990 0009-3130/20/5606-0990

2020 Springer Science+Business Media, LLC

Chemistry of Natural Compounds, Vol. 56, No. 6, November, 2020

ACYL LIPIDS AND LIPOPHILIC AND PHENOLIC

COMPOUNDS FROM RARE PLANT SPECIES

E. S. Bogdanova,

V. N. Nesterov,

L. M. Kavelenova,

R. R. Sarvarova,

S. V. Saksonov,

and O. A. Rozentsvet

Fifteen species of rare calciphyte plants growing in Samara Oblast were studied. The most variable of the

studied quantitative parameters (acyl lipids, pigments, phenolic compounds) were lipids and phenolic

compounds, in contrast to pigments and fatty acids.

Keywords: acyl lipids, calciphytes, photosynthetic pigments, phenolic compounds.

Rare plant species show consistent trends toward contracted numbers and/or areas, which gives them high scientific

value. The flora of the middle Volga region comprises such species, many of which represent ecologically specialized plant

groups. In particular, such plants include calciphytes, which have adapted to specific soil and climatic conditions and inhabit

locations with exposed limestone, chalk, clays, and other carbonate rocks [1].

The chemical composition of the plants depends largely on the environmental conditions (temperature, humidity,

etc.), plant species, development phase, and localization in certain organs [2].

The goal of the work was to study the qualitative and quantitative compositions of acyl lipids, fatty acids (FA),

photosynthetic pigments, and phenolic compounds (PC) of rare plants growing in Samara Oblast.

A total of 15 species from 11 families were studied: Artemisia salsoloides Willd., Anthemis trotzkiana Claus

(Asteraceae); Pimpinella titanophila Woronow, Bupleurum falcatum L. (Apiaceae); Krascheninnikovia ceratoides (L.)

Gueldenst. (Chenopodiaceae); Onosma volgensis Dobrocz. (Boraginaceae); Gypsophila volgensis Krasnova (Caryophyllaceae);

Ephedra distachya L. (Ephedraceae); Astragalus zingeri Korsh., Hedysarum grandiflorum Pall. (Fabaceae); Linum flavum L.,

Linum uralense Juz. (Linaceae); Polygala sibirica L. (Polygalaceae); Reseda lutea L. (Resedaceae); Cotoneaster laxiflorus

J. Jacq. ex Lindl. (Rosaceae). All plants were calciphytes with respect to ecology and rare and/or threatened species with

respect to nature conservancy status [3].

Lipids were isolated from fresh leaves by extraction with CHCl

–MeOH. Each lipid class was analyzed separately

by high-performance thin-layer chromatography (HPTLC). The acyl-containing lipids isolated from the calciphyte leaves

were dominated by glycolipids (GL, ~51% of total lipids). The GL included monogalactosyl diacylglycerins (MGDG),

digalactosyl diacylglycerins (DGDG), and sulfoquinovosyl diacylglycerins (SQDG) (Table 1).

The GL composition of most species obeyed the trend DGDG > MGDG > SQDG. The quantitative dominance of

DGDG over MGDG was apparently related to the specific habitat conditions of the calciphytes. Leaves of P. sibirica and

B. falcatum had the highest amounts of GL with 11.8 and 9.5 for DGDG; 9.6 and 6.7 for MGDG, and 6.0 and 6.2 mg/g of dry

mass for SQDG, respectively. These parameters for the other species averaged 3.0 for MGDG, 4.1 for DGDG,

and 2.1 mg/g of dry mass for SQDG. The phospholipids (PL) included phosphatidylcholine (PC), phosphatidylethanolamines

(PE), phosphatidylinositols (PI), phosphatidic acids (PA), phosphatidylglycerols (PG) and diphosphatidylglycerins (DPG).

The dominant PL classes in most studied species were PC (0.8–4.0 mg/g of dry mass) and PG (0.4 up to 3.0 mg/g of dry

mass), the relative contents of which in the total PL reached 57.3%. For example, the PC contents in leaves of C. laxiflorus and

L. flavum were 67.8%. The fractions of PG in A. zingeri and R. lutea were 47.1 and 36.1% of total PL. The very low PE

contents in A. salsoloides, C. laxiflorus, L. flavum, L. uralense, and R. lutea of ≤ 3.0% of total PL were interesting.

1) Institute of Ecology of River Basin, Samara Federal Research Center, Russian Academy of Sciences,

10 Komzina St., Tolyatti, 445003, e-mail: [email protected]; 2) S. P. Korolev Samara National Research University,

34 Moskovskoe Shosse St., Samara, 443086, e-mail: [email protected]. Translated from Khimiya Prirodnykh Soedinenii,

No. 6, November–December, 2020, pp. 854–857. Original article submitted March 2, 2020.

DOI 10.1007/s10600-020-03211-y

991

Neutral lipids (NL) included acyl-containing lipids such as triacylglycerins (TAG), diacylglycerins (DAG), and free fatty

acids (FFA). The amounts of TAG among them were 0.6–6.8 mg/g. DAG varied from 0.4 to 2.3 mg/g; FFA, from 0.2 to 4.5 mg/g

of the dry mass. TAG accumulated most in C. laxiflorus and E. distachya; FFA and DAG, in P. titanophila and P. sibirica.

The fatty acid (FA) composition was determined after methanolysis of the total lipid extract. FAs with 12–24 C atoms

were detected using gas chromatography (GC) (Table 2). Saturated FAs (SFAs) of all plants were dominated by palmitic

(C16:0), the quantitative contents of which varied from 19.1 to 30.0% of total FAs. The next FAs in decreasing order were

stearic (C18:0) and arachic FA (C20:0). Species differences were observed in the SFA contents. For example, A. zingeri had

a high content of lauric acid C12:0 (1.7%); L. uralense, C18:0 FA (8.3%). A characteristic feature of the FA composition of

E. distachya, O. volgensis, and A. salsoloides was a high content of saturated long-chain FAs C20:0, C22:0 (behenic), and

C24:0 (lignoceric). Unsaturated FAs (USFAs) were represented by oleic C18:1n9, linoleic C18:2n6, and

-linolenic C18:3n3.

The plants A. salsoloides, A. zingeri, B. falcatum, E. distachya, and P. titanophila had identical contents of C18:2n6 and C18:3n3 FAs.

TABLE 1. Acyl-containing Lipids in Calciphyte Leaves, mg/g of Dry Mass

Compound class

Plant

MGDG DGDG SQDG PC PE PG PI PA DPG TAG DAG FFA

I 1.2 3.1 2.0 1.3 0.05 0.4 0.6 1.4 – 2.0 1.2 0.6

II 0.8 2.0 2.0 1.3 0.1 0.7 0.7 0.6 0.1 1.1 1.4 Tr.

III 4.2 5.8 3.8 2.8 0.5 2.4 0.1 0.4 0.2 1.2 1.0 1.2

IV 6.7 9.5 6.2 3.6 0.5 1.5 0.6 – 0.1 3.3 2.0 0.8

V 3.4 4.8 1.8 4.0 0.08 1.5 0.3 – – 6.8 1.9 Tr.

VI 1.8 2.9 0.6 0.8 0.3 0.7 0.4 2.8 0.08 4.9 0.4 Tr.

VII 3.5 4.0 1.7 0.2 0.05 0.3 0.7 3.7 – 2.1 0.2 2.3

VIII 3.7 3.4 2.3 2.9 0.7 2.2 1.0 – 0.2 1.5 1.4 1.1

IX 7.0 6.9 2.0 3.4 1.0 3.0 1.4 0.7 0.2 2.2 1.1 0.9

X 4.6 7.9 4.2 2.3 0.03 0.8 0.1 – 0.07 1.0 0.7 0.4

XI 1.1 2.1 1.2 1.1 0.06 0.6 0.2 – – 0.2 0.2 0.2

XII 0.7 1.8 1.2 1.7 0.03 0.7 0.5 – – 1.7 0.7 2.5

XIII 3.5 5.0 2.1 2.7 0.6 0.9 1.6 1.3 0.2 2.5 2.1 4.5

XIV 9.6 11.8 6.0 3.9 0.9 2.2 1.2 0.2 0.1 2.3 2.3 4.4

XV 6.4 7.3 4.2 1.5 0.08 1.5 0.5 – – 1.0 1.3 3.6

______

I, A. salsoloides; II, A. trotzkiana; III, A. zingeri; IV, B. falcatum; V, C. laxiflorus; VI, E. distachya; VII, G. volgensis; VIII,

H. grandiflorum; IX, K. ceratoides; X, L. flavum; XI, L. uralense; XII, O. volgensis; XIII, P. titanophila; XIV, P. sibirica; XV,

R. lutea.

TABLE 2. Fatty Acid Contents in Calciphyte Leaves, % of ΣFA

Saturated FA Unsaturated FA

Plant

12:0 16:0 18:0 20:0 22:0 24:0 16:1n9 18:1n9 18:2n6 18:3n3

Other FA

I – 25.2 2.4 2.8 2.3 5.8 1.7 11.5 18.8 28.5 1.0

II 0.3 32.5 2.5 1.3 1.3 0.3 1.6 6.8 20.5 25.7 7.2

III 1.7 25.5 5.1 2.4 1.0 0.4 3.1 8.1 4.2 45.2 3.3

IV 1.0 23.9 0.3 0.8 0.5 0.5 2.0 6.2 29.7 26.9 8.2

V 0.5 28.7 3.5 1.4 0.8 0.3 2.9 8.7 10.3 40.6 2.3

VI 0.3 25.7 4.2 2.4 3.2 2.2 1.2 19.8 16.5 16.9 7.6

VII 0.6 19.1 3.3 2.5 0.8 0.6 1.8 13.8 12.8 42.2 2.5

VIII 0.4 25.4 5.3 3.5 1.7 0.6 2.5 10.9 11.8 36.3 1.6

IX 0.5 19.6 1.7 1.6 2.0 1.4 1.8 17.9 11.2 38.7 3.6

X 0.3 26.5 4.0 2.2 1.0 0.8 2.0 4.7 12.0 42.8 3.7

XI 0.6 30.0 8.3 2.9 1.7 1.1 2.4 3.7 13.0 34.1 2.2

XII 0.2 25.1 3.9 5.1 2.8 1.8 3.5 15.8 5.2 34.0 2.6

XIII 0.6 25.3 1.5 0.6 0.9 1.9 2.0 6.7 23.8 28.8 7.9

XIV 0.1 24.5 5.0 1.0 0.2 0.5 1.9 3.7 14.3 46.8 2.0

XV 0.2 28.8 3.7 1.9 0.9 0.2 2.5 4.5 8.0 46.7 2.6

______

Designations are the same as in Table 1.

992

The amounts of photosynthetic pigments chlorophylls a and b (Cl a, Cl b) and carotenoids (Car) were determined

after extraction from fresh leaves by cold Me

CO (90%). The contents of total Cl calculated per dry mass varied in the range

1.7–4.6 mg/g; of Car, 0.4–1.2 mg/g (Table 3). High contents of pigments were characteristic of L. flavum, P. sibirica, and

R. lutea. Small quantities of Cl were observed in E. distachya (1.7 mg/g); of Car, in A. trotzkiana (0.4 mg/g of dry mass).

All studied species were heliophytes and long-day plants. Sun-loving plants are known to contain less chlorophyll than

shade-tolerant species. The ratio of Cl a and Cl b was >3 for heliophytes and ~2.3 for shade-tolerant plants [4]. The studies

showed that the Cl a/b ratio varied in the range 2.4–4.1, i.e., this parameter was low in most studied calciphyte species except

for B. falcatum.

PC were extracted from dry plant matter by distilled H

O. The PC contents according to various data can vary

considerably from 20.0 to 190.0 mg/g of dry mass [3, 5]. Our previous work showed that plants growing in an arid climate

accumulated large quantities of PC, up to 51.7 mg/g [6]. The present work showed that the contents of water-soluble PC in

leaves of calciphytes varied from 17.5 to 132.5 mg/g of dry mass. The highest contents were found in A. salsoloides and

C. laxiflorus (Table 3). PC were not observed in leaves of L. uralense.

TABLE 3. Contents of Pigments and Phenolic Compounds in Calciphyte Leaves, mg/g of Dry Mass

Plant

ΣChlorophylls

Carotenoids Chlorophylls

Chlorophyll/carotenoid Phenolic compounds

I 2.8 0.6 2.5 4.6 132.5

II 2.4 0.4 2.4 6.0 25.9

III 4.4 0.9 3.3 5.1 47.6

IV 4.6 0.9 4.1 5.1 51.9

V 4.6 0.8 2.8 5.8 88.7

VI 1.7 0.4 3.2 4.0 20.0

VII 2.4 0.6 2.8 4.2 57.0

VIII 4.9 0.9 3.0 5.2 26.5

IX 3.1 0.6 3.0 4.8 17.5

X 6.2 0.9 3.4 6.8 27.5

XI 3.5 0.6 2.9 6.1 –

XII 3.8 0.6 2.8 5.8 37.3

XIII 3.8 0.8 2.8 4.6 60.0

XIV 5.4 1.2 2.8 4.5 27.4

XV 5.2 0.8 2.9 6.2 54.9

______

Designations are the same as in Table 1.

Fig. 1. Ratio of lipids, pigments, and PC in calciphyte leaves: Lu,

L. uralense; Kc, K. cerotoides; Ps, P. sibirica; Lf, L. flavum; Hg,

H. grandiflorum; Bf, B. falcatum; Ed, E. distachya; Rl, R. lutea;

At, A. trotzkiana; Pt, P. titanophila; Ov, O. volgensis; Cl, C. laxiflorus;

Az, A. zingeri; Gv, G. volgensis; As, A. salsoloides.

100

Lu Ks Ps Lf Hg Bf Ed Rl At Pt Ov Cl Az Gv As

- Lipids

- Pigments - PC

993

Figure 1 shows ratios of measured groups of chemical compounds in the studied plants. Thus, L. uralense was

characterized by a high percent lipid content; A. salsoloides, the lowest content. However, A. salsoloides had the maximum

amount of PC as compared with the other species. Lipids, especially PE, and PC were the most variable of the studied

parameters (coefficient of variation >60%). Photosynthetic pigments and FAs were the least variable (coefficients of variation

30.8% and 9.7–17.0%, respectively). Thus, the results established that the quantitative contents of acyl lipids, pigments, and

PC depended considerably on the plant species.

EXPERIMENTAL

Plant material was collected in the middle of the day in June 2018 in areas of 20 × 20 m. Analyses used leaves from

15–20 plants. Three independent biological samples were taken from the combined biomass and frozen in liquid N

GL were separated by HPTLC on plates with an aluminum backing (10 × 10 cm, 5–17 μm; Sorbfil, Russia) using

solvent system Me

CO–C

–H

O (91:30:8, by volume). PL were separated using two-dimensional HPTLC on glass plates

(6 × 6 cm; Lenkhrom, Russia) using solvent system CHCl

–MeOH–C

–NH

OH (130:60:20:12, by volume) in the first

direction and CHCl

–MeOH–C

–Me

CO–AcOH (140:60:20:10:8, by volume) in the second direction. NL were separated

by one-dimensional HPTLC on plates (10 × 10 cm) with an aluminum backing with sequential use of solvent systems

toluene–hexane–HCOOH (70:30:0.5, by volume) and hexane–Et

O–HCOOH (60:40:1, by volume) [7]. The obtained

chromatograms were detected by spraying with H

solution (10%) in MeOH followed by heating at 180°C for 15 min.

The amounts of GL, PL, and NL were determined using densitometry (Sorbfil, Russia). The standards for constructing

calibration curves were MGDG, PC, and tripalmitate (Sigma-Aldrich, USA).

FA were analyzed as their methyl esters. Methanolysis used refluxing HCl solution (5%) in MeOH for 1 h followed

by cooling. The FA methyl esters were extracted by hexane, purified by TLC, and analyzed on a Chromatec Crystal 5000.1

chromatograph (Russia) in isothermal mode using a capillary column (105 m × 0.25 mm; Restek, USA). The column temperature

was 180°C; vaporizer and detector, 260°C; carrier gas (He) at flow rate, 2 mL/min.

The contents of photosynthetic pigments in the Me

CO (90%) extract were determined by spectrophotometry

at λ 662, 645, and 470 nm. The concentrations of Cl a, Cl b, and Car were calculated by the Lichtenthaler method [8].

Water-soluble PC were extracted by distilled H

O from air-dried plant matter. The PC contents were determined

using Folin–Ciocalteu reagent according to the published method [9] in the drop modification suggested by the manufacturer

(Panreac Quimica, Spain). A calibration curve was plotted using gallic acid.

The tables give arithmetic averages of three biological and three analytical replicates and their standard errors.

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