Ajuga turkestanica (Rgl.) Briq. (Lamiaceae) is an endemic semi-bush plant that grows in a narrow range in Surkhandar’ya and Kashkadar’ya Regions of the Republic of Uzbekistan, primarily on clayey and rocky slopes of the foothills and mid-mountain band [1]. Ecdysteroids [2], iridoids [3], and carbohydrates [4] of the air-dried aerial part of the plant collected during flowering have been studied. Drugs and biologically active additives with adaptogenic, lactostimulating, tonic, and bile-stimulating activity are based on ecdysteroids and the iridoid glycosides harpagide and 8-O-acetylharpagide from A. turkestanica [5, 6]. The lipid composition of ecdysten production wastes from the air-dried aerial part of A. turkestanica, almost half the mass of which consisted of free fatty acids (FFA), was reported [7].

In continuation of research on A. turkestanica lipids [8], we studied lipids from air-dried roots, fresh aerial part (AP), and seeds of this plant. Seeds were collected in 2010 in Surkhandar’ya Region, RUz; roots and the aerial part, also there in 2011 during flowering. Flowers were separated from the AP and studied separately. Lipids were extracted from the roots according to Folch [9]; from the AP and flowers, by the same method but after preliminary double deactivation of biomass enzymes by hot isopropanol [10]. First, free lipids (FL) from seeds were extracted by benzine. Bound lipids (BL) were isolated from the remaining pulp. The resulting extracts were purified of non-lipid impurities. Table 1 presents the results.

Table 1 Lipid Contents in Separate Organs of Ajuga turkestanica
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Table 1 shows that the flowers contained two times more lipids than roots and the AP in this vegetative phase. Seeds contained comparable amounts of FL and BL. Their total mass (5.4%) was greater than the lipid yield from other plant organs. The oil content of A. turkestanica seeds (i.e., the content of FL, 2.9%) was four and more times less than that of seeds from other representatives of the Lamiaceae family [11].

Aerial Part. Total lipids from the AP were dark green in color and contained 1528 mg% chlorophyll pigments according to spectrophotometry. These lipids were separated into separate groups and classes by column chromatography (CC) over silica gel with elution of neutral lipids (NL) by CHCl3, glycolipids (GL) by acetone, and phospholipids (PL) by MeOH. As a result, four fractions each of NL and GL and one PL fraction were isolated. Complex fractions of NL and GL were rechromatographed to obtain narrower fractions or separate lipid classes. The composition of each fraction was established using analytical TLC. Constituents were identified by qualitative reactions and comparison with model compounds and the literature. The carotinoid content in NL was determined photocolorimetrically and was 500 mg% of the lipid mass. Table 2 presents the composition of total lipids from the AP of A. turkestanica. Almost half (49%) of the mass of total lipids was GL; about a third (30.4%), NL; and 20.7%, PL.

Table 2 Groups and Classes of Total Lipids from the Aerial Part of Ajuga turkestanica
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Neutral Lipids from the AP. The principal NL constituents were hydrocarbons, fatty-acid esters (FAE), and sterols (18.9% total). The isoprenoid hydrocarbon squalene was identified by TLC on Silufol using system 1, where it had R f 0.59 and was detected after treatment of the plate with H2SO4 (50%) at 110°C as a pink spot [10].

Carotenes absorbed in the UV spectrum with λmax (benzine, nm): 426.42, 450.61, 475.83; xanthophylls, with λmax (benzine, nm): 419.96, 442.86, and 471.43 [12].

The presence in the NL of FAE with sterols, triterpenols, and polyprenols was confirmed by analytical TLC on Silufol plates using solvent system 2, comparison with a known analogous NL class, and qualitative reactions and hydrolysis products. Forcing alkaline hydrolysis of FAE produced FA and unsaponified substances. FA were treated with diazomethane. The compositions of the resulting methyl esters were determined by GC (Table 3). TLC on Silufol using systems 3 and 4 identified in the FAE unsaponified substances the indicated isoprenoid and cyclic alcohols.

Table 3 Composition of Fatty Acids of Lipids from Fresh Aerial Part of Ajuga turkestanica During Flowering, % of LFA
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The FFA fraction with polyprenols was treated with diazomethane. FA methyl esters were separated from polyprenols by preparative TLC (PTLC) using system 3 and were analyzed by GC (Table 3).

TLC over silica gel using system 5 identified in the main GL fraction (Table 2) esters of sterylglycosides (SGE), monogalactosyldiacylglycerins (MGDG), traces of sterylglycosides (SG) [13], and chlorophyll derivatives. The presence in fraction 2 of sterylglycosides and cerebrosides (CB) was confirmed by qualitative reactions and the TLC mobility of spots of the compounds using the same solvent system [14]. CB were separated from this fraction by PTLC over silica gel using solvent system 5 in order to determine the FA composition. Digalactosyldiacylglycerins (DGDG) were practically the only constituents of GL fraction 3. According to the chromatographic mobility on TLC using system 5, GL fraction 4 contained sulfolipids [13]. This fraction was purified by PTLC over silica gel using solvent system 6 for subsequent isolation and analysis of the FA.

The qualitative composition of PL from the AP of A. turkestanica was established using two-dimensional TLC over silica gel and systems 7 and 8. The principal constituents of this lipid group gave a positive reaction for phosphorus with Vaskovsky reagent [10] and were phosphatidylcholines (PC) with minor amounts of phosphatidylinositols (PI) and phosphatidylethanolamines (PE). Analysis of the PL by one-dimensional TLC using system 7 with subsequent treatment of the plate with Vaskovsky reagents and storage in air for 10-15 min showed that the blue color of the PC and PI spots changed to brown. We assumed that the PL fraction contained iridoids [3] and/or ecdysteroids [2] characteristic of A. turkestanica.

TLC on Silufol using solvent system 9, which separates iridoids and ecdysteroids [15], detected two spots in the PL that were raspberry-colored if the plate was treated with acidic vanillin solution. The spots had R f values 0.19 (harpagide) and 0.34 (8-O-acetylharpagide) with the latter dominating. The PL were also separated in this system. Spots of model PI and PC had the same mobility as harpagide and 8-O-acetylharpagide. The iridoid content in AP lipids was 1.4% (Table 2).

Trace amounts of ecdysterone, which gave a blue spot upon treatment of the plate with acidic vanillin solution but was not detected by Vaskovsky reagent, were found in addition to iridoids in the PL with R f 0.5 under these same TLC conditions. Lipids of flowers, roots, and FL and BL of seeds were analyzed qualitatively by TLC on silica gel and Silufol analogously to AP lipids.

Flowers. Paraffinic hydrocarbons and FAE with triterpenols, sterols, and polyprenols were detected in the NL. GL included sterylglycoside esters, sterylglycosides, cerebrosides, MGDG, DGDG, and sulfolipids. PC, PI, and 8-O-acetylharpagide were identified in PL of flowers.

Roots. FAE with triterpenols and sterols, FFA, triterpenols, and sterols were found in NL from roots. The GL contained esters of sterylglycosides and sulfolipids. Traces of PL were observed. Other identified lipophilic constituents were two iridoids with noticeable dominance of 8-O-acetylharpagide and flavonoids. The total iridoid content in the total lipid extract of roots was 2.57% (of the extract mass). This was almost twice that in the AP extract.

Seeds. FL of seeds had the same NL constituents as those of flowers but also contained FFA, triacylglycerins, and xanthophylls. BL had the same NL composition as FL with the exception of xanthophylls. GL included constituents that were identified in flowers. The PL contained PC, PE, and PI in addition to iridoids and minor amounts of ecdysterone that were coextracted with the polar lipids.

Lipids of acyl-containing classes and groups were hydrolyzed in order to establish the FA composition. The isolated FA were converted to methyl esters (ME) by treatment with diazomethane. The FAME were analyzed by GC on a moderately polar phase. Saturated FA were identified using a ME model mixture 12:0–20:0; unsaturated FA, based on a comparison of their retention times (RT) with those of acids isolated from natural sources and literature data [16]. Table 3 presents the FA composition of lipids from the AP of A. turkestanica. Table 4 lists the FA from total lipids of other plant organs.

Table 4 Composition of Fatty Acids of Total Lipids from Vegetative and Generative Organs of Ajuga turkestanica, % of LFA
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Table 3 shows that FAE, FFA, and PL had the richest variety of FA. Among them, saturated even acids of the series 10:0–24:0 with 16:0 dominating were detected; of the odd series, 15:0 and 17:0. Unsaturated acids of these lipids were represented by 16:1, 18:1, 18:2, and 18:3. Half or more of total acids in FAE and FFA was a mixture of 18:1 and 18:3. The similarity of the FA compositions of FFA and DGDG in addition to CB and PL was interesting despite that fact that these classes belonged to different lipid polarity groups (NL, GL, PL). These classes in the first and second instances had very similar contents of 16:0 and (18:1 + 18:3) acids. However, differences were observed in the level of 18:2 acid. The MGDG and SGE fractions had characteristically the highest (76.9%) total unsaturated FA whereas saturated FFA dominated (64.0%) the sulfolipids.

The qualitative FA composition of lipids from other plant organs (Table 4) was similar to that of the AP and included 5–12 components. Roots showed the largest variety of FA in lipids (12 acids). The acid composition of seed lipids (five constituents) was simpler. Greater than half (57%) of the total FA mass in roots was 16:0 and 18:2. The 15:0 acid was found only in FA of roots and flowers. The levels of 20:0, 22:0, and 24:0 acids were also higher.

Greater than half the FA mass (55%) in the AP of A. turkestanica consisted of total unsaturated acids 18:1 and 18:3. FA of flowers were enriched in saturated components with 16:0 dominating (40%). The fraction of 18:1 and 18:3 was slightly less (35%).

Although the qualitative compositions of FA from FL and BL were similar in seeds, distinct differences occurred in the contents of the individual constituents. Thus, the level of 18:2 in FL acids was almost twice that in BL; of 16:0, 1.8 times less than in BL. The FL typically had the highest total unsaturated FA.

Experimental

GC of FAME was carried out on an Agilent 6890 N instrument with a flame-ionization detector using a capillary column (30 m × 0.25 mm) with HP-5 stationary phase, He carrier gas, and temperature programmed from 60 to 270°C. The carotinoid content was determined on a KFK-2-UKhL 4.2 photocolorimeter as previously described [17]; iridoids, on the same instrument according to the literature [3]; chlorophylls, by spectrophotometry on an SF-46 instrument [18].

TLC analysis of lipids was performed on silica gel and Silufol plates using solvent systems heptane:benzene (9:1, 1); hexane:Et2O (95:5, 2; 4:1, 3; 3:2, 4); CHCl3:Me2CO:MeOH:AcOH:H2O (65:20:10:10:3, 5); CHCl3:MeOH:NH4OH:H2O (48:14:1:1, 6); CHCl3:MeOH:NH3 (65:35:5, 7); CHCl3:MeOH:AcOH:H2O (60:20:1:1, 8); and CHCl3:MeOH:Me2CO (6:2:1, 9). Solvent systems 1–4 were used to separate NL; 5 and 6, GL; and 7 and 8, PL. Polyprenols had R f 0.41 in solvent system 4. This agreed with the literature [19].

Spots of NL compounds were detected in I2 vapor and by spraying plates with H2SO4 (50%) with subsequent heating; of GL, with α-naphthol solution (0.5%) and H2SO4 (50%) [10]; CB, on a separate plate were also detected by bromthymol blue solution (0.04%) in NaOH solution (0.01 N) [14]; PL, by Vaskovsky and Dragendorff reagents and ninhydrin solution [10]; iridoids, by a mixture of alcoholic vanillin (0.1%) with H2SO4 (0.5%) and heating [3].

Flavonoids had R f 0.89 for TLC on Silufol using solvent system 9 and comparison with a model sample of quercetin. They appeared as yellow spots upon detection with acidic vanillin solution.

Lipids were saponified by methanolic KOH solution (10%) upon refluxing for 1 h. The ester fraction was hydrolyzed using methanolic KOH solution (30%). FAME were prepared by treatment of an Et2O solution with diazomethane.

FL from air-dried seeds were extracted by benzine (bp 75–80°C) in a Soxhlet apparatus for 18 h. BL were extracted from the remaining pulp according to Folch by a CHCl3:MeOH (2:1, v/v) mixture and were purified of impurities by washing with CaCl2 solution (0.04%).