Effect of insect tea on D‐galactose‐induced oxidation in mice and its mechanisms

Abstract Insect tea is a traditional Chinese drink that contains abundant bioactive substances. In this study, the preventive effect of Insect tea on D‐galactose‐induced oxidation in mice was studied. The serum, liver, and spleen of mice were measured by biochemical and molecular biological methods, which showed that Insect tea could increase the biochemical indexes of the thymus, brain, heart, liver, spleen, and kidney in mice with induced oxidative damage. Insect tea can increase the levels of SOD (superoxide dismutase), GSH‐Px (glutathione peroxidase), and GSH (glutathione) and decrease the levels of MDA (malondialdehyde) in the serum, liver, and spleen of mice with oxidative damage. Pathological observation also confirmed that Insect tea could inhibit oxidative damage of the liver and spleen tissue caused by D‐galactose in mice. Further molecular biological experiments also showed that Insect tea could upregulate the mRNA and protein expression of Cu/Zn‐SOD (cuprozinc‐superoxide dismutase), Mn‐SOD (manganese superoxide dismutase), CAT (catalase), HO‐1 (heme oxygenase‐1), Nrf2 (nuclear factor‐erythroid 2 related factor 2), γ‐GCS (γ‐glutamylcysteine synthetase), and NQO1 (NAD(P)H dehydrogenase [quinone] 1) in the liver and spleen of oxidized mice. Insect tea has a good preventive effect on D‐galactose‐induced oxidation in mice, and the effect is better than vitamin C, an antioxidant. Insect tea is rich in isochlorogenic acid A, quercetin, rutin, hesperidin, neochlorogenic acid, and cryptochlorogenic acid. The combination of these bioactive substances has good antioxidant effects. Thus, Insect tea is a functional food with a good antioxidant effect that has value for future development and utilization.


| INTRODUC TI ON
Insect tea is a type of special tea beverage that is produced in China; Insect tea is different from ordinary tea, as it is healthy or medicinal tea. It is a special tea beverage made by insects that is excreted in insect droppings after eating tea leaves (Li & Zhou, 2005). Hydrillodes morose Butler, Nodarianiphona Butler, Aglossa dimidiate Haworth, Herculia glaucinalis L., and Fujimacia bicoloralis Leech have been found to produce Insect tea (Figure 1). The larvae of these insects feed on the leaves of Kuteng tea, Chemical Tree Baije, Trifolium pratense, and other plants and excrete droppings. After collection, the series of processed products represent caterpillar tea products (Jiang, 2000).
Insect tea contains 17 mineral elements, including K, Mg, Ca, Na, Fe, Mn, and Zn, among which approximately 10 elements are essential trace elements in the human body, and the contents of Fe, Zn, Ca, and Mg are higher than the levels of some famous teas (Yang & Li, 2011). Additionally, different Insect teas are rich in crude protein, crude fiber, fat, tea polyphenols, caffeine, sugar, vitamins, and amino acids (Guo, Xu, Wen, Huang, & Wang, 2008;Zhou, Feng, Zhu, & Zhao, 2015). Insect tea, as a traditional drink and Chinese medicine, has the functions of reducing and eliminating fever, detoxifying, strengthening the stomach, and helping digestion. It has good effects on diarrhea, epistaxis, gingival bleeding and hemorrhoid bleeding (Feng, Luo, & Zhao, 2013).
Oxygen stress occurs gradually and causes persistent damage to individuals. Oxidative stress will lead to and aggravate many diseases, including hypertension, type 2 diabetes, atherosclerosis, and dementia (Buford, 2016;Chard et al., 2017;Kitada, Ogura, & Koya, 2016). Excessive redox-active free radicals can cause oxidative damage of biological macromolecules, leading to oxidative stress in the body, accompanied by the occurrence and development of random oxidative stress, increased production of hydrogen peroxide by mitochondria, and increased oxidative damage in the body (Rao, 2009). Redox regulation is an important issue in the study of oxidative stress. Maintaining redox balance and regulating redox-related genes are new strategies to alleviate oxidative stress (Hohensinner et al., 2018). D-galactose is a commonly used senescent agent in research that can be used to establish an oxidative stress animal model. A small amount of D-galactose can be converted into glucose and will participate in metabolism, but a large amount of D-galactose will lead to the disorder of cell metabolism, changes in the activity of oxidase in tissues and cells, and the production of many superoxide anions and oxidative products, resulting in oxidative damage to the structure and function of biological macromolecules and ultimately leading to oxidative stress (Li et al., 2016). The oxidation model of D-galactose was established to verify the antioxidant effect of antioxidant active substances. It has been gradually applied to the research and development of antioxidant health products.
Studies have shown that natural foods have strong antioxidant and free radical scavenging abilities due to their structural characteristics. Phenolic hydroxyl structures, especially those in catechol or pyrogallol, are easily oxidized quinone structures. They have a strong ability to capture free radicals such as reactive oxygen species, including lipid free radicals produced by oxidation, which can reduce or prevent oxidation in tissues (Li, Xia, Yang, & Zhong, 2013).
In animal and human clinical studies, natural foods have good antioxidant effects and can protect the body from oxidative stress-induced damage (Carluccio et al., 2003;Li et al., 2013). Particularly, for SOD, GSH-Px, and CAT, which are important antioxidant enzymes in the body, natural foods can effectively increase their vitality in the body and play an antioxidant role (Sharma et al., 2016).
In this study, effective active substances were extracted from Insect tea and applied to mice with D-galactose-induced oxidative damage. The effects of Insect tea extract on the serum and tissues of mice with D-galactose-induced oxidative damage were observed.
The mechanism of Insect tea extract in preventing oxidation was F I G U R E 1 Production process of Insect tea elucidated by detecting oxidation-related genes, which provided a theoretical basis for further human research and the industrial development of Insect tea extract.

| Extraction of Insect tea
Five hundred grams of Insect tea was crushed into a powder, and 50 ml of 5% ethanol solution (volume ratio) was added for extraction (90°C, 30 min). The extract was amalgamated twice and repeated and then was subjected to rotary evaporation to obtain Insect tea (Zhang et al., 2018).

| Animal oxidation experiment
Forty SPF-grade 6-week-old ICR mice (Chongqing Medical University) were fed for one week, and then, the mice were divided into four groups: normal, model, Insect tea, and vitamin C. Each group consisted of 10 mice, five male and five female. During the first four weeks, the mice in the normal and model groups were only fed with diet and drinking water. The mice in the Insect tea group and vitamin C group were fed with Insect tea extract and vitamin C at a daily concentration of 100 mg/kg by gavage. Four weeks later, D-galactose was injected intraperitoneally into each mouse of the model, Insect tea and vitamin C groups at a concentration of 120 mg/kg per day for 6 weeks. At the same time of D-galactose injection, the mice in the Insect tea group and vitamin C group received intragastrically administered Insect tea and vitamin C at the same concentration for 6 weeks; after 24 hr of fasting, all the mice were sacrificed (Li et al., 2018). Blood was taken from the heart and liver for follow-up experiments. The indexes of the thymus, brain, heart, liver, spleen, and kidney were measured. The organ index was equal to the organ mass (g)/body mass of mice (kg) ×100.

| Determination of the MDA content and SOD and GSH-Px activities in serum and liver tissues
The obtained mouse plasma was centrifuged at 730 × g for 10 min, and then, the upper serum layer was taken. The content of MDA and the activities of SOD and GSH-Px in the serum were determined by using commercial kits (Nanjing Jiancheng Bioengineering Institute). A 10% homogenate was prepared from the liver of mice and was centrifuged at 730 × g for 10 min. The supernatant was taken to determine the MDA content and SOD and GSH-Px activities in liver tissue using commercial reagents (Nanjing Jiancheng Bioengineering Institute).

| Pathological observation of liver and spleen tissues
Next, 0.5-cm 2 liver and spleen tissues from the mice were removed and fixed in 10% formalin solution for 48 hr. The skin, liver, and spleen tissues were dehydrated, rendered transparent, waxed, embedded and sectioned, and then were stained with H&E. The morphological changes in the tissues were observed under an optical microscope (BX43, Olympus).

| Quantitative PCR (qPCR) assay
The liver and spleen tissues of mice were crushed, and then, RNAzol was used to extract total RNA from the tissues. The total RNA concentration was diluted to 1 μg/μl, and 5 μl of this diluted total RNA solution was removed and subjected to reverse transcription to obtain cDNA. Next, 2 μl of DNA template, 10 μl of SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.), and 1 μl each of forward and reverse primers (Table 1) were mixed and reacted at 95°C for 60 s, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 35 s; finally, the DNA was detected at 95°C for 30 s and 55°C for 35 s, and GAPDH was used as an internal reference. The 2 −ΔΔCt method was used to calculate the relative expression of genes (SteponePlus, Thermo Fisher Scientific, Inc.) (Zhang et al., 2018).

| Western blot analysis
Samples of liver and spleen adipose tissue (100 mg) and 1 ml of RIPA (Thermo Fisher Scientific, Inc.) were homogenized at 2,190 × g at 4°C for 5 min and were centrifuged at 2,190 × g 4°C for 15 min. The intermediate protein layer solution was extracted. The BCA protein quantitative kit (Thermo Fisher Scientific, Inc.) was used to quantify the protein. The samples of each group were diluted to 50 µg/ml, and then, the diluted protein was mixed with sample buffer at 4:1 and heated at 100°C for 5 min. Next, acrylamide, resolving buffer, stacking buffer, distilled water, 10% APS, and TEMED (Thermo Fisher Scientific, Inc.) were proportionally mixed to form an SDS-PAGE separating and concentrating glue, which was poured into the running rubber board for use. The prestained protein ladder and samples were placed into the sample hole of the rubber sheet, and then, the SDS-PAGE glue containing protein was subjected to vertical gel electrophoresis for 50 min. The PVDF membrane (Thermo Fisher Scientific, Inc.) was activated by methanol for 1 min, and then, the transmembrane was sealed for 1 hr by 1 × TBST solution containing 5% skim milk. After closure, the PVDF membrane was washed with 1 × TBST, and the primary antibody was incubated at 25°C for 2 hr. After washing five times with 1 × TBST, the secondary antibody was incubated at 25°C. Finally, Supersignal West Pico PLUS was used to spray the PVDF film, followed by placement in iBright FL1000 (Thermo Fisher Scientific, Inc.) for observation (Li et al., 2018).

| Statistical analysis
The serum and tissue determination experiments of each mouse were performed three times in parallel, and then, the mean values were taken. Next, SAS 9.1 statistical software was used to analyse the data. One-way ANOVA was used to analyse whether significant differences occurred among groups of data at the level of p < .05. Table 2 reveals that the indexes of thymus, brain, heart, liver, spleen, and kidney of normal mice were the highest, while the index of organs of model mice is the lowest. Insect tea extract and vitamin C could significantly (p < .05) increase the organ index of oxidative damage-induced mice. At 100 mg/kg, the effects of Insect tea extract were significantly (p < .05) better than those of vitamin C. Therefore, Insect tea can inhibit the decline in the organ index caused by tissue atrophy. The organ weight and organ index of animals are indicators to study the state of animal body. The changes in the organ weight and organ index can reflect the state of the organism subjected to oxidative stress. When the organism is under oxidative stress, the thymus and brain will atrophy more obviously than other organs (Tang & He, 2013). The liver and kidney are the main metabolic organs in mice. Decreases in their weight and organ index directly affect the metabolic ability of the organism. At the same time, the liver is one of the immune organs of animals. A change in the liver index indicates that the immunity of the organism is also affected (Khan, Singer, & Vaughan, 2017). The spleen is related to the cellular immune system in the body and plays an important role in immune mechanism. A decline in the quality of the spleen indicates organ atrophy, which can reduce its immune function. Therefore, determining the spleen organ index can directly reflect the structural changes in organs and their functions, which has important reference value to evaluating the success of the establishment of an oxidative stress mouse model (Manini, 2010). The results of this study also showed that D-galactose could reduce the visceral index of mice induced by oxidative damage. Insect tea could alleviate the decrease in the visceral index of mice induced by D-galactose, thus preventing the oxidative damage in mice, and the effect was better than that of vitamin C.

Gene Name Sequence
Cu/Zn-SOD Forward: 5'-AACCAGTTGTGTTGTCAGGAC-3'   MDA is a lipid peroxide formed by oxidation. The level of MDA in vivo also directly reflects the degree of oxidation (Hosen, Islam, Begum, Kabir, & Howlader, 2015). Based on these results, it can be seen that Insect tea can regulate oxidative regulation factors in serum.      (Kosenko et al., 2017). CAT is an antioxidant enzyme that mainly exists in erythrocytes and some tissue cells, as well as in mitochondria and the cytoplasm (Selvaratnam & Robaire, 2016). In the process of normal oxidative respiration, organisms constantly produce ROS. As a highly active molecule, it contains unpaired electrons.  (Pawlak et al., 1998). In this study, Insect tea can significantly increase the Cu/Zn-SOD, Mn-SOD, and CAT expression in mice, thereby effectively slowing down the oxidation caused by D-galactose. Mean values with different superscript letters (a-d) in the same row are significantly different (p < .05). Vitamin C: mice treated with 100 mg/kg of vitamin C; Insect tea: mice treated with 100 mg/kg of Insect tea extract. and enhance the tolerance of cells to oxidative stress. Therefore, regulating the Nrf2/NQO1 signaling pathway is an effective means to exert the antioxidant activity of active substances (Jiang et al., 2017). In this study, Insect tea can upregulate the expression of oxidized Nrf2, further enhancing the expression intensity of HO-1, gamma-GCS, and NQO1, and playing a protective role in mice with D-galactose-induced oxidative damage and preventing oxidative stress. The effect is better than that of vitamin C, the commonly used oxidant.

| Constituents of insect tea
Insect tea contained neochlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, hesperidin, and quercetin ( Figure 8) at 0.90, 2.04, 6.43, 1.64, 1.63, and 1.59 mg/g, respectively. Phenols are natural antioxidants because they have more active phenolic hydroxyl group, which can provide hydrogen and free radicals to react to form inert products or more stable free radicals, thus interrupting or slowing down the chain reaction of free radicals (Wang et al., 2011). The results showed that the antioxidant activity of polyphenols and flavones in some natural foods is much better than that of vitamin C.
The difference in the antioxidant activity of polyphenols and flavones in natural foods is not only due to the molecular structure but also to the difference in stereoconformation (Shen, Jin, Yang, & Zhao, 2000;Yilixiati & Li, 2012 (Zhang & Wang, 2007). Rutin is a flavonoid compound and a strong oxidant for scavenging free radicals. It can terminate the chain reaction of free radicals, inhibit the peroxidation of polyunsaturated fatty acids on biofilms, remove lipid peroxidation products, and protect the integrity of biofilms and subcellular structures.
Rutin plays an important role in the body (Zhang, Liu, Song, & Chen, 2003). Hesperidin is easily oxidized and destroyed, is often used as a natural antioxidant in the food industry, has antimicrobial activity, and prevents the skin from oxidative damage caused by ultraviolet radiation (Abolaji, Babalola, Adegoke, & Farombi, 2017). Quercetin is a 5-hydroxyflavone with double bonds between the two and three sites in its molecule and two hydroxyl groups at the 37 and 47 sites.
Quercetin can be used as a free group acceptor in metal chelation or oxidation of oils and fats. Additionally, quercetin can be used as an antioxidant of oils and ascorbic acid. It has also been gradually applied in functional foods with antioxidant activity (Zheng et al., 2017). Insect tea contains active substances with a strong antioxidant effect. This study also confirmed that the antioxidant effect of Insect tea at the same concentration is much stronger than that of vitamin C. Insect tea can play a preventive role in animal oxidation by containing active ingredients.

| CON CLUS IONS
This study showed that Insect tea could prevent D-galactose-induced oxidation in mice, making the indexes of the serum, liver, and spleen in mice with D-galactose-induced oxidative damage similar to those observed in normal state mice, and increased levels of vitamin C with the same concentration of strong antioxidants were found. The research further confirmed that Insect tea contains abundant bioactive substances and needs further applications and study. Additionally, Insect tea can be used as a high-quality food and medicine homologous resource rich in antioxidants. The types of effective substances in Insect tea have also been verified, and the mechanism of action has been preliminarily clarified in this study. However, further human studies are needed to confirm its mechanism more accurately, and the relationship and binding effect of various active substances need to be further studied.

CO N FLI C T O F I NTE R E S T
The authors of this manuscript state that they do not have conflict of interest to declare.

E TH I C A L A PPROVA L
The protocol for these experiments was approved by the Ethics

Committee of Chongqing Collaborative Innovation Center for
Functional Food (201901001B, Chongqing, China).