| Genipin,a kind of iridoid natural material,has rich research value,whice can be prepared by enzymatic hydrolysis of geniposide with β-glucosidase or microbial fermentation of gardenia.In the field of edible pigments,gardenia blue pigment,a natural blue pigment,can be produced through the reacted of genipin and amino acids.For many natural pigments,gardenia blue pigment becomes a hot research spot,owing to its good solubility,stability and strong coloring ability.In addition,genipin is also the main medicinal ingredients of Chinese herbal medicine gardenia,and it has a significant effect in the anti-inflammatory,cholagogic,liver-protective,reducing blood fat,etc.In recent years,many research confirmed that genipin shows certain effect in anti-tumor,its non-toxic dose can effectively inhibit breast cancer,liver cancer,cervical cancer and other cancer cell proliferation and migration.In this paper,we investigated the process of preparation of genipin from gardenia through enzymolysis.Then,the related application of genipin were further explored.Including the optimization of the synthesis of gardenia blue pigment with amino acid and genipin,moreover the apoptosis of colon cancer cells induced by genipin was explored.The specific research contents and results are as follows:(1)Ultrasound method was used to extract geniposide from gardenia,and the optimum extraction conditions were as follows: liquid ratio was 15: 1,ultrasonic time was 22 min,ethanol concentration was 44%,ultrasonic temperature was 44 ℃,and extraction times selected 1 times,respectively.Furthermore,it is found that the relative standard deviation(RSD)value of 3.08% between our experimental value and the predicted value of the response surface model,which proves that the model is well fitted,and the optimum-technological conditions are stable and reliable.(2)Separation and purification of crude geniposide was carried out by macroporous adsorption resin and alumina column chromatography to obtain exquisite geniposide with purity up to 95%;Next,enzymatic hydrolysis of geniposide to produce genipin with β-glucosidase was carried out.The optimum conditions were as follows: the pH value of reactant solution was 4.5,the hydrolysis time was 2h,the hydrolysis temperature was 40 ℃,and the ratio of β-glucosidase to geniposide was 16:1.The flat yield of genipin can reach 92.87% by this method.Further through the extraction,recrystallization to obtain genipin which could be purified to 97%.(3)The synthesis of gardenia blue pigment was carried out by using the microwave synthesis technique.The detail optimized microwave synthesis process was as follows: the microwave temperature was 100 ℃,the ratio of arginine to genipin was 2:1,and the synthesis time was 20 min,the color value of gardenia blue pigment can reach 83.8 by this mehod.Furthermore the pH,light and thermal stability of gardenia blue pigment were studied.The results displayed that gardenia blue pigment was very sensitive to high temperature and strong light.With the increase of temperature and the intensity of light,the degradation rate of gardenia blue pigment gradually accelerated,and the strong acid and alkali environment also accelerated the degradation of gardenia blue pigment.Finally,both the photodegradation and thermal degradation of gardenia blue pigment were found to be in accordance with the second-order kinetics model.(4)By MTT assay,we found that the proliferation of HCT116 colon cancer cells were significantly inhibited by genipin,and the inhibitory effect showed high levels of time-dependent and concentration-dependent,further by DAPI fluorescence staining,AnnexinV-FITC/PI double staining and the detection of ROS and MMP in colon cancer cells after intervention with genipin.The results showed that genipin significantly induced apoptosis of colon cancer cells,and this apoptosis process was associated with ROS-mediated mitochondrial apoptosis pathway. |
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