INTRODUCTION:

Quinoa (Chenopodium quinoa Willd.) is a dicotyledonous plant of the Chenopodiaceae family. Quinoa is one of the oldest crops in the Andean region of South America, grown for about 7000 years.¹ Quinoa is not grains like typical grains (monotone plants). It's more like a fruit. It is also often called a pseudo-seed plant because of its unusual composition. Quinoa grains and sprouts are used as food. The use of quinoa in the world is rapidly expanding. Whereas previously it could only be purchased at organic or health food stores, now it can be found in almost every grocery store.

Quinoa grains have a high protein content (15 percent on average) of high biological value. This means that they have a well-balanced amino acid composition (close in amino acid ratio to cow's milk casein). The content of sulfur-containing amino acids (cysteine and methionine) in quinoa grains is significantly higher in comparison with the concentration of these amino acids in other cereal plants.^2,3 This phenomenon is probably related to the fact that this crop originally grew and cultivated in areas with volcanic soil.⁴ The solubility of quinoa proteins can be enhanced by enzymatic hydrolysis. This determines its prospects for use as an ingredient in beverage mixtures⁵. Quinoa grains are highly nutritious food product; possess wide range of biological properties and was chosen by FAO as one of the crops that able to provide food security in the future.^6-8

The main carbohydrate of quinoa grains is starch. It is about 58.1-64.2% dry matter of grain. Quinoa grains are rich in vitamins, trace elements, mono- and polyunsaturated fatty acids.^9-10 The microelement composition of quinoa grains is mainly calcium, magnesium, iron, copper, and zinc. Iron compounds in quinoa have high solubility and digestibility.¹¹ The vitamins are α-carotene and niacin. In addition, thiamine, folic acid (78.1mg per 100g), vitamin C (16.4 mg per 100g), vitamins B₂, A, and E. are contained in significant quantities in quinoa. The fat content of quinoa is 14.5% on average, of which 70% is poly and monounsaturated fatty acids (linoleic and oleic acids are about 38.9% and 27.7% respectively). The presence of tocopherols in quinoa reduces the risk of oxidation of unsaturated fatty acids.¹² Quinoa contains a large number of secondary metabolites (fatty acids, flavonoids, terpenoids, and phytoecdysteroids). The high content determines a wide range of biological activity: antidiabetic, antitumor, antimicrobial, anti-inflammatory, and immunomodulating.^1,13-15

Phytoecdysteroids are interesting to study. These are secondary metabolites first discovered in the whole quinoa plant in 1984¹⁶. Phytoecdysteroids are polyhydroxylated steroids, insect molting hormones, and secondary plant metabolites. They protect plants from insects and nematodes.^17-19 The pharmacological activity of phytoecdysteroids is very wide. They exhibit adaptogenic properties, accompanied by increased productivity, improved cognitive functions, and anxiolytic effect. Also, taking phytoecdysteroids reduces glycemia, cholesterolemia. Phytoecdysteroids have an anabolic effect. They find prophylactic use in osteoporosis and other diseases.^20-24 Phytoecdysteroids are found in more or less quantities in many wild plant species. However, they are almost absent or contained in very small amounts in cultivated agricultural plant species. In addition to quinoa, spinach, several plants of the Chenopodiaceae family^25,26, and champignons (“food plants”) also contain phytoecdysteroids.²⁷ The main phytoecdysteroid, 20-hydroxyecdysone (30μg/g), was found in quinoa seeds.^15,28 Several minor compounds (3-9μg/g) such as makisterone A, 24-epi-makisterone A, 24 (28)-dehydromakisterone A, and 20,26-dihydroxyecdysone have also been detected. It was later shown that quinoa flour contains the unique ecdysteroid kancollasterone.²⁹

Other important secondary metabolites in quinoa are polyphenolic compounds. About 29 species of phenolic acids have been identified in quinoa. Two large groups can be distinguished among them: acids structurally similar to benzoic acid and acids structurally similar to cinnamic acid. The following acids of the benzoic acid group have been identified in quinoa grains: 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, gallic acid, 1-O-galloyl-β-D- glycoside, protocatechuic acid, syringic acid, vanilla acid, vanilla acid glycosyl ester, vanilla acid 4-O-glycoside, vanillin. Cinnamic acid analogs are identified in quinoa grains. These include caffeic acid, chlorogenic acid, cinnamic acid, o-coumaric acid, p-coumaric acid, p-coumaric acid glycoside, 8,5'-diferulic acid, ferulic acid, isoferulic acid, 4'-geranyloxyferulic acid, rosmarinic acid, sinapinic acid.

The main aglycones of flavonoids identified in quinoa are kaempferol and quercetin. In addition to them, aglycones of acacetin, myricetin, daidzein, and genistein were also identified. Flavones, flavonols, flavanones, flavonols, and isoflavones stand out structurally among flavonoids in quinoa. The following flavones have been identified: orientin and vitexin. Among flavonols: kaempferol and its derivatives, myricetin, quercetin and its derivatives, rutin. Among the flavanones: hesperidin, neohesperidin, naringin. Among the flavanols: catechin, epicatechin, epigallocatechin. Among isoflavones: biochanin A, daidzein, genistein, prunetin, puerarin.^30-33 All compounds have a wide range of biological effects, including antioxidant, anti-inflammatory, antitumoral, protective, neuroprotective, etc.

The objective of the study is to develop an effective method of extraction from quinoa grains in the laboratory to obtain a functional food ingredient (FIU) enriched with phytoecdysteroids and polyphenols.

MATERIALS AND METHODS:

We used commercial black quinoa grains pre-milled in a laboratory blender (“FimarFRI, Italy”) and sieved through a sieve to select flour fraction with particle diameter less than 0.35mm. Rectified ethyl alcohol (National Standard, GOST R 51723-2001, ethyl alcohol drinking 95%) was used as an extractant.

Extraction:

Extraction was performed with water-alcohol solutions at ethanol concentrations of 0, 20, 40, and 60% at 24-26℃. 1000ml of extractant was added to 25g of quinoa grinding grains. Then it was thermoregulated for 60 min with constant stirring using a top-drive stirrer. The resulting mixture was centrifuged for 30 min (BEKMANJ-6B centrifuge) at 4000rpm. The supernatant was selected. 300ml of the corresponding extractant was added to the sludge. Then the extraction was repeated. The supernatants obtained were combined.

Membrane treatment:

The obtained extract was subjected to ultrafiltration in a tangential flow at a membrane filtration unit based on the ASF-018 filter holder (VLADISART, RF) through a membrane with a pore diameter of 10 kD with a collection of low-molecular fraction. The collected low-molecular fraction (LMF) was concentrated by reverse osmosis method on the unit with the filter “URF-1812” roll membrane. Ethanol was removed on the rotary evaporator using an alcoholic extractant.

Hydrophobic sorbent purification:

Additional purification of LMF was carried out on a preparative column with a hydrophobic sorbent C18 (4.5×9cm). Aqueous extract of LMF was applied on the column, washing off the complex of polyphenols and phytoecdysteroids, which was trapped by sorbent 75% ethyl alcohol. Then the alcohol was removed on the rotary evaporator. The final dry product was obtained from its aqueous solution by lyophilization (lyophilic drying LS-500, PRINTEKH production, RF).

Figure 1 shows scheme for obtaining FPI from quinoa grains.

Milled and sifted quinoa grains (less than 0.35 mm)

Water or aqueous alcohol extraction for 1 hour at 25℃

Centrifugation

Reextraction

Ultrafiltration (10 kDa), reverse osmosis

Rotary alcohol removal

Chromatographic purification on a C₁₈ column

Rotary alcohol removal, lyophilization

FFI enriched with phytoecdisteroids and polyphenols

Figure 1: Scheme for obtaining FFI.

Methods of biologically active substances analysis:

Phytoecdysteroids at each stage of FFI production were determined by two methods: HPLC with a spectrophotometric detector and HPLC with a mass spectrometer (HPLC-MS). The main criteria for choosing the optimal method for further research were the accuracy of the method and simplicity. 20-hydroxyecdysone (98%, manufactured by Scientific GmbH, Germany) was used as a standard sample.

Analysis of the content of 20-hydroxyecdysone by HPLC with a spectrophotometric detector:

20-hydroxyecdysone was determined by reverse phase HPLC (Phenomenex Luna C₁₈(250×4.6mm, 5μm) with a precolumn with pretreatment on a polyamide sorbent according to the procedure³⁴with minor modifications). The geometric parameters of the sample preparative column have been changed. The flush was carried out with the sorbent being dried by blowing. The determination of the final volume was carried out by weighing the combined flush on an analytical balance. Conditions: elution rate – 0.75ml/min; UV detector (UV/VIS) – 151, 243nm; mobile phase A – 20% acetonitrile, mobile phase B – 60% acetonitrile; elution program – linear-gradient B from 0 to 30%, 15 min. Figure 2 shows an HPLC chromatogram of a standard sample 20E and dry FFI.

Figure 2: Chromatograms 20E (10μg /ml) and FFI.

Analysis of 20-hydroxyecdysone by HPLC-MS:

The 20E content was determined by HPLC-MS using 1100 chromatograph with 6410 mass detector (Agilent Technologies). Column: Poroshell 120 EC-C18 3.0×50 mm, 2.7μm (Agilent Technologies). Gradient elution was performed with a mixture of 0.1% solution of formic acid in water (eluent A) and acetonitrile (eluent B) with a flow rate of 0.4ml/min according to the following scheme: 0 min – 5% eluent B, 5 min – 27% eluent B, 5.5 min – 90% of eluent B, 8.5 min – 90% eluent B, 9.5 min – 5% eluent B, 13.5 min – 5% eluent B. The following parameters of the mass detector were selected: atomizing gas pressure of 2.8 bar; drying gas temperature 350°C; flow rate of drying gas 10 l/min; polarity is positive; capillary voltage 4000 V; voltage on the fragmenter 98 V; recorded mass transitions in the MS/MS mode 481.3 → 445.4 with a collision energy of 8 eV (used for quantitative analysis), 481.3 → 371.4 with a collision energy of 12 eV (used for qualitative confirmation). Additional dilution of the extract before analysis was carried out with a 50% solution of methanol in water to a concentration falling in the range from 0.01 to 5μg/ml (calibration range). The sample was then vortexed and centrifuged with a relative centrifugation force of 18,407 g for 10 min. After centrifugation, it was poured into a vial for HPLC analysis. Figure 3 shows a typical HPLC-MS chromatogram of a finished FFI sample and a standard 20E sample.

Figure 3: Chromatogram A - standard sample 20E (0.5μg/ml); Chromatogram B is a sample of dry FFI obtained by extraction with 40% ethanol.

Determination of total polyphenols:

Total polyphenols were determined spectrophotometrically by the method of Folin-Ciocalteu^35. which is often used in the analysis of plant materials and medicinal plant raw materials^36-42. 2.0 ml of 10% Folin-Ciocalteu solution was added to 400μl of the sample and incubated for 5 min. Then 1.6ml of 7.5% Na₂CO₃ was added and the samples were incubated for 1 hour in a dark place. The optical density of the solutions was measured at a wavelength of 765nm using a SpectroQuest 2800 spectrophotometer (UNICO, USA). Gallic acid was determined in solutions using a calibration curve obtained using the standard of gallic acid (97.5%, Sigma, USA).

Determination of the total content and profile of individual flavonoids:

The flavonoid profile was determined using an Agilent 1200 liquid chromatography system with a diode array spectrophotometric detector (DMD) and an Agilent 6410 Triple Quadrupole triple quadrupole mass spectrometric (MS) detector. HPLC-DMD conditions: stationary phase – Phenomenex Luna C₁₈ column 150×4.6mm (5μm); mobile phase A – 0.1% solution of formic acid in water, phase B – 0.1% solution of formic acid in acetonitrile; gradient elution: 0 min – 15% B, 5 min – 20% B, 20 min – 30% B, 40 min – 60% B, 41–50 min – 15% B; the elution rate of 0.5ml/min; column temperature 40ºС; sample volume 5µl; detection at λ = 350nm (flavonol glycosides) and λ = 370nm (flavonols); spectra were recorded in the wavelength range of 200-400nm. MS conditions: electrospray ionization in the registration mode of positive ESI/MS⁺ and negative ESI/MS^– ions; the capillary voltage is +3500 V and -2500 V, respectively; dryer gas flow 9 l/min; temperature 325ºC; spray pressure 0.27 kPa. Data processing was performed using MassHunter Workstation Software Qualitative Analysis Version B.02.00. The flavonoid content was calculated by the external standard method. Commercially available pure substances rutin (≥94%, Sigma-Aldrich, USA), kaempferol-3-glucoside (≥95%, PhytoLab, Germany), and isoquercitrin (≥94%, HWI ANALYTIK GMBH, Germany) were used as standard samples. The arithmetic average of the results of three parallel measurements was taken as the final result. A typical chromatogram of the obtained FFI (20% aqueous-alcoholic extractant) is presented in Figure 4. Identification of flavonoids was carried out according to retention times, UV, and mass spectra compared with published data^31-43.10 individual flavonol glycosides (Table 1), the main of which were quercetin-3-O-(2,6-di α-L-rhamnopyranosyl)-β-D-galactopyranoside, quercetin-3-O-β-D-apiofuranosyl-(1→2)-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-galactopyranoside and kaempferol-3-O- (2,6-di-α-L-rhamnopyranosyl) -β-D-galactopyranoside, found in quinoa extracts. Phenolic acids, such as isomers of unidentified derivatives of ferulic acid with m/z 692/690 [M + H]⁺/[M - H]^–, as well as a small amount of free ferulic acid are found in quinoa extracts.

Figure 4: Chromatogram of FFI (20% alcohol) at λ = 350nm. The peak numbers in the chromatogram correspond to the numbers of flavonoids in Table 1.

Table 1: Retention parameters, absorption maxima and masses of flavonoids in quinoa extracts

no	Flavonoid	Rt, min (±0,2 min)	λ_max, nm (±2 nm)	ESI/MS⁺	Detectable ion	ESI/MS^-	Detectable ion
1	Quercetin-3-O-β-D-apiofuranosyl- (1 → 2) -O-α-L-rhamnopyranosyl- (1 → 6) -β-D-galacto-pyranoside + hexose	13.8	255, 266, 354	905.4, 773.3, 611.2, 465.2, 303.2	[M + H]⁺, [M – apiose * + Н]⁺, [M – apiose – galactose + Н]⁺, [M – apiose – galactose – rhamnose]⁺, [M – apiose – galactose – rhamnose – hexose]⁺	903.3	[M – H]^-
2	Quercetin-3-O- (2,6-di-α-L-rhamnopyranosyl) -β-D-galactopyranoside	14.4	256, 268, 354	757.4, 611.3, 465.2, 303.2	[M + H]⁺, [M – rhamnose + Н]⁺, [M – 2 rhamnose + Н]⁺, [M - 2 rhamnose – galactose + H]⁺	755.4	[M – H]^-
3	Quercetin-3-O-β-D-apiofuranosyl- (1 → 2) -O-α-L-rhamnopyranosyl- (1 → 6) -β-D-galactopyranoside	15.1	256, 268, 354	743.3, 611.3, 465.2, 303.2	[M + H]⁺, [M – apiose + Н]⁺, [M – apiose – rhamnose + Н]⁺, [M – apiose – rhamnose – galactose]⁺	741.2	[M – H]^-
4	Kaempferol-3-O- (2,6-di-α-L-rhamnopyranosyl) -β-D-galactopyranoside	16.1	266, 348	741.4, 595.3, 449.2, 287.2	[M + H]⁺, [M – rhamnose + Н]⁺, [M – 2 rhamnose + Н]⁺, [M - 2 rhamnose – galactose + H]⁺	739.2	[M – H]^-
5	Kaempferol-3-O-β-D-apiofuranosyl- (1 → 2) -O-α-L-rhamnopyranosyl- (1 → 6) -β-D-galactopyranoside	17.1	258, 264, 352	727.3, 595.1, 449.2, 287.2	[M + H]⁺, [M – apiose + Н]⁺, [M – apiose – rhamnose + Н]⁺, [M – apiose – rhamnose – galactose]⁺	725.2	[M – H]^-
6	Quercetin-3-O-β-D-apiofuranosyl- (1 → 2) -β-D-galactopyranoside			597.2, 465.1, 303.2	[M + H]⁺, [M – apiose + Н]⁺, [M – apiose – galactose + Н]⁺	595.2	[M – H]^-
7	Isorhamnetin-3-O- (2,6-di-α-L-rhamnopyranosyl) -β-D-galactopyranoside			771.3, 625.2, 479.2, 317.2	[M + H]⁺, [M – rhamnose + Н]⁺, [M – 2 rhamnose + Н]⁺, [M - 2 rhamnose – galactose + H]⁺	769.2	[M – H]^-
8	Quercetin-3-O-β-D-apiofuranosyl- (1 → 2) -β-D-glucopyranoside	17.3	256, 264, 356	597.1, 465.2, 303.2	[M + H]⁺, [M – apiose + Н]⁺, [M – apiose – glucose + Н]⁺	595.2	[M – H]^-
9	Quercetin-3-O-β-D-apiofuranosyl- (1 → 2) -β-D-glucuronide	18.0	256, 266, 354	611.3, 479.2, 303.2	[M + H]⁺, [M – apiose + Н]⁺, [M – apiose – glucuronic acid + Н]⁺	609.2	[M – H]^-
10	Quercetin-3-O-β-D-glucuronide	21.0	256, 266, 356	479.2, 303.2	[M + H]⁺, [M – glucuronic acid + Н]⁺	477.1	[M – H]^-

Table 2: The yield of 20E hydroxyecdysone according to the stages of the isolation process

No	Extraction stage	Yield (%) relative to the raw extract	The composition of the extractant (% alcohol in water)
No	Extraction stage	Yield (%) relative to the raw extract	0	20	40	60
1	Quinoa grains	100.0	100.0	100.0	100.0	100.0
2	Ultrafiltrate 10kD	85.8	85.8	88.6	77.1	74.0
3	Concentrate after reverse osmosis	73.3	73.3	73.0	70.3	83.3
4	After C18 and rotary evaporator	68.3	68.3	69.4	67.6	67.9
5	Dry product	52.2	52.2	56.1	54.5	56.2

Table 3. The content of 20E in dry FFI from quinoa grains

No	The composition of the extractant (% alcohol in water)	20Е. mg/g		Concentration degree 20E relative to quinoa content in grain
No		HPLC-UV	HPLC-MS
Product 1	0	35.5±1.8	34.9±0.8	139
Product 2	20	36.9±1.8	36.4±0.8	143
Product 3	40	37.2±1.9	35.7±0.8	140
Product 4	60	30.8±1.5	31.4±0.7	123

Table 4: Content of flavonoids (g/100g)

Quinoa flavonoids	Total content	1*	2*	3*	Concentration rate flavonoids
Quinoa grains	0.0946±0.002	0.0357±0.0006	0.0296±0.0006	0.0173±0.002	–
Product 1	13.90±9.36	5.15±0.14	4.49±0.12	2.51±0.07	147
Product 2	23.00±0.48	8.66±0.18	6.67±0.14	4.41±0.09	243
Product 3	25.15±0.52	9.41±0.20	7.29±0.15	4.38±0.10	266
Product 4	21.41±0.60	8.15±0.19	6.36±0.13	3.75±0.08	226

* Notes:

1 – Quercetin-3-O-(2,6-di-α-L-rhamnopyranosyl)-β-D-galactopyranoside

2 – Quercetin-3-O-β-D-apiofuranosil-(1→2)-O-α-L-rhamnopyranosil-(1→6)-β-D-galactopyranoside

3 – Kaempferol-3-O-(2,6-di-α-L-rhamnopyranosyl)-β-D-galactopyranoside

Table 5: Profile of flavonoids in water extraction and % yield relative to the original extract.

S. No	Faction	Total content	1*	2*	3*	yield %
1	Original Extract	26.04±0.78	9.92±0.24	8.18±0.19	5.21±0.12	100.0
2	Ultrafiltration 10kD	22.25±0.56	8.60±0.21	6.92±0.16	4.68±0.11	85.5
3	Concentrate after reverse osmosis	17.35±0.45	6.81±0.18	5.54±0.14	3.28±0.09	66.6
4	Purified preparation after C18 and rotor evaporator	13.90±0.36	5.15±0.14	4.49±0.12	2.51±0.07	53.4

* Notes: