ( وحايض انكهىسوجٍُك وفصم ٪80انًٍثاَىلبىاسطت حى رنك باخز عٍُاث يٍ انُباث انجاف واسخخالصهاوقذ . و االوساق انًسخزسع فً انعشاق فً كم يٍ انجزوس ثى حى حشخٍص انًشكباث انًفصىنت باسخخذاو طٍف االشعت .عانٍت األداء انسائهتكشوياحىكشافٍا يع طبقت انشقٍقتال يشكباحها بكشوياحىكشافٍا باالضافت انى يقاسَخها يع انًىاد انقٍاسٍت باسخخذاو كشوياحىكشافٍا انطبقت انشقٍقت وكشوياحىكشافٍا فىق انبُفسجٍت واالشعت ححج انحًشاء . عانٍت االداء انسائهت . االرلطيون ، حامض هيذروكسي سيناميك ، ليغنان ، دراسة كيميائية :الكلمات المفتاحية Introduction Actium lappa (common name: Burdock) is a flowering plant that belongs to the family Asteraceae (Compositae). With the advancement of different state-of-the-art analytical techniques, more active ingredients of Actium lappa have been identified over the last decade (1) . The literature furnishes numerous data on their anti- inflmmatory, hepatoprotective, antitumor, antimicrobial, antifungal, anti-aging and hypoglycemic effects (2) . The main active ingredients isolated from this herb are: Caffeic acid (3, 4 – Dihydroxy - cinnamic acid); Chlrogenic acid (1S,3R,4R,5R)-3-{[(2Z)-3-(3,4 dihydroxyphenyl) prop -2 – enoyl] oxy }- 1, 4 ,5- trihydroxycyclohexanecarboxylic acid; Arctiin (3R,4R)-4-[(3,4-dimethoxyphenyl)methyl]-3- {[3-methoxy-4-[(2S, 3R, 4S, 5S, 6R) -3, 4, 5-trih ydroxy -6- (hydroxymethyl) oxan -2-yl ] oxyphenyl ] methyl}oxolan-2-one; Arctigenin (3R,4R)-4-[(3, 4-dimethoxyphenyl) methyl]-3- [(4- hydroxy -3- methoxy phenyl) methyl]-2- tetrahydrofuranone (3) . Dong WH. et al. ( 2006) (4) Study the condition for extraction of arctiin from fruits of Arctium lappa using supercritical fluid extraction, they found that optimal extraction conditions were: pressure 40 MPa, temperature 70 degrees C, using methanol as modifier carrier, while Liu S. et al.(2005) (5) isolated and identified arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC- ESI/MS. 1 Corresponding author E-mail:alduha_sun@yahoo.com. Received: 18/7/2012 Accepted:12/12/2012 http://www.botany.hawaii.edu/Faculty/Carr/aster.htm http://www.ncbi.nlm.nih.gov/pubmed?term=Dong%20WH%5BAuthor%5D&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=Liu%20S%5BAuthor%5D&cauthor=true&cauthor_uid=15881114 Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 19 Ferracane R. et al. (2010) (6) were analyzed phytochemical compounds by liquid chromatography coupled to electro spray tandem mass spectrometry (LC/MS/MS) in negative mode. Caffeic acid Chlorogenic acid Arctiin Arctigenin Experimental Plant Materials The plant sample was collected from the department of medicinal plants, College of Agriculture, University of Baghdad. Identified by Dr.Saged Auda Mohammad , head of medicinal plant department. The plant leaves and roots were air dried in the shade at room temperature, coarsely powdered by mechanical grinder and weighed. Chemicals All reagents and solvents used were analytical grade. Standards used to identify the main active constituents (Arctiin, Arctigenin, Caffeic acid and Chlorogenic acid) were obtained from China (Cheng du Biopurify phytochemicals Ltd.).UV was done in methanol. Instruments Electrical sensitive balance: Sartorius/ Germany, Ultraviolet light : Desaga Heidelberg/ Germany, Rotatory evaporator: Buchi Rotatory evaporator, Chiller: Ultratemp 2000 , Water bath: Memmert/Germany, HPLC: Water/Irland, FTIR: Shimadzo FT-IR-84005 IR Spectrometer, HPTLC:Eike Reich/CAMAG – Laborator. Extraction of plant 100 g of dried milled leaves and roots were extracted separately in a soxhlet apparatus with 80% methanol (3:1 solvent/plant ratio), for 6 hours. The extracts obtained were evaporated to dryness at 45 o C under vacuum using rotary evaporator (7) . General phytochemical screening by chemical tests The different parts of the plant have been screened for the occurrence of saponines, flavonoids, tannins and alkaloids by the following chemical reactions A- Ferric chloride test for tannins. B- Froth test for saponin. C- Conc H2SO4 with ammonia for flavonoids. D- reagent for alkaloids (8) . Isolation and identification Mayer ' s of active constituents Separation of the main active constituents from both leaves and roots of Arctium lappa L. were carried out using Preparative TLC: PLC plates, 20x20cm and 1mm thickness of silica gel GF254 developed in solvent system (CHCl3- MeOH 80:20) together with standard references http://www.ncbi.nlm.nih.gov/pubmed?term=Ferracane%20R%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://upload.wikimedia.org/wikipedia/commons/c/c3/Kaffees%C3%A4ure.svg Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 20 The chromatogram was visualized by UV lamp (at 254 nm and 366 nm) (9) . Identification of active constituents was done by: 1-Matching with standards by TLC using the following mobile phases: S1: Chloroform: aceton: formic acid (75,16.5, 8.5), S2: Ethanol:acetic acid (85:15), S3: Chloroform:Methanol (80:20) (10) 2-High Performance Liquid Chromatography (HPLC): HPLC analysis was done using C18 – column (150 × 4.6 mm) with specific conditions for each compound:  HPLC conditions for Arctiin, Arctigenin and Caffeic acid: The mobile phase is methanol: water (60:40) , flow rate: 0.5 ml/min at 280 nm (11) .  HPLC conditions for Chlorogenic acid: The mobile phase is acetonitrile: acetic acid: water (15: 0.5:85), flow rate 1ml/min at 320 nm (12) . 3- Fourier transform infrared spectroscopy (FTIR) in KBr disk. 4-High performance thin layer chromatography (HPTLC): the mobile phase is ethanol: acetic acid (85:15), the list of standards: Arctiin std., Arctigenin std., Caffeic acid std., Chlorogenic acid std., then Leaf sample and Root sample. Results and Discussion The preliminary phytochemical investigation revealed the presence of saponins, flavonoids, tannins in both parts of the plant, while the alkaloids are absent as shown in table( 1). Table 1: The results of the general screening by chemical tests. Identification for each isolated compound depend on : 1- Accurate measurement of Rf values in TLC as shown in table 2 and figure 1 Table 2: Rf values of some plant constituents and their standards in different developing solvent systems in TLC. TLC Rf (Isolated compound) S1 S2 S3 TLC Rf (Standard) S1 S2 S3 Compounds 0.66 0.67 0.72 0.67 0.68 0.71 Arctiin 0.85 0.80 0.94 0.86 0.82 0.92 Arctigenin 0.35 0.49 0.46 0.36 0.48 045 Caffeic acid 0.03 0.04 0.03 0.02 0.03 0.04 Chlorogenic acid Figure 1 :Thin layer chromatograms of both root and leaf sample with four references standard on silica gel GF254, developing in S1 solvent system and detection by UV light at 254 nm & 366 nm.[ 1:Std. Arctiin , 2: Std.Arctigenin , 3:Std. Caffeic acid , 4: Std.Chlorogenic, R: root extract, L: leaf extract.]. The plant sample Plant part Saponin Alkaloid Flavonoid Tannin Actium lappa L. Root + - + + Leaf + - + + Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 21 2-High performance thin layer chromatography HPLC HPLC analysis can be carried out for qualitative analysis by comparison of retention times of analyzed samples and authentic standards at identical chromatographic conditions .the results are shown in Figures (2, 3) and table 3. HPLC of std. Arctigenin HPLC of std. Arctin HPLC of std. Caffeic acid HPLC of methanolic Leaf extract HPLC of methanolic Root extract Figure 2: HPLC analysis of std. Arctiin, std. Arctigenin, std.Caffeic acid and methanolic extract of leaf &root sample. Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 22 HPLC of std. Chlorogenic acid. Methanolic Root extract Methanolic Leaf extract Figure 3: HPLC analysis of std. chlorogenic acid and methanolic extract of root & leaf sample. Table 3: The retention time of each isolated compound with that of reference standards 3-FT-IR spectroscopy IR spectroscopy is most frequently used in phytochemical studies as a ' finge printing ' device, for comparing a natural with a synthetic reference standard .Such comparisons are very important in the complete identification of many types of plant constituents as shown in table (4 ). Rt of compounds in leaf extract Rt of compounds in root extract Rt of standard Compound 5.074 5.231 5.063 Arctiin 8.616 8.734 8.770 Arctigenin 2.850 2.707 2.650 Caffeic acid 9.541 9.818 9.889 Chlorogenic acid Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 23 Table 4: IR absorption bands of isolated compounds ( in cm -1 ). 4-HPTLC analysis HPTLC was carried out for further identification of main active constituents present in methanolic extract of both leaves and root of Actium lappa, by measuring the Rf values and UV spectrum :The results obtained are shown in figure 4. All tracks at wavelengtth 254 nm Arctiin std. Arctigenin std. Chlorogenic acid Caffeic acid std. Figure 4: HPTLC analysis of methanolic extract with reference standards. Aproximate positions of characteristic bands. Compound 3470(OH), 1780(C=O),1594,1520(Ar-H), 1080, 1030, 1020(β-glycopyranoside), 2930,2850, 1465, 1420,1262, 670. Arctiin 3470(OH), 1770(C=O), 1610, 1520, 3030(Ar-H), 1465, 1390 , 1270. Arctigenin 3433, 3234, 2910, 1645, 1620, 1448, 1278 , 1217 , 1120, 974,576 Caffeic acid 3421, 2929, 1697, 1635, 1456, 1398 ,1278,1182, 812. Chlorogenic acid Iraqi J Pharm Sci, Vol.22(1) 2013 Active constituents in Arctium lappa L. 24 End Rf values recorded for standards in HPTLC which have small peak area, are excluded because these are due to impurities. All chromatographic data coincide with that reported and shown by the standards and so as the obtained UV & FTIR results. This confirms the presence of arctiin, arctigenin, caffeic acid & chlorogenic acid in both leaves & root of Arctium lappa. Conclusion The Iraqi plant , Actium lappa, is a good source for many phenolic compounds with antioxidant activity. 80% methanol is the best solvent suitable for the extraction of the main active constituents from both root and leaf of the plant .The same constituents were obtained from both parts, however; the results showed that there was a slight different in the amount of each plant constituents. This study confirms the presence of Chlorogenic acid, Caffeic acid, Arctiin and Arctigenin in Actium lappa plant cultivated in Iraq. References 1. James A.Duke; Mary Jo Bogenschutz- Godwin “Hand Book of Medicinal Herbs ” , second Edition, 2002; by CRC. 2. SheldonHendlerPH.D.M.D.; David Rorvik, “ PDR for Nutritional Supplements ” Second Edition, 2008(11): 156-159. 3. Joanne Barnes; Linda A. Anderson and J.D. Phillipson, “ Herbal Medicines ” , 2007; Sep. 4. Dong WH; Liu B., “Study on condition for extraction of arctiin from fruits of Arctium lappa using supercritical fluid extraction”, Zhongguo Zhong Yao Za Zhi. 2006 (31): 1240-1276. 5. Liu S, Chen K; Schliemann W; Strack D,“ Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC- ESI/MS”, Phytochem Anal. 2005(16): 86- 90. 6. Ferracane R, Graziani G, Gallo M, Fogliano V, Ritieni A., “ Metabolic profile of the bioactive compounds of burdock (Arctium lappa) seeds, roots and leaves”, J Pharm Biomed Anal. 2010(51): 399-404. 7. Fabio Bahls Machado; Rafael Eidi Yamamoto; Karine Zanoli.; “ Evaluation of the Antiproliferative Activity of the Leaves from Arctium lappa by a Bioassay-Guided Fractionation ” ,Molecules, 2012(17): 1852- 1859. 8. FabriciaSPredes; AnaLTG Ruiz; JoaoE Carvalho; Mary A Foglio; “ Antioxidant and invite anti proliferative activity of Arctium lappa root extracts ” , Complementary and Alternative Medicine , 2011(10): 1186- 1472. 9. Long-Ze Lin; James M Harnly; “ Identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized LC-DAD-ESI/MS profiling method ” , Journal of Agricultural and Food Chemistry 2008(56): 10105- 10114. 10. Liu S et al. “ Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromotography in combination with HPLC-ESI/MS ” , Phytochemical Anal 2005(16): 86-89. 11. Xiao Wang; Fuwei Li; Qinglei Sun; Jingpeng Yuan; Ting Jiang; Chengchao Zheng ; “ Application of preparative high- speed counter-current chromatography for separation and purification of arctiin from Fructus Arctii”, Journal of Chromatography A 2005 (10): 247-251. 12. Rosalia ferracane; Giulia Graziani; Monica Gallo; “ Metabolic profile of the bioactive compounds of burdock (Arctium lappa)seeds,root and leaves”, Journal of pharmaceutical and Biomedical Analysis 2010(51): 399-404. http://www.amazon.com/s/ref=ntt_athr_dp_sr_2?_encoding=UTF8&field-author=David%20Rorvik&search-alias=books&sort=relevancerank http://www.ncbi.nlm.nih.gov/pubmed?term=Dong%20WH%5BAuthor%5D&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=Liu%20B%5BAuthor%5D&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed/17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=Liu%20S%5BAuthor%5D&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=Chen%20K%5BAuthor%5D&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=Schliemann%20W%5BAuthor%5D&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=Strack%20D%5BAuthor%5D&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed/15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=Ferracane%20R%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=Graziani%20G%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=Gallo%20M%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=Fogliano%20V%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=Fogliano%20V%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=Ritieni%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed/19375261 http://www.ncbi.nlm.nih.gov/pubmed/19375261