Specific Composition of Lecithins in Ginseng Root Cell Culture

Pharmaceutical Chemistry Journal Vol. 38, No. 2, 2004 MEDICINAL PLANTS SPECIFIC COMPOSITION OF LECITHINS IN GINSENG ROOT CELL CULTURE V. P. Pchelkin,1 M. A. Shatalova,1 V. D. Tsydendambaev,1 A. M. Nosov,1 and A. G. Vereshchagin1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 38, No. 2, pp. 34 – 36, February, 2004. Original article submitted February 4, 2003. As is known, lipids belonging to various classes are the main components of membranes in the living cell. Each lipid is composed of a set of molecules of several types with different combinations of fatty acid residues. The properties of various membranes are determined both by the particular composition of these fatty acids and by the relative content of the base molecules of various types [1]. The specific composition of glycerophospholipids is most exhaustively studied for the storage organs of plants in vivo, where the content of lipids of this group is usually higher by one order of magnitude than in hydrated tissues. Investigations of the composition of these lipids in the somatic cells of plants are still rare [2]. This situation is even more pronounced with respect to all objects cultivated in vitro. Indeed, there is still a very small body of quantitative data on the specific composition of acyl-containing glycerophospholipids even in the medicinal plants most widely used and most significant in therapeutic practice. This statement also refers to the main class of such lipids — lecithins (phosphatidylcholines). This study is devoted to establishing the specific composition of lecithins in a preparation isolated by quantitative extraction from the standard sample of a two-week suspension cell culture of the root of ginseng, Panax ginseng Mey (Araliaceae family). fixed in 100 ml of boiling isopropyl alcohol, after which the sum of lipids of ginseng root cells was isolated by stepwise extraction with a yield of 98.6 % [4]. Subsequent calculations were performed taking into account the percentage water content in the raw biomass of the initial cell culture. As a rule, the initial sample (12 g of raw biomass) corresponded to 0.7 g of a dry substance of the cell biomass containing about 79 mg of esterified fatty acids. Phosphatidylcholines (9.9 mg) isolated from an aliquot of the ginseng root extract were separated from the lipids of other types by preparative TLC and subjected to enzymatic hydrolysis by phospholipase C from Clostridium perfringens in 0.1 M Tris buffer (pH 7.3) [5, 6]. A mixture of 1,2-diacyl-sn-glycerins (7.5 mg, 97.9%) obtained upon this reaction was extracted with purified chloroform and fractionated by absorption TLC on silica gel impregnated with silver nitrate [7]. The absolute amounts of etherified fatty acids in the aliquot of phosphatidylcholines and in separate fractions of 1,2-diacyl-sn-glycerins (containing 0.5 to 3 mg of etherified fatty acids) were determined with respect to margaric acid (50 mg) added to the samples. This compound is missing from the natural lipids of ginseng and, hence, can be used as the internal standard [8]. Upon methanolysis of the mixtures, the total fractions of methyl ethers of fatty acids separated from the esterification products were separated into components by gas chromatography under standard conditions [7, 8]. Methyl ethers of individual fatty acids were identified by their relative retention times [8]. The fatty acid composition was calculated by the internal normalization method (for peak areas S ) on an IBM XT computer (Great Britain) using Nelson 3000 program package (Nelson Analytical, USA). These data were used for the automated calculation of the absolute content and the statistical specific composition of lipids. These calculations were performed in terms of TurboBasic, Fortran 77 (Version 3.31) EXPERIMENTAL PART The experiments were performed with a suspension cell culture of the root of ginseng, Panax ginseng C. A. Mey (strain IFR-Zh1, No. 3) from the All-Russia Collection of Higher Plant Cells [3]. The cell culture was grown in 0.5-liter flasks under standard conditions [3]. An accurately weighed amount (~12 g) of a two-week raw biomass was 1 Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia. 93 0091-150X/04/3802-0093 © 2004 Plenum Publishing Corporation 94 V. P. Pchelkin et al. TABLE 1. Fatty Acid Composition of Phosphatidylcholines in a Two-Week Suspension Cell Culture of Ginseng Root and in Hydrolysis Products (Diacylglycerins) of free 1,2-diacyl-sn-glycerins obtained by soft enzymatic hydrolysis of the initial phosphatidylcholines had virtually the same fatty acid composition, which indirectly confirmed the proximity of the two classes of lipids with respect to the qualitative specific composition. The total content of phosphatidylcholines in a ginseng root cell culture, calculated for the total amount of free 1,2-diacyl-sn-glycerins isolated upon the enzymatic hydrolysis, was 24.9 mM per gram of dry biomass. The concentration of phosphatidylcholines in the initial culture (1.07 mg per gram of raw tissue) was higher than that usually observed in most plant tissues with the same degree of hydration [2]. For the comparison, the content of phosphatidylcholines in the cell cultures of carrot and tobacco does not exceed 0.15 and 0.49 mg/g (raw tissue), respectively [12, 13]. On the other hand, the value for ginseng is slightly below that for a rauwolfia cell culture [13]. The content of the main individual phosphatidylcholines in the ginseng cell culture is very close to that reported for peanut fruits [9]. Therefore, the culture under consideration exhibits no sharp differences from other plants in this respect. Comparative data on the specific composition of the given class of lipids are presented in Table 2. As can be seen from these data, there are nine individual species for which only the fatty acid composition (rather than the positions of fatty acid residues in the molecules) have been established. Esterified fatty acids, mol.% Class of lipids Phosphatidylcholines 1,2-Diacyl-sn-glycerins palmitic stearic oleic linolic linolenic 30.4 32.3 1.8 1.6 8.1 7.1 58.4 58.1 1.2 0.9 and 8086 Object Linker (Version 3.04, 1985) (Microsoft, USA). RESULTS AND DISCUSSION The results of determination of the fatty acid composition of the initial phosphatidylcholines in 1,2-diacyl-sn-glycerins isolated from ginseng root cell cultures are presented in Table 1. As can be seen from these data, the main fatty acid components of these phosphatidylcholines are linolic (cis, cis-octadeca-9,12-dienoic, 18 : 2), palmitic (hexadecanoic, 16 : 0), and oleic (cis-9-octadecenoic, 18 : 1). The initial phosphatidylcholines also contain significant amounts of stearic (octadecanoic, 18 : 0), and linolenic (cis, cis, cis-octadeca-9,12,15-trienoic, 18 : 3) acids. The preparation TABLE 2. Specific Composition of Phosphatidylcholines in Some Plants (Percentage of the Total)* Phosphati dylcholine types 16:0\16:0 18:0\16:0 16:0\18:1 18:0\18:1 18:1\18:1 16:0\18:2 18:0\18:2 18:1\18:2 18:2\18:2 16:0\18:3 * Cell cultures Seeds ginseng carrot [12] tobacco [13] rauwolfia [13] sunflower [11] cotton [6] rice [10] 2.4 9.4 0.2 1.1 6.4 4.9 – 0.3 0.5 0.6 50.9 35.3 2.9 3.0 6.8 9.5 27.8 34.1 2.1 0.7 – 5.2 – – 2.2 3.2 – – – 0.6 43.3 27.9 – – 11.8 8.6 25.1 37.8 – – – 14.1 – 3.0 11.5 6.6 – 0.7 – 0.8 48.0 26.2 – 2.8 3.0 6.2 8.2 12.2 8.1 1.2 – 14.2 – 4.0 – 1.6 traces 0.2 2.1 traces 73.7 40.6 9.9 5.7 – 2.3 12.1 29.1 0.6 0.1 0.4 1.1 0.4 1.1 3.7 3.2 1.8 1.6 2.3 2.4 15.6 14.1 7.6 6.8 20.3 20.9 44.6 45.6 – – 3.1 5.5 0.4 0.7 14.8 10.9 1.2 0.7 5.6 5.5 23.3 22.1 1.4 1.5 17.6 22.1 23.7 22.1 – – – 3.8 – – 18.5 16.8 – – 19.6 18.7 20.3 14.5 – – 28.9 32.3 12.7 13.9 – – Potato tuber [2] Soya bean sprout [2] Pea leaves [2] – 7.0 – – – – – – – – 35.5 32.1 6.9 4.7 – – 31.8 37.5 10.1 3.0 0.4 4.0 – 1.3 3.2 3.0 0.4 0.5 0.4 0.6 26.8 25.0 5.4 3.9 9.2 9.2 35.1 32.0 5.9 1.3 – 7.9 – 1.0 – 1.7 1.5 0.1 – 0.1 28.0 27.0 1.3 1.7 3.2 2.9 21.2 23.0 16.0 5.7 Upper values gives experimental data; lower values are calculated using the fatty acid composition according to the theory of statistical distribution of fatty acid residues in the molecules of glycerophosphocholines [7]. Specific Composition of Lecithins in Ginseng Root Cell Culture This number is much smaller than it might have been expected according to the theory of statistical distribution of the fatty acid residues in the molecules of phosphatidylcholines. All these nine individual types are characteristic of the storage organs of widespread cultivated plants such as sunflower, cotton, and rice. On the other hand, only five of these nine types are always found in phosphatidylcholines of the hydrated tissues of vegetative organs of some other cultivated plants (potato, soya bean, and peas [2]). As for the qualitative specific composition, phosphatidylcholines of the ginseng root cell culture are generally very close to the same class of lipids and possess a set of fatty acids analogous to that isolated previously from the storage organs of other plants [6, 10, 11]. Moreover, this composition for phosphatidylcholines of the ginseng root cell culture differs only very slightly from that typical of the other hydrated tissues of different plants [2]. In addition, it should be noted that rice grains contain five individual types of phosphatidylcholines [10], while the analogous tissues of potato contain only six such types [2]. The main types of phosphatidylcholines of the ginseng root cell culture are palmitoyl-linoleoyl and -dilinoleoyl phosphatidylcholines, the content of which accounts for about 80 mol.% of the total sum for all types. These phosphatidylcholines are quite typical of the cell cultures of carrot, tobacco, and rauwolfia [12, 13]. The concentrations of these individual types determined in our experiments for phosphatidylcholinesof the ginseng root cell culture sharply differ from the values anticipated according to the theory of statistical distribution of the acyl residues in the molecules (Table 2). For palmitoyl-linoleoyl phosphatidylcholines of ginseng, the interval of differences between the experimental and statistical concentrations exceeds 15 mol.%. Nevertheless, the sum of fractions of palmitoyl-linoleoyl and -dilinoleoyl phosphatidylcholines for the statistical composition was also below 80 mol.% and quite close to the experimental sum. 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