Quinoa seeds (Chenopodium Quinoa): Nutritional value and potential biological effects on hyperglycemic rats

2.1 Source of quinoa seeds

Quinoa seeds were purchased from Desert Research Center, Cairo, Egypt.

2.2 Chemicals and kits

All chemicals utilized in the experiment were of analytical quality and had been purified prior to use. Folin-Ciocalteau reagent and caffeic acid were obtained from Gerbsaure Chemical Co. ltd., Germany and Sigma-Aldrich (St. Louis, MO, USA), respectively. Kits for aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine, bilirubin, and uric acid were purchased from Borhringer Ingelheim GmbH, Ingelheim, Germany.

2.3 Experimental rats

Twenty-four (n = 24) healthy male rats weighing 100–150 g and aged 7–9 weeks were procured from Cairo university, Egypt. Every-four rats were kept in plastic cages at room temperature (25 ± 2 °C) with 12-hour light/dark cycles. Rats were isolated for a week to adapt to lab conditions before being fed a standard commercial pelleted meal (Lab Diet No. 500; PMI Feeds Inc, St. Louis, MO, USA) with a chemical composition of fat, 6.0 %,24 % protein, 5.7 %, 8.0 % ash fiber, and 58 % carbohydrate, as well as water was available.

2.4 Nutritional value

Moisture, protein, carbohydrates, lipids, fibers, and ash contents of quinoa seeds were estimated as a standard method AOAC (2016) and expressed as g/100 g on a dry basis. Carbohydrate was calculated by difference.

2.5 Fatty acids composition

The quantitative measurements of fatty acids in quinoa seed oil were performed from the lipid extraction of the section previous using a capillary gas chromatograph (HP 6890) and expressed in proportional area percent. Once the extraction was carried out, it was methylated following the method AOAC (2016). In a 100 ml flask, 0.1 g of the quinoa seed oil is dissolved in heptane (2 ml) with methanolic potash solution (0.2 ml, 2 N) to trans esterified into carboxylic acid methyl esters by shaking. A gas chromatograph with a DB-23 (5 % - cyanopropyl–methyl poly siloxane) capillary column (60mx 0.32 mm X0.25 m film thickness) and flame ionization detector used to identify the obtained methyl esters of fatty acids. The retention periods of carboxylic acid methyl esters were compared to a known carboxylic acid reference combination. An integrator calculated peak areas directly.

2.6 Determination of total polyphenolic and total flavonoids

The total polyphenol levels in quinoa seeds were evaluated according to Žilić et al., (2012) using caffeic acid as standard and presented results calculated as mg caffeic equivalent/kg of quinoa (mg CE/kg as ppm). Total flavonoids content was assessed by aluminum chloride test (Kim et al., 2006), results were calculated as mg quercetin equivalent/kg of quinoa (mg QE/kg as ppm).

2.7 Minerals content

The minerals content was estimated as a standard method (AOAC 2016) by using atomic absorption (NC.9423–400-30042 England).

2.8 Amino acid analysis

Acid hydrolysate of the quinoa seed by 6 N HCl used to determine amino acids as professional method AOAC (2016), by using an amino acid analyzer (LC 3000 amino acid analyzer, high-performance system, a product of LC biochrom Eppdrop, Germany).

2.9 Diabetes type 2 induction

Streptozotocin injections are used to induce overnight fasting lab rats with type 2 diabetes by intraperitoneal injection of a single dose (60 mg/kg) of streptozotocin (Sigma-Aldrich Crop, St, Louis, MO, USA). Food and water are freely available in injected rats. Within four days, diabetes mellitus developed and stabilized in streptozotocin-treated rats. Diabetic rats were defined as those rats with a fasting blood glucose level greater than 300 mg dL and considered as model rats with diabetes.

2.10 Experimental nutrition design

The rats were randomly spilt into 3 groups, eight rats per group. The control rats in group 1, diabetic rats in group 2, and diabetic quinoa seed treated at concentrations (25 %) in group 3.

2.11 Analysis of blood serum

After four weeks for rats, the water is anesthetized with diethyl ether and the rats are prevented from eating for 8 h. Blood samples are drawn from the orbital veins without the use of heparin. The level of glucose in the serum of rats measured by the method of Barham and Trinder (1972). The activities of Aspartate aminotransferase AST (E. C. 2. 6. 1. 1), Alanine aminotransferase ALT (E. C. 2. 6. 1. 2), and Alkaline phosphatase ALP (E. C. 3. 1. 3. 1) were determined using the procedures illustrated by Kachmar and Moss (1976), Bergmryer and Harder (1986), Varley et al. (1980) respectively. Bilirubin, creatinine, and uric acid were measured by the technique of Goryachkovskiy (1998). The cholesterol levels were measured using the procedures described by Assmann (1979).

2.12 Statistical analysis

The average ± SD of the triplicates was recorded for all analyses, with regard to the biological analyses that were estimated on the serum of rats, analyzed by Duncan’s multiple range test at p < 0.05 by using SPSS Inc. program version15 (2006). Differences (one-way ANOVA) were considered significant at a p-value below 0.05.

3.1 Chemical composition

Proximate chemical composition and minerals content of quinoa seeds are recorded in Table 1. Chemical composition revealed that the percent of moisture, protein, fat, fiber, ash and calculated carbohydrates were 11.20 %, 13.64 %, 5.89 %, 6.7 %, 3.1 % and 70.67 % respectively. Nascimento et al., (2014) found that the values of protein, fat, fiber and ash in quinoa seeds were 12.1, 6.3. 10.4 and 2.01 %, db, respectively. Bhargava, et al., (2006) reported that the fat level of quinoa was 6.31 %, db. Current results satisfied with Sciarini, et al., 2020 and Repo-Carrasco et al., 2010 about the protein content (11 to 22 %) in quinoa seeds. Quinoa is characterized by its high mineral content; calcium content was 650 mg/100 g seeds. Sodium content was 176 mg/100 g quinoa seeds. Potassium content was 830 mg/100 g whereas phosphorus content was 122 mg/100 g quinoa seeds. Iron content was 14.50 mg/100 g. Zinc found in quinoa seeds at level of 45 mg/100 g. Magnesium and manganese are found in sufficient amounts at 326 and 194 mg/100 g respectively, and in bioavailable forms to maintain a balanced human diet.

Our statistics result is consistent with that disclosed by Gordillo-Bastidas, et al., (2016) especially for iron (9.47 mg/100 g db), calcium (87 mg/100 g dm), magnesium (362 mg/100 g db), and potassium (907 mg/100 g db). Thus, we have confirmed what El Sohaimy et al., (2018) concluded, which is quinoa was perfectly equilibrated minerals specially (K, Na, Mg, Ca and Fe). A slight difference between the quinoa content of (K) and obvious difference of (Ca, P, Fe, Zn, and Mg) in the current study and that recorded by Stikic et al., (2012) for quinoa grower in north-west of Serbian may be due to the difference in the variety and cultivation conditions.

Quinoa (Chenopodium quinoa) are pseudo cereal grains rich in micronutrients including phytochemicals. The data in Table 2 shows the most essential phytochemicals in quinoa seeds (ppm), and the total polyphenols present in quinoa seeds were 435.0 ppm. According to (liu, et al., 2021), phenolic compounds are largely found in the seed coat of quinoa seeds and make up most of the secondary plant metabolites that lead to several functional properties. Quinoa contains more phenols than whole cereals and considered the highest phenols concentration of ferulic acid and quercetin in quinoa seeds (Gordillo-Bastidas et al, 2016). Recently, researchers are interested in studying the biological effects of phenolic materials because of their association with fibers, Suriano et al., 2018 and liu et al., 2021 found that the highest rates of α-glucosidase and α-amylase inhibition of bound phenols after digestion, which contributes to lowering blood sugar. Repo-Carrasco-Valencia et al., (2010) determined total polyphenols and total flavonoids in quinoa and found that the polyphenols were 131.8 ± 10.3 mg 100 g⁻¹ and flavonoids were 62.07 ± 5.1 mg 100 g⁻¹. Quinoa is characterized by its high content of total tocopherol (vitamin E), as its concentration reached 520 ppm, the most common tocopherol in quinoa seeds were γ -tocopherol 350 ppm followed by α-tocopherol 55 ppm then β-tocopherol at the concentration of 18.9 ppm. The tocopherols content of quinoa is essential and acts as an antioxidant at the level of the cell membrane, defending the membranes' fatty acids against oxidative stress. Values of phytochemical content in quinoa seeds indicate that quinoa can serve as a natural antioxidant and necessary to avoid the chronic and degenerative diseases (liu et al., 2021).

3.2 The amino acid compositions of quinoa seeds

Data in Table 3 carried out the amino acid composition of quinoa seeds. Obtained data illustrated that the total essential amino acid of quinoa seed was 4.88 g/100 g sample, and the total non-essential amino acids was 8.24 g/100 g. Quinoa is rich in lysine (0.43 mg/100 g quinoa) and threonine (0.40 mg/100 g quinoa), which are the most often deficient amino acids in cereal. The present results are comparable to described by Starzyńska-Janiszewska et al., (2016), regarding the lysine content of quinoa. Therefore, quinoa has a high nutritional value and belongs to the category of complete proteins (Okon, 2021). Plus, it contains a relatively complete and balanced formula of essential amino acids, which makes it complementary to most cereals, even some legumes (Okon, 2021).

Table 4 illustrates the results acquired from the present study regarding fatty acids. The fat contents showed a varied composition of fatty acids, most of them linoleic acid (omega-6 polyunsaturated fatty acids) as the highest fatty acid (56.31 %) found in quinoa seeds, followed by oleic acid (22.00 %) then α-linolenic acid (9.20 %). In general, total saturated fatty acids is low in quinoa seeds (9.60 %), whereas monounsaturated fatty acids are found in 24.54 %, but the highest fatty acid content was for polyunsaturated fatty acids (65.61 %). The results of linolenic acid in quinoa are close to those obtained by Rosero et al. (2013) 44.5–56.4 %, confirmed that linoleic acid was present in abundance in the polyunsaturated fatty acids found in different types of quinoas. Regarding to α-linolenic acids, Rosero et al., (2013) and Nasir et al., (2015) found that quinoa contained between 3.8 and 8.8 % confirming the results of current study. On the other hand, Gordillo-Bastidas et al., (2016) scored different value with the current results for unsaturated fatty acids (24.0–27.7 % oleic acid, 38.9–57 % linoleic acid and 4 % α-linolenic acid).

With regard to the vitamins content in quinoa seeds which are essential compounds for human health, quinoa seeds have significantly higher content of numerous vitamins compared to the most common cereals such as wheat, barley and rice. The results of Table 5 showed that every 100 g of quinoa seeds contains: 55.04 mg of vitamin A in the form of carotenoids, 15.06 mg of ergocalciferol, 10.2 mg of vitamin C. The concentration of vitamin B1 was 1.5 mg/100 g, vitamin B2 was 2.17 mg/100 g and the niacin reached to 1.1 mg/100 g which is vital for body health. Also, every 100 g of quinoa seeds contains 5.6 mg of vitamin B5, 0.32 mg of pyridoxine, 6.5 mg of folic acid and 0.23 mg of vitamin B12, which is not found in cereals. Bhargava et al. (2006), reported that the quinoa flour has 23.5 g folic acid (B9), 0.61 mg pantothenate (B5), and 0.2 mg pyridoxine (B6) in terms of a 100 g flour. El Sohaimy et al. (2018) confirmed that quinoa flour is high in vitamin C (1.93 mg/ Kg), B3 (0.15 mg/ Kg), B6 (11.22 mg/ Kg), and B12 (0.09 mg/ Kg), which are the essential nutrients required for the metabolism of practically all known living organisms. Thus, eating quinoa prevents scurvy and cold infection.

3.3 Serum ALT, AST and ALP level

The level of AST, ALT and ALP enzymes are essential liver function assessment indications. Statistically increases were reported in the level of serum AST, ALT and ALP in diabetic rats of group 2 (14.85 ± 0.71, 15.00 ± 0.63and 75.12 ± 4.65 (IUL^-1)) respectively, compared with normal rats of group1. On the other hand, the levels of ALT, AST, ALP was statistically unchanged in diabetic rats treated with quinoa seeds compared with control rats (Table 7). The elevated serum transaminases might be attributed to cell damage caused by high level of glucose blood (group 2) induced oxidative stress and inflammation. In the present study, high level of glucose blood (group 2) induced increases in AST, ALT, and ALP activities were reduced by Chenopodium quinoa seeds (group 3) without significant differences compared with the control (group 1). These results are in the line with others found by Cao et al., (2020), who shown that feeding quinoa for 8 weeks improved liver tissue and the level of transaminases such as ALT and AST in rats fed a high-fat diet. Perhaps due to the abundance of proteins, minerals, dietary fibers, essential amino acids, and bioactive compounds in quinoa seeds, which protect the liver from oxidative stress caused by high blood glucose levels. Betacyanins, rutin,quercetin, and other flavonoids also present in quinoa, according to earlier studies. These compounds were proven to have an antiinflammatory effects, perform as an antioxidative and established to decrease lipid peroxidation, hepatic lipid accumulation, inflammation and oxidative stress, (Zhao et al. 2018; Abdel-Wahhab et al., 2021).

3.4 Serum total lipids, total cholesterol, ldl-Cholesterol and HDL- Cholesterol

In all experimental groups, blood total lipids, total cholesterol, ldl-cholesterol and HDL-cholesterol were measured (Table 8). In diabetic rats of group 2, blood total lipids, ldl-Cholesterol and total cholesterol, levels were significantly elevated, whereas HDL-Cholesterol levels were reduced, compared to control rats of group 1. Otherwise, no significant effect was observed in these parameters between the rats in group 3 and the control group. The Quinoa seeds (Chenopodium quinoa) showed hepato protective effects induced damage lab rats.

The present results indicated that most of the fourth week of quinoa seed intake among diabetic rats had a hypoglycemic effect but caused the best changes with respect to cholesterol, LDL and HDL levels. Streptozocin-induced diabetes leads to some alterations in the level of metabolic enzymes in relation to insulin, and consequently leads to hyperglycemia. Diabetes induces half of the irrational modifications in plasma lipids. However, lipoproteins, such as partial body tissues especially the lungs, play a huge role in the intentional fair fatty acids in the blood, and subsequently the oxidation and metabolic modifications of these fatty acids in combination with unwanted cholesterol,molecules, and phospholipid synthesis, yet the secretion of some proteins in plasma. HDL is a particular transport system of lipids that reverses the transportation of cholesterol by eliminating it from tissues (Maranhão et al., 2018). In addition, an increase in the levels of LDL cholesterol and triglycerides. Yanardag et al., (2002) illustrated that the high levels of glucose in diabetic rats may increase triglycerides, VLDL and LDL, do not directly reduce HDL. Furthermore, quinoa has a high fiber content, which binds to bile acid and increases cholesterol degradation (Escudero, et al., 2006) as well as the fermentation of fiber in the colon produces short chain fatty acids and reduces cholesterol synthesis in the liver (Graf et al., 2015; Aniess et al., 2020). Cao et al., (2020) showed anti-hyperlipidemia benefits in rats treated with a high-fat diet after eating quinoa for 8 weeks.

3.5 Serum total bilirubin, uric acid and creatinine

Table 9 shows the blood total bilirubin, uric acid and creatinine levels in all experimental groups.

High creatinine levels show a person with kidney failure, as Barakat and Mahmoud (2011) have documented, due to higher cholesterol. When compared to normal group 1 diabetic rats, serum total bilirubin, uric acid,creatinine and levels were statistically improved in group 2 diabetic rats. In the serum of group 3 diabetic rats, minor differences in total bilirubin, creatinine, and uric acid levels were detected. Certain quinoa vitamins, minerals and fiber are necessary because of their function as antioxidant substances in the membranes of renal cells, such as selenium, magnesium, folic acid and tocopherols (Altunkaynak et al., 2008). The most obvious limitation of the present study is that,some procedures were not available in the laboratory, thus we transfer some samples to other laboratory to complete the analysis.