Separation, Purification And Structure Elucidation Of Phenolic Compounds From Canarium Album L.

Chinese olive (Canarium album L.), a valuable resource for traditional medicine and food use, possesses some pharmacological functions such as anti-bacterium, anti-virus, anti-inflammation and detoxification. Previous study has showed that Chinese olive fruit is rich in phenolic compounds which were closely related to its organoleptic and pharmacological characteristics. However, up to date, reports on separation and identification of phenolic compounds from C. album were terribly scarce, and phenolic profile of C. album was still uncertain. Therefore, the purpose of the present work was to extract, isolate and purify the phenolic compounds from Chinese olive fruit, and elucidate their structures by chromatographic and spectrometric techniques, which will be of great significance for further utilization and pharmacological investigation of Chinese olive.First, chemical components of Chinese olive fruit cultivated from Fujian Minhou area were analysed. The results indicated that Chinese olive fruit pulp contained protein 1.7 g/100g and fat 1.1 g/100g. The main saccharides of C. album fruit were sucrose (635 mg/100g), fructose (232 mg/100g), glucose (52 mg/100g), raffinose (30 mg/100g) and maltose (7 mg/100g). The organic acids of fruit were malic acid (537 mg/100g), citric acid (64 mg/100g), tartaric acid (34 mg/100g), quinic acid (28 mg/100g), oxalic acid (24 mg/100g), fumaric acid (14 mg/100g) and acetic acid (11 mg/100g). The phenolic content of fruit pulp determined by modified Folin-Ciocalteus method was 1.5 g/100g, obviously higher than Mediterranean olive (Olea europaea L.) and other common fruits, while Chinese olive stone presented low phenolic content (0.2 g/100g). C. album kernels had relatively high fat (52.8%), protein (29.5%), iron and calcium contents. The kernel proteins were rich in essential amino acids. The kernel oils were rich in unsaturation fatty acids (73.3%), which showed that C. album kernels had great potential for use as food resources.Extraction of phenolic compounds from Chinese olive fruit was investigated by solvent digestion, supersonic-assisted and microwave-assisted methods. The results showed that 60~70% (V/V) acetone was the best solvent for phenolic extraction, but 60% (V/V) ethanol was preferable in industry application. The optimum conditions for solvent digestion were as follows: leaching time 1 h,temperature 20~30℃, material/solvent ratio 1:20. The optimal power and extraction time for supersonic assisted extraction were 80 w and 25 min, while that for microwave assisted extraction were 340 w and 30 s. The phenolic yields of three methods differed insignificantly (P>0.05), and their phenolic compositions were almost same. The extraction rate was up to 90%, and Chinese olive crude extracts containing 34% phenolics was obtained. In comparation with solvent extraction method, supersonic-assisted and microwave-assisted method were more rapid and efficient for extracting phenolics from Chinese olive fruit.Purification of phenolic crude extract from Chinese olive on macroporous resin was studied. Through static adsorption and desorption tests, AB-8 resin was chosen for the separation of phenolics due to its higher adsorption and desorption capacity. Then, dynamic adsorption and desorption experiment was carried out on an AB-8 resin packed column (Φ2.5 cm×30 cm) to obtain optimal separation parameters. The results revealed that the largest adsorption capacity of AB-8 was achieved when initial phenolic concentration was 10 mg/ml, feed flow rate was 2 ml/min and feed volume was 9 bed volume (BV). The saturated resin was first washed with 3.5 BV of water to remove impurities, then desobed with 2.5 BV of 90% aqueous ethanol at flow rate of 1 ml/min. The desorption ration of phenolics from resin was up to 95%, and the purity of phenolic in crude extracts was increased from 34% to 86%, higher than that patent reported (76%). The recovery of phenolics was about 74%.The phenolic compounds in crude extract from Chinese olive fruit were separated and purified by silica gel column, AB-8 macroporous resin column, polyamide column, TSKgel Toyopearl HW-40 column and thin layer chromatography, and the relative contents of phenolic compositions in crude extract were determined by HPLC. The results indicated that sixteen phenolic compounds were obtained from Chinese olive fruit, and by UV,IR,HPLC-ESI-MS,1D NMR (1H-NMR,13C-NMR,DEPT-135) and 2D NMR (1H-1H COSY,HMQC,HMBC) spectrometric techniques, phenolic compositions in crude extract were elucidated as followings: gallic acid (28.6%), HHDP hexose ester isomers (Ⅹ14.3%,Ⅻ11.6%), HHDP alkyl derivatives (Ⅴ4.1%,Ⅳ1.7%), methyl gallate (3.8%), brevifolin-carboxylic acid (3.2%), isocorilagin (3.1%), kaempferol-3-O-β-D-glucopyranoside (2.9%), 3-O-galloyl quinic acid butyl ester (2.6%), hyperin (2.3%), phenolic acid with Mw 348 (Ⅰ2.1%), 3,3′-dimethoxy-2,2′,4,4′-tetrahydroxy- diphenylformic acid (1.5%), ethyl gallate (1.3%), amentoflavone (1.1%) and sinapic acid (0.6%). Among them, 3-O-galloyl quinic acid butyl ester was one new phenolic compound, remaining compounds except gallic acid and hyperin were first identified in C. album...