摘要
茶叶是我国具有悠久历史和深刻文化底蕴的特色经济作物,也是当前世界三大饮料中最具有生命力,最受消费者欢迎的文明健康饮料。茶叶中含有丰富的化学成分,包括多酚类、咖啡因、茶色素、茶多糖、茶皂素和茶氨酸等多种对人体健康有益的天然产物,具有抗氧化、抗突变和防癌抗癌等广泛的生物学活性,其开发应用前景也十分广阔。
本文基于绿茶中的主要功效成分茶多酚所具有的防癌与抗癌作用,采用现代生物活性物质提取与分离技术,获得纯度较高的茶多酚,并以两种水溶性壳聚糖为载体,通过离子凝胶法制备纳米茶多酚,优化纳米茶多酚的制备工艺,筛选纳米茶多酚的最佳粒度和包封率,并分析纳米粒的粒径分布和体外表征。采用生物学前沿技术和手段,建立人肝癌HepG2肿瘤细胞培养模型以及肿瘤动物模型,研究纳米茶多酚的抗肿瘤作用,通过显微镜和透射电镜的形态学观察、酶标仪以及流式细胞术等方法,探讨纳米茶多酚的抗肿瘤作用机理。
主要研究结果如下：
1.绿茶采用超微加工技术可以显著提高茶多酚的有效溶出。本研究结果显示,绿茶原料经超微碎粉后茶多酚的溶出量由原来的156.34±4.22 g/kg提高到272.67±9.56 g/kg。茶多酚的分离纯化主要采用有机溶剂法与大孔树脂法相结合进行,获得的茶多酚纯度为93.75%,产品得率达到6.14%。高效液相色谱分析表明,纯化的茶多酚与有机溶剂提取的粗多酚相比,儿茶素的含量较高,且咖啡因的含量有所降低。
2.以羧甲基壳聚糖和壳聚糖盐酸盐为载体,利用分子自组装技术在溶液中制备纳米茶多酚,通过Box Behnken试验设计和响应曲面法优化纳米茶多酚制备工艺,获得粒径较小且包封率较高的纳米茶多酚。动态光散色仪检测结合透射电镜观测显示,纳米粒形态不规则,但分布较为均匀,其粒径大多在200 nm-400 nm范围内,Zeta电位值为30 mV左右,在溶液中呈现相对稳定的胶体状态。以壳聚糖制备的纳米粒是一种优良的缓释载体,纳米茶多酚在溶液的显示了较强的缓释性能,在PBS为7.4的缓冲溶液中纳米茶多酚的体外缓释时间可达48 h。纳米茶多酚经冷冻干燥后室温下保存,稳定性良好。
3.主要通过溶血性试验、细胞毒性试验和急性毒性试验研究纳米茶多酚的体内外安全性。结果显示,纳米茶多酚无溶血和凝集现象；对L929细胞的相对增殖度为(100.03±0.06)%,毒性分级结果显示为无毒性。急性毒性试验显示,最大灌服剂量为3 g/kg BW纳米茶多酚以及2.55 g/kg BW勺空载纳米粒均对小鼠无明显影响。由此表明,纳米茶多酚以及壳聚糖纳米粒载体在此剂量范围内均为安全的,对机体无毒性。
4.体外抗肿瘤作用结果表明,纳米茶多酚(0.5 mg/mL-1.0 mg/mL)对人肝癌细胞HepG2有明显的生长抑制作用,并呈剂量与时效关系。透射及扫描电镜结果表明,纳米茶多酚作用于人肝癌HepG2细胞后,细胞变小,胞膜皱缩,细胞核固缩,致密浓染并伴有核型的不规则变化,出现核碎裂等凋亡的典型特征,表明纳米茶多酚可明显诱导人肝癌细胞HepG2细胞凋亡。
5.流式细胞术检测纳米茶多酚对HepG2细胞周期与细胞凋亡影响,结果表明,浓度为1.0 mg/mL勺纳米茶多酚处理HepG2细胞24 h后,早期凋亡及晚期凋亡的细胞分别占总检测数的23.83%及3.30%,明显高于未给药对照组2.44%和2.89%,相同浓度的纳米茶多酚可使HepG2细胞的细胞周期阻滞在G0/G1期(对照组为0.62%,给药组为5.54%),阻断细胞周期向S期进行,阻止细胞增殖,诱导细胞凋亡。
6.通过建立小鼠H22肝癌移植瘤模型,并结合体内抗氧化实验及病理技术,测定小鼠移植瘤体积、抑瘤率以及瘤块组织形态学变化,研究纳米茶多酚的体内抗肿瘤功效及初步探讨其作用机制。试验结果显示,纳米茶多酚对小鼠H22肝癌移植瘤具有显著的体内抗肿瘤作用,6.0、12.0 mg/kg剂量纳米茶多酚处理后抑瘤率分别达到51.50%和58.17%；体内抗氧化实验结果表明,纳米茶多酚可显著提高荷瘤小鼠血清T-AOC活性,同时降低肝脏H202含量以及SOD活性；病理检测结果表明,纳米茶多酚使肿瘤组织周边结缔组织增多,呈现细胞凋亡特征。
Tea has a long history and a deep cultural heritage as a kind of characteristic economic crops in China. At the same time, it also is the most civilized and healthy beverages in current three welcome drinks by consumers in the world.Tea is rich in a lot of chemical components, including polyphenols, caffeine, tea pigment, tea polysaccharide, tea saponin, theanine and other natural products beneficial to human health, such as antioxidant, anti-mutation and anti-cancer biological activity, and its development prospects are also very bright.
As the major effective ingredients green tea polyphenols possessed the antitumor activities. Tea polyphenols with high purity was prepared from green tea using the advanced extraction and separation technology of bioactive substances. Then, chtosan loaded tea polyphenols nanoparticles (CS-TP NPs) were prepared by ionic gelation method using two water-soluble chitosans as carriers of tea polyphenols. The preparation conditions were optimized and selected to obtain best size and encapsulation efficiency, and analyzed the size distribution of nanoparticles and in vitro characterization.
The models of human hepatocarcinoma cell HepG2 and tumor animal were established by advanced biological technology and means to study antitumor activity of the CS-TP NPs. The methods of morphological observation by microscopy and transmission electron microscopy, microplate reader and flow cytometry were used to analysis the antitumor mechanism of CS-TP NPs.
The main research results were as follows:
1. Extraction in advance using superfine comminuted processing technology can significantly improve the effective dissolution of tea polyphenols. The results of this paper showed that comparison with raw materials, the dissolution volume of tea polyphenols in superfine comminuted green tea from the original of 156.34±4.22 g/kg increased to 272.67±9.56 g/kg. Separation and purification of tea polyphenols using organic solvent combined with macroporous resin to obtain a purity of 93.75% polyphenols, the product yield was 6.14%. HPLC analysis showed that compared to crude polyphenols, the purified polyphenols has a high content of catechins and low caffeine.
2. Based on carboxymethyl chitosan and chitosan hydrochloride as a carrier, CS-TP NPs was preparated in solution by molecular self-assembly techniques, and optimized through the Box-Behnken experimental design and response surface methodology to obtain tea polyphenols nanoparticles with smaller particle size and high encapsulation efficiency. Detection with dynamic light scattering and observation by transmission electron microscopy (TEM) showed that the nanoparticles had an irregular shape, however, the distribution was more uniform, and the range of particle size was mostly in the 200 nm～400 nm. Zeta potential value was about 30 mV, and it present relatively stable colloidal state in solution. Nanoparticles prepared with chitosan was an excellent delivery carrier, and CS-TP NPs showed a strong sustained release in solution, the release time of polyphenols in nanoparticles was up to 48 h in PBS 7.4 buffer solution in vitro. CS-TP NPs were stable in room temperature when they were freeze-dried.
3. The safety of CS-TP NPs was systematically evaluated by haemolyticus test, cytotoxicity test and acute toxicity experiment in vitro and in vivo. The results showed nanoparticles didn't emerge haemolysis and conglomeration. The relative growth rate of L929 cells in CS-TP NPs was (100.03±0.06)%, and the toxicity grading results showed the non-toxic. Acute toxicity test showed that the maximum dose 3 g/kg BW of CS-TP NPs and 2.55 g/kg BW of the empty nanoparticles all have no effect on mice. It is suggested that CS-TP NPs and chitosan blank nanoparticles were safe and non-toxic to organism in this dose range.
4. The results of anti-tumor effect in vitro showed that CS-TP NPs (0.5 mg/mL～1.0 mg/mL) significantly inhibited the growth of human hepatoma cell HepG2 with a dose and time dependent relationships. TEM and scanning electron microscope (SEM) results showed that HepG2 cell performed the typical apoptosis character of cell lessening, cytomembrane collapse, karyopyknosis, inregular changes of caryotype and nuclear fragmentation after addition of CS-TP NPs, which indicated CS-TP NPs could induce the apoptosis of human hepatocarcinoma cell lines HepG2.
5. The effect of CS-TP NPs on cell cycle and cell apoptosis of HepG2 was measured with flow cytometry. After the treatment for 24 h at 1.0 mg mL-1, the apoptotic cells at the early and late stage accounted for 23.83% and 3.30% of the total counted cells, significantly higher than the control of 2.44% and 2.89%, respectively. Under the same concentration, CS-TP NPs could keep the cell cycle of HepG2 cells to G0/G1 stage (control 0.62%, treatment 5.54%), block cell cycle to S phase, inhibit the proliferation and induce the apoptosis of the cancer cells.
6. Mice transplanted with H22 liver tumor were established to research the antitumor effect of CS-TP NPs. The tumor volume, inhibition rate and morphological changes in the block in mice were detected and the antioxidant activity and pathological technology were utilized to detect the antitumor mechanism in vivo. The results showed that CS-TP NPs had significant antitumor effect on mice transplanted with H22 liver tumor in vivo. Mice were treated with 6.0 mg/kg and 12.0 mg/kg CS-TP NPs respectively, the tumor growth inhibition rate were 51.50% and 58.17%. Antioxidant results in vivo show that CS-TP NPs could significantly increase the activities of GSH-Px and T-AOC in mice serum, and decrese the content of H2O2 and SOD in liver. Results of tumor pathological section indicated that the connective tissues in the surrounding of tumor were increased after treatment with CS-TP NPs, and presented cell apoptosis state.
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