Nutritional compositions in young leaves and stem from quinoa (Chenopodium quinoa Willd.) grown in Korea

The leaves and stem of quinoa (C. quinoa) were obtained by sowing and cultivating the quinoa seed which is gained from National Institute of Crop Science, Rural Development Administration located in Pyeongchang. The quinoa cultivated in a farm situated on the Gimpo, northwestern Gyeonggi. Leaves and stem were collected from 50 quinoa aged 1-month year old (height, 20-25 cm), and we removed roots and washed three times with cold water, immediately. Leaves and stem were divided and chopped into slices using a chopper (TC-8B, Techon Co., Bucheon, Korea). Then, all samples stored in a deep freezer set to the temperature of −70°C. Various kinds of standards such as free sugars, organic acids, fatty acid methyl ester, β-carotene, vitamins B₁, B₂, B₃, B₅, B₆, and B₁₂, ascorbic acid, and α-, β-, γ-, and δ-tocopherol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The standards for analyzing mineral content were obtained from AccuStandard (New Haven, CT, USA).

Quinoa leaves and stem were determined and compared for proximate composition and dietary fiber content according to instructions on the Association of Official Analytical Chemists (AOAC, 2005). The moisture content was determined by air oven method, and the moisture content (%) was calculated as follows: (fresh weight - dry weight) / sample weight × 100. The crude protein content was analyzed according to the Kjeldahl method. The crude lipid content was determined by Soxtec extraction system (Soxtec 1043, Foss Tecator AB, Höganäs, Sweden), and the crude ash content was evaluated according to the direct muffle-furnace method, and the ash content (%) was calculated as follows: (before incinerated weight - after incinerated weight) / sample weight × 100. The crude fiber content was measured using a fiber extractor (FIWE 6, Velp Scientifica, Usmate, Italy). The carbohydrate content (%) was calculated as follows: 100 - the moisture, crude ash, crude protein, and crude lipid content (%). Measurement of the dietary fiber content was performed by a simplified enzymatic-gravimetric method.

The contents of free sugar and organic acid in leaves and stem were measured using Agilent 1100 series liquid chromatography (Agilent Technologies, Palo Alto, CA, USA). Sample 5 g were blended with distilled water of 50 mL and centrifuged at 11,300 ×g for 20 min using a centrifuge (Supra-21K, Hanil, Incheon, Korea). The supernatant was filtered with a 0.45 μm filter in order to obtain the pure extracts. This filtrate was used as to sample. To analyze the fructose, glucose, sucrose, maltose, and lactose, we infused the sample of 20 μL into the high-performance liquid chromatography installed with a carbohydrate analysis column (4.6×250 mm, Waters Co., Milford, MA, USA). Five free sugar were separated with an isocratic elution at a flow rate of 1.0 mL/min and a mobile phase of an acetonitrile (80): water (20) mixture (v/v). The peaks were detected with a refractive index (RI) detector and each compound were identified by comparison with standards and the content was calculated with peak area. The sample of 20 μL were injected into the high-performance liquid chromatography installed with a Shodex Rs Pak KC-811 column (8.0×300 mm, Shodex, Tokyo, Japan), in order to analyze the organic acids (malic acid, citric acid, oxalic acid, acetic acid, formic acid, and succinic acid). Six organic acids were separated using a mobile phase of 0.1% H₃PO₄ and an isocratic elution at a 1.0 mL/min as flow rate. Each peak was detected with an ultraviolet (UV) detector and each compound were identified by comparing the standards and the content was calculated with peak area.

Minerals evaluated in leaves and stem of quinoa were calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), zinc (Zn), and selenium (Se). Each sample was pre-treated by the following procedures. Leaves and stem were dissolved in a 90% HNO₃/H₂O₂ mixture and then digested using a microwave digestion system for 30 min, respectively. The solution obtained from each sample was filtrated with a filter paper. This filtrate was used for analyzing. Seven elements (Ca, Cu, Fe, K, Mg, Na, and Zn) were evaluated using an inductively coupled plasma (ICP) spectrometer (Perkin Elmer Co., Shelton, CT, USA) and another elements (Se) was analyzed by ICP-mass spectrometry. The operating conditions of the ICP spectrometer were as follows: 1.4 kW reflected power, 10 L/min argon plasma gas flow rate, 0.2 L/min auxiliary gas flow rate, and 0.55 L/min nebulizer gas flow rate. The operating conditions of the ICP-mass spectrometry as follows: 1.4 kW reflected power, 9.6 V lens voltage, 18 L/min plasma flow rate, 1.5 L/min auxiliary gas flow rate, and 0.92 L/min nebulizer gas flow rate.

Fatty acids content and composition were analyzed according to the method of Jang et al. (2016). Each sample 10 g were obtained with organic solvent, and the 25 mg extracted fat was dissolved in 0.5 N NaOH-methanol of 2 mL and changed to its fatty acid methyl esters using 14% BF₃-methanol of 2 mL. Gas chromatography (Agilent 6890N/5975 MSD series, Avondale, PA, USA) installed with an SP^TM 2560 column (100 m×0.25 mm, Supelco Inc., Bellefonte, PA, USA) was used for analyzing the fatty acid contents. The column oven temperature was increased sequentially by 170°C to 245°C. Carrier gas was helium (He) and it’s a flow rate was 0.75 mL/min. The injector temperature was 250°C and the flame ionization detector (FID) temperature was 285°C. A FID conversion factor was used for calculating the fatty acid contents.

Vitamins evaluated in leaves and stem of quinoa were β-carotene, vitamin B complex, vitamin C, and vitamin E, and they were determined according to the AOAC method (2005).

Samples for analyzing the β-carotene were dissolved in ethanol 30 mL and 10% pyrogallol 1 mL. Then, KOH 3 mL was added to the sample solution and heated for 30 min. The sample was cooled and 30 mL distilled water were added for obtaining the ether layer. And then, the ether layer was dehydrated and evaporated. The dehydrated product and n-hexane 20 mL were compounded, and its was used as the sample. The sample was injected into an Agilent 1100 series HPLC system installed with a Nova-Pak silica column (3.9×150 mm, Waters Co., Milford, MA, USA). The operating conditions were following as: n-hexane and isopropyl alcohol (99:1, v/v) of mobile phase, 1.0 mL/min of flow rate, and 20 μL of injection volume.

Samples for analyzing the vitamin B complex were extracted with 75 mM ammonium formate solution (pH 7.0) 20 mL for 1 h. The extracts were centrifuged in order to separate the supernatant and precipitate for 15 min, and the supernatant was obtained. The obtained supernatant was filtered with a Millipore 0.45 μm membrane filter. The filtrate was used as the test sample, and its injected into an HPLC-tandem mass spectrometer (Agilent 1200 series, Agilent Technologies, Santa Clara, CA, USA) connected with a Luna C₁₈ column (3.0×150 mm, Phenomenex, Torrance, CA, USA). The operating conditions were following as: the mobile phase A was 5 mM ammonium dissolved in distilled water, and mobile phase B was 5 mM ammonium dissolved in methanol. They were eluted through the gradient system as follows: 0 min (90% A, 10% B); 0-8 min (55% A, 45% B); 8-15 min (90% A, 10% B). In addition, the samples were analyzed by the conditions such as 0.3 mL/min flow rate and 35°C temperature.

Samples for analyzing the vitamin C were prepared adding 5% meta-phosphoric acid. The extracts were filtered through a Millipore 0.45 μm membrane filter, and was injected into an Agilent 1200 series HPLC system connected with a Shiseido Capcell Pak C₁₈ column (4.6×250 mm, Shiseido, Tokyo, Japan). The operating conditions were following as: 0.05 M KH₂PO₄ and acetonitrile (99:1, v/v) of mobile phase, 0.9 mL/min of flow rate, and 254 nm of UV detector wavelength.

The extraction of samples for analyzing the vitamin E were conducted in the same way as that for β-carotene. However, flow rate of mobile phase was 0.5 mL/min.

All experiments except for dietary fiber were carried in triplicate or more, and the statistical analysis of results were performed using the Statistical Package for Social Sciences statistics 25.0 (SPSS Inc., Chicago, IL, USA). In order to compare the between samples data of the two groups (leaves and stem), independent sample t-test was used.

The proximate composition in leaves and stem of quinoa (C. quinoa) differed significantly (p⟨0.01, Table 1). The moisture accounts in the majority of proximate composition in leaves and stem (88.95% and 92.72%, respectively). The moisture content of stem higher than that of leaves, and which was significantly different (p⟨0.001). Quinoa leaves exhibited significantly higher levels of crude ash, crude protein, crude lipid, and carbohydrate than those of the quinoa stem (p⟨0.01). However, the crude fiber content (1.13%) of leaves showed significantly lower levels than that (2.10%) of stem (p⟨0.001). The dietary fiber content of leaves and stem were 3.32% and 3.10%, respectively. Briefly, the dietary fiber was exhibited higher in quinoa leaves than stem.

These results are exhibited similar tendency with that of a previous study of Datura innoxia, in which the D. innoxia stem showed a higher moisture compared to the its leaves (Ayuba et al., 2011). In addition, the stem of Epiphyllum oxypetalum had a higher moisture content than that of leaves (Ingale and Sajid, 2015). However, the studies of several scholar were demonstrated that leaves exhibited a higher moisture than stem (Anyasor et al., 2014; Asuk et al., 2015; Kpomah and Odokwo, 2020; Lohdip et al., 2015). According to the study of Lohdip et al. (2015), the plant belonging to Chenopodium sp. showed a higher ash, crude fat, and carbohydrate content in leaves than stem, while crude fiber exhibited a higher in stem than leaves. These results were very much like our results. A vast majority of reports verified that the proximate composition including dietary fiber content can vary from growth conditions such as season, soil composition, climatic conditions (Jang et al., 2011; Rohloff et al., 2015). Therefore, this study suggests that the proximate composition including dietary fiber content of quinoa can depend on various factor and their different parts.

The free sugar content in leaves and stem of quinoa is given in Table 2. Fructose, glucose, sucrose, maltose, and lactose were analyzed but most free sugar had not detected in samples. Only fructose and glucose were detected in stem, and they contents were 44.25 mg% and 107.62 mg%, respectively. That is to say, the content of glucose was twice times or more of the content of fructose. Free sugars are closely related to sweetness, and intensity of sweetness depends on the kind of free sugar and amount (Nam et al., 2018). For example, the free sugar content was similar with the trend of °Brix (Lee et al., 2013), and Moskowitz (1970) was reported that the sweetness of glucose was only ∼50% of the that of sucrose, and fructose has a sweetness 60-70% higher than sucrose. Therefore, it can be said that stem of quinoa has a higher sweet taste than leaves and its sweetness originate from fructose and glucose. According to several research (Ackerson, 1981; Guy et al., 1992), a part of free sugars and its synthesis are related to metabolic adaptations from the low temperature, and the water stress in plant was showed to increase the content of free sugar. Moreover, the free sugar content varied on climate conditions such as temperature, relative humidity, the amount of light, and the amount of rainfall (Anderson, 1986). Consequently, it is forecasted that the intensity of sweetness by the amount and composition of free sugar may differ according to the quinoa part and also that their amount and composition of free sugar will vary between cultivation condition.

Acetic acid was the most abundant organic acid in leaves and stem of quinoa, with their higher levels determined in leaves (428.40 mg%) (Table 3). Malic acid, oxalic acid, formic acid, and succinic acid were not detected in leaves and stem, and citric acid was detected but that is very small amount with 3.57 mg% and 6.41 mg%, respectively. Briefly, citric acid content of stem was exhibited a significant higher level than that of leaves (p⟨0.001).

In the metabolic pathway of animals and plants mitochondria, organic acids such as citric acid, oxalic acid, and acetic acid play a central role. For example, citric acid and oxalic acid are involved in diverse metabolic pathway including detoxification of metal substances and resistance for anaerobic stress, and acetic acid of plant participates in the tricarboxylic acid cycle, one of the energy metabolisms (Mucha et al., 2005; Yang et al., 2014). In addition, the level of organic acid in plant also can be varied according to cultivars as well as between individual part, and the growth condition such as soil, climate. Moreover, the previous study reported that the accumulation of organic acids is increased by environmental stress (Lopez-Bucio et al., 2000). Therefore, it is expected that the level of organic acid contained in quinoa had been varied by various factors. Sensory characteristics including flavor, color, and texture are affected by organic acids, and tissue and biological stability also closely related in organic acids (Chung et al., 2000). For this reason, organic acids are being widely used as additives in the vegetable or fruit beverages, drinks, and smoothie (Pereira et al., 2013). Therefore, it is likely that leaves and stem of quinoa can be effectively applied in a variety of food filed, even though the level of organic acids are low.

Table 4 presents the mineral content of quinoa according to its different two parts. All elements except for copper were identified in quinoa, and the major minerals were calcium, potassium, magnesium, and sodium. Particular, potassium was verified as having the highest level in leaves and stem of quinoa; its content in two parts was determined as 750.65 mg% and 869.15 mg%, respectively. Potassium content of quinoa stem higher than that of quinoa leaves and this was exhibited a significant difference (p⟨0.001). However, calcium, iron, magnesium, sodium, zinc, and selenium were showed the significant higher in leaves than stem (p⟨0.001). Notably, calcium and magnesium contents of leaves were roughly six times than those of stem. The content of zinc and selenium in leaves and stem was trace as the 0.89 mg%, 0.09 mg% (leaves), 0.23 mg%, 0.05 mg% (stem). These results similar with results of other studies that among minerals contained in quinoa, amaranth, spinach, moringa leaves, Chenopodium album sprouts, and Stinging nettle sprouts potassium was highest (Edelman and Colt, 2016; Pathan et al., 2019; Pradhan et al., 2015). In addition, the potassium content results of quinoa according to tissue is consistent with what reported by Park et al. (2011). Park et al. (2011) demonstrated that potassium content in stem of Cucumis sativus L. higher than leaves. Park et al. (1993) reported that the mineral content in leaf and stalk of Brassica juncea is similar or the mineral content in leaf exhibited higher than those of stem, among others calcium and magnesium content exhibited the big difference. It was very similar to our results. The minerals absorbed from plant roots are transported to the stems or leaves, or some mineral is stored in the roots (Nam et al., 2018). Accordingly, the mineral contents in plant are known to vary differ according to the various tissue. In addition, the mineral contents can be affected by the growth season, harvest season, soil conditions, and the quantity or variety of fertilizer (Mitchell, 1936). Therefore, it is considered that distribution of minerals in quinoa tissues may completely different on various factors.

Most fatty acids were not identified in leaves and stem of quinoa, and only palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2, n-6), and linolenic acid (C18:3, n-3) (Table 5). All fatty acids were exhibited as significant higher levels in leaves than in stem (p⟨0.001), and the content difference of fatty acid ranges from between 2.5 times and 6.8 times. Especially, linolenic acid (C18:3, n-3) contained as highest level was detected only in leaves (176.09 mg%) but no identified in stem. For leaves, the unsaturated fatty acid (UFA) content was higher than the saturated fatty acid (SFA) content; its content of UFA and SFA was determined as 276.81 mg% and 85.85 mg%, respectively. However, the UFA and SFA in stem was showed 15.52 mg% and 24.68 mg%, it was verified a result opposite to result in leaves. Briefly, SFA content in stem was roughly 1.5 times high than UFA content. According to Choi and Lim (2014), depending upon the extracts, but most extracts from Euphorbia Supina of a therophyte contained high n-3 fatty acid. Also, another study revealed that there was linolenic acid (C18:3, n-3) of high content in leaves of plant (Pascual-Villalobos and Lopez, 2010). Chung et al. (1996) demonstrated that leaves of pine sprouts contained higher SFA than UFA and stem of pine sprouts showed higher UFA than SFA. Briefly, these results opposite to our results. However, they reported that SFA and UFA contents of pine needles was different according to sampling times. Therefore, we are expected to be the fatty acid content and composition of plant showed completely different by environment, climate, condition, etc. as well as sampling times. Most of the fatty acids in plant are contained in the seed of the plant, and the leaves and stem contain very small amounts, so related studies are insignificant. However, Petrov et al. (2016) reported that the plant contained high fatty acid will have a crucial role in the regulation of resistance to low-temperature stress in the tissue of Yakut horses. Consequently, the fatty acids of plant are considered that to be important as food for animals as well as have sitological value.

Vitamin C (ascorbic acid) and E (tocopherol) was the major vitamin in leaves of quinoa and all vitamin except for vitamin B₁₂ (cyanocobalamin) were identified in leaves (Table 6). Quinoa leaves contained the highest level of vitamin E (2.25 mg/100 g), and vitamin C was next highest (2.02 mg/100 g). In addition, many vitamin B₁ (thiamine) and vitamin B₂ (riboflavin) were detected in leaves with 293.35 μg/100 g and 256.54 μg/100 g, respectively. For stem of quinoa, β-carotene, vitamin B₁, vitamin B₂, and vitamin E were the major vitamin. It has only a small amount of vitamin B₅ (pantothenic acid) and vitamin B₆ (pyridoxine) with 6.44 μg/100 g and 3.22 μg/100 g, respectively, and also the content of vitamin B₃ (niacin) was minute amounts. Vitamin B₁₂ and vitamin C were not detected in stem. Overall, quinoa leaves were contained higher level of vitamin than quinoa stem, and its difference was significant (p⟨0.05). In particular, the content difference of vitamin B₁, vitamin B₂, and vitamin B₆ ranged from 5.1 times to 6.8 times. Vitamin A and E are known to be a biological compound with anticancer, antioxidant, anti-hypertension, and anti-inflammation properties and crucial role in the tissue of human. In addition, vitamin B₁ and B₂ content in plant has a potential to improve nutrition for human health (EI Sohaimy et al., 2015). Especially, vitamin B₁ have the resistance to bacterial, viral, and fungal infections in vegetable crop (Ahn et al., 2005). Thus, the quinoa leaves rich in vitamin A, E, and B₁ are expected to be a useful for human and have excellent resistance to various plant diseases.