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Oxidative stress refers to the state of oxidative imbalance in the body and is also the main cause
of liver damage.
When oxidative stress occurs, the production of excess oxygen free radicals can cause damage to cells, resulting in damage
to oxidative stress.
Studies have shown that reactive oxygen species (ROS) are products of the normal metabolism of oxygen and an important substance that induces oxidative stress, and excessive ROS can affect the stability of the genome and lead to a variety of chronic diseases
.
Purple calyx jade hairpin, also known as purple flower jade hairpin, is a perennial root herb of the genus Hosta of the lily family (Liliacea), and its whole grass or root can be medicinally used and edible
.
At present, studies have confirmed that the flavonoids, saponins and polysaccharides in the hosta have certain anti-inflammatory and bacteriostatic activities, but there are few studies
on the antioxidant mechanism of root polysaccharides (HVRP) of the jade hairpin.
Jinshan Wu, Huimin Liu* from College of Food Science and Engineering, Jilin Agricultural University, and Hongzhang Liu* from College of Life Sciences, Jilin Agricultural University, aimed to explore the effect of HVRP on oxidative stress damage induced by tert-butyl hydrogen peroxide (t-BHP) in HepG2 cells, analyze the antioxidant mechanism of HVRP at the cellular level, and provide a theoretical reference
for the application of HVRP in the field of functional foods.
1.
Single-factor test results
The effects of material-liquid ratio, extraction temperature and extraction time on the extraction rate of polysaccharides are shown in Figure 1, and the extraction rate reaches the maximum when the material-liquid ratio is 1:30.
With the increase of extraction temperature, extraction first increased and then decreased, and the extraction rate reached the maximum at 80 °C.
With the extension of extraction time, extraction first increased and then decreased, and the extraction rate was the largest
at 2 h.
When the extraction temperature exceeds 80 °C and the time exceeds 2 h, the polysaccharide will decompose under the influence of continuous high temperature, resulting in a decrease
in the extraction rate.
2.
Response surface optimization test analysis results
Response surface experimental design and result analysis
According to the results of Table 3, the multiple regression fitting of the experimental data was performed by Design-Expert software, and the quadratic multiple regression model equation was obtained: Y=24.
44+1.
21A+0.
46B+0.
55C+1.
35AB-2.
77AC+0.
78BC-3.
15A 2-0.
55B 2-2.
12C2
。 The statistical analysis results of quadratic model regression are shown in Table 4, the regression model is highly significant (P<0.
000 1), the misfit term P=0.
879 6, the coefficient of determination R<b14>2=0.
993 4, the corrected R2Adj shows that the regression equation fits well, the experimental method has high confidence, and the model can be used to analyze and predict the extraction conditions
of polysaccharides.
It can be seen from the F value that the influence of each factor on the extraction rate of HVRP is extraction temperature> feed-liquid ratio > extraction time
.
Determination and verification of optimal extraction processes
The optimal extraction process conditions for HVRP were 83.
63 °C, extraction time of 2.
92 h, material-liquid ratio of 1:30.
60, and the predicted extraction rate was 24.
9%.
According to the actual situation, the extraction temperature was adjusted to 84 °C, the extraction time was 3 h, the material-liquid ratio was 1:31, and the HVRP extraction rate was 23.
9% and the error was 1%, indicating that the feasibility of the model was high
.
3.
Physical and chemical properties of HVRP
The extraction rate of HVRP after defatting and deproteinization treatment was 9.
29%.
The mass fraction of reducing sugars in HVRP was 11.
17% (111.
66 mg/g), the protein mass fraction was 0.
6% (5.
96 mg/g), and the content of uronic acid was high, and the mass fraction was 12.
31% (123.
12 mg/g).
The results showed that the mass fraction of uronic acid in the polysaccharide in Northeast hosta was determined by carbazole method as 20.
36%~52.
76%; The mass fraction of uronic acid in lily polysaccharides reached 12.
89%.
4.
Analysis results of HVRP structural characteristics
It can be seen from Figure 2A that the infrared spectrum of HVRP has one strong absorption peak at 3 422 cm-1, which is caused by O-H telescopic vibration.
The absorption peak at 2 916 cm-1 was C—H telescopic vibration; The absorption peaks at 1 745, 1 634 and 1 446 cm-1 were caused by C=O symmetric and asymmetric tensile vibrations, indicating the presence of uronic acid in HVRP, which was consistent with the physical and chemical analysis results.
The absorption peak at 1 088 cm-1 was caused by the ether bond of the pyran ring, indicating that HVRP may be pyranoses; Absorption peaks at 813, 614 cm-1 indicate that they may contain α sugars
.
As shown in Figure 2B, with the increase of NaOH concentration, the maximum absorption wavelength of HVRP and Congo red reagent continued to decrease, and the maximum absorption wavelength was not redshifted compared with Congo red, and the continuous decrease of the maximum absorption wavelength may be because HVRP is randomly coiled conformation in aqueous solution, and hydrogen bonds are continuously destroyed
by alkaline solution.
From this, it is judged that HVRP does not have a triple helix conformation
.
5.
In vitro antioxidant activity of HVRP
It can be seen from Figure 3 that with the increase of HVRP mass concentration, its antioxidant activity first increased and then stabilized, showing a certain dose-effect relationship, when the mass concentration of HVRP was 100 μg/mL, the DPPH radical scavenging rate was 63.
93%, the hydroxyl radical scavenging rate was 70.
31%, and the superoxide anion radical scavenging rate was 83.
45%.
HVRP exhibits strong antioxidant capacity in vitro, and its antioxidant activity may be related to
the presence of certain uronic acids in HVRP.
6.
HepG2 cell survival rate
The intracellular toxicity of HVRP was detected by MTT method, as shown in Figure 4A, 0~100 μg/mL HVRP was not toxic to the growth of HepG2 cells, and when the mass concentration was 200 μg/mL, the viability of HepG2 cells was inhibited
.
Therefore, 5, 25, 100 μg/mL were selected for cell experiments
.
In order to investigate the mechanism of HVRP on oxidative damage in hepatocytes, t-BHP was used to establish a model
of oxidative damage.
As shown in Figure 4B, HepG2 cell viability decreased
significantly with increasing t-BHP concentration.
Fitted by GraphPad Prism 7.
0 software, the semi-inhibitory concentration (IC50) of t-BHP action was (217.
52±20.
94) μmol/L
.
Therefore, t-BHP at a concentration of 220 μmol/L was selected to establish a model
of cellular oxidative damage.
7.
Effect of HVRP on oxidative damage to the antioxidant capacity of cells
65 times
that of the normal group.
HVRP treatment significantly reduced ROS levels, which were 1.
27 times
higher than those in the normal group in the HV100 group.
As shown in Figure 5B~F, compared with the model group, HVRP can significantly or very significantly reduce the MDA content, significantly or significantly improve the SOD, CAT and GPx activity and increase the GSH content
.
The above results show that HVRP can help cells resist oxidative damage by increasing the activity of antioxidant enzymes in oxidative damage
cells and reducing ROS levels.
8.
Effect of HVRP on gene expression of Keap1/Nrf2/ARE signaling pathway in oxidative damage cells
It can be seen from Figure 6 that compared with the model group, HVRP treatment can significantly upregulate the expression of GST, HO-1, NQO1, JNK, Keap1 and Nrf2 genes in the oxidative stress pathway, and when the mass concentration of HVRP treatment is 25 μg/mL, the expression of the above genes is significantly upregulated, and the expressions of GST, HO-1 and NQO1 downstream genes of antioxidant signaling pathway are 4.
36, 4.
67 and 5.
28 times
that of the model group, respectively.
9.
Effect of HVRP on the expression of Keap1/Nrf2/ANE signaling pathway proteins in oxidative damage cells
。 Nuclear transfer of Nrf2 is a key step in inducing NQO1 and HO-1 expression, and HVRP treatment leads to a dose-dependent decrease in the level of Nrf2 in the cytoplasm and a dose-dependent increase in the level of Nrf2 in the nucleus, indicating that HVRP can promote nuclear transfer of Nrf2 (Figure 7B).
Compared to the model group, HVRP treatment increased the levels of kinase p-JNK/JNK upstream of the Nrf2/ABOVE pathway (Figure 7C).
The above results are similar to those in the literature [32], suggesting that JNK phosphorylation may promote Nrf2 nuclear transfer, thereby regulating the expression
of phase II detoxification enzymes NQO1 and HO-1 downstream of the Nrf2/ANE pathway.
Conclusion
In this experiment, the HVRP extraction process was optimized, and the optimal extraction conditions were as follows: extraction temperature 84 °C, extraction time 3 h, material-liquid ratio 1∶31, and the HVRP extraction rate under these conditions was 23.9%.
In vitro antioxidant experiments showed that HVRP had strong free radical scavenging activity
.
The study of antioxidant mechanism proves that HVRP protects HepG2 cells from t-BHP-induced oxidative damage by activating the oxidative stress Keap1/Nrf2/
AREing pathway.
In summary, this study can provide a scientific basis
for the development and application of HVRP as a natural antioxidant in functional foods.
01 Corresponding author
Professor Liu Hongzhang, a member of the Communist Party of China, Ph.
D.
, is a doctoral supervisor of the College of Life Sciences, Jilin Agricultural University, and has served as the head of the Department of Horticulture, the dean of the College of Chinese Materia Medica, the secretary of the Party General Branch of the College of Biotechnology, and the dean of the College of
Life Sciences.
Jilin Province Key College and Jilin Province Key Discipline Leader, Jilin Province Excellent Teaching Team Leader, Jilin Province Characteristic Professional Leader, National Excellent Video Open Course "Changbai Mountain Characteristic Plant Resources" and Jilin Province Excellent Course "Biological Resources Science" host, Jilin Province Fruit Variety Approval Committee and Changchun Horticultural Society Chairman and other positions
。 He founded the doctoral authorized discipline of "crop resources science", and mainly established the first first-level science discipline "biology" master's authorized discipline of Jilin Agricultural University and built it into a key discipline in Jilin Province; He mainly teaches undergraduate courses "Biological Resources Science" and "Introduction to Modern Agriculture and Technology" and postgraduate courses such as "Biological Experiment Design and Statistical Analysis" and doctoral students' "Crop Resources Science" and "Biological Resources Diversity and Its Protection", mainly engaged in the research of characteristic horticultural plant resources and biotechnology in Changbai Mountain, presided over more than ten projects of the Ministry of Science and Technology, the Ministry of Agriculture and the Department of Science and Technology of Jilin Province, published more than 150 academic papers, presided over the selection and breeding of 8 new varieties, and trained more than 80 master's and doctoral students.
Edited 12 textbooks and books; He has won 7 first, second and third prizes of Jilin Province Science and Technology Progress Award and 5 second and third prizes of Jilin Province Teaching Achievement Award, and was rated as Jilin Province Advanced Individual in Teacher Morality, Changchun City Pacesetter of Teacher Virtue and Famous Teacher
of the 7th Jilin Province Higher Education School.
D.
, and a master's supervisor
of the College of Food Science and Engineering, Jilin Agricultural University.
Mainly engaged in the research of molecular nutrition and food chemistry, he has achieved a series of innovative results
in the isolation and extraction, functional research and delivery of natural plant active ingredients 。 He has presided over 6 national, provincial and ministerial projects such as the sub-projects of the National 13th Five-Year Key R&D Plan and the Jilin Provincial Natural Science Foundation Project, published more than 40 papers in domestic and foreign academic journals such as Journal of Agriculture and Food Chemistry, Food Chemistry, Food & Function, Nutrients, Food Control, Food Science, etc.
, and won 8 provincial and ministerial scientific research awards.
Associate editor of 1 textbook
.
He has served as a reviewer
for the Journal of Agricultural and Food Chemistry, Food Research International, Phytomedicine, Food & Function, Journal of Functional Foods, etc.
02 First author
Wu Jinshan, a member of the Communist Party of China, is a 2020 master's student at the College of Food Science and Engineering, Jilin Agricultural University, with research interests in food science and functional food
.
As the first author, he published an SCI paper in the International Journal of Biological Macromolecules (JCR2 area), and won many honorary titles such as postgraduate scholarships and outstanding graduate cadres
.
This article "Protective Mechanism of Hosta Polysaccharides on Cellular Oxidative Stress Damage" is from Food Science, Vol.
43, No.
17, 2022, pp.
138-146, authors: Wu Jinshan, Huang Rong, Liu Shuying, Liu Huimin, Liu Hongzhang, Liu Jingsheng
.
DOI:10.
7506/spkx1002-6630-20210602-026
。
