Fatty acid composition of lipids of Iris sibirica

Summary

Different Iris species are a rich source of secondary metabolites and they are widely used due to their medicinal properties, i.e. such as antibacterial, cytotoxic, hepatoprotective, antiplasmodial, and immunomodulatory effects. Determination of the fatty acid composition is necessary to create complex phytopreparations on their basis, and also, it is important for understanding of the adaptive capabilities of the plant. In our study, a comparative analysis of fatty acids composition of total lipids of the leaves and rhizomes of Iris sibirica was carried out by the gas chromatography-mass spectrometry method. The degree of unsaturated fatty acids and the activity of acyl-lipid desaturases were determined by the Lyons method. 14 fatty acids were identified in the leaves of I. sibirica, their content being 6.1 mg/g; 18 acids were found in the rhizomes, their content being 6.9 mg/g. Among the saturated fatty acids palmitic acid dominates (C16:0), and among the unsaturated ones, it is linoleic (C18:2ω6) and α-linolenic (C18:3ω3) acids. The content of the unsaturated fatty acids in the leaves is higher (45%) due to a high content of the polyunsaturated fatty acids, as well as the reduced share of the saturated and the monounsaturated acids, compared to the rhizomes (the content of the unsaturated fatty acids is 40%), which causes a higher value of the double bonds index (1.10), and the coefficient of unsaturation (0.81), which indicate the relative cold resistance of the plants. By contrast, in the rhizomes the concentration of the saturated fatty acids increases and the level of the unsaturated fatty acids reduces. The fatty acids composition of the leaves and rhizomes of Iris sibirica was established for the first time.

Key words:
Iris sibirica • fatty acids • acyl-lipid desaturases • gas chromatography-mass spectrometry

Introduction

Analysis of the fatty acid composition of lipids is one of the components of the determination of the medicinal quality of plants. Fatty acids are important for the normal functioning of the human body. Polyunsaturated fatty acids are the structural elements of phospholipids and lipoproteins of cell membranes, they are part of the connective tissue and the membranes of nerve cells, they take part in the transportation and oxidation of cholesterol, provide the elasticity of blood vessels and regulate many processes in the body¹⁾.

The unsaturated fatty acids of oleic acid (ω-9) can be synthesized by combining the reactions of elongation (extension of the chain) and desaturation (formation of the extra double unsaturated bonds), due to the presence of Δ9-desaturase system. However, the human body cannot synthesize linoleic (ω-6) and α-linolenic (ω-3) acids due to a lack of Δ12 and Δ15 desaturases^{2, 3)}. These acids are essential or indispensable and must come from food because they are necessary for the synthesis of other polyunsaturated fatty acids of ω-6 and ω-3 type.

Acyl lipids play an important role in plants life having bactericidal and fungicidal properties, they perform the protective function; stimulate the growth of plants; they are the storage compounds, providing the host with energy. Lipids actively change the metabolism during the autumn period and increase the resistance of plants to low temperatures^{4, 5)}.

In the study of plant lipids, growing under the conditions of abiotic stress, it is important to identify the qualitative and quantitative composition of fatty acids, with the aim of creation the complex plant-based preparation, as well as for understanding their role in metabolism and plant adaptation.

The Iridaceae family contains approximately 1,800 species in 80 genera^{6, 7)}. The genus Iris is the most extensive and comprises about 200 species, distributed in the most of the Northern Hemisphere, 16 species grow in Ukraine⁸⁾, and about 40 species grow in the territory of the neighbouring states⁹⁾. Species of the genus Iris were introduced into the culture as ornamental plants and they take one of the first places in the world among the flower crops in the amount of varieties. All irises are perennial grasses with annual inflorescence and perennial shortened vegetative shoots, which are rhizogenic and which are immersed in the soil or crawling on the surface. For research we have chosen the Iris sibirica (series Sibiricae (Diels) Lawrence), section Limniris (Tausch) Spach em. Rodion) of the Iridaceae Juss. Family⁶⁾. The plant is unpretentious and easily breeds by seeds.

Analysis of the literature showed that the plants of the Iris genus contained various biologically active substances: flavonoids^{10, 11)}, isoflavones^{12, 13)}, xanthones^{14, 15)}, carboxylic acids¹⁶⁾, terpenoids, ether oil, and tannins; however, the fatty acid composition has not been almost examined. Information about the use of the polyunsaturated fatty acids as pharmacological agents is rare in the literature. It is known that the oil extract of the rhizomes of I. pseudacorus is used in the treatment of non-healing wounds, burns, cuts, erosions, as analgetic, haemostatic and anti-burn agents¹⁷⁾. The antibacterial activity of lipophilic extracts from the rhizomes and leaves of I. pseudacorus¹⁸⁾, I. hungarica and I. sibirica¹⁹⁾ by diffusion in agar and serial dilutions are also established. The antibacterial, antifungal activity of the chloroform and ethyl acetate extracts of the rhizomes of I. germanica has been found, and the petroleum extract showed antibacterial, antifungal and insecticidal activity^{12, 20)}. The antioxidant and anticholinesterase activities of the chloroform extract from the rhizomes of I. albicans have been established²¹⁾.

The fatty acids composition of I. pseudacorus, I. pseudacorus f. alba²²⁾, I. hungarica²³⁾ was previously studied by us. For the productive cultivation of species and varieties of irises, it is necessary to examine the composition of the fatty acid as one of the indicators that is responsible for the biochemical processes in the cells.

The aim of this study was a comparative analysis of the fatty acid composition of total lipids, the determination of the degree of unsaturation of fatty acids and the activity of acyl-lipid desaturases of the leaves and rhizomes of I. sibirica.

Experimental part

Plant material

The objects of the examination were the leaves and rhizomes of I. sibirica, which were prepared in N. N. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine, Kiev, in the latter half of September 2015. I. sibirica from the collection of nursery of the Botanical Garden is the introduced view from the seeds that were received by hortus botanicus and collected in the phase of mass flowering. The systematic belonging of plant was established by one of the authors. Analysis and estimation of the results were carried out with the air-dry raw materials. Voucher specimens have been deposited in the Herbarium of the Pharmacognosy Department and Botany Department, National University of Pharmacy, Kharkiv, Ukraine.

Analysis of methyl esters of fatty acids

Analysis of methyl esters of fatty acids was carried out by gas chromatography with mass spectrometric detection (GC/MS)^{24, 25)}. The internal standard (solution 50.0 µg of tridecane in hexane) and 1.0 ml methylating agent (14% of BCl₃ in methanol, Supelco 3-3033) were added to the weight of the raw materials (50.0 mg) contained in a 2.0 ml vial. This mixture was kept in a hermetically closed vial for 8 hours at 65 ºC. At this time fatty oil was fully extracted and hydrolyzed into its constituent of the fatty acids and their methylation was done. The reaction mixture was drained off from a precipitation of the plant material and was diluted with 1.0 ml of distilled water. Methyl esters were extracted with 0.2 ml of methylene chloride, carefully shacked up for several times within an hour, then the obtained extract was chromatographed.

Chromatographic conditions

A sample injection (2.0 µl) was carried out into the chromatographic column in the splitless mode within 0.2 minutes. The contents of methyl esters of the fatty acids were determined on an Agilent Technologies 6890 chromatograph with a mass spectrometric detector 5973. A capillary column HP-INNOWAX was used for splitting (30 m × 0.25 mm × 0.50 µm). Mobile phase: helium, gas flow rate is 1.2 ml/min. Temperature of a heater of the sample injection is 250 ºC. Temperature of a furnace is programmable from 50 to 320 ºC with the rate of 4 degree/min.

Identification of the components

Individual components were identified by comparison of their mass spectra using both “NIST-MS Library 05” and “Wiley GC-MS Library 2007” ^26,27). The substance content was estimated relating to the internal standard. Calculation of the components content С (mg/kg) was carried out by the formula:

where P₁ – the peak area of the tested substance; P₂ – the peak area of the standard; 50 – the mass of the internal standard (μg), injected into the sample; m – the sample mass (g).

The relative component content was defined in per cent from their total amount.

Determination of the degree of unsaturation of fatty acids and the activity of acyl-lipid desaturases

The double bonds index (DBI) for the assessment of unsaturation of fatty acids (FA) was estimated by the method of Lyons³⁾:

where – P_j is the content of each unsaturated FA (%) and n_j is the number of double bonds in its molecule. The unsaturated coefficient was estimated as the total content of unsaturated (UFA) – to – saturated acids (SFA) ratio:

The activity of acyl-lipid -, ω6 and ω-3 membrane desaturases^{2, 29)}, catalyzing the introduction of double bonds into the carbon chains of oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids, were determined as steroyl- (SDR), oleyl- (ODR) and linoleyl- (LDR) desaturase ratio, which were estimated as the content (% from total FA) of the components C18, according to the equations:

where C18:0, C18:1, C18:2 and C18:3 is the percentage of the total acids of stearic, oleic, linoleic and linolenic acids, respectively.

Statistical analysis

The results were the mean ± SD of three parallel measurements. All statistical comparisons were made by means of Student’s t-test, values of p < 0.05 were regarded as significant. Statistical analysis of the results obtained was carried out by the method of the smallest squares according to the SPhU monograph “5.3.N.1. Statistical analysis of the results of chemical experiment” (2015). For the calculations and statistical analysis of the obtained data Microsoft Office Excel was used.

Results

The GC/MS analysis of the fatty acid composition of total lipids has showed that in the leaves of I. sibirica there were 14 fatty acids (FA), their total content being 6.1 ± 0.11 mg/g; in the rhizomes 18 FA have been identified, their content being 6.9 ± 0.12 mg/g. Lengths of carbon chains were from 12 to 24 atoms (Figs. 1–2, Table 1).

The submitted data have shown that in the leaves and rhizomes of I. sibirica the dominant saturated acids were palmitic (C16:0) (in the leaves, 29.11%; in the rhizomes, 34.78%) and lauric (C12:0) acids (16.11% and 10.90%, respectively). In addition to these acids, saturated fatty acids with 14, 15, 17, 20, 22, 23, 24 carbon atoms have been identified. The content was not more than 1–2% of each of them. An exception was stearic acid (C18:0), its content being 3.70% of the total acids in the leaf and 6.57% in the rhizomes. The content of the unsaturated acids in the leaf was 45%, which was by 5% higher than in the rhizomes, by reducing the content of palmitic, lauric acids and myristic (C14:0) it was absent in the leaf.

Among the unsaturated fatty acids of lipids, monoenoic, dienoic, trienoic acids were identified, forming ω9, 2ω6, 3ω3 of the biosynthetic families of the fatty acids with cis-orientation of double bonds in the chain. The highest percentage of ω6 dienoic linoleic (C18:2ω6) acid was obtained in the rhizomes (27.65%) and a smaller content was detected in the leaves (18.92%). The highest content of ω3 trienoic α-linolenic (C18:3ω3) acid (22.37%) was determined in the leaves of I. sibirica, at the same time, in the rhizomes α-linolenic acid (2.07%) was in the least amount.

Discussion

The aim of this study was a comparative analysis of the component composition of fatty acids of the leaves and rhizomes of I. sibirica by the chromatography-mass spectrometry method. Besides, analysis of composition of unsaturated acids is of scientific interest for the determination of the adaptive capacities of the plant⁴⁾.

In the leaves of I. sibirica in the unsaturated fraction the content of monoenoic acids was decreased almost by 3 times by oleic acid (C18:1ω9), because of their active conversion into polyenoic acids, their content being increased from 30% in the rhizomes to 42% in the leaves. The content of monoenoic acids has been 2.83% and 10.28% for the leaves and for the rhizomes, respectively. Among monoenoic ω9 acids in the rhizomes, oleic (5.63%), palmitooleic (C16:1ω7; 1.77%) and 10-octadecenoic (C18:1ω10; of 2.88%) acids have been identified. In the leaves the content of oleic acid has been 1.78%, and palmitooleic acid, 1.05%.

From dienoic fatty acids, where in the synthesis ω6 desaturase takes a part, linoleic (C18:2ω6) acid similarly has been identified, its content in the leaves and the rhizomes of Iris being 18.92% and 27.65%, respectively. It is known²⁸⁾ that the low content of oleic acid with a high level of linoleic and linolenic acids can further the activation of R-genes (resistance genes) and the development of hypersensitivity reactions. Stark comparison in the content of these acids can be seen in the leaves.

Trienoic acids in the rhizomes have been represented only by α-linolenic (C18:3ω3) acid in a small amount of 2.07%. At that time, in the leaves the content of α-linolenic acid has been increased by 10 times (22.37%). It should be noted that in the leaves hexadecadienoic acid (C16:3ω3; 0.7%) appears, which plays a large part in the determination of the cold resistance of plants, and promotes the process of photosynthesis at low temperatures²⁹⁾. The biosynthesis of dienoic and trienoic acids 18:2ω6, 18:3ω3 and 16:3ω3 plays a defining role in the adaptation of plants to hypothermia. These polyunsaturated fatty acids regulate the “fluidity” of cell membranes in a wide temperature range, influence the resistance of plants to difficult environments^{4, 28)}.

The unsaturated coefficient characterizes the change in membrane fluidity due to the changes in the saturated – to – unsaturated fatty acids ratio in the membrane lipids. An increase in the unsaturated coefficient in the leaves was provided by increasing the fraction of the polyunsaturated fatty acids, which was very important for the maintenance by membranes of the liquid-crystalline state and increasing the resistance of leaves to hypothermia³⁰⁾. In the rhizomes the unsaturated coefficient was lower than in the leaves, it was probably connected with reducing their physiological activity and the cell membrane reacted to gel and they almost were not in effect.

It is known³¹⁾ that a level C>20 of fatty acids in the vegetative organs of the higher plants is usually less than 1–2%. However, according to the literature data³²⁾, an increased content of the level C20 of fatty acids in the vegetative organs of the plants indicates that the plant has adapted to adverse environmental conditions. The fraction C20-24 of fatty acids in the rhizomes was 4% and in the leaves, 4.85%. This confirms that I. sibirica has adapted to the conditions of our latitude⁶⁾.

The degree of unsaturation of lipids is best characterized by the double bonds index (DBI)³⁾. As you can see, the DBI in the leaves of Iris (1.10) is higher than in the rhizomes (0.72), by all appearances, this is due to the mechanisms of survival of the leaves and their adaptation to the action of the negative temperatures as of autumn-winter period. Irises belong to the long term vegetative plants that do not have pronounced period of winter dormancy, because the foliage in the central part of foliate fascicle remains green⁶⁾.

When the temperature reduces, the fluidity of membranes decreases and as the result, there is an enhancement of the synthesis of enzymes of desaturases in the cell and also the quantity of polyunsaturated fatty acids is increased. As the result, the membranes assume the liquid-crystalline state and their fluidity restores^{1, 30, 33)}. Therefore, the activity of desaturases is the determinant in the adaptation of plants to low temperatures. For Iris sibirica, the activity of steroyl- (SDR), oleyl- (ODR) and linoleyl- (LDR) desaturases is different. The value of desaturase ω9 (SDR) in the leaf is smaller, perhaps it is due to the fact that in the rhizomes the content of short-chain fatty acids is higher, and they contribute to the maintenance of membrane fluidity at the required level, i.e. the reaction of the underground organs to the adaptation were faster^{2, 29)}. The value of ODR for the leaf has been determined as high – 0.96, and it has revealed that oleic acid was actively converted by acyl-lipid ω6-desaturases into linoleic acid. On the other hand, the reaction of ω-3 desaturase (LDR) that causes the introduction of a third double bond is different: in the rhizomes it was only 0.07, while in the leaf this value was 0.54, due to the appearances of hexadecatrienoic acid and the content of α-linolenic acid increased.

The differences in the fatty acid composition in the leaves and rhizomes of Iris show different regulatory mechanisms in the plant. By the adaptation of the leaf of Iris to cold snap the main role belongs to ω-3 desaturase as the increase of its activity leads to the transformation of linoleic acid to linolenic. Adaptation of the rhizomes is achieved by increasing the activity of ω6 desaturase that is responsible for the biosynthesis of linoleic acid and of ω-3 desaturase in the less degree.

In addition, it should be noted that fatty acids are known for their antioxidant, antifungal, anti-inflammatory and immunomodulatory properties³⁴⁾, they are involved in metabolism, they have a positive effect in digestion, and create favourable conditions for the life activity of beneficial intestinal microorganisms^{1, 5)}. In recent years, the research investigated fatty acids and the results obtained showed that they have significant sedative and hypnotic effects³⁴⁾, as well as antibacterial, antitumour, antimycobacterial and antiviral activities^{35, 36)}. In our previous papers¹⁹⁾, we reported the results of the antimicrobial activity screening of the dry extracts of the rhizomes and leaves of I. sibirica which at the concentration of 1% inhibited the growth of gram-positive and gram-negative bacteria and fungi. So, the pharmacological activity of Iris plants is due to the composition of their biologically active compounds.

Conclusions

As a result of the experiment, it has been established that the leaves and rhizomes of I. sibirica have a different fatty acid composition. The content of the saturated fatty acids in the rhizomes is prevalent, and in the leaves the fraction of unsaturated (in particular the polyenoic) fatty acids is larger due to polyenoic ones; it confirms the value of the coefficient of unsaturation and the double bonds index. When the temperatures decrease, the membrane of the rhizomes become gel state and the biochemical processes slow down. The aboveground part adapts to weather conditions due to the increased content of polyunsaturated fatty acids. In addition, the experimental data show that for a harvest of the raw materials it is better to select the period of early autumn, when the biologically active substances in the organs of the plant accumulate. This is the first report on the determination of the fatty acids from the leaves and rhizomes of I. sibirica by GC/MS. The composition of fatty acids also gives the prerequisites to use I. sibirica for medical purposes.

Conflicts of interest: none.

Received September 19, 2017

Accepted November 6, 2017

Olga Mykhailenko, PhD (Pharmacy)

V. Kovalyov

S. Kovalyov

E. Toryanik

National University of Pharmacy

4-Valentunivska St., 61168 Kharkiv, Ukraine

e-mail: zolya85@gmail.com

T. Osolodchenko

Head of the Laboratory of Biochemistry of Microorganisms and Nutrient Media

Institute of Microbiology and Immunology named after I. I. Mechnikov of the National Academy of Medical Sciences of Ukraine, Kharkiv, Ukraine

Y. Buidin

Head of Department of the Ornamental plants, Senior Researcher National Botanical Garden n.a. M.M. Gryshko of NAS of Ukraine, Kyiv, Ukraine