Isolation and Identification of Phenolic Compounds from Dianthus orientalis Wildly Grown in Iraq

The plant Dianthus Orientalis that belongs to the Caryphyllaceae family is one of the useful plants in Iraq. Its seeds are commonly used for toothache. This project provides the first comprehensive research done in Iraq and the world to study the phytochemicals and the methods of extraction and isolation of active constituents from Dianthus orientalis wildly grown in Iraq. The plant was harvested from Penjwin in AL-Sulaymaniyah city, Iraq in September 2019.The whole plant were washed carefully, dried in shade area for two weeks, and milled in a mechanical grinder to a coarse powder. The plant was defatted by maceration with hexane for 7days and dried after that extracted by cold extraction methods using 80% methanol solvent for 9 days then fractionation with chloroform, ethyl acetate and n-butanol to separate the active constituents according to the change in polarities. The chloroform, ethyl acetate fractions were used for identification and isolation of phenolic compounds by TLC, PTLC, HPLC and LC/mass, FTIR. Results of the phytochemical screening exposed the presence of, phenols in the plant extract. The phenolic compound (vanillic acid, coumaric acid, cinnamic acid, genistein, oleuropein) were separated and purified by PTLC. The isolated compounds were subjected to several chemical, chromatographic and spectral analytical techniques for their identification such as TLC, HPLC, FTIR and LC/mass.


Introduction
Dianthus L. is annual or perennial herb belongs to Angiosperm's family Caryophyllaceae, subfamily Caryophyllideae and tribe Caryophyllideae (1) . The family Caryophyllaceae is well known for ornamental flowering plants (2) . The unusual characteristic of the family is appearance of stable and endurable foam when parts of the plants are put into water and shaken. This behavior is due to the occurrence of high amount of saponins in the family (3) .

Collection of plant materials
Dianthus orientalis were obtained from Penjwin in AL-Sulaymaniyah city, Iraq in September 2019.The plant was identified and authenticated by Dr.Karzan Aumar Kadir /Department of Biology /College of Sciences/ University of Sulaimani The plant were washed thoroughly, dried under shade, and ground in a mechanical grinder to a coarse powder.

Extraction
The whole plant coarse powder 250gram was macerated with normal hexane for one week in conical flask 2000ml with shaking many times in the shade and then filter it, and then the organic layer was taken and dried in the shade the defatted powdered plant material was soaked in 1500ml methanol, with occasional shaking, at room temperature. After 3 days, the methanol soluble materials were filtered off and this method is repeated for three times (extraction will done in 9 days).The filtrate was evaporated to dryness under vacuum using rotary evaporator. A dark browngreenish residue was obtained. The residue twenty grams was suspended in 500ml water and partitioned successively with chloroform, ethyl acetate, and n-butanol (3x500ml) for each fraction. The first two fractions dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.

Isolation of phenolic compounds from the ethyl acetate fraction and chloroform fraction by preparative layer chromatography (PLC):
One gram of each fraction dissolve in 3 ml of methanol and applied on 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 solvent system (chloroform: acetone :formic acid)(75:16:1). The band had been scrapped off, eluted with methanol and then filtered; the filtrate evaporated to dryness, the band that separated from ethyl acetate fraction was symbolized as E1.

Isolation from the chloroform fraction by preparative layer chromatography (PLC):
Four bands were isolated from chloroform fraction utilizing the same procedure applied to the ethyl acetate fraction and using the same mobile phase (chloroform: acetone: formic acid) (75:16:1) The compounds were isolated from chloroform fraction were symbolized as C1, C2, C3, C4.

Identification of the isolated phenolic derivatives from ethyl acetate and chloroform fraction of Dianthus orientalis
The Compound that symbolized as E1, C1, C2, C3, C4 was identified by several methods including chemical, chromatographic, and spectral methods as: Spraying with 5% ethanolic KOH on TLC plate HPLC analysis HPLC technique (Knuaer, Germany) was applied for the detection of different constituents found in the ethyl acetate, chloroform fractions as flavonoids and phenolic acids, and for identification of the isolated compounds from Dianthus orientalis.The mobile phase contains 1% aq. Acetic acid solution (Solvent A) and acetonitrile (Solvent B), the flow rate was adjusted to 1 ml/min, the column was thermostatically controlled at 280 ˚C and the injection volume was kept at 20 μl. A gradient elution was performed by varying the proportion of solvent B to solvent A as shown in the table (1). The HPLC chromatograms were detected using a photo diode array UV detector at three different wavelengths (272, 280 and 310 nm) flow rate 1ml|min (17) .

FTIR
Identified chemical bands in molecules. IR spectra range of scanning was 4000-400 cm -1 LC/MS: Analytical LC-MS was performed using Agilente/System Joined to an Applied Biosystems API 2000 mass spectrometer .Mobile phase solvents acetonitrile and water A column of 0.19mm external diameter (75μm I.D.) and 200mm length was packed with Thermo Scientifice Hypersil Gold C18 with 5μm particle size. Samples were run under the following conditions: m/z range was 250 to 10001, 200K resolution, and dynamic exclusion set to 1 with a limit of 90 seconds.

A.Ferric chloride test
Positive due to formation of dark brown color B.NaOH Positive due to formation of yellow color .

3.Saponin test
Positive due to froth formation.

4.alkaloid test: Dragendorff's reagent
Positive due to formation of orange-brown precipitate

Identification of E1
Spraying with 5%ethanolic KOH on TLC plate give yellow colored spot. HPLC of isolated E1: the HPLC chromatogram of standard vanillic acid and isolated cpd E1were shown in figure (4), spectrum of std vanillic acid and isolated cpd E1 and as shown in figures (5).

FTIR
IR spectrum of isolated cpd E1 was showed in the figure (6) and interpretation of the bands in the table (3)

LC/mass
Analytical LC-MS was performed using an Agilent System joined to an Applied Biosystems API 2000 mass spectrometer. The LC-MS chromatogram of the isolated compounds E1 as in figure (7) .   In planeC-H bending 1110 C-O-C stretching 809 Out of plane C-H Aromatic bending 679 Out of plane C=C Aromatic bending

Figure 7. LC/MS chromatogram of isolated compound E1
All these data coincide with that reported for vanillic acid therefore compound E1 could be vanillic acid with mwt 168.1Gram/mol.   130 Figure 13.IR spectrum of isolated cpdC1  (14), spectrum of standard genistein and isolated cpd C2 was shown in figures (15).      Out of plane C-H Aromatic bending All these data coincide with that reported for cinammic acid therefore C3cpd could be cinammic acid

Conclusion
The following points were pinched based on prior findings; 1. Phytochemical screening of Dianthus orintalis widely grown in Iraq demonstrates the presence of various phytochemicals, which were separated from plant according to differences in their chemical nature. 2. The phenolic compounds: vanillic acid, coumaric acid, genistein, cinammic acid, and oleuropein were isolated from the plant. 3. isolated phenolic acids were identified by TLC, preparative TLC, HPLC,IR LC/Mass