Phytochemical Investigation for the Main Active Constituents in Arctium lappa L . Cultivated in Iraq

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.


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 antiinflmmatory, hepatoprotective, antitumor, antimicrobial, antifungal, anti-aging and hypoglycemic effects (2) .The main active ingredients isolated from this herb are: Caffeic acid (  (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.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.

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.

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 H 2 SO 4 with ammonia for flavonoids.D-reagent for alkaloids (8) .

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).

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 R f values and UV spectrum :The results obtained are shown in figure 4.

Chlorogenic acid
End R f 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 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.

Figure 4 :
Figure 4: HPTLC analysis of methanolic extract with reference standards.

-
Accurate measurement of R f values in TLC as shown in table 2 and figure1