1. INTRODUCTION
⌅Metabolic syndrome (MS) is of high prevalence worldwide and represents a public health problem with elevated costs. The syndrome has not been fully understood in terms of its multifaceted origin, although a sedentary life style and unbalanced dietary pattern might be the most common factors in its development (Lemieux and Després, 2020Lemieux I, Després J-P. 2020. Metabolic syndrome: Past, present and future. Nutrients 12 (11), 3501. doi: https://doi.org/10.3390/nu12113501.; Bovolini et al., 2021Bovolini A, Garcia J, Andrade MA, Duarte, JA. 2021. Metabolic syndrome pathophysiology and predisposing factors. Int. J. Sports Med. 42 (3), 199–214. doi: https://doi.org/10.1055/a-1263-0898.). MS is an aggregate of clinical conditions including abdominal obesity, hypertriglyceridemia, high density lipoprotein-cholesterol, high blood glucose, hypertension, and elevated inflammatory cytokines and is often associated with fatty liver. Therefore, MS is linked to an increased risk of chronic diseases, especially cardiovascular diseases (CVDs) and type 2 diabetes (Lemieux and Després 2020Lemieux I, Després J-P. 2020. Metabolic syndrome: Past, present and future. Nutrients 12 (11), 3501. doi: https://doi.org/10.3390/nu12113501.; Bovolini and García, 2021Bovolini A, Garcia J, Andrade MA, Duarte, JA. 2021. Metabolic syndrome pathophysiology and predisposing factors. Int. J. Sports Med. 42 (3), 199–214. doi: https://doi.org/10.1055/a-1263-0898.). Current therapy for MS is limited to the individual management of hypertriglyceridemia, hypertension, and hyperglycemia, in addition to controlling diet with regular exercise. The beneficial effects of nutraceuticals in MS are worth investigating. Nutraceuticals are bioactive ingredients from foods that possess prevention or treatment of one or more chronic diseases. Quinoa seeds might be a good source of nutraceuticals which may have health benefits towards MS. Quinoa is called a pseudo-cereal from a botanical point of view, due to its exceptional balance between protein, oil, fibers and carbohydrates, in addition to the presence of phytochemicals and variable bioactive constituents. Therefore, quinoa is considered a functional food that may lower the risk of chronic diseases (Vega-Gálvez et al., 2010Vega-Gálvez, A, Miranda M, Vergara J, Uribe E, Puente L, Martínez EA. 2010. Nutrition facts and functional potential of quinoa (Chenopodium quinoa willd.), an ancient Andean grain: a review. J. Sci. Food Agric. 90 (15), 2541–2547. http://dx.doi.org/10.1002/jsfa.4158.). It is an important source of minerals, vitamins, polyphenols, phytosterols, flavonoids, vitamin E and omega-6 fatty acids with possible nutraceutical benefits (James, 2009James LE. 2009. Quinoa (Chenopodium quinoa Willd.): composition, chemistry, nutritional, and functional properties. Adv. Food Nutr. Res. 58, 1–31. Doi: https://doi.org/10.1016/S1043-4526(09)58001-1.). In a previous work, quinoa alcoholic extracts (Hydrophilic extract) were shown to prevent steatohepatitis and CVDs (Al-Okbi et al., 2021Al-Okbi SY, Abd El Ghani S, Elbakry HFH, Mabrok HB, Nasr SM, Desouky HM, Mahmoud KF. 2021. Kishk Sa′eedi as a potential functional food for management of metabolic syndrome: A study of the possible interaction with pomegranate seed oil and/ or gum Arabic. J. Herbmed. Pharmacol. 10 (3), 319–330. doi: https://doi.org/10.34172/jhp.2021.37.). Therefore, in the present study, the health benefits of the other compartment of quinoa, the lipophilic extract, were investigated. Due to the reported presence of the aforementioned anti-inflammatory, hypolipidemic, and antioxidant constituents in the lipophilic compartment of quinoa, it was hypothesized that the petroleum ether extract of quinoa might have potential beneficial effect towards MS. Hence, the objective of the present research was to study the lipotropic activity (promoting the removal of fat from the liver), the lowering effect on postprandial glucose, the antioxidant and the correction of dyslipidemia by petroleum ether extracts from two quinoa varieties in a rat model of MS. Investigations of the histopathological changes in the liver and heart in MS rats and the consequent effect of quinoa extracts were among the aims of the present study. This research also aimed at associating biological activity with the content and profile of α-tocopherol and fatty acids, respectively, in both quinoa varieties.
2. MATERIALS AND METHODS
⌅2.1. Materials
⌅Two varieties of wild Chenopodium quinoa seeds from the Amaranthaceae family were studied in the present work: quinoa 1, which was obtained from the Agriculture Research Center, Giza, Egypt; and Hualhuas, which was supplied from the International Potato Center (CIP), Lima, Peru.
2.2 Methods
⌅2.2.1. Preparation of petroleum ether extracts from two Chenopodium quinoa varieties
⌅The quinoa seeds of the two varieties were separately washed with water and dried in a hot air oven at 40 ºC. Known weights of both seed varieties were extracted with a continuous extraction apparatus (soxhlet) by petroleum ether (40-60). After complete extraction, the solvent was evaporated at a temperature not exceeding 40 °C and the obtained extracts were used in the present study.
2.2.2. Determination of α-tocopherol and fatty acid methyl esters in the petroleum ether extracts of the two quinoa varieties.
⌅The samples were saponified according to Lee et al. (2012)Lee YY, Park HM, Lee CK, Kim SL, Hwang, T, Choi MS, Kwon Y, Kim WH, Kim SJ, Lee SC, Kim YH. 2012. Comparing extraction methods for the determination of tocopherols and tocotrienols in seeds and germinating seeds of soybean transformed with OsHGGT. J. Food Comp. Anal. 27 (1), 70–8. for the determination of α-tocopherol. The samples (2 g) were added to 15 ml of ethanol containing 6% pyrogallol in a saponification vessel and mixed by vortex for 30 seconds. After sonication for 5 min, 5 ml of potassium hydroxide (30%) were added and the vessel was flushed with nitrogen gas for 1 min. The contents were heated at 70 °C for 50 min in a shaking water bath. After the samples had been cooled for 5 min in an ice bath, 20 ml of sodium chloride (2%) were added, and mixed by vortex for 30 seconds. The mixture was extracted three times with 50 ml n-hexane: Ethyl acetate extraction solvent (80:20, v/v containing 0.1 g/L BHT). The extracts were collected, diluted to a final volume of 50 ml, filtered through a 0.2 mm filter and analyzed by HPLC. Agilent Technologies 1100 series liquid chromatography equipped with degasser, quaternary pump, auto sampler, diode-array and florescence detectors were used. The analytical column was Eclipse XDB-C18 (150 X 4.6 µm; 5 µm). Alpha-tocopherol was identified and quantified by comparison with a known standard.
Methyl esters were prepared via trans-esterification of the fatty acids using a methanol sulfuric acid and chloroform mixture according to Indarti et al. (2005)Indarti E, Majid MIA, Hashim R, Chong, A. 2005. Direct FAME synthesis for rapid total lipid analysis from fish oil and cod liver oil. J. Food Compost. Anal. 18 (2–3), 161–170.. Twenty mg of the oil were weighed into clean, 10-ml screw-cap glass tubes, to which 4 ml fresh solution of a mixture of freshly prepared methanol, concentrated sulfuric acid and chloroform (1.7/0.3/2 v/v/v) was added. The tubes were covered and trans-esterification was run at 100 °C for 30 min. On completion of the reaction, the tubes were brought to room temperature. Then, 3 ml distilled water were added to the mixture and thoroughly mixed by vortex for 1 min. After the formation of two phases, the lower phase containing the fatty methyl esters was transferred to a clean glass tube and dried with anhydrous Na2SO4. Fatty acids were analyzed by GC after the addition of a known volume of chloroform. The assessment of the methyl ester was carried out by injecting 2µl into a Hewlett Packard HP-system 6890 gas chromatograph equipped with FID. A HP-5 capillary column (30 m x 0.32 mm i.d.; 0.25 µm film thickness) was used to separate the different methyl esters. The chromatographic analysis conditions were as follows: initial temperature 70 ºC, held for 1 min, then raised to 120 ºC at a rate of 40 ºC /min, held for 2 min, then the temperature was finally raised to 220 ºC at a rate of 4 ºC /min and then held for another 20 min. The injector and detector temperatures were 250 ᵒC and 280 ºC, respectively. Identification of the fatty acid methyl esters was carried out by direct comparison of the retention times of each of the separate compounds to standards of the fatty acid methyl esters analyzed under the same conditions. Quantification was based on peak area integration.
2.2.3. Animals
⌅Adult male albino rats of body weight ranging from 70 to 80 g (age: 5-6 weeks) were used in the present study. The animals were purchased from the Animal House of National Research Centre, Cairo, Egypt. The rats were housed in stainless steel cages (2 rats/ cage); water and food were given ad-libtium with a 12-hour light/dark cycle. The animal experiment was carried out according to the Medical Research Ethics Committee, National Research Centre, Cairo, Egypt, and followed the recommendations of the Health Guide for Care and Use of Laboratory Animals.
2.2.4. Diets
⌅Two diets were prepared -- a balanced diet and a high fructose-high fat diet (HFHFD). The balanced diet was composed of 10% protein supplemented from casein, 10% sunflower oil, 70.5% starch, 5% cellulose, 1% vitamin mixture and 3.5% salt mixture. The HFHFD was prepared from 10% protein supplemented from casein, 20% sheep tallow, 63.5% fructose, 2% starch, 1% vitamin mixture and 3.5% mineral mixture. The high fructose-high fat diet was prepared in accordance with Al-Okbi et al. (2021)Al-Okbi SY, Abd El Ghani S, Elbakry HFH, Mabrok HB, Nasr SM, Desouky HM, Mahmoud KF. 2021. Kishk Sa′eedi as a potential functional food for management of metabolic syndrome: A study of the possible interaction with pomegranate seed oil and/ or gum Arabic. J. Herbmed. Pharmacol. 10 (3), 319–330. doi: https://doi.org/10.34172/jhp.2021.37. with some modification for the induction of metabolic syndrome (MS).
2.2.5. Preparation of the extracts in suitable form for rat dosing
⌅The extracts were emulsified separately in water using tween 80 at 10% from the extract weight and the whole mixtures were mixed using vortex to be ready for dosing the test groups through a gastric tube. Similar quantities of water and tween 80 were mixed by vortex (representing the vehicle) to be given to the control groups.
2.2.6. Design of the animal experiment
⌅Four groups of rats were assigned, each of 8 rats: control-fed balanced diet (NC), control-fed HFHFD (MSC) and two test groups fed the HFHFD and treated by either quinoa 1 or Hualhuas petroleum ether extract at a daily oral dose (500 mg/kg) for a month. The rats in the NC and MSC groups were given a daily oral dose from the vehicle. Body weight and food intake were measured once weekly during the experiment. An oral glucose tolerance test (OGTT) was carried out after 4 weeks. After an overnight fast, blood was taken from the tails of all the rats for the determination of fasting blood glucose (zero time) using a Glucometer. Quinoa 1 and Hualhuas extracts were given to the rats in groups 3 and 4, respectively, orally at 500 mg/kg rat body weight; while the two control groups were given the vehicle. All the rats, including the two control groups, were given 1 g glucose/kg rat body weight in solution form (Al-Okbi et al., 2018Al-Okbi SY, Hussein AMS, Elbakry HFH, Fouda KA, Mahmoud KF, Hassan MF. 2018. Health benefits of fennel, rosemary volatile oils and their nano-forms in dyslipidemic rat model. Pakistan J. Biolog. Sci. 21 (7), 348–358. doi: https://doi.org/10.3923/pjbs.2018.348.358.). Blood glucose was assessed after 1/2 hr, 1hr, 2 hrs and 4 hrs from glucose intake. The rats continued feeding on the same diets and doses for an extra week. At the end of the experiment, nutritional parameters represented by total food intake, body weight gain and feed efficiency ratio (body weight gain/total food intake) were calculated. Blood samples were taken by heart puncture from fasted anesthetized rats and collected in heparinized tubes. The tubes were centrifuged and the plasma was separated. Anesthesia was carried out by an intraperitoneal injection of the pentobarbital (50 mg/Kg rat body weight). Plasma malondialdehyde (MDA) as a determinant of lipid peroxidation and total antioxidant capacity (TAC) was determined as an indicator of oxidative stress and antioxidant status, respectively, as previously assayed (Satoh, 1978Satoh K. 1978. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chim. Acta 90 (1), 37–43. doi: https://doi.org/10.1016/0009-8981(78)90081-5.; Koracevic et al., 2021Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. 2001. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 54 (5), 356–61. doi: https://doi.org/10.1136/jcp.54.5.356.). Plasma lipids represented by total cholesterol (TC), triglycerides (TGs), low density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined in accordance with previous methods (Watson, 1960Watson D. 1960. A simple method for the determination of serum cholesterol. Clin. Chim. Acta. 5 (5), 637–643. doi: https://doi.org/10.1016/0009-8981(60)90004-8.; Megraw et al., 1979Megraw R, Dunn D, Biggs H. 1979. Manual and continuous flow colorimetry of triglycerols by a fully enzymatic method. Clin. Chem. 25 (2), 273–284. doi: https://doi.org/10.1093/clinchem/25.2.273.; Schriewer et al., 1984Schriewer H, Kohnert U, Assmann G. 1984. Determination of LDL cholesterol and LDL apolipoprotein B following precipitation of VLDL in blood serum with phosphotungstic acid/MgCl2. J. Clin. Chem. Clin. Biochem. 22 (1), 35–40. doi: https://doi.org/10.1515/cclm.1984.22.1.35.; Burstein et al., 1970Burstein M, Scholnick HR, Morfin R. 1970. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J. Lipid Res. 11 (6), 583–595.). The ratio of TC/HDL-C was calculated as cardiovascular disease risk factor. Rats were dissected after euthanasia through cervical dislocation under anesthesia. Livers and hearts were instantaneously removed from the dissected rats; a part of the liver was analyzed for total liver lipids according to the procedure of Cequier-Sànchez et al. (2008)Cequier-Sànchez E, Rodrìguez C, Ravelo G, Zàrate R. 2008. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. J. Agric. Food Chem. 56 (12), 4297–4303. Doi: https://doi.org/10.1021/jf073471e.. Another part of the liver was kept with the hearts in 10% formalin for histopathological examination (Bancroft and Layton, 2019)Bancroft JD, Layton C. 2019. The Hematoxylins and Eosin. In SK Suvarna, C Layton, JD Bancroft [Ed.] Bancroft′s Theory and Practice of Histological Techniques. Elsevier, Vol. 8. p. 126-38..
2.2.7. Statistical analysis
⌅The results were expressed as the mean±SE. Normality of the data was confirmed by the Kolmogorov-Smirnov test, thus the parametric test of one-way analysis of variance ANOVA, followed by the Tukey test using the SPSS statistical program were applied. In all cases p ≤ 0.05 was used as the criterion of statistical significance.
3. RESULTS
⌅3.1. Fatty acids profiles and α-tocopherol in the petroleum ether extracts of quinoa
⌅The fatty acid profiles are compiled in Table 1. It could be noticed that linoleic acid (omega-6) was the major fatty acid in both quinoa1 and hualhuas (46.895 and 58.800%, respectively). The second predominant fatty acid was oleic, a monounsaturated fatty acid, which showed 28.808 and 16.210% in quinoa1 and hualhuas, respectively. Linolenic acid (omega-3) demonstrated a higher level in hualhuas (8.981) compared to quinoa1 (1.261). Total unsaturated fatty acids were 76.964 and 83.991 in quinoa1 and hualhuas, respectively; while total saturated fatty acids were 11.533 and 10.239. The ratio Omega-3: Omega-6 was shown to be 1:37 and 1:7 in quinoa1 and hualhuas oils, respectively.
| Quinoa variety | ||
|---|---|---|
| Quinoa 1 | Hualhuas | |
| Fatty acids | ||
| C14:0 (Myristic) | 0.426 | - |
| C16:0 (Palmitic) | 10.570 | 10.239 |
| C18:0 (Stearic) | 0.537 | - |
| C18:1 (Oleic) | 28.808 | 16.210 |
| C18:2 (Linoleic; omega-6) | 46.895 | 58.800 |
| C18:3 (Linolenic; omega-3) | 1.261 | 8.981 |
| Total unsaturated fatty acids | 76.964 | 83.991 |
| Total saturated fatty acids | 11.533 | 10.239 |
| Omega -3: Omega- 6 | 1: 37 | 1: 7 |
| Alpha-tocopherol (µg/g) | 787.638 | 81.490 |
Table 1 demonstrates the levels of alpha-tocopherol in the petroleum ether extracts of both quinoa varieties. It was observed that quinoa 1 contained a very high level of alpha-tocopherol (787.638 µg/g) compared to Hualhuas (81.490 µg/g)
3.2. Results of the biological experiments
⌅Glucose tolerance curves are shown in Figure 1. Insignificant changes in fasting blood glucose were noticed when all groups were compared to the NC group. The blood glucose levels of the MSC group of the different times intervals demonstrated significantly higher levels compared to the CN group. Rats treated with quinoa extracts exhibited a significant decrease in blood glucose throughout the determined intervals when compared to the MSC group, except for the group given the hualhuas extract after 4 h from glucose administration where the reduction was insignificant. All the levels of blood glucose in the rats given quinoa extracts during the glucose tolerance curve demonstrated insignificant changes from those of the NC group, except the group given hualhuas on 2- and 4- hour intervals, where they exhibited a significant increase.
The different plasma lipids, MDA, TAC and liver fat of the different groups are compiled in Figure 2. Significant increases in TG, TC, LDL-C and TC/HDL-C with significant reduction in HDL-C were manifested in the MSC group compared to the NC group. All lipid parameters showed significant improvements after treatment with quinoa extracts in comparison to the MSC group, except for only TG, in the case of the group being given the quinoa 1 extract. The rats treated with quinoa 1 extract showed a significant reduction in TC and LDL-C compared to those treated with hualhuas. The plasma MDA of the MSC group showed a significant elevation compared to the NC group. Both test groups demonstrated significant reductions in MDA compared to the MSC group. The plasma MDA of the group treated with quinoa1 exhibited a significant increase in comparison to the NC group; while hualhuas showed insignificant changes. The MSC group showed a significant reduction in TAC compared to the NC group. The groups treated with quinoa extracts demonstrated an insignificant increase compared to the MS control and a significant reduction compared to the NC group. Total liver fat of the MSC group demonstrated a significant increase compared to the NC group. The group treated with the quinoa 1 extract showed a significant reduction in total liver fat compared to the MSC group while demonstrating an insignificant change from the NC group. The Hualhuas extract-treated group only showed an insignificant reduction in total liver fat compared to the MSC group, while demonstrating a significant elevation in comparison to the NC group.
Figure 3 exhibited the nutritional parameters of different groups. Final body weight, body weight gain, total food intake and food efficiency ratio in the MSC group demonstrated insignificant changes in comparison to the NC group. The petroleum ether extracts of quinoa produced significant decreases in all nutritional parameters compared to either the MSC or the NC group, except for total food intake in the case of the petroleum ether extract of hualhuas, which showed an insignificant change.
The histopathological changes in the liver of the different experimental groups are seen in Figure 4. It can be observed that the MSC group showed great degeneration and vacuolation of hepatic cells, with deposition of fatty globules. The MSC group manifested slanted hepatic sinusoids together with focal necrosis. Inflammation was observed with the infiltration of mononuclear inflammatory cells. The presence of necrobiosis, pyknotic nucleus and deep homogenous eosinophilic cytoplasm, in addition to kupffer phagocytic cell activation were also noticed in the MSC group. The group treated with quinoa 1 demonstrated mild to moderate histopathological changes compared to the MSC group; while rats given hualhuas did not produce any improvement in liver histopathology compared to the MSC group.
Figure 5 shows the histopathological changes in the heart in the different experimental groups. The NC group showed a normal appearance of the heart. The heart muscle of the control group with metabolic syndrome showed dispersed fragmentation together with muscle myofiber necrosis, nuclei pyknosis, and striation loss with interstitial edema. The longitudinal section of the myocardium of the group treated with the quinoa 1 extract exhibited minute focal necrosis, mild fragmentation and edema of the myofibers. The cross section of the myocardium of the rats given hualhuas quinoa extract presented moderate myofiber necrosis accompanied by nuclei pyknosis, together with loss of striation, fragmentation and interstitial edema.
4. DISCUSSION
⌅The demonstrated significant elevated glucose tolerance, dyslipidemia (high TG, TC, LDL-C and TC/HDL-C with low HDL-C), high MDA, low total antioxidant and elevated liver fat in addition to histopathological changes in the liver and heart in rats fed on HFHFD compared to the NC group indicated the induction of MS, which agreed with a previous study, (Al-Okbi et al., 2021Al-Okbi SY, Abd El Ghani S, Elbakry HFH, Mabrok HB, Nasr SM, Desouky HM, Mahmoud KF. 2021. Kishk Sa′eedi as a potential functional food for management of metabolic syndrome: A study of the possible interaction with pomegranate seed oil and/ or gum Arabic. J. Herbmed. Pharmacol. 10 (3), 319–330. doi: https://doi.org/10.34172/jhp.2021.37.). Elevated TGs, TC and LDL-C, along with a reduction in HDL-C were reported as risk factors for cardiovascular diseases (CVDs) especially on elevation of TC/HDL-C as could be seen on feeding HFHFD in the present study. Treatment with the extracts produced variable improvements in biochemical parameters and histopathology with a reduction in body weight compared to the MSC group. The therapeutic efficiency of both quinoa extracts resides in significantly reducing plasma TG, TC and LDL-C with the elevation of HDL-C. The reduction of TC/HDL-C on administration of the extracts pointed to the inhibition of cardiovascular disease risks. Liver fat was significantly reduced due to treatment with quinoa 1, which indicated the potential reduction of fat synthesis, elevated fat breakdown and /or lipotropic effect of such extract. The reduction of glucose tolerance and body weight might indicate the potential anti-diabetic and anti-obesity effects of the extracts, respectively. The reduction in body weight gain without affecting food intake in the case of treatment with the hualhuas extract might propose the presence of bioactive constituent with increasing energy expenditure. However, the reduction in food intake on administration of the quinoa 1 extract might point to the anorexigenic effect of such extract. The reduction of MDA on administration of the extracts revealed inhibition of lipid peroxidation with subsequent reduction of oxidative stress. The anti-MS effect of the extracts might be ascribed to the presence of alpha-tocopherol, omega-3 and omega-6 fatty acids as determined in the present study and other bioactive constituents reported previously. Gamma and α-tocopherol were identified in quinoa (Pereira et al., 2019Pereira E, Encina-Zelada C, Barros L, Gonzales-Barron U, Cadavez V, Ferreira ICFR. 2018. Chemical and nutritional characterization of Chenopodium quinoa Willd (quinoa) grains: A good alternative to nutritious food. Food Chem. 280, 110–114. doi: https://doi.org/10.1016/j.foodchem.2018.12.068.; Tang et al., 2016Tang Y, Li X, Chen PX, Zhang B, Liu R, Hernandez M, Draves J, Marcone MF, Tsao R. 2016. Assessing the fatty acid, carotenoid, and tocopherol compositions of Amaranth and Quinoa seeds grown in Ontario and their overall contribution to nutritional quality. J. Agric. Food Chem. 64 (5), 1103–10. doi: https://doi.org/10.1021/acs.jafc.5b05414.). In addition, phytosterols and carotenoids reported in previous studies in quinoa (Tang et al., 2016Tang Y, Li X, Chen PX, Zhang B, Liu R, Hernandez M, Draves J, Marcone MF, Tsao R. 2016. Assessing the fatty acid, carotenoid, and tocopherol compositions of Amaranth and Quinoa seeds grown in Ontario and their overall contribution to nutritional quality. J. Agric. Food Chem. 64 (5), 1103–10. doi: https://doi.org/10.1021/acs.jafc.5b05414.; Vega-Gálvez et al., 2010Vega-Gálvez, A, Miranda M, Vergara J, Uribe E, Puente L, Martínez EA. 2010. Nutrition facts and functional potential of quinoa (Chenopodium quinoa willd.), an ancient Andean grain: a review. J. Sci. Food Agric. 90 (15), 2541–2547. http://dx.doi.org/10.1002/jsfa.4158.) might participate in such health benefits. The total phytosterols were reported to range from 9.4 to 12.2 g/kg (Shen et al., 2022Shen Y, Zheng L, Peng Y, Zhu X, Liu F, Yang X, Li H. 2022. Physicochemical, antioxidant and anticancer characteristics of seed oil from three Chenopodium quinoa genotypes. Molecules 27, 2453. https://doi.org/10.3390/molecules27082453.). Trace amounts of α- and β-tocotrienols were found in quinoa (Tang et al., 2015Tang Y, Li X, Chen PX, Zhang B, Hernandez M, Zhang H, Marcone MF, Liu R, Tsao R. 2015. Characterisation of fatty acid, carotenoid, tocopherol/tocotrienol compositions and antioxidant activities in seeds of three Chenopodium quinoa Willd. genotypes. Food Chem. 174, 502–508. doi: https://doi.org/10.1016/j.foodchem.2014.11.040.). Carotenoids, mainly trans-lutein and zeaxanthin, were identified for the first time in quinoa seeds (Tang et al., 2015Tang Y, Li X, Chen PX, Zhang B, Hernandez M, Zhang H, Marcone MF, Liu R, Tsao R. 2015. Characterisation of fatty acid, carotenoid, tocopherol/tocotrienol compositions and antioxidant activities in seeds of three Chenopodium quinoa Willd. genotypes. Food Chem. 174, 502–508. doi: https://doi.org/10.1016/j.foodchem.2014.11.040.). The antioxidant activities of lipophilic extracts were reported to be positively correlated with total carotenoids and total tocopherols (Tang et al., 2015Tang Y, Li X, Chen PX, Zhang B, Hernandez M, Zhang H, Marcone MF, Liu R, Tsao R. 2015. Characterisation of fatty acid, carotenoid, tocopherol/tocotrienol compositions and antioxidant activities in seeds of three Chenopodium quinoa Willd. genotypes. Food Chem. 174, 502–508. doi: https://doi.org/10.1016/j.foodchem.2014.11.040.). Hypercholesterolemia was reported to produce a reduction in antioxidant enzymes (glutathione and catalase) causing damage to the oxidative defense system of the cell. Such changes lead to reactive oxygen species that lead to high oxidative stress (Nwichi et al., 2012Nwichi SO, Adewole EK, Oada DA, Ogidiama O, Mokobia OE, Farombi EO. 2012. Cocoa powder extracts exhibits hypolipidemic potential in cholesterol-fed rats. Afr. J. Med. Med. Sci. 41, Suppl 39–49.). Therefore, the hypocholesterolemic effect seen in the present study together with the reported antioxidant effect of quinoa could provide potential protection from CVDs.
It was reported that bread made from quinoa when consumed on a daily basis for a short term could modify glucose tolerance with a minimal effect on CVD risk biomarkers (Li et al., 2018Li L, Lietz G, Bal W, Watson A, Morfey B, Seal C. 2018. Effects of quinoa (Chenopodium quinoa Willd.) consumption on markers of CVD Risk. Nutrients 10 (6), pii: E777. doi: https://doi.org/10.3390/nu10060777.). In support of the previous studies, quinoa was shown to have potential in improving glucose tolerance (Mithila and Khanum, 2015Mithila MV, Khanum F. 2015. Effectual comparison of quinoa and amaranth supplemented diets in controlling appetite; a biochemical study in rats. J. Food Sci. Technol. 52 (10), 6735–4671. doi: https://doi.org/10.1007/s13197-014-1691-1. and Gabrial et al., 2016Gabrial SG, Shakib MR, Gabrial GN. 2016. Effect of pseudocereal-based breakfast meals on the first and second meal glucose tolerance in healthy and diabetic subjects. Open Access Maced. J. Med. Sci. 4 (4), 565–573. doi: https://doi.org/10.3889/oamjms.2016.115.). Previous studies (Mithila and Khanum, 2015Mithila MV, Khanum F. 2015. Effectual comparison of quinoa and amaranth supplemented diets in controlling appetite; a biochemical study in rats. J. Food Sci. Technol. 52 (10), 6735–4671. doi: https://doi.org/10.1007/s13197-014-1691-1.) demonstrated a reduction in body weight gain and food intake in rats fed quinoa, which agreed with the present study.
The oil content in quinoa ranges from 4.6 to 7.2%, (AAFRD, 2005AAFRD—Alberta Agriculture, Food and Rural Development. Quinoa—The Next Cinderella Crop for Alberta; el Hafid, R., Aitelmaalem, H., Driedger, D., Bandara, M., Stevenson, J., Eds.; Technical Report; Alberta Agriculture, Food and Rural Development (AAFRD): Edmonton, AB, Canada, 2005. p. 18.; Koziol, 1993Koziol MJ. 1993. Quinoa: A potential new oil crop, in Janick J, Simon JE (Eds.) New crops. Wiley, New York, pp. 328-336.). The present work showed that the oil content in quinoa was 4.48 and 5.20% for quinoa 1 and hualhuas, respectively, which is similar to the abovementioned values. The two known essential fatty acids to humans are α-linolenic [Omega-3 (ω-3)] and linoleic [Omega-6 (ω-6)]. The essential fatty acids are metabolized in the body to long-chain fatty acids of 20-22 carbon atoms. Linolenic acid is metabolized to eicosapentaenoic (EPA) and docosahexanoic acid (DHA), while linoleic acid is metabolized to arachidonic acid. EPA and DHA are known for their anti-inflammatory and hypolipidemic effects (Al-Okbi et al., 2020Al-Okbi SY, Mohammed SE, Al-Siedy ESK, Ali NA. 2020. Fish oil and primrose oil suppress the progression of Alzheimer’s like disease induced by aluminum in rats. J. Oleo Sci. 69 (7), 771–782. doi: https://doi.org/10.5650/jos.ess20015.). Linoleic acid is the most predominant fatty acid in quinoa as can be seen from the present study. In a previous study, palmitic acid, oleic acid, linoleic acid and linolenic were shown to be present in quinoa at 10.66, 24.7, 62.47 and 2.19%, respectively (Altuna et al., 2018Altuna JL, Silva M, Álvarez M, Quinteros MF, Morales D, Carrillo W. 2018. Ecuadorian quinoa (Chenopodium quinoa willd) fatty acids profile. Asian J. Pharm. Clin. Res. 11 (11), 209–211. doi: https://doi.org/10.22159/ajpcr.2018.v11i11.24889.). In another study, Peruvian quinoa was shown to contain 50.2% linoleic acid, 26% oleic acid and 4.8% linolenic acid (Repo-Carrasco et al., 2003Repo-Carrasco R, Espinoza C and Jacobsen SE. 2003. Nutritional value and use of the Andean crops quinoa (Chenopodium quinoa) and kaniwa (Chenopodium pallidicaule). Food Rev. Int. 19, 179–189. https://doi.org/10.1081/FRI-120018884.). The present study showed that both varieties are rich in linoleic acid but only hualhuas contains appreciable amounts of linolenic acid (8.981%), while the variety quinoa 1 only contained a very low percentage (1.261). The fatty acid profiles of quinoa varieties in the current study somehow fall within the ranges of fatty acids reported by Altuna et al. (2018)Altuna JL, Silva M, Álvarez M, Quinteros MF, Morales D, Carrillo W. 2018. Ecuadorian quinoa (Chenopodium quinoa willd) fatty acids profile. Asian J. Pharm. Clin. Res. 11 (11), 209–211. doi: https://doi.org/10.22159/ajpcr.2018.v11i11.24889. and Repo-Carrasco et al. (2003)Repo-Carrasco R, Espinoza C and Jacobsen SE. 2003. Nutritional value and use of the Andean crops quinoa (Chenopodium quinoa) and kaniwa (Chenopodium pallidicaule). Food Rev. Int. 19, 179–189. https://doi.org/10.1081/FRI-120018884. with only a higher value for linolenic in the hualhuas variety. The unsaturated fatty acids in quinoa in the present study are extremely high, nearly 7-8 times the saturated fatty acids. The ratio of omega-3/omega-6 fatty acids in quinoa was 1/6 (Shen et al., 2022Shen Y, Zheng L, Peng Y, Zhu X, Liu F, Yang X, Li H. 2022. Physicochemical, antioxidant and anticancer characteristics of seed oil from three Chenopodium quinoa genotypes. Molecules 27, 2453. https://doi.org/10.3390/molecules27082453.), which somehow is equal to that determined in the present study concerning hualhuas (1/7), which more or less reflects a good ratio of health benefits as anti-inflammatory.
Polyunsaturated fatty acids, represented by ω-3 and ω-6, have several positive effects on cardiovascular disease, improved insulin sensitivity and as anti-inflammatory, although a balance in the ratio ω-3/ ω-6 must be achieved (Bibus and Lands, 2015Bibus D, Lands B. 2015. Balancing proportions of competing omega-3 and omega-6 highly unsaturated fatty acids (HUFA) in tissue lipids. Prostaglandins Leukot Essent Fatty Acids. 99, 19–23. doi: https://doi.org/10.1016/j.plefa.2015.04.005.). All unsaturated fatty acids present in quinoa could be protected by the presence of vitamin E, identified in the present study, and which acts as a natural antioxidant which makes them more stable and less likely to become rancid, guaranteeing a longer shelf-life (Koziol, 1993Koziol MJ. 1993. Quinoa: A potential new oil crop, in Janick J, Simon JE (Eds.) New crops. Wiley, New York, pp. 328-336.). The content of vitamin E in quinoa is important for the human body since this vitamin acts as an antioxidant at the cell membrane level, protecting the fatty acids of the cell membranes against damage caused by free radicals. In a previous report, α-tocopherol was demonstrated to range from 2.6 to 5.37 mg/100 g quinoa (Vega-Galvez et al., 2010Vega-Gálvez, A, Miranda M, Vergara J, Uribe E, Puente L, Martínez EA. 2010. Nutrition facts and functional potential of quinoa (Chenopodium quinoa willd.), an ancient Andean grain: a review. J. Sci. Food Agric. 90 (15), 2541–2547. http://dx.doi.org/10.1002/jsfa.4158.). These levels are very low compared to the α-tocopherol in quinoa varieties in the present study. The total tocopherol content in three varieties of quinoa ranged from 117.29 to 156.67 mg/kg and mainly consisted of γ-tocopherol (Shen et al., 2022Shen Y, Zheng L, Peng Y, Zhu X, Liu F, Yang X, Li H. 2022. Physicochemical, antioxidant and anticancer characteristics of seed oil from three Chenopodium quinoa genotypes. Molecules 27, 2453. https://doi.org/10.3390/molecules27082453.)
5. CONCLUSIONS
⌅Petroleum ether extracts from both quinoa varieties showed the potential to prevent MS in rats via improving dyslipidemia, glucose tolerance, cardiovascular risks and oxidative stress along with reducing body weight. Both extracts produced significant reduction in body weight gain, MDA and TC/HDL-C. Rats treated by either quinoa extract exhibited a significant decrease in blood glucose throughout the determined intervals when compared to the MSC group, except for the group given the hualhuas extract after 4 h from glucose administration, where the reduction was insignificant. Quinoa 1 extract possesses lipotropic effects through reducing liver fat. Also, the quinoa 1 extract was superior in reducing plasma TC, LDL-C and histopathological changes in the liver and heart; while hualhuas was more efficient in improving plasma TGs. The bioactivity of quinoa extracts might be attributed to the presence of alpha-tocopherol and omega-3 and omega-6 fatty acids as determined in the present study. The strengths of the present study reside in highlighting the beneficial effects of the petroleum ether extracts of two quinoa varieties on MS and the associated changes in the liver and heart as well as the possible bioactive constituents in the extracts represented by fatty acids and vitamin E. However, the weaknesses of the study might be the lack of detailed investigations into the different bioactive phytochemicals in the extracts. Therefore, future research could be addressed for the identification, isolation or fractionation of other different bioactive components present in such extracts which might play a role in protection from MS. In a prospective study, the extracts should be clinically evaluated in humans.