"Jufeng" grape is one of the main cultivated varieties in China. Because of its large grain, large meat, moderate acidity and sweetness, it is popular with consumers. "Jufeng" grape is mainly processed into juice and wine. The by-product "Jufeng" grape skin formed in its processing and production process contains a large number of anthocyanins (mainly malvacaine), polyphenols and other active ingredients. The grape skin of "Jufeng" is usually abandoned, which causes environmental pollution and resource waste. "Jufeng" grape skin is the main raw material of natural pigment, but the research and development of this high-quality natural pigment resource is less, and the standard products related to malvacaine are scarce and expensive. Therefore, it is necessary to establish a simple method for the separation and purification of anthocyanins.
Xue Hongkun and Liu from school of engineering, Northeast Agricultural University Chai and Liu Cheng Hai et al. Used AB-8 macroporous resin -SephadexLH-20 dextran gel technology to separate and purify anthocyanins from 'Kyoho' grape skin and obtain a single high purity anthocyanin component. The structure of anthocyanins obtained after purification was identified by ultra performance liquid chromatography with three quadrupole time of flight mass spectrometry, and the anti-tumor activity of anthocyanin components was investigated.
1. Yield of anthocyanin from 'Jufeng' grape skin
According to formula (1), the yield of pigment I, III and IV are (0.108 ± 0.011)%, (0.053 ± 0.007)% and (0.038 ± 0.005)% respectively, while the theoretical yield of anthocyanin is 0.21% ~ 0.25% according to the content of "Jufeng" grape anthocyanin. Therefore, the purification and separation method is feasible in the preparation efficiency.
2. Results of UV-Vis analysis
After the sample 1 was separated and purified by Sephadex LH-20 gel, the pigment solution of "Kyoho" grape skin pigment was separated into 4 different color pigments by gel column. They were pigment I (pale pink), pigment II (Orange), pigment III (purple red) and pigment IV (pink) respectively, and the color bands were scanned by ultraviolet visible spectrum in the range of 200~700 nm. There was no UV absorption peak near 280 nm and 520 nm for pigment II. The results showed that pigment II was a non anthocyanin substance and no further study was conducted. However, the maximum absorption peaks of pigment I, III and IV are all around 280 nm and 520 nm. It can be seen that pigment I, III and IV are anthocyanins, and pigment III has a weak absorption peak at 305 nm, indicating that pigment III may be acylated anthocyanins.
3. HPLC analysis results
At 520 nm, six anthocyanins were detected in sample 1, and the peak time was in the range of 13-22 min. The peak time of 13.69 min and 21.84 min were the main components of anthocyanin in sample 1. The relative contents of the two components were 38.15% and 47.62%, respectively. The peak time of the other four components was 15.51, 16.99, 19.05 and 20.04 min respectively, and their relative contents were 2.57%, 4.08%, 4.05% and 3.53% respectively. Four anthocyanin components were detected in sample 2, the peak time was 13.63 min and 21.86 min, the relative content was 27.58% and 69.11%, the peak time of the other two peaks was 17.06 min and 19.09 min, the relative content was 2.08% and 1.23%, respectively. Comparing the components of anthocyanins in sample 1 and sample 2, it is found that the proportion of anthocyanins obtained by different purification methods is quite different.
4. Analysis results of triple quadrupole time of flight mass spectrometry by ultra high performance liquid chromatography
The results showed that the retention time of pigment I was 2.57 min. MS and MS2 information showed that the molecular ion peak [M +] was 595.4. Two fragment ions were detected in MS as M / Z 449.1 and M / Z 287.2, respectively. M / Z 287 is the characteristic ion of cyanidin. The fragment ([m-146] +) corresponds to the fragment of rhamnosyl lost in anthocyanin, while the fragment ([m-146-162] +) of M / Z 287 corresponds to the fragment of hexose lost in M / Z 449.
The retention time of pigment III was 4.87 min. MS and MS2 information showed that the molecular ion peak [M +] was 801, and the fragment ion M / Z was 331.1, 493.2 and 639.1, respectively. M / Z 331 is the characteristic ion of Mallow pigment, M / Z 493 is the hexose of Mallow pigment, M / Z 639 is the hexose of Mallow pigment, M / Z 639 is the hexose of Mallow pigment, and the fragment ([m-146] +) is the fragment obtained after the removal of a molecule of water by coumaric acid. The percentage of a440 nm / a λ Max is 13.2%. It is speculated that pigment III is 3,5-hexose.
The retention time of pigment IV was 6.35 min. the information of MS and MS2 showed that the molecular ion peak [M +] was 493 and the fragment ion was m / Z 331.1. M / Z 331 is the characteristic ion of Mallow pigment. The lost fragment 162 may be glucose and galactose. According to the literature, pigment IV is mallow-3-galactoside.
5. Inhibitory effect of anthocyanin on the growth and proliferation of HepG2 hepatoma cells and A549 lung cancer cells
The survival rate of HepG2 hepatoma cells and A549 lung cancer cells treated with three anthocyanins decreased significantly with the increase of the concentration of three anthocyanins (P < 0.05). When the concentration of anthocyanin was 50 mg / ml, the survival rates of HepG2 hepatoma cells and A549 lung cancer cells were (90.01 ± 2.03)%, (85.11 ± 1.86)%, (91.02 ± 1.55)% and (90.03 ± 1.43)%, (83.99 ± 1.89)%, (91.98 ± 1.52)% respectively, while when the concentration of anthocyanin was 600 At μ g / ml, the survival rates of HepG2 hepatoma cells and A549 lung cancer cells were (51.98 ± 0.87)%, (46.05 ± 0.92)%, (58.05 ± 0.84)% and (47.97 ± 0.89)% respectively (42.04 ± 0.82)%, (53.12 ± 0.91)%, the survival rates of HepG2 hepatoma cells and A549 lung cancer cells in the former were 38.03%, 39.06%, 32.97% and 42.06%, 41.95% and 38.86% lower than those in the latter, respectively.
6. Effect of anthocyanin on the invasion of HepG2 and A549 lung cancer cells
Compared with HepG2 hepatoma cells and A549 lung cancer cells control group, three anthocyanins treated HepG2 hepatoma cells and A549 lung cancer cells, found that the number of transmembrane cells decreased significantly, and the invasion rate decreased significantly. According to formula (3) (as shown in the figure below), the invasion rates of control group, pigment I, pigment III and pigment IV to HepG2 hepatoma cells and A549 lung cancer cells were 100%, (55.81 ± 1.69)%, (19.65 ± 1.24)%, (34.16 ± 0.97)% and 100%, (48.18 ± 1.62)%, (14.57 ± 0.79)% and (34.12 ± 1.02)% respectively. The analysis data showed that Three anthocyanins can inhibit the invasion of HepG2 and A549 lung cancer cells in the order of pigment III > pigment I > pigment IV. The results of this study are consistent with those of MTT experiment.
conclusion
After purification by AB-8 macroporous resin and Sephadex Sephadex LH-20, the pigment extracts from grape skin of Kyoho grape were obtained with 4 components. Among them, pigment II was non anthocyanin, pigment I, pigment III and pigment IV were respectively IV, -3-, rutin and mallow. The yield and purity of 5-diglucoside-coumarinyl and mallow-3-galactoside were (0.108 ± 0.011)%, (0.053 ± 0.007)%, (0.038 ± 0.005)% and 98.52%, 96.84% and 91.36%, respectively. However, after purification by AB-8 macroporous resin and polyamide resin, the components of anthocyanin were quite different under the same HPLC conditions Further study. To explore the effect of pigment I, III and IV on antitumor activity, it was found that three anthocyanins could significantly inhibit the proliferation and invasion of HepG2 hepatoma cells and A549 lung cancer cells, and significantly increase the apoptosis of cancer cells. The sequence of antitumor effect of three anthocyanins was pigment III > pigment I > pigment IV.
In this paper, the separation, purification, structure identification and antitumor activity of anthocyanins from "Jufeng" grape skin are from 41 volumes, 5 issues, 39-48 pages, food science, 2019. The authors are Xue Hongkun, Tan Jiaqi, Liu Chai and Liu Chenghai. DOI:10.7506/spkx1002-6630-20190115-165。 Click below to read the original text.
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Revised / edited by: Yuan Yi; editor in charge: Zhang Ruimei pictures from Baidu pictures and original articles