Tocopherols and flavonoíds of SOS-7 halophyte

Halofito es una semilla oleaginosa codificada como SOS-7 (semilla oleaginosa Salicomia, séptimo año de selección). Los tocoferoles del aceite de halofito SOS-7 fueron determinados directamente en el aceite usando cromatografía líquida de alta presión acoplada a detector fluorescente. Se encontró que el aceite contenía 710 ppm de tocoferoles totales. Los tocoferoles alfa, beta, gamma y delta, se encontraron a niveles de 38.2,1.0, 58.7 y 2.1%, respectivamente. Nueve glicósidos flavonoides fueron aislados e identificados de las semillas y se encontró que pertenecen a la clase flavonol dentro de los flavonoides. Estos flavonoles fueron identificados como: quercetina-3,7-diglucosido, quercetina-3-glucosido-7-galactosido, quercetina-3-soforosido, quercetina-3-glucosido, quercetina-3-galactosido, isorannetina-3, 7-di-glucosido, isorannetina-3-glucosido, kampferol-3, 7-diglucosido y kampferol-3-glucosido.


INTRODUCTION
The world-wide demand for vegetable oils has greatly increased, this led to the search for new seed oils.SOS-7 Halophyte, is a new oil seed crop, as climatic tolerant, has been successfully tried in desert areas and irrigated with sea water (1).In continuation to the previous studies in this laboratory (2) (3) (4) on characterizing halophyte, SOS-7 oil produced from locally cultivated halophyte plant, it was advisable to carry out further studies dealing with tocopherols and flavonoids to complete the picture of the seed constituents.
Tocopherol content of an oil is of equal interest to fatty acid composition in evaluating the quality of the oil.Tocopherols are the best-known and most universally distributed antioxidants, constitute fat-soluble vitamin E. Four forms, alpha, beta, gamma and delta tocopherols (alpha-T, beta-T, gamma-T and delta-T) (Fig. 1) are found in vegetable oils.
Gamma-T has been found to be a more effective antioxidant than beta-T which is in turn effective than alpha-T (5).
The term flavonoid is applied to compounds with a structure based on flavone (2-phenyl chromone).Flavone consists of two benzene rings (A and B) joined together by a gamma pyrone ring (Fig. 2).The structures, commonly found in nature, are flavan-3 ols (cathechins), flavonols, flavanones, flavanonols and flavan-3.4-diols.Flavonoid compounds are widely distributed in higher plants.They were isolated from different parts of plants with variations In the types of compounds found according to various anatomical tissues of any plant (6).It has been reported that flavonoids have antioxidant effect on unsaturated fatty acids (7).As far as one knows, nothing has been reported about the flavonoids of SOS-7 halophyte in the current literature.

Tocopherols (T)
The oil was extracted from the ground seeds using CHCl3/MeOH (2:1, v/v) and the solvent was evaporated under vacuum.Standard tocopherols (St.T), alpha-T , beta-T, gamma-T and delta-T were purchased from E. Merk, Germany.

High pressure liquid Chromatography (HPLC)
Tocopherol constituents were determined directly in the oil using Toyo Soda-CCPM, high pressure liquid chromatograph, coupled to Hitachi-650-10-S fluorescence detector with an exitation and emmission wave lengths of 290 and 325 nm respectively.The analysis was carried out under the following operating conditions : column, 150 x 4.6 mm packed with silica gel YMC-A-012 SIL; solvent, hexane: isopropyl alcohol (100: 0.5 v/v) at flow rate of 1-2 ml/min; sample size, 20 microlitre (1-1.5% oil in hexane) and chart spead, 5mm/min (8).The amounts of the resolved tocopherol components were estimated from the measured areas under the appropriate peaks (PA) in comparison with those of the standard tocopherol solutions (St.T) according to the following equation (8) (9) (10).P.A. of St.T. X mg/ml (St.T)

Flavonoids
The solvents, n-hexane, chloroform, ethanol, methanol and water were successively used in extracting the different constituents from the ground seeds.The extracts were concentrated under reduced pressure and applied on Whatman paper No. 1 to determine their flavonoid constituents.A two dimensional chromatographic technique was used with solvent systems n-butanol/acetic acid/water (BAW 4:1:5, v/v/v) and 15% acetic acid in the first and second directions respectively.Paper chromatography showed the absence of flavonoids in n-hexane and chloroform extracts (negative results with AICI3 spray reagent).The other extracts gave positive results with AICI3 spray reagent and they all had the same flavonoid compounds.The three extracts (ethanol, methanol and water) were combined and concentrated under reduced pressure.The concentrated extract was applied on a cellulose column, and eluted with ethanol containing increasing amounts of water.The collected fractions were further separated into nine single components by applying elution technique on Whatman paper No.S.The isolated compounds were inves-tigated according to the standard methods of identification (11) (12) (13) (14).
All the authentic samples (markers) of the flavonol compounds used in this paper were separated from the egyptian flora and identified by the researchers in the laboratory of Plant Taxonomy and Egyptian Flora, National Research Centre, Cairo.Standard sugars were purchased from BDH Chemicals Ltd., England.

Tocopherols
Tocopherols are important antioxidants which also function as vitamin E. It is known that tocopherols are superior radical chain-breaking antioxidants (15) compared to synthetic ones.Several methods have been developed for the analysis of tocopherols in vegetable oils.It was found that HPLC analysis of tocopherols is a sensitive and selective method.In the present work, HPLC analysis of crude SOS-7 halophyte oil, using a fluorescence detector indicates that this oil is rich in tocopherols and contains as much as 710 ppm total tocopherols .Four tocopherol isomers, alpha-T (38.2%), beta-T (1.0%), gamma-T (58.7%) and delta-T (2.1%) were detected.Their relative retention times were 3.35, 5.65, 6.0 and 10.4 min respectively.From these results it can be seen that SOS-7 halophyte oil is rich in gamma-T which is superior as antioxidant (5).

Flavonoids
All the nine isolated compounds appeared as brown spots under UV light which changed to yellow on exposure to ammonia vapour.Complete acid hydrolysis of the nine compounds (1-9) gave quercetin, kaempferol and isorhamnetin as aglycones together with glucose and galactose as sugar moities which means that these compounds belong to the 0-flavonol glycosides.Acid hydrolysis also showed that compounds 1,2,3,6 and 8 are disaccharide glycosides while compounds 4, 5, 7 and 9 are monosaccharide glycosides .The Rf values, chemical and UV data of the isolated compounds are recorded in Tables I, II and III respectively .
UV data (Table III) show that compounds 1, 2, 6 and 8 were occupied in positions 3 & 7. Occupation of position 3 was confirmed by the stability of the UV spectrum after addition of NaOMe.However, position 7 was confirmed to     Further investigation was carried out on compound 3 by H2O2 oxidation which gave rise to a disaccharide.This disaccharide was confirmed to be sophorose after co-chromatographed with authentic sugar samples.

Compounds
From the above data it can be concluded that compounds 1, 2, 3, 6 and 8 could be identified as quercetin-3,7diglucoside, quercetin-3-glucoside-7-galactoside, quercetin-3-sophoroside, isorhamnetin, 3-7-diglucoside and kaempferol-3-7-diglucoside.be occupied by the absence of the shift in band II with sodium acetate compared with that of methanol and also with the absence of a small peak at 320 nm with NaOMe.

Monosaccharide Glycosides (Compounds 4, 5,7 and 9)
The results of the chemical investigation, complete and mild acid hydrolysis, of compounds 4, 5, 7 and 9 are recorded in Table II.It can be noticed that glycosides 4 and 5 gave quercetin as an aglycone and glucose and galactose, respectively, as sugar moieties on complete acid hydrolysis.However, compounds 7 and 9 gave isorhamnetin and kaempferol as aglycone moities respectively and glucose as sugar moiety.The types of the aglycones and sugars were confirmed by co-chromatographing the products of hydrolysis with authentic samples using different solvent systems (Table II).
Table III shows the UV data of compounds 4, 5, 7 and 9 and the data are in agreament with those reported for the flavonol monosaccharide glycosides.
From all the results discussed above, it can be generally concluded that SOS-7 halophyte is rich in natural antioxidants namely, tocopherols and flavonoids. .-Aca-