Separation and Identification of Phenolic Acid from Borago officinalis (F:Boraginaceae) Cultivated in Iraq

139 Separation and Identification of Phenolic Acid from Borago officinalis (F:Boraginaceae) Cultivated in Iraq Ashwaq T. Kareem and Maha N. Hamad *Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq  Abstract The plant Borago officinalis, which belongs to the Boraginaceae family and Celebrated as borage, is one of the useful medicinal plants cultivated in Iraq. It was used in olde medicine in Iraq, Irane, Syria and Europe for management of various diseases. It is commonly used as a tonic, tranquilliser, management of cough,sore throat , pneumonia,urinary tract infection, rheumatoid arthrites antioxidant, and anticancer. This project provides the first comprehensive research done in Iraq to study the phytochemicals and the methods of extraction and isolation of active constituents from Borago officinalis cultivated in Iraq. The plant was harvested in spring from AL-Rifai, Nassiriyah city/ IRAQ in February 2019. The aerial parts were washed carefully, dried under dark for two weeks, and milled in a mechanical grinder to a fine powder.The plant was extracted by cold extraction methods using 85% methanol solvent for three days then Fractionation with petrollium ethere, chloroform, ethyl acetate and n-butanol(n.b) to separate the active constituentse according to the change in polarities. The ethyle acetate fraction and n-butanol fraction were used for identification and isolation of phenolic compounds by TLC,PLC,HPLC and LC/mass. Results of the phytochemical screening exposed the presence of, phenols, tannins, fatty acid, in the plant extract. The phenolic acid(Sinapic acide, Rosmarinic acides, Caffeic acid) were separated and purified by PLC.The isolated compounds were subjected to several chemical ,chromatographic and spectral analytical techniques for their identification such as TLC, HPLC,UV andLC/mass.


Introduction
Borage (Borago officinalis L.) is an annual plant belonging to the family. Boraginaceae. It originates from Western regions of Mediterranean area and grows nearly in whole America, Europe, Canada and Iran (1,2) . The plant grows during November to January and reaches a height of 70 to 100 cm (3,4) Its stem is shielded with hairs that secrete a strong smell nearly the aroma of fresh cucumbers while on the tops of the shoots there are the starshaped inflorescences which initially are pink, later turn blue, seldom white (5,6) .
Aerial partse have been used in old medicine in Iraq as atonic, tranquillizer, management of cough, pneumonia, sore throat, swelling and inflammatory diseases. The leaves and flower possess biological activities for cancer and heart diseases prevention (7) and have antibiotic properties (8) , condense cardiovascular diseases (9) and provide benefits for improving healthe due to their various biological events (10) .
Results reported by previous studies shown the presences of phenolics acid, flavonoids (Quercetin, Myricetin, Luteolin and Rutin) and isoflavonoid besides, the dominant individual fatty acids of methanolic extract as Oleic acid which is an unsaturated fatty acid (omega-9), linoleic fatty acids (omega-6i) and Hexadecanoic acid. Thet methanolic extract was more biologically active than ethanolic extract (11) .

Plant collection
Borago officinalis plant was harvested from AL-Rifai, Nassiriyah city/ IRAQ in February 2019. The aerial plant was driede in the shadow for two weeks and powdered. The plant was identifiede and authenticated by Prof. Dr Israai Mohammed Department of Biology /College of Sciences/ University of Baghdad.

Extraction and fractionation of the different active constituent
Two hundred and fifty grams of the powdered plant material was soaked in 1500ml, with 85% methanol and shaking, at roome of temperature. After three days, the methanol soluble materials was filtered off. The filtrate was evaporated until dryness unders vacuum using a rotatory evaporator. A dark greenish residue was obtained. Then suspended in 500ml water and partitioned successively with petroleum ether (B.p. 30-60 ℃), chloroform, ethyl acetate, and n-butanole (3×500ml) for each fraction the first three fractions dried over sodium sulfate anhydrous, filtered, and evaporated to dryness (13,14) . The scheme of fractionation is shown in (Figure 2). (14 ) Identification of phenolic compounds in Borago officinalis plant extract 1. Preliminary phytochemicals showing of the phenolic compounds using a methanolic extract from the plant using a NaOH test, lead acetate, and ferric chloride test (15,16) . 2. Isolation and purification of phenolic compounds from the fractions ethyl acetate and n.butanoleei by preparative layer chromatography. Isolation of phenols was done by using preparative TLC; 2 grame of each fraction dissolve in 10 mlof methanole and applied to 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 S1 solvent system. The band had been scrapped off, eluted with methanol and then filtered, the filtrate evaporated to dryness, in a vacuum as shown in Figure 3 and 4. 3. Thin-layer chromatography. In this qualitative identification, a ready-made aluminium plates of silica gel of 245 with developing solvent systems were used for detection the plant phenols in fractions ethyl acetate and n.butanoleei with standards, as listed in Table (1).

Borago officinalis plant active constituents
In this studyi, cold extraction method was done by 85% absolute methanol to extract the active constituenti depend on the nature of these active constituents.Each 250 g of plant extract yielded 32 g residue

Preliminary qualitative phytochemicale analysis
The results of the phytochemical analysis of polyphenol in methanolic crude extract given in (  Table 2). The isolation of phenols were done by using preparative TLC, in jar contained the S1 solvent system. As in Figure 3

Figure4
. Preparative thin-layer chromatography plate for n.b fraction on silica gel GF254 developed in the S1 system, detection by UV light at 366nm. Table 3. TLC profile of isolated compound (cpd) number 1 compared with standard rosmarinic acide using the mobile phase solvents ( S1, S2, S3).

Solvent system
Rf of standard Rosmarinic acid..

HPLC analysis
The result gained from HPLC analysis method. 1. The retention time of Rosmarinic acide standard match with a retention time of isolatedi compound number 1 and UV spectrum of separated compound number 1 match with UVs spectrum of Rosmarinic acide standard as in Figure 14and 15 and Table 6 . 2. The retention time of the isolated compound number 2 match with a retention time of Sinapic standard and UV spectrum of the separated compound number 2 match with standard Sinapic acide as in Figure 16 and 17 and Table 6. 3. The retention time of the isolated compound number 3 match with a retention time of Caffeic acidi standard and UV spectrum of the separated compound number 3 match with Caffeic acid standard as in Figure 18 and 19 and Table 6.      Retentiontime of Caffeic acid standard. Retention time for isolated cpdnumber3. 10 10

LC/mass
The result gained from LC/mass.