| As a class of natural material, which is rich in various biological activities, plant polyphenol links closely with human life. It has effected on human digestion, nutrition, health and has been widespreadly used in the area of food, drug and cosmetic industry.In this paper, the extraction, isolation, anti-tumor activity and anti-bacterial activity of the polyphenols from fresh Laminaria japonica Aresch were studied systematically, and some characteristics, mechanism and application were also investigated. This work may provide primary data for further researching on the relationship between the structure and function of phlorotannins, as well as provide a new way for high-value utilization of kelp and exploiting some new anti-tumor medicine and food antiseptic.This study includes six parts of content as follows:1. Selection of measuement method of phlorotannins on exudation rate The kelp could be synthetically utilized to extract phlorotannins and other by-products. Based on comparison study on the two methods of determination phlorotannins, ferrous tartrate colorimetry and Folin-Denis method, ferrous tartrate colorimetry with PG as reference material was chosen because of its facility and credibility. Phlorotannins content of fresh kelp was slightly higher than dry kelp under the same extraction conditions.2. Determination of the optimal conditions for extraction of phlorotanninsAdopt fresh kelp as materials, ultrasonic and microwave extraction as pretreatment, extraction rate of phlorotannins as target, on the base of the single factor experiment and orthogonal test, the optimal extraction conditions of kelp polyphenols were researched. The results show that optimal extraction conditions of kelp polyphenols from Laminaria japonica Aresch are the ratio of sample weight to the solvent volume 1:7(g/mL), 85% ethanol, temperature 70℃, extraction times 2, extraction time 4h, and the exudation rate of kelp polyphenols is 2.08%.3. Investigation on anti-tumor activity and anti-bacterial activity of the crude extracts of brown algae phlorotanninsThe anti-tumor activity of crude extract of phlorotannins for human lung adenocarcinoma cell (A549), murine leukemic cell (P388), human hepatocellular carcinoma cell (BEL-7402) and human cervical cancer cell (Hela) had been determined respectively by MTT method in vitro. The results showed crude extracts has anti-tumor activity and the inhibitory rate were 27.40±4.71%, 43.44±1.86%, 30.20±1.16%, 29.68±2.61%, respectively at the concentration of 100μg/mL. The result of the microscope detection showed that the morphologic features of tumor cells were changed from the normal control group. Dosage-effect relationship showed the half-maximal inhibitory concentration (IC50) for P388 cell and BEL-7402 cell were 120μg/mL and >200μg/mL respectively. Moreover, the cytotoxic effect to P388 and BEL-7402 seems to be dose-dependent and time-dependent and the inhibitory rate for P388 was superior to that of BEL-7402.The anti-bacterial activity of crude extracts of phlorotannins had been determined by flat plate slotting to inhibit growth method in vitro, respectively. The results of bacteriostatic experiment showed that 2 species of gram-positive cocci and 5 species of gram-negative bacilli were inhibited by crude extract which had wide antimicrobial performance. Dosage-effect relationship was dose-dependent and the minimal inhibitory concentration (MIC) was 10μg/mL～40μg/mL. Meanwhile, the inhibitory effect of crude extract to Escherichia coli, Pseudomonsa aeruginosa was relatively obvious and fungi such as Penicillium, Candida albicans were also inhibited.4. Isolation and purification process of the extract crudesThe crude extracts could be divided into 4 kinds of bioactive fractions (A1, A2, B1, B2) after extraction with organic solvent, adsorption and desorption with AB-8 macroporous resin, column chromatography with sephadexLH-20. All bioactive fractions were compared in each purification process. Screening results of antitumor and antibacterial activity showed A2 > A1> B2 > B1 and A2 > B2 > A1 > B1, respectively. And the inhibitory rate of fraction A2 was 61.96±7.02% and 40.47±8.70% to BEL-7402 and P388 respectively at the concentration of 70.42μg/mL, and antibacterial activity was also increased compared to front parts.5. Primary research on the physical and chemical characteristics of phlorotanninsThe physical and chemic characteristics of phlorotannins such as dissolubility, light refraction, characteristic color reaction, pH stability, heat stability and spectral feature were determined primaryly. The results showed that the dissolubility of phlorotannins in CH3OH, CH3CH2OH, C2H5COOC2H5 were better than that in H2O, CH3Cl, C2H5OC2H5. Refractive index of phlorotannins in different solvents was C2H5COOC2H5 > CH3CH2OH > CH3COCH3 > CH3OH > H2O > CH3Cl = C2H5OC2H5. Characteristic color reaction showed kelp phlorotannins had the commonness of phenols. Otherwise, kelp phlorotannins had pH stability, thermal stability between pH 2.0～11.0 and 40℃～100℃. Ultraviolet absorption spectra of component A2 was similar to PG and phloroglucinol dehydrate. IR spectras of fraction A1, fraction A2 fraction B2, PG and pyrogallol had obvious expansion and vibration of phenol hydroxyl, aromatic rings. Kelp phlorotannins compound is preliminarily deduced as similar structure of PG or pyrogallol.6. Primary discussion of anti-tumor activity and anti-bacterial activity mechanism of phlorotanninsThe change of cell morphology was observed by fluorescent microscopy, and scavenging ability of free radicals and carcinogenic agents were discussed. At the same time, we analysed cell apoptosis, cell cycle by flow cytometry and detected UV absorption. The results showed that microscope detection the morphologic features of P388 and BEL-7402 tumor cells were changed from the normal control group after treated 48h. The scavenging rate of·OH, O2·-, NO2–were 75.37±5.68 %, 55.48±4.35%, 50.51±6.46%, respectively. The anti-tumor activity of phlorotannins was related with the cell apoptosis and cell proliferation cycle, especially obvious of A2 apoptosis peak, but the molecular mechanisms remain unclear. Ultraviolet absorption of the system containing phlorotannins and bacteria was increased at 280nm, especially for G-. |
PDF Full Text Request