Separation , Characterization and Anticoagulant Activity of Coumarin and its Derivatives Extracted from Melilotus officinalis

Coumarin is a phenolic natural product extracted from yellow clover Melilotus officinalis by maceration in ethanol solvent. Crude extract was analyzed by high performance liquid chromatography then divided into two portions, first one characterized by Fourier transform infrared spectroscopy (FTIR) and investigated for anticoagulation activity by prothrombin time (PT) while the second portion subjected for fractionation using column chromatography (CG) and fractions were characterized by FTIR, each fraction was detected as anticoagulant agent. As results, coumarin crude extract eluted with retention time (7.6 min.) as standard coumarin by HPLC, molecular spectrums of fractions obtained by column chromatography were measured using FTIR. Anticoagulation activity of crude extract results were dose dependent pattern .We concluded that M. officinalis is a good source for coumarin and coumarin derivatives as shown in (HPLC) analysis while using column chromatography (CG) and (FTIR) considered as valuable techniques for separation and characterization.

Many years ago, plants were consumed for medicinal purposes which using as herbal or alternative homemade remedies, either as extracts, or as purified potential fractions with biological activities.The world health organization (WHO) establishes that 80% of human beings in growing counties (65% of the world's population) remain confidences in using bioactive derivatives utilized from plants (Karakas et al., 2012).M. officinalis (yellow sweet clover) is being as one of important therapeutic plants which were utilized since long times ago by several herbalists and physicians as a digestive, eye inflammation, hemorrhoids, stomach ulcer and disorders.Extract of dried petals of M. officinalis took part in pharmaceutical effects for the symptomatic treatment of venous-lymphatic in-sufficiency (Mahajan and Gajare, 2012).Coumarin considered as the major active compound in the M. officinalis crude extract and appear to function in various capacities in different plant defense mechanisms against insect herbivores and fungi (Murray et al., 1982) , in addition, this component has biological properties due to obstacle blood clot formation in the vessel (Klein, 2011).In this study, two techniques were used in analysing and detecting the presence of coumarin compound in crude extract ; High performance liquid chromatography technique (HPLC) by pass a pressurized liquid "referred to mobile phase" and a sample mixture through out a column contain adsorbent particles, causing the separation of the sample compounds because they have various degrees of interaction with the absorbent; while the other technique was Fourier Transform Infrared Spectroscopy (FTIR) briefly by a moving mirror inside the apparatus changes the distribution of infrared light that passes through the interferometer.The signal directly recorded, named an "interferogram", represents light output as a function of mirror position.An apparatus records this raw data into the final result.Anti-coagulant activity measured by prothrombin time kit (PT).

Preparation of plant extract
Leaves of M. officinalis were collected from gardens of Baghdad University, (10) gm of dried powdered plant sample defatted with (100) ml hexan for (3) hours.Then extracted in (100) ml of ethanol by maceration and crude extract evaporated at 40 0 C by using incubator and resultant was collected and stored at 4 0 C until use (Szewczyk and Koka, 2012).

Detection by High performance liquid chromotochraphy analysis
Chromatographic separation of coumarins was performed by apparatus Shimadzu LC-10 system with LC-10AD pump, UV-variable wave length detector (at 203 nm) .Sample passes through a column C-18, (5ìm, 250 mm x 0.6 mm).Separations were carried out with isocratic mode, acetonitrile: water (40:60; v/v) utilized as a mobile phase at a flow rate of (1) mL min-1; with an injection volume of 20 ìL, The HPLC system was operated at room temperature (23 ± 10C).The sample was injected with concentration 10mg / 1 ml while standard coumarin as 1mg/ml (Celeghini, et al., 2001).

Separation of coumarin derivatives by column chromatography
Separated active ingredients were added as three volumes of ether to one volume of crude extract in separating funnel then shaked very well , two layers obtained as upper ether layer and lower aqueous layer.
Column (3.1 x5) packed with suspension of silica gel and methanol, after the stability of the gel the column were washed with methanol then added ether layer to the column.Washed with solvent system consist of diethyl ether with 10% acetic acid.Fractions were collected as 1 ml for each tube with flow rate 12ml/hr.

Fourier transform infrared spectroscopy (FTIR) analysis
Chemicals that used in this study were purchased from Merck, BDH, Sigma Aldrich and Fluka companies.Melting point have been estimated using digital stuart scientific SMP3 melting point apparatus.FTIR spectra were established by Shimadzu FTIR-8400 Fourier transform infrared spectrophotometer using KBr discs in the (4000-600) cm -1 spectral range (Sliverstien and Webster, 1998).

Prothrombin Time assay procedure
A test tube contains 0.1 ml of plasma was kept in incubator for 3 to 5 min at 37°C after that 0.2 ml of Biolabo reagent (biolabo SA, France) was added to the test tube after warming the reagent at 37°C.Vary concentrations of M. officinalis coumarin extract were added separately to the test tube, the coagulation time or prothrombin time (PT) was estimated in seconds by stopwatch in addition to recording coagulation time of +ve and -ve control.Each test concentration was repeated three times and data average used.Normal saline used instead of extract in -ve control while EDTA as a +ve control (Govindappa et al., 2015

HPLC analysis
M. officinalis extract was analyzed by HPLC apparatus and coumarin compounds eluted retention time at 7.66 min.which were closed to retention time of Standard coumarin which appeared at 7.63 min.(Fig. 1 a & b).
As showed in (Fig. 1 a), the concentration of coumarin components in the plant extract is low due to small peak of eluted compounds compared with high peak of standard coumarin in (Fig. 1 b) although the concentration used of M. officinalis extract was 10 mg/ml while the concentration standard coumarin prepared with 1mg/ml.

Fourier transform infrared spectroscopy (FTIR) characterization of crude extract
M. officinalis crude extract confirmed the presence of phenolic and amino coumarin derivatives (7-hydroxy coumarin and 6-amino

Anti-coagulant activity
In this study, anti-coagulant activity of the crude extract was investigated by prothrombin time kit and results were showed by table (1) below:-Prothrombin time of treated blood (46 Sec., 140 Sec.and No-clot) with different concentration of M. officinalis extract (25, 50 and 75 µl) respectively, showed anti-coagulation activity by dose-dependent manner compared with the negative control which was 14 seconds while treated blood with EDTA as +ve control prevent clot formation.
Normally, blood should be stay as a fluid inside the vascular system while forming clot The results indicated that Dicourmal or bis 4-hydroxy coumarin gave the higher anticoagulant activity with (2800 sec.)due to presence of methoxyl group so the modification of 4-hydroxy coumarin by adding methoxyl group will increase anticoagulation effect (Venugopala, et al., 2013& Mathur andArora, 1963).dicumarol, inhibits the clotting mechanism by interaction the production process of an important factors which were produced by liver and necessary for vitamin K activity (Asif, 2015).
Present data shows that the following compounds (6-amino coumarin, mixture of 6-amino coumarin and 6-cyano coumarin, and 6-carboxylic coumarin ) compounds gave valuable activity with time as (935, 890 and 875 Sec.respectively) due to presence of nitrogen, cyano and carboxyl substituents in 6-position.
Coumarin derivatives consuming orally is effective treatment for a wide range of clot dependent disorders.Investigations have shown that oral consuming treatment is medicinally reactive against intravascular clot formation in patients suffering from thromboembolism and atrial fibrillation disorders in blood stream; this is prevention stroke and mortality in cases of operations of myocardial and heart valves.Otherwise, anticoagulant medications using with cases suffering from heart disorders dependent rheumatism due to inhibiting variety proteins causing activation clot formation.Continuously treated with heparin causing osteoporosis beside uncomfortable situation for the patients so the coumarins are used instead of heparin (Goodman & Gilman's, 2006).

CONCLUSION
Coumarins naturally found in M. officinalis consider as important phenolic compounds due to their clinically and biologically applications.HPLC proved as a useful tool for quality detection while FTIR is valuable for control molecular characterization of these compounds.
Coumarins shows significant anticoagulant potential, especially purified coumarin derivatives compounds.

Table 1 .
Anticoagulant activity of M. officinalis

Table 2 .
Anticoagulant activity of Coumarin and coumarin derivatives separated from M. officinalis extract by column chromotochraphy