Background[1]
Hydroxytyrosol has anti-inflammatory effects, dilates blood vessels and antibacterial effects, can prevent atherosclerosis, and can reduce the incidence of certain cancers (such as breast, prostate, endometrium, digestive tract); because hydroxytyrosol has It has strong antioxidant activity and the effect of reducing the incidence of cancer, so it has great medical prospects. Research has found that various extracts from olives have certain antibacterial properties. Through comparison, it was found that low concentrations of hydroxytyrosol can have a significant inhibitory effect on Gram-negative bacteria. This is because hydroxytyrosol can penetrate The bacterial cell mucosa decomposes bacterial mucopeptides and causes damage to the bacterial mucosa. The antibacterial activity of oleuropein is significantly weaker than that of hydroxytyrosol. This may be because the glycoside structure of oleuropein is connected to the hydroxyl group of hydroxytyrosol. Structurally, it is difficult to penetrate bacterial mucosa and shows weak antibacterial properties.
Preparation[2]
Method 1:
Preparation of hydroxytyrosol and its rapid nitration method:
(1) Extraction-Extraction Parallel: Weigh a certain amount of sun-dried olive pomace powder, and use a mixed biphasic solvent integrating extraction-extraction. The ratio of olive pomace to solvent is 1:10~ 50 (w/v), the extraction temperature is room temperature about 20-25°C, the extraction time is 1 to 60 minutes, and the ultrasonic power is 100 to 1100W. Perform ultrasonic extraction-extraction, and then use a separatory funnel to collect the upper layer of the solution. Ethyl acetate layer. The above-mentioned biphasic polar solvent is a mixture of ethanol-water-ethyl acetate.
(2) Column separation: The collected liquid in (1) is concentrated by rotary evaporation at 25-50°C and 150 rpm to obtain a certain amount of concentrated liquid, which is separated by silica gel resin, first adsorbed for 5-10 minutes, and then used with an appropriate proportion of Elute with a polar mixed solvent and collect 6 to 8 column volumes of the target collection solution.
(3) Rotary evaporation: Rotate the target collection liquid to dryness at 25-50°C, and the content of hydroxytyrosol can reach more than 90%.
(4) Nitrolation: Collect concentrated hydroxytyrosol and mix it with NaNO2 dissolved in weak acid. At room temperature, with microwave assistance, react for 1 to 8 minutes for rapid nitration to synthesize 4-nitro. Hydroxytyrosol.
The reaction formula of the rapid nitrolation of hydroxytyrosol to synthesize 4-nitrohydroxytyrosol in the present invention is as follows:
(5) Drying: Extract the reaction solution finally obtained in (4) with ethyl acetate, add anhydrous Na2SO4 to remove water, and perform rotary evaporation and concentration at 25-50°C, and vacuum drying to obtain 4 – The yield of nitrohydroxytyrosol can reach more than 90%, and the content can reach more than 95%.
The biphasic mixed solvent used in the parallel extraction-extraction described in step (1) is an ethanol-water-ethyl acetate mixture, with a volume ratio of 1:1:1 to 2:3:5.
The polar mixed solvent in step (2) is chloroform-methanol, with a ratio of 9:1 to 1:1.
The rotary evaporation conditions in step (3) are: the temperature is 40~90°C, the vacuum degree is 0.06~0.09Mpa, and the rotation speed is 100~300rmp.
The molar ratio of hydroxytyrosol and NaNO2 used in the nitration process in step (4) is 1:2~2:1, the microwave power is 100~1000W, and the stirring speed is 100~500rpm.
In step (5), the number of ethyl acetate extractions is 3 to 5 times, 60 to 80 mL each time. The vacuum drying conditions are: temperature 25 to 50°C, vacuum degree 0.06 to 0.1Mpa, time 12 to 18 hours.
Method 2:
Weigh 2.5kg of p-hydroxyphenylethyl alcohol, dissolve it in 100kg of 0.1M pH=6.5 phosphate buffer, add 5kg of ascorbic acid, add 1L of enzyme solution (specific activity 500U/mg), stir and react at 50°C for 60 minutes. The reaction solution passes through the nanofiltration membrane, and the dialysate passes through the styrene resin column (about 100kg resin). Wash with water until the effluent is free of hydroxytyrosol spots and is detected by TLC. The water wash is concentrated to 50kg and extracted 5 times with 100kg ethyl acetate. , the ethyl acetate liquid was concentrated to recover the solvent, and 2.5kg of light yellow viscous substance was obtained, with a hydroxytyrosol content of 88.3%.
Method 3:
Take the purified polyphenol oxidase solution, embed it in chitosan and cross-link it with glutaraldehyde to form, and measure its activity of 50U/mg; take 5kg of p-hydroxyphenylethyl alcohol and dissolve it in 2000L 0.05mol/L phosphate buffer (pH value 6.5), add 10kg ascorbic acid, stir to dissolve, add 1kg immobilized enzyme, stir and react at 50°C for 60 minutes, the resulting reaction is also centrifuged to recover the immobilized polyphenol oxidase, and the reaction solution is passed through nanofiltration membrane, the dialysate passes through a styrene resin column (about 200kg of resin), and is washed with water until the effluent is free of hydroxytyrosol spots using TLC. The water wash is concentrated to 100kg, and extracted 5 times with 100kg of ethyl acetate. The ethyl acetate solution The solvent was concentrated and recovered to obtain 4 kg of light yellow viscous substance with a hydroxytyrosol content of 91.2%.
Main reference materials
[1] Xu Chao, Gao Junchao, Zhang Jian, & Jiang Shende. (2010). Research on the synthesis process of hydroxytyrosol. Fine Chemicals, 27(12), 1209-1212.
[2] Jin Yuanbao, Dang Jianzhang, Dai Jianguo, Jingang, & Yu Huahong. (2011). Research progress on separation and purification methods of hydroxytyrosol. Food Science and Technology (7), 174-177.
[3] Zhang Xiaomei. (2008). Study on the cellular and molecular mechanisms of the biological effects of hydroxytyrosol. (Doctoral dissertation, Dalian Medical University).
