Phytochemical Study of some Flavonoids Present in t h e Fruits of Two Ammi L . Species Wildly Grown in Iraq

Ammi species belong to the family Umbellifereae that provide a host of bioactive compounds (mainly coumarins and flavonoids) of important biological activities, like prevention and treatment of heart and vascular disease and some types of cancer. Literature survey revealed that there was no study concerning Ammi flavonoids in Iraq. Ammi majus and Ammi visnaga, which are wildly grown in Iraq, were chosen for this study. This study concerned with extraction, identification, isolation, and purification of some biologically important flavonols quercetin and kaempferol from the fruits of Ammi majus and Ammi visnaga. Extraction of these flavonols was carried out using 85% methanol and 90% ethanol. Identification of these flavonols quercetin and kaempferol was done using thin Layer chromatography (TLC) where different solvent systems had been tried. Ultra violet (UV) Light and iodine vapor where used for detection. This identification was further augmented by using high performance Liquid chromatography (HPLC) and then these flavonols were isolated and purified. The most suitable extraction, isolation and purification procedures of flavonols were fully described in this study. The identification of isolated flavonols (quercetin & kaempferol) was carried out using melting point (M.P.), Thin Layer chromatography (TLC), and infrared spectroscopy (IR).This study confirms the presence of quercetin and kaempferol in Ammi majus & an Ammi visnaga fruit, the percentage of quercetin was higher in Ammi visnaga than Ammi majus, while the percentage of kaempferol was higher in Ammi majus than Ammi visnaga.


Introduction
Ammi is a genus of 3-6 species of flowering plants in the Apiaceae Family; They are native in southern Europe, northen Africa and south west Asia . (1,2)Ammi majus and Ammi visnaga are one of the most important medicinal plant species in the world ( figure 1 , figure 2 ). (3)In Iraq Ammi majus usually found in fields and gardens and by the side of channels, often as weed of cultivation.It's collected from Kut, Baghdad, Hawija and many other areas.While Ammi visnaga are widely distributed in Erbil, Mousl, Baghdad, Sulaimania and Kirkuk in north of Iraq . (4)orresponding author E-mail : dr.ksaour@yahoo.comReceived : 13/10/2009 Accepted : 13/2 / 2010

Figure (1) photography of Ammi majus figure (2) photography of Ammi visnaga
Epidemiological data, clinical investigations, and animal studies provide strong evidence that the main active constituents of this genus are: Coumarin and their derivatives, flavonoids, volatile oil and fixed oil . (5)All the flavonoids described in Ammi species can be classified into flavonols (quercetin , kaempferol , isorhamnetine ) and flavones (apigenin , luteolin , chrysoeriol ) these types of flavonoid have been show to be powerful antioxidant and free radical scavengers. (6- 8)Among these flavonoids : quercetin which is a flavonol type of flavonoids that has been shown to help prevent the development of a variety of condition related to inflammation and free radical damage, including arthritis, allergies, macular degeneration, heart disease, gout, and various forms of cancer . (9,10)The other flavonol found in these plants is the kaempferol, which is a strong antioxidant and help to prevent oxidative damage of our cells, lipids and Deoxyribonucleic acid (DNA).It seems to prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein and the formation of platelets in the blood.Studies have also confirmed that Kaempferol acts a chemopreventive agent which means that it inhibits the formation of cancer cell (9,11) Materials and methods

A. Plant materials
The plant materials (dried ripe fruits) of Ammi majus L. (Apiaceae) was collected during the months of March and April from local fields about 2 Km south of Kut.While the fruits of Ammi visnaga (Apiaceae) were collected from the botany garden in the College of Pharmacy, Uuniversity of Baghdad.(Pharmacognosy department).Both of them were identified by the department of pharmacognosy, College of Pharmacy, University of Baghdad and authenticated by National Iraqi Herbarium, Botany Directorate at Abu -Ghraib.A 50 gm of powdered fruits of Ammi majus and Ammi visnaga were packed in a thimble of soxhlet extractors.500 ml of petroleum ether (b.p 40 -60 c 0 ) was used in a soxhlet extractor for three hours to get rid of lipids and fat. (9)The defatted powdered fruits after drying over night were extracted with 500 ml of 85% methanol for 12 hours by use of soxhlet apparatus for flavonoids extraction as free and glycosides. (6,7)The methanolic extract was then filtered and then a portion had been taken and kept aside for further work.The remaining portion of filtrate was evaporated under reduced pressure at a temperature not exceeding 40 °C (F1).The methanolic extract (F1) residue was weighted and subjected for identification and purification procedures.To continue the extraction process, the previously extracted plant materials (fruits) of both Ammi majus and Ammi visnaga were dried at room temperature, and then were extracted again with 500 ml of 90 % ethanol for 12 hours by use of reflux apparatus for extracting the remaining flavonoids. (6,7).The contents of the flask were filtered while hot and the ethanolic extract was allowed to cool at room temperature.A portion of ethanolic extract had been taken and kept aside for further work.The remaining portion of ethanolic extract was concentrated under reduced pressure to dryness (F2) and the dried extract (F2) was weighted and subjected for identification and purification procedures.Later on, equal volumes of methanolic extract and ethanolic extract of both Ammi majus and Ammi visnaga were mixed together to give methanolic ethanolic extract which also concentrated under reduced pressure to dryness (F3).The dry extract (F3) was weighted and subjected for identification and purification procedures.Figure (3 and 4)

C. Isolation and purification of quercetin and kaempferol
After locating of quercetin and kaempferol of the extract in comparison with standards, preparative thin layer chromatograghy was done to isolate and purify them.The portion of mixture methanolicethanolic extract (F3) was used to obtain the final product by applying it as a concentrated solution in arrow of spots using capillary tube and the standard sample was applied in one side of the plate.the mobile phase used was S 1 = chloroform : Aceton : formic acid ( 75 : 16.5 : 8.5 ) the separated compound appear as a band identified using u.v light detection method.The band corresponding to the standard was scrapped out and collected in a beaker and eluted with gentle heating and filtered.Then the filtrate was evaporated to dryness under reduced pressure to give yellow precipitate.The precipitate then recrystallized using hot ethanol and maintained for TLC and measuring melting point and Infra red spectrum.

D. Qualitative and quantitative estimation of flavonoid using HPLC technique
Qualitative and quantitative estimations of quercetin and kaempferol were done by using Knauer/Germany High Performance Liquid Chromatography (HPLC) in which identifications were made by comparism of retention time obtained at identical chromatographic conditions of analyzed samples and authentic standards.The HPLC conditions are listed in the following table (1):

Results and Discussion
Three extraction portions were obtained from the experimental work in which methanolic extract (F1), ethanolic extract (F2) and the third extract portion, which is a mixture of methanolicethanolic extract (F3).
Results showed that the third extract portion was the best, because the amount of both extract and flavonoid were higher than the two other extract portions.As shown in (table 2).

Identification of Flavonoids by TLC
TLC of the extracts (F1,F2,F3) obtained from dried ripe fruits of Ammi majus and Ammi visnaga , confirms the presence of quercetin and kaempferol in all extraction portions in comparison with standards.As represented in table (3) and figure (5) .

Isolation and Quantitative determination of quercetin and Kaempferol by preparative TLC
From the investigation of the fruits extracts (fractions) of Ammi majus and Ammi visnaga on TLC plates, it was found that quercetin present in both fruits extracts but higher in Ammi visnaga while kaempferol also present in both fruits extracts but higher in Ammi majus.The percentage of both quercetin and kaempferol were obtained by weighing of the isolated compounds as shown in table (4)

Characterization of the isolated kaempferol and quercetin TLC
Both isolated compounds appeared as a single spot having the same color and R f value as that of reference standards.

Measuring melting points
The isolated compounds were identified to be quercetin and kaempferol from their sharp melting point.Since one of these compound showed a melting point of 314 -316 c 0 compared to quercetin melting point 316 c 0. The other compound showed a melting point of 275 -276 c 0 compared to melting point 276 -278 c 0 for standard kaempferol.

IR.
The IR spectra of each isolated quercetin and kaempferol were recorded as KBr disc using IR spectra photometer BUCK scientific model 500, gave identical results were compared with authentic standard samples; which confirm that our isolated compounds are quercetin and kaempferol, (17) as shown in figures (6-7).

HPLC analysis
Generally, the percentage of quercetin and kaempferol is higher in methanolicethanolic extract than in methanolic extract and the methanolic extract contains higher amount of quercetin and kaempferol than ethanolic extract.In addition, Ammi majus had shown different percentages of quercetin and kaempferol than Ammi visnaga.Since the percentage of quercetin in Ammi majus was lower than the percentage of quercetin in Ammi visnaga in all extracts.While the percentage of kaempferol in all Ammi majus extracts was higher than the percentage of kaempferol in Ammi visnaga extracts.The result indicates that the HPLC method was efficient for qualitative identification and quantitative determination of quercetin and kaempferol.as show in table (5) and figures (8-13).

Conclusions
Phytochemical investigation of Ammi majus and Ammi visnaga fruits, grown in Iraq revealed the presence of important group of medicinal natural products belong to flavonoid derivatives.Quercetin and kaempferol were isolated and identified in Ammi majus and Ammi visnaga fruits by using simple and reproducible TLC and HPLC method.The flavonoid, quercetin and kaempferol are found in the fruits of Amm majus and Ammi visnaga, were quercetin present in large quantities in the fruits of Ammi visnaga than that of Ammi majus, while kaempferol present in the fruits of Ammi majus in large quantities than in Ammi visnaga.

Figure 4 :
Figure 4 : Schematic procedure for the extraction method of flavonoids from Ammi visnaga fruits.