Amaranthus retroflexus L. (Amaranthaceae) is a herbaceous annual weed with a natural areal in North America. The species has been introduced in other regions. It is an aggressive weed of grain, vegetable, and industrial crops [1]. A. retroflexus inhabits the European part of Russia, Siberia, southern Europe, the Mideast, Asia Minor, Mongolia, and northern Africa. It is often encountered in Uzbekistan in crops in irrigated and non-irrigated fields [2]. Indigenous people of North and South America, Asia, Africa, and Europe have used leaves and seeds of A. retroflexus as food for many centuries [3].

Aqueous infusions of A. retroflexus herb are used in folk medicine of various countries for diarrhea, intestinal colic, and chronic uterine inflammation and as a hemostatic for serious hemorrhaging. The decoction of the flowering tops is used to treat goiter [4]. The aqueous extract of plants collected during flowering and dried possesses protisticidal and bactericidal properties [5]. The plant has demonstrated the ability to accumulate Pb, Zn, Cu, and other heavy metals in the subterranean and aerial organs, owing to which this wild amaranth species has been proposed for phytoremediation of waste waters and contaminated soils [6].

The following is known about the biological activity of plant lipids. The extract of cotton leaves containing polyprenols, phytosterols, and tocopherols exhibits growth-stimulating activity for wheat, corn, tomatoes, and cucumbers. Preliminary treatments of the seeds of these crops with an aqueous emulsion of the biostimulator at doses of 1–10 g/ton accelerates development of the plants and increases their drought resistance [7]. The preparation Uchkun was designed based on this extract [8]. A complicated mixture of saturated (14:0–34:0) and unsaturated fatty acids (FAs) of the C18–20 series (18:1 and 18:2 dominated), phytosterols, polyprenols with an impurity of triterpenes from Siberian fir Abies sibirica was patented as an agent with fungicidal properties than increased the harvest of grain, legume, and vegetable crops [9]. The growth- and immune-stimulant of agricultural crops KLIPS with fungicidal activity was developed based on total saturated and unsaturated FAs of the C20–C34 series, β-sitosterol, tocopherol, and phytol from this same plant [10].

Chemical data on lipids from seeds and leaves of A. retroflexus are sporadic and incomplete. Seeds of A. retroflexus growing in central Yakutia were reported to contain 3.5% lipids with 6.4% squalene [11]. Representatives of the family Amaranthaceae are known to differ from plants of other families by a higher content in the seed lipids of squalene, a lipophilic compound with high physiological activity [12].

Eight free FAs (12:0, 14:0, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:0) and six phospholipids (PL, lyso-phosphatidylcholine, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acyl-lyso-phosphatidylethanolamine, and N-acylphosphatidylethanolamine) were identified in seed lipids of A. retroflexus growing in Georgia [13].

Linoleic (18:2, 19.0%) and linolenic FAs (18:3, 3.71%) were found in the lipophilic fraction of the aqueous EtOH extract of the leaves [14]. Lyophilized leaves of A. retroflexus grown in central Yakutia contained 6.06% total lipids, of which 1.93% were neutral lipids (NL), 2.48%, glycolipids (GL), and 1.64% PL [15]. Eighteen FAs with chain lengths from 12:0 to 23:0 with 18:3 (35.4%), 16:0 (31.9%) and 18:2 (18.0%) FAs dominating were identified by us in total lipids from leaves.

Climate factors (temperature variations, water availability, habitat, etc.) are known to have a considerable effect on the lipid composition of amaranth [14, 16], which varies under the influence of these factors over a broad range for various varieties and species, including A. retroflexus [17].

The goal of the present work was to study the constituent and FA compositions of NL and PL from seeds and the aerial part of A. retroflexus growing in Uzbekistan and to determine the biological activity of the separate lipid components on wheat and cucumber crops under salt stress.

Representatives of the family Amaranthaceae characteristically have higher squalene contents in the NL than plants of other families. Squalene is an isoprenoid hydrocarbon with high physiological activity, the presence of which in lipids is the subject of much research [14].

Seeds. The contents of moisture and volatiles in seeds were determined. NL and PL were isolated. The refractive index and acid number of the NL were determined. NL were found to contain unsaponified substances (US), squalene, and free FAs. PL were separated by column chromatography (CC) over silica gel into GL and PL. The yields of the lipid groups were established gravimetrically. Table 1 presents the results.

Table 1. Characteristics of Amaranthus retroflexus Seed Lipids
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Table 1 shows that the content of total lipids in the seeds was 7.78%. The contents of squalene and NL in A. retroflexus fell in the range found in seeds of many other amaranth species [18]. The NL had a high percentage (8.12%) of US, which reflected the level of lipophilic compounds without acyl substituents (hydrocarbons including carotenoids and squalene, fatty alcohols, phytosterols, triterpenols, etc.).

The qualitative compositions of NL, US, GL, and PL were established by TLC on silica gel and Silufol plates. NL from seeds contained paraffinic hydrocarbons, squalene, FA esters with phytosterols and triterpenols, triacylglycerides (main component), free FAs, phytosterols, and triterpenols according to TLC using solvent systems 1–5. The US were found to include paraffinic hydrocarbons, squalene, triterpenols, and phytosterols using solvent systems 2–5. The composition of the GL (TLC, system 6) comprised sterylglycoside esters with FAs (Rf 0.96), monogalactosyldiacylglycerides (Rf 0.75), sterylglycosides (Rf 0.57, main component), digalactosyldiacylglycerides (Rf 0.28), and an unidentified constituent (Rf 0.31).

PL (TLC, system 7) were represented by phosphatidylcholines (Rf 0.34, main constituents), phosphatidylethanolamines (Rf 0.46), phosphatidic acid (Rf 0.15), and phosphatidylinositols (Rf 0.17).

Alkaline hydrolysis of NL, GL, and PL isolated FAs. Their methyl esters were prepared and purified of impurities by preparative TLC using system 4. Methyl esters of FA (MEFAs) were analyzed by GC. Table 2 presents the results.

Table 2. Compositions of Fatty Acids in Neutral Lipids, Glycolipids, and Phospholipids from A. retroflexus Seeds, GC, % of Acid Mass
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The identification of a FA peak with RT 18.139 (Table 2) as trans-18:1n9 was confirmed by Ag+-TLC and IR spectroscopy. IR spectra of starting NL and isolated MEFAs were typical for these lipid groups. However, a weak band for vibrations of an isolated trans-olefinic group was observed at 973 cm–1 [19]. A spot with Rf 0.65 was detected after separation of the MEFAs on Ag+-TLC. It corresponded to the spot for a model sample of the methyl ester of elaidic acid (Rf 0.67). Elaidic acid was identified previously in seed lipids from the cultivated species A. viridis (1.07%) and A. tricolor (1.15%) [20]. It was observed for the first time by us in seed lipids of wild amaranth A. retroflexus.

The methyl esters of cis-18:1n9 and 18:3 were not separated under the used GC conditions. The presence of linolenic acid 18:3 was established by Ag+-TLC by comparing the chromatographic mobility of a spot with Rf 0.50 with that of the ME of 18:3 from linseed oil (Rf 0.52).

Table 2 shows that PL from seeds contained 14 FAs that were esterified; NL and GL, 16 each. The dominant FAs were 18:2n6 and total cis-18:1n9 and 18:3n3. The fraction of 16:0 acid was rather high (15.93–22.43%). Total unsaturated FAs were higher in NL and lower in PL. Elaidic acid (trans-18:1n9) was detected in minor amounts in all lipid groups. Elaidic acid was not previously found in seed GL and PL from representatives of this family.

A comparison of the results with the literature [11] showed that seeds of A. retroflexus of Uzbekistan origin, in contrast to seeds of plants from central Yakutia, contained twice the total lipids (6.98 and 3.5%, respectively) but less squalene (by 1.4 times). Lipids of wild amaranth afforded 14–16 identified FAs (Table 2), in contrast to 8 FAs from A. retroflexus growing in Georgia [13].

Aerial Part. Lipids (total lipids 3.88%) from the air-dried aerial part (leaves and stems) collected during fruiting were extracted analogously to the seed lipids. The yields of NL and PL were 2.75% and 1.13% of the raw-material mass, respectively. The squalene content in NL was determined. US (12.8%) were isolated from NL. The carotenoid content (174.55 mg%) in them was determined. PL were separated by CC over silica gel into GL and PL groups. Their contents were determined gravimetrically. Table 3 presents the results.

Table 3. Compositions of Fatty Acids in Neutral Lipids, Glycolipids, and Phospholipids from the Aerial Part of A. retroflexus, GC, % of Acid Mass
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The qualitative compositions of NL, GL, and PL from the aerial part of A. retroflexus were identical to the analogous lipids from A. caudatus seeds [21] except for the absence in GL of an unidentified constituent with Rf 0.31. Total PL from A. retroflexus were dominated by GL (0.8%), which were 2.5 times greater than PL (0.32%). It is noteworthy that the contents of US and squalene (3.15%) were high in the aerial part of A. retroflexus while squalene occurred in amounts up to 0.77% in leaves of 45 amaranth genotypes [18]. FAs isolated from the lipids were analyzed by GC analogously to seed FAs. Table 3 presents the results. Table 3 shows that lipids from the aerial part consisted of 14 (GL) or 15 (NL, PL) constituents, the composition of which was like the set of seed FAs. NL and PL were dominated by acid 18:2n6 (62.28 and 34.59%, respectively); GL, 16:0 (37.12%). Elaidic acid in lipids from the aerial part were identified for the first time in the family Amaranthaceae. The total unsaturation of the FAs was greater in NL. The saturated–unsaturated ratio of total FAs in GL was 1:1. Acid 26:0 was identified in this lipid group.

According to the results, leaves and stems of A. retroflexus growing in Uzbekistan contained 1.5 times less total lipids with NL dominating (NL–PL ratio 2:4). Leaves of the plant from central Yakutia during the same development phase not only had more total lipids but also PL (3.6 times, PL–NL ratio 2:1) [15]. The FA compositions of these two samples differed by the sets and contents of individual constituents. Total lipids from leaves of Yakutia amaranth contained 18 identified FA with chain lengths from 12:0 to 23:0 and were dominated by acids 18:3 and 16:0. FAs from leaves of Uzbekistan amaranth included 16 constituents with acid 18:2 (62.3%) dominating.

Biological Activity of A. retroflexus Lipids. Salination is known to cause osmotic stress in plants that results in destruction of cellular metabolism and suppresses growth and development of cultivated plants [22]. The effect of soil salination is evident in the early development of the plants. Preliminary treatment of plant seeds with growth regulators is one method for diminishing the negative effects of salt. The growth rate of sprouts and biomass accumulation are the main parameters of resistance to salination.

The protective effects of NL, total FAs obtained from NL, methyl esters of total FAs, and US isolated from A. retroflexus seed NL on the growth and development of wheat and cucumber under salt stress were studied. Wetted seeds were cultivated under NaCl salination conditions. The resistance parameters were the lengths of the aerial and root parts and the accumulation of mass by fresh and dried sprouts.

US and FAs at a concentration of 0.0001% and MEFAs at a concentration of 0.001% were found to have positive effects on the growth of wheat sprout roots. Their lengths were practically at the same level (6.69–6.72 cm) and exceeded the control by an average of 13.0% (Table 4). Table 4 shows that MEFAs at a concentration of 0.001% had the greatest effect on the growth of wheat stems. The length was greater than that of the control by 25.4%. This parameter was practically at the level of Uchkun preparation, where the root length was greater than that of the control by 21.6%; stems, by 26.4%. NL at a concentration of 0.001% and US at 0.0001% were slightly less active.

Table 4. Effect of Preliminary Treatment of Wheat Seeds with A. retroflexus Seed Lipids on Sprout Growth and Mass Under Salt Stress
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The length of the aerial parts in these versions exceeded that of the control by 12.8%. US at a concentration of 0.0001% were even less active (length greater than that of the control by 7.4%).

The study of the effects of the substances on biomass accumulation showed that the fresh weight was greatest for wheat seeds treated with US and MEFAs at a concentration of 0.001%. All sprouts were heavier than the control by 44.1 and 34.3%, respectively. FAs at 0.0001% were less active (fresh weight greater than that of the control by 28.4%).

Both studied concentrations of US had positive effects on the sprout dry weight. The sprout masses were greater than that of the control by 57.1 and 42.8%. FAs at a concentration of 0.001% stimulated accumulation of dry weight also by 57.1%. The MEFAs at the studied concentrations were active on the same level and greater than the control by 35.7%. The fresh weight in tests with the reference preparation exceeded that of the control by 33.3%; dry weight, by 78.6%.

NL at a concentration of 0.001% had negative effects on the accumulation of fresh and dry biomass. The fresh weight was lower than that of the control by 43.1%; of the dry weight, by 64.3%. NL at a concentration of 0.0001% increased the fresh weight by 8.8%; dry weight, by 21.4%.

US at a concentration of 0.0001% were highly active for roots of cultivated cucumber sprouts. Their length exceeded that of the control by 80.0% (Table 5).

Table 5. Effect of Preliminary Treatment of Cucumber Seeds with A. retroflexus Seed Lipids on Sprout Growth and Mass Under Salt Stress
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The parameters for treatment with MEFAs were slightly lower. Treatment of seeds at a concentration of 0.001% gave root lengths greater than that of the control by 68.5%; at 0.0001%, by 59.8%. FAs at a concentration of 0.001% stimulated root growth by 54.8%.

FAs had a stimulating effect on the growth of cucumber stems under salt stress. Preliminary treatment of cucumber seeds with FAs at a concentration of 0.001% increased the growth of stems by 66.9% over the control; at 0.0001%, by 90.9% (Table 5). These were better parameters than those for Uchkun preparation. MEFAs at a concentration of 0.0001% were less active. The stem lengths were longer than that of the control by 53.5%.

FAs at a concentration of 0.0001% had the greatest effect on the accumulation of fresh and dry mass of cucumber sprouts. The fresh weight was greater than that of the control by 28.0%; dry weight, by 23.5%. These also were superior to the parameters for Uchkun preparation. A high yield of dry mass that exceeded that of the control by 14.7% was observed for the effect of FAs at a concentration of 0.001%. MEFAs at a concentration of 0.001% also increased the biomass. The fresh weight exceeded that of the control by 11.2%; dry weight, by 8.8%.

Like for wheat, NL had an inhibitory effect on the fresh and dry weights of sprouts. NL at a concentration of 0.001% gave parameters lower by 53.9 and 67.7%, respectively; at 0.0001%, by 20.9 and 34.4%.

Thus, lipid constituents of US, FAs, and MEFAs from A. retroflexus seeds possessed stress-protective activity under salt stress conditions. Preliminary treatment of wheat and cucumber seeds with these substances at concentrations of 0.001 and 0.0001% inhibited the effects of salt stress. This was evident in activation of sprout growth and accumulation of fresh and dry mass. NL did not exhibit protective effects.

EXPERIMENTAL

IR spectra of MEFAs were taken from films on a PerkinElmer Model 2000 FTIR spectrometer. GC analysis of MEFAs used an Agilent 6890N chromatograph. Carotenoid contents were analyzed by a photoelectrocalorimetric method under the published conditions [23]. FAs were identified by GC using previously published data [24]. Quantitative analysis of squalene used HPLC on a Shimadzu LC-20 instrument with a Supelco column (150 × 4.6 mm) with C18 stationary phase and particle size 5 μm and UV detection of squalene at 210 nm [25]. The mobile phase was a MeOH–MeCN (1:1) mixture. The mass concentration of squalene was established from the sum of peak areas using an internal standard of squalene (Sigma-Aldrich). Ripe seeds and aerial parts of plants during fruiting were collected in Tashkent Region in 2019.

NL were extracted from ground seeds by exhaustive extraction with hydrocarbons (bp 72–80°C) in a Soxhlet apparatus [26]; from the ground aerial part, by standing (3×) with hydrocarbons for 24 h. The contents of free FAs in the NL were calculated from the acid number [27]. PL were extracted by CHCl3–MeOH (2:1) as before [28]. Non-lipid impurities were removed from the extract using aqueous CaCl2 solution.

CC of PL used a glass column (2-cm diameter) packed with Chemapol silica gel of particle size 100/160 mesh with a sample–sorbent ratio of 1:60 (by weight). The NL were eluted by CHCl3; GL, Me2CO; PL, MeOH. Analytical TLC of NL, US, GL, and PL was performed on L5/40 silica gel plates (Chemapol) and Silufol plates; Ag+-TLC of MEFAs, on L 5/40 silica gel plates with added (30%) AgNO3 in benzene.

Preparative TLC of MEFAs used solvent system 4. MEFA bands on the sorbent were detected by I2 vapor, scraped from the plates, and desorbed from the silica gel by multiple elutions with CHCl3. The eluates were combined. The CHCl3 was evaporated in a rotary evaporator. The purified MEFAs were dissolved in hexane and analyzed by GC.

The solvent systems were 1) hexane–Et2O–AcOH (7:3:0.1); 2) hexane–Et2O (6:4); 3) 7:3; 4) 8:2; 5) heptane–C6H6 (9:1); 6) CHCl3–Me2CO–MeOH–AcOH–H2O (65:20:10:10:3); 7) CHCl3–MeOH–NH4OH (conc.) (65:35:5) [28]. Lipids and lipophilic substances were identified based on known qualitative reactions, literature data, and comparisons of chromatographic mobilities with those of model samples [squalene (Sigma-Aldrich, USA), phosphatidylcholine (Lipoid S80, Lipoid GmbH, FRG)].

FAs and US were isolated from lipids by alkaline hydrolysis as before [27]. FAs were methylated using freshly prepared diazomethane.

Biological Tests. Solutions were prepared by treating a weighed portion of the tested lipids with aqueous Tween 80 solution (0.1%, Schuchardt, Munich, Germany) and stirring vigorously. The resulting solution was diluted with H2O to concentrations of 0.001 and 0.0001% [29]. The studied substances were used for preliminary treatment of wheat (Triticum aestivum L.), variety Tatiana, and cucumber seeds (Cucumis sativus L.), variety Orzu.

Biological activity was studied using wheat seeds wetted with lipids at concentrations of 0.001 (10 mg/L) and 0.0001% (1 mg/L) for 18 h; cucumbers, for 6 h [30].

The controls were seeds wetted with H2O. The reference preparation was the biostimulant Uchkun. Seeds (10 ea) were placed into Petri dishes, treated with NaCl solution (1%, 5 mL), and cultivated for 5 d in a thermostat at 25°C. The activity was evaluated from the linear growth of the aerial and subterranean organs of sprouts and from their fresh and dry weights. The test results were processed mathematically by dispersion analysis using the OriginPro computer program [31].