Long-term feeding of sand rice (Agriophyllum squarrosum seed) can improve the antioxidant capacity of mice

PnShu Zhao, Lian Shi, Xia Yan, ChaoJu Qian, WiJia Zhao, Yan Chan,XiaoYu Yin, XinK Fan, TinZhou Fan,c, YuQiu Liao,c, ShanShan Zhou,XiaoFi Ma,＊

a Key Laboratory of Ecological Safety and Sustainable Development in Arid Lands,Northwest Institute of Eco-Environments and Resources,Chinese Academy of Sciences,Lanzhou, Gansu, 730000, China

b Key Laboratory of Stress Physiology and Ecology in Cold and Arid Regions of Gansu Province,Northwest Institute of Eco-Environments and Resources,Chinese Academy of Sciences, Lanzhou, Gansu, 730000, China

c University of Chinese Academy of Sciences, Beijing, 100049, China

d Key Laboratory of Inland River Ecohydrology,Cold and Arid Regions Environmental and Engineering Research, Northwest Institute of Eco-Environments and Resources,Chinese Academy of Sciences, Lanzhou, Gansu, 730000, China

e School of Life Sciences, Nantong University, Nantong, Jiangsu, 226019, China

f Institute of Qinghai-Tibetan Plateau Research, Southwest Minzu University, Chengdu, Sichuan, 610041, China

g Shanxi Institute of Science and Technology, Faculty of Environmental Science and Engineering, Jincheng, Shanxi, 048000, China

Keywords: Sand rice Functional food Long-term effects Health evaluation Antioxidant capacity

ABSTRACT Unhealthy eating habits with sugar-rich and high-fat foods can bring nutritional imbalance and other chronic diseases.At present, there is an increasing demand in regulating human sub-health through functional foods.Agriophyllum squarrosum (L.) Moq., a pioneer plant native to sand dunes with ecological restoration function, is a traditional food plant of sand regions and Mongolian folk medicine.Numerous studies have confirmed that A.squarrosum seed,also called sand rice,has high nutritional value.However,the long-term health effects of eating sand rice remain unclear.To better explore and evaluate its long-term effects,hereby,ICR mice were fed with sand rice flour at different ratios(control,60%and 95%)for 100 days.Results show that the growth and blood glucose level curve of these experimental mice were lower and more stable than that of the control.In addition,mice fed pure sand rice ingredient(95%)gained more stable body weight and blood glucose after 28 days,which revealed that sand rice is a food with comprehensive nutritional value and rarely negatively impacts the body growth performance.Our data also demonstrates that,the content of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)provided the lowest level in the blood of mice with a diet of pure sand rice ingredient,0.48,4.14,0.91,3.03 mmol/L,respectively.Also,T-SOD and GSH-PX activity provided a high level in mice with pure sand rice gradient,which significantly increased by 36%(T-SOD)and 19%(GSH-PX)compared to the control.These results suggest that long term intake of the pure sand rice ingredients is more helpful for stabilizing blood lipid and improve antioxidant capacity and beneficial to people suffering from chronic disease.This study supplies the first animal evidence to support the claim that sand rice is a promising functional food with comprehensive nutrition in the future.We are confident that,with increasing demands of daily sand rice usage, planting A.squarrosum in the vast desert regions will further increase the local economy and ecological restoration when coping with global warming.

1.Introduction

With economic development and certain sub-health problems(obesity, hypertension, diabetes, hyperlipidemia, cardiovascular and cancer),in conjunction with unhealthy eating habits(Aghajanpour et al.,2017; Verger et al., 2021), people realize that functional foods can perform a similar effect as certain drugs,but without the negative effects(Karelakis et al., 2019).Also, global climate anomalies have brought dramatic changes in crop distribution areas, resulting in a food crisis(FAO, http://www.fao.org/3/a-i3866e.pdf).Therefore, screening and developing functional foods that can adapt to climate change and be consumed over a longer period in arid and sandy areas are of double significance in coping with a food crisis and solving human sub-health problems.

Agriophyllum squarrosum (L.) Moq.is an edible annual herbaceous plant of the Amaranthaceae family widely distributed in mobile sand and semi-fixed sand dunes of Central and East Asia,which provide value for the ecology, food and pharmaceutical industry (Xu et al., 2018; Zhao et al.,2021).A.squarrosum is also an excellent pioneer resource plant for windbreaks and sand fixation due to characteristics of drought, heat,barren and sand burial resistance (Zhao et al., 2014).The seeds contain more protein,crude fiber and metal elements,lesser carbohydrates than the world-recognized crop quinoa (Chen et al., 2014; Yin et al., 2021;Wang et al.,2009),whose ingredients are consistent with the proportion recommended for the human body.Historically, A.squarrosum seeds were used as a military food source (Gao, 2002).Because of its greasy taste,delicious,high nutritional value,people in sandy areas usually use its seed for various foods(Zhao et al.,2016a).In addition,the seeds have been used in Mongolian folk medicine as an antidiabetic, diuretic,analgesic, and antipyretic agent (Gong et al., 2012) to prevent chronic and degenerative diseases, which make it suitable for pharmacological development.From the perspective of seed metabolites, nutritional analysis and palatability(Chen et al., 2014;Yin et al., 2021;Zhou et al.,2021a,2021b),it meets all the requirements of a functional food without other nutritional supplements.However,the edible value of sand rice is basically based on the analysis of its nutritional composition and medicinal ingredients.

As the mouse genome is extremely similar to the human genome, it offers a model and has particular significance for the treatment and effect evaluation of human physiology and diseases (Waterston et al., 2002).Previous studies have found that quinoa seed, a functional food, has antioxidant, anti-hypertensive and anti-diabetic properties through feeding experiment in rats (Pa′sko et al., 2010a, 2010b; Arneja et al.,2015; Alghamdi, 2018).Subsequently, experiments on human dietary requirements further confirmed that quinoa seed is beneficial from the standpoint of metabolism, cardiovascular and gastrointestinal health of high-risk consumers,such as children and the elderly(Navruz-Varli and Sanlier, 2016; Vilcacundo and Hernandez-Ledesma, 2017).In contrast,there has been no research on the impact of long-term consumption of sand rice on humans or animals.Whether sand rice can also be used as functional food for long-term consumption in the daily diet of some special groups (diabetes, obesity, hyperlipidemia) as quinoa or even better than quinoa seed is open for debate.

In the present study,non-targeted metabolomics analysis was used to detect sand rice compounds,and health indicators were used to valuate comprehensive nutrients and medicinal value of sand rice Meanwhile,ICR mice, which has characteristics of strong adaptability, strong physique, strong fecundity, rapid growth rate, and good experimental repeatability,was feed with different ratios of sand rice flour for 100 days to explore the nutritional and medical effects of sand rice.This study will provide benificial solutions for the health concerns of long-term consumption of sand rice and meet the people's needs for nutrition and health.This also includes the development of functional food markets,which will not only trigger development of the local economy by directing desertification control in sand areas,but also mitigate the food crisis due to climate change.

2.Materials and methods

2.1.Plant material

Sand rice was harvested in Wuwei City, Gansu Province (WW,37°54′10.98′′N,102°54′04.20′′E,1,530 m),China in 2019,and stored in the laboratory of Northwest Institute of Eco-Environment and Resources,Chinese Academy of Sciences.After grinding,edible grade sand rice flour was stored at 4°C for diet preparation.

2.2.Non-targeted metabolomic analysis

100 mg of dry sand rice was used to perform GC/TOF-MS analysis in Shanghai Biotree biotech Co., Ltd.(Shanghai, China), and repeat three times.The handling and testing methods of sand rice are described in detail in Hao et al.(2019).

2.3.Animal grouping and treatment

Four-week male ICR mice(18–22 g)were housed in a 12-h light/dark cycle room with 50%–80% humidity and 18–25°C temperature.After three days of acclimatization with the same control diet,18 mice of the same size were randomly divided into three groups including control,60% (A60) and 95% (A95) ingredient of A.squarrosum seed (sand rice)flour.Six mice in each group were locked in two cages and fed with 30 g food(Table 1)every day and provided with a constant supply of water for 100 days.

Table 1Diet formulation.

2.4.Determination index and method

Body weight and blood glucose were measured weekly by an analytical balance (ME204T/02, Mettler Toledo, Switzerland) and contour TS blood glucose meter(Bayer HealthCare LLC,USA),respectively.Before the blood glucose test, ICR mice were kept fasting for 12 h, but drink water freely.Naturally spilled blood from the tail vein was collected for the blood glucose test.Each test of six ICR mice was repeated three times.To measure four indices of blood lipid,fresh mouse blood was stored in a refrigerator at 4°C for at least 8 h until coagulated.After centrifuging 4,000 rpm for 10 min, 200 μL serum was used to measure the content of triglyceride (TG), total cholesterol (TC), lowdensity lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol(HDL-C),respectively,in the laboratory of Affiliated Hospital of Nantong University,China.

2.5.Detection of antioxidant capacity in liver tissue

Three-hundred mg of liver tissue from the control, A60, and A95 mice, respectively, were homogenized in appropriate amount of icenormal saline (0.9% NaCl) with a homogenizer.Then, antioxidant activity in liver tissue was detected using the following kits; BCA protein concentration purchased from Beyotime Biotechnology Co., Ltd.(Shanghai, China), malondialdehyde (MDA,Cat number: E-BC-K025-S),total superoxide dismutase (T-SOD, Cat number: E-BC-K019-S), and glutathione peroxidase (GSH-PX, Cat number: E-BC-K096-S) purchased from Elabscience Biotechnology Co., Ltd.(Wuhan,China).

2.6.Statistical analysis

SPSS Statistics 17.0 and GraphPad Prism software were used to analyze health indicators.Pearson correlation coefficient(PCC)was used to evaluate the correlation between blood glucose level and body weight.One-way analysis of variance(ANOVA)was used to evaluate significant difference between feeding groups, and values are presented as mean± SEM.

3.Results

3.1.Nutritional and medicinal composition of sand rice

A total of 433 known metabolites were detected from three replicates in sand rice.To study the potential nutrition and pharmacological activity, composition and content of metabolites, we addressed 25 metabolites of relatively high content,including three carbohydrates,one fiber,five amino acids, two fatty acids, twelve organic acids, and two flavonoids(Table 2).

Table 2Main nutritional and medicinal metabolites of sand rice.

The carbohydrate, fiber, amino acids and fatty acids are important nutritional components of sand rice.These compounds provide the raw materials for synthesis of essential nutrients such as proteins, fats and vitamins.Its medicinal value is largely attributed to twelve organic acids and two flavonoids.Twelve organic acid metabolites were identified with variations ranging from l-malic acid to ferulic acid in sand rice.Among them, five phenolic acids, including protocatechuic, gallic, pcoumaric, vanillic and ferulic acid, have the effect of antioxidants for prevention of heart disease.Two flavonoids, naringenin and neohesperidin, were also identified, which have antioxidant, hypoglycemic and other extremely important properties.

3.2.Variation curve of body weight and blood glucose level in mice after feeding sand rice ingredients

In the 100 days feeding trial, we found all ICR mice had a normal diet, moved quickly, glossy coat color and none of the symptoms found in septic mice(Supplemental Figure).The body weight curve shows that before 28 days,the weight of A60 and A95 mice increased more rapidly than control.Also, the growth curve of A60 mice was higher than that of control and A95 and there was no significant difference between the control and A95 mice (Fig.1a).After 28 days, we found an overall lower body weight curve of A95 mice than the control and A60 mice.Interestingly, 49 days later, the body weight of A60 and A95 miceslowed down gradually, while that of control still increased at a fast rate.

Fig.1.Variation curve of body weight(a)and blood glucose level(b).Values are presented as mean±SEM for six mice(Note:control,A60 and A95 represent mice fed with a normal diet without adding sand rice flour, and adding 60% and 95% ingredient of sand rice flour, respectively.)

The blood glucose values of mice in all experimental groups were within the normal range and no more than 7 mmol/L(Fig.1b).Before 28 days,the blood glucose decreased while weight increased under different treatments,which show a negative correlation between body weight and blood glucose level(Table 3).Thus,we speculate that mice are prone to hypoglycemia due to rapid growth.PCC had a maximum value of 0.93 in mice fed with A60, which shows a strong negative correlation between blood glucose and body weight.28 days later,the trend of blood glucose level in A60 and A95 mice was relatively more stable than the control mice.These results shows that sand rice ingredients could regulate and stabilize blood sugar,and further influence blood glucose metabolism in ICR mice.

Table 3PCC between blood glucose level and body weight.

3.3.Variation of blood lipid content in mice after feeding sand rice ingredients

To reflect the metabolism of lipids in the body, TG, TC, LDL-C and HDL-C were determined in the 100-day feeding of ICR mice.Contents of the four indices were within normal blood lipid range.Also, there was some variation of each blood lipid index in A60, A95 and control.The content of TG decreased while sand rice ingredients increased(Fig.2a),the content of TC and HDL-C were highest in A60, while lowest in A95(Fig.2b, d).The content of LDL-C in A60 was almost close to control,while decreased heavily in A95 (Fig.2c).Also, the four indices were lowest in A95,0.48,4.14,0.91,3.03 mmol/L,respectively.

Fig.2.Variation of blood lipid content in mice.a,b,c,d are the contents of TG,TC,LDL-C and HDL-C,respectively.Values are presented as mean±SEM for 6 mice(Note: control, A60 and A95 represent mice fed with the normal diet without adding sand rice flour, and adding 60% and 95% ingredient of sand rice flour,respectively.There was no significant difference between the three different treatments (control, A60 and A95), so "＊" was not marked in the figure.)

3.4.Antioxidant capacity of mice adding sand rice ingredients to diet

To detected antioxidant capacity of mice liver tissue, MDA, T-SOD and GSH-PX were tested(Fig.3).MDA content significantly decreased by 42% in A60 (p < 0.05) and 31% in A95 (p < 0.01) in comparison to control (Fig.3a), and no significant difference between A60 and A95,which suggests that ingredients of sand rice can effectively reduce the toxic effect of free radicals on ICR mice.T-SOD activity increased in accordance to the proportion of sand rice ingredients(Fig.3b),about 600 in A60,800 in A95.The significant difference of T-SOD activity indicates high oxidation resistance of liver cell in ICR mice.

Fig.3.Variation of antioxidant capacity in mice.(a)Content of MDA.(b,c)Activity of T-SOD and GSH-PX.Values are presented as mean±SEM for 6 mice.“＊"and"＊＊"indicate significant differences at p<0.05 and p<0.01 respectively(Note:control,A60 and A95 represent mice fed with the normal diet without adding sand rice flour, and adding 60% and 95% ingredient of sand rice flour, respectively.)

The variation pattern of GSH-PX activity is basically consistent with T-SOD activity.The more sand rice flour added the higher GSH-PX activity (Fig.3c).GSH-PX activity in A95 was significantly higher than the control group (p < 0.01).There was little difference between A60 and control suggesting small mixing effect between sand rice ingredients and other food.In addition,T-SOD and GSH-PX activity were the highest and significantly increased by 36%(T-SOD)and 19%(GSHPX) in comparison to control, which indicates that pure sand rice ingredients are more beneficial for improving the antioxidant resistance of mice liver.

4.Discussions

4.1.The nutritional and medicinal value analysis of sand rice

Many beneficial compounds including carbohydrates, fibers, amino acids, fatty acids, organic acids and flavonoids were validated in sand rice.Leucine, valine and proline are listed in Table 2，which may contribute much to the content of 23.2% protein (Chen et al., 2014).They also can provide energy, regulate blood glucose levels and other amino acids metabolism (Layman, 2003; Macotela et al., 2011).There was no difference in the content of leucine and valine between sand rice and quinoa seed (Zhao et al., 2023).Quinoa seed does contain higher proline than sand rice, which may be related to better adaptability of A.squarrosum to a stress environment(Zhao et al.,2016b).In our study,linoleic acid and o-Hydroxyhippuric acid 2,were high.Those fatty acids are healthy substitutes for saturated animal fats (Marshall, 2011; Flock and Kris-Etherton, 2011).There were three main carbohydrates(Table 2) detected in sand rice, and the total carbohydrate content was 54%, which is lower than 67% of quinoa seed (Zhao et al., 2023).Research has reported that phenolic acids and flavonoids are mainly concentrated in the seed coat of A.squarrosum, and its flavonoids content are significantly higher than that of peanut, corn, wheat, rice and other crop seeds(Xu et al.,2018).Our study found that l-malic acid has the highest concentration of organic acids, which is an important intermediate product in the internal circulation of the human body.1-malic acid is also widely used in food additives and developed in medicine with many effects (Table 2).Other organic acids and two flavonoids were also rich in sand rice, and have many functions for regulating health (Table 2).In addition, glucose-1-phosphate and protocatechuic acid are two compounds with high content in sand rice.However,according to previous research,they were not detected in the metabolome of rice and wheat, which can make up for the nutritional deficiency of wheat and rice as staple foods (Zhao et al., 2016b).Although these sand rice metabolites can regulate body health, further molecular or physiological studies are needed to continue confirming their beneficial effects.No matter what the existing research results have shown, the content and composition of major nutrients in sand rice could better meet the daily dietary and nutritional needs of people at different ages than quinoa seed.

4.2.The health benefit of sand rice

The variation curve of body weight(Fig.1a)and blood glucose level(Fig.1b) in mice were analyzed after feeding sand rice ingredients.In addition, blood lipids (Fig.2) and antioxidant capacity (Fig.3) of mice were also studied to explore the health benefits of sand rice.We obtained good growth curves for A60 and A95 mice than control(Fig.1a).These results further support the comprehensive nutrients of sand rice (Chen et al.,2014).Interestingly,our experiments found A60 mice grew faster than A95 before 28 days(Fig.1a),which suggests that a mixture of sand rice is more helpful for children than pure sand rice due to nutritional requirements during the rapid growth of children(Lama et al.2020).28 days later, we also found A95 mice had an overall lower body weight curve than the control and A60 mice,which suggests that pure sand rice is better for weight control of adults.In addition,investigating the relationship between blood glucose and body weight found that the blood glucose level of mice at an early stage is negatively correlated to body weight (Table 3).This phenomenon shows that dietary sand rice ingredients could regulate and stabilize blood glucose metabolism in ICR mice while gaining weight.Also, mice fed with A60 shows that their negative correlation is more significant, which may be that mice grew faster at an early stage and were prone to hypoglycemia.

Numerous studies have found that blood glucose and lipid metabolism interact with each other (Wang et al., 2017).Thus, monitoring blood lipid levels can help to detect abnormal blood glucose levels earlier, so timely adjustment and treatment can effectively prevent the risk of cardiovascular disease (CVD), atherosclerosis, obesity, diabetes mellitus and hypercholesterolaemia(Hobbs et al.,2016;Trautwein et al.,2018).In this paper,we found that the four indices of blood lipid(TG,TC,LDL-C and HDL-C)were not significantly changed after the sand rice flour feeding trial (Fig.2).In contrast, there were subtle differences under different treatments.Previous research has demonstrated that the level of HDL-C is negatively correlated with the occurrence of coronary heart disease (Qi et al., 2021).However, TC, TG and LDL-C were positively correlated with blood glucose levels (Wang, 2016).In our study,compared with the control and A60, A95 had the lowest TG, TC and LDL-C suggesting that pure sand rice ingredients can control blood lipid metabolism.This is of great significance for lowering blood glucose to prevent various chronic diseases.Similar results were found in experiments with quinoa seed fed to rats (Pa′sko et al.2010a).

The excessive accumulation of ROS in cells can destroy the oxidation system and cellular constituents,thus leading to diseases such as diabetes,atherosclerosis, coronary heart disease (CHD), osteoporosis and cancer(Ray et al.,2012).The important antioxidant enzymes(MDA,T-SOD and GSH-PX) are extremely critical for keeping the system healthy and maintaining cellular active (Krishnamurthy and Wadhwani, 2012).Compared with the control group,MDA,GSH-PX and T-SOD of the sand rice flour feeding group were significantly changed(Fig.3).T-SOD is the only enzyme to eliminate superoxide ions (O2-) as hydrogen peroxide(H2O2)in animal cells,and H2O2can be destroyed by GSH-PX into H2O,thereby eliminating ROS damage (Forman and Zhang, 2021).Our ICR mice feeding trial determined that MDA content decreased(Fig.3a),while the contents of GSH-PX and T-SOD increased significantly(Figs.3b,3c),which shows that the degree of free radical attack of reaction cells was weakened,and the ability of the body to remove ROS was enhanced after eating sand rice flour(Cui et al.,2021).This study further supported that sand rice can reduce free radicals and have high oxidation resistance(Mathew et al.,2015;Xu et al.,2018).Similar effects were obtained in rats fed with quinoa seed(Pa′sko et al.,2010b).Furthermore,compared with the control and A60,mice fed with A95 had stronger antioxidant capacity.This phenomenon indicates that pure sand rice is an excellent anti-oxidation food.Its antioxidant capacity is mainly attributed to natural antioxidant active substances,including twelve organic acids and two flavonoids listed in Table 2.These compounds can scavenge ROS and significantly reduce the risk of chronic diseases(Zhao et al.,2016),which is consistent with previous research(Xu et al.,2018).In addition,studies also show that flavonoids can significantly decrease MDA and increase SOD and GSH-PX levels of mice organs(including serum,liver,kidney and heart)(Dong et al.2021).This is why the antioxidant capacity of mice liver was enhanced after prolonged feeding of sand rice.Our study also identified five phenolic acids(protocatechuic,gallic,p-coumaric,vanillic and ferulic acids)of sand rice，which can inhibit the formation of free radicals and inactivate them through human diet experiments (Shahidi and Ambigaipalan, 2015; de Oliveira et al., 2018).Also, l-malic acid might induce feedback resistance mechanisms and significantly improve antioxidant capacity(Zhang et al.,2020).In our study,the benefit of sand rice was proven by health evaluation in ICR mice from A60 and A95,especially the value of increased antioxidant capacity.In the future, experiments with volunteers will be needed to further confirm these findings.

Above all, sand rice, containing comprehensive nutrients and antioxidant substances, is a very good functional food for diabetes, coronary heart disease (CHD), cancer other sub-health problems in human populations.Thus, sand rice is promising in the field of food and medicine.As a food plant, our research team has be fortunate in domesticating a variety of A.squarrosum with larger seed (1.3 g per 1,000 seeds), spike (5 cm length), and high yield (more than 100 kg/acre), which is 3–5 times better than that of wild types (unpublished data).As a medicinal plant, the high accumulation of beneficial metabolites is inseparable from harsh environmental conditions in the desert.Also, high altitude is conducive to the accumulation of organic acids and flavonoids (Zhou et al., 2021a, 2021b), while other environments are favorable to the accumulation of functional metabolites,where the expression of related genes are induced(Zhou et al.,2021b).Our findings suggest that the cultivation and nutritional value of A.squarrosum is benifical for populations in desert ecosystems during times of global warming.

5.Conclusion

Through detailed data from metabolome,body weight,blood glucose level, blood lipid and antioxidants, our study reveals that sand rice is a comprehensive functional food with beneficial nutrients and various antioxidant ingredients.The vast desert offers great potential for A.squarrosum cultivation, and makes it a promising functional food to meet requirements of human health and further to open up the functional food market, to drive the ecological restoration of desert areas and to cope with global warming.

Acknowledgements

This research was financially supported by the Biological Resources Programme, Chinese Academy of Sciences (Grant No.KFJ-BRP-007-015), the National Natural Science Foundation of China (Grant No.31901079 and 32171608), Jiangsu Provincial Key Research and Development Program (Grant No.18YF1NA123), Key Laboratory of Highway Construction and Maintenance Technology in the Loess Region of Shanxi Transportation Research Institute (Grant No.BRZD 1809) and Lanzhou Talent Innovation and Entrepreneurship Project(Grant No.2021-RC-76).

Appendix A Supplementary figure