Bioactive lipids in gintonin-enriched fraction from ginseng
a b s t r a c t
Background: Ginseng is a traditional herbal medicine for human health. Ginseng contains a bioactive ligand named gintonin. The active ingredient of gintonin is lysophosphatidic acid C18:2 (LPA C18:2). We previously developed a method for gintonin-enriched fraction (GEF) preparation to mass-produce gin- tonin from ginseng. However, previous studies did not show the presence of other bioactive lipids be- sides LPAs. The aim of this study was to quantify the fatty acids, lysophospholipids (LPLs), and phospholipids (PLs) besides LPAs in GEF.
Methods: We prepared GEF from white ginseng. We used gas chromatography-mass spectrometry for fatty acid analysis and liquid chromatography-tandem mass spectrometry for PL analysis, and quantified the fatty acids, LPLs, and PLs in GEF using respective standards. We examined the effect of GEF on insulin secretion in INS-1 cells. Results: GEF contains about 7.5% linoleic (C18:2), 2.8% palmitic (C16:0), and 1.5% oleic acids (C18:1). GEF contains about 0.2% LPA C18:2, 0.06% LPA C16:0, and 0.02% LPA C18:1. GEF contains 0.08% lysophos- phatidylcholine, 0.03% lysophosphatidylethanolamine, and 0.13% lysophosphatidylinositols. GEF also contains about 1% phosphatidic acid (PA) 16:0-18:2, 0.5% PA 18:2-18:2, and 0.2% PA 16:0-18:1. GEF- mediated insulin secretion was not blocked by LPA receptor antagonist.
Conclusion: We determined four characteristics of GEF through lipid analysis and insulin secretion. First, GEF contains a large amount of linoleic acid (C18:2), PA 16:0-18:2, and LPA C18:2 compared with other lipids. Second, the main fatty acid component of LPLs and PLs is linoleic acid (C18:2). Third, GEF stim- ulates insulin secretion not through LPA receptors. Finally, GEF contains bioactive lipids besides LPAs.
1.Introduction
Ginseng, the root of Panax ginseng Meyer, has been used as a tonic for human health [1]. Ginseng contains diverse bioactive medicinal components. Ginsenosides were the first ingredients of ginseng to be isolated. Ginsenosides are glycosides derived from triterpenoid dammaranes with 30 carbon atoms. Each ginsenoside has a common hydrophobic four-ring steroid-like backbone and has carbohydrate moieties at the carbon-3, carbon-6, or carbon-20 positions [2]. About 30 ginsenosides have been isolated from ginseng by changing the processing conditions such as pressure and temperature. Ginsenosides exhibit a variety of in vitro andin vivo pharmacological effects in biological systems [3,4]. Acidic polysaccharides were identified next. They consist of a complex polymer chain of monosaccharides rich in L-arabinose, D-galactose, L-rhamnose, D-galacturonic acid, D-glucuronic acid, and D-galactosylresidue linked together through glycosidic bonds, resulting in complex macromolecular architectures [5]. Although the structures of acidic polysaccharides are not clearly understood, it is known that they regulate immune systems [5].Recent studies found that ginseng also contains a novel phos- pholipid (PL) complex with ginseng proteins such as ginseng major latex-like protein 151, also called gintonin. Further study revealed that the functional component of gintonin is lysophosphatidic acid(LPA, 1-acyl-2-hydroxy-sn-glycero-3-phosphate) and that gintonin functions as an exogenous ginseng-derived G protein-coupled LPA receptor ligand [6,7].
Thus, gintonin is distinguished from previ- ously identified ginseng saponins and acidic polysaccharides, because the representative functional component is ginseng- derived lipid ligand [7]. LPA is a simple lysophospholipid (LPL; Fig. 1A) composed of glycerol 3-phosphate (G3P), fatty acids, and phosphates (Fig. 1) [8]. LPA is mainly synthesized in plants by glycerol 3-phosphate acyltransferase (GPAT) transferring fatty acids such as linoleic (C18:2), palmitic (C16:1), or oleic acid (C18:1) to the sn-1 or sn-2 positions of G3P [8] (Fig. 1C). In biological systems, LPA also plays an intermediate role in syntheses of phosphatidic acid (PA) and other PLs [8] (Fig. 1B, 1C). Interestingly, ginseng contains an extraordinarily high concentration of LPAs, especially LPA C18:2, about 10-fold greater than that in other herbal medicines and foodstuffs [9]. We previously developed a simple method for mass production of gintonin-enriched fraction (GEF) from ginseng using ethanol and water, and demonstrated that GEF exhibits various in vitro and in vivo physiological and pharmacological effects [10e 14]. However, although we obtained qualitative evidence that GEF contains other lipid components along with LPAs [10], until now these bioactive lipids related to LPAs and other PL components of GEF have not been identified and quantified.
We analyzed representative bioactive fatty acids, LPLs, and PLs in GEF using gas chromatography-mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Here, we report that GEF contains bioactive lipid components closely related to LPAs. The main fatty acid compositions of GEF, in quantitative order, are linoleic (C18:2), palmitic (C16:0), and oleic (C18:1) acids. The main PL components of GEF, in quantitative or- der, are PAs, LPAs, lysophosphatidylinositol (LPI), lysophosphati- dylcholine (LPC), and trace amounts of other LPLs. In experiments using non-neuronal cells, we also found that GEF contains other active ingredient for insulin secretion besides LPAs. The present report also discusses the possible contributions of fatty acids, PLs, and LPLs in GEF-mediated physiological and pharmacological roles in biological systems.
2.Materials and methods
Four-year-old Korean White ginseng was purchased from a local ginseng market. 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphate (LPA C14:0), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (LPA C16:0), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate (LPA C18:0),1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA C18:1), 1- myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC C14:0), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (lyso- phosphatidylethanolamine [LPE] C16:0), 1-oleoyl-2-hydroxy-sn- glycero-3-phosphoethanolamine (LPE C18:1), 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoinositol (LPI C16:0), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(10-myo-inositol; LPI C18:1),1,2-dimyristoyl-sn-glycero-3-phosphate (PA 14:0-14:0),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA 16:0-18:1),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate (PA 16:0-18:2),1,2-dilinoleoyl-sn-glycero-3-phosphate (PA 18:2-18:2), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (phosphati- dylcholine [PC] 14:0-18:0), 1-palmitoyl-2-linoleoyl-sn-glycero-3- phosphocholine (PC 16:0-18:2), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (PC 18:2-18:2), and 1,2-dipalmitoyl-sn-glycero-3-phospho-(10-myo-inositol; phos-phatidylinositol [PI] 16:0-16:0)were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1- Linoleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA C18:2) was purchased from Echelon (Salt Lake City, UT, USA). 1-Linoleoyl-2- hydroxy-sn-glycero-3-phos-phatidyicholine (LPC C18:2) was pur- chased from Larodan (Solna, Sweden).
Fatty acids and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Insulin assay enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shibayagi Co. Ltd. (Shibukawa, Gunma, Japan). RPMI 1640 Medium and all other materials for cell culture were purchased from ThermoFisher Scientific Korea (Gangnam-gu, Seoul, South Korea). Ki16425 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). All other reagents used were purchased from Sigma-Aldrich.GEF was prepared according to a previously described method [10]. Briefly, 1 kg of 4-year-old ginseng was ground into small pieces (>3 mm) and refluxed with 70% fermentation ethanol eight timesfor 8 h at 80◦C each. The ethanol extracts (350 g) were concentrated.Ethanol extract was dissolved in distilled cold water at a ratio of 1e 10 and stored in a cold chamber at 4◦C for 24 h. The supernatant and precipitate produced by water fractionation, after the ethanolextraction of ginseng, was separated by centrifuge (3000 rpm, 20 min). The precipitate was lyophilized after being centrifuged. This fraction was designated GEF and had a yield of 1.3%.Free fatty acid (FFA) analysis using GC-MS was performed at the Korea Basic Science Institute (Western Seoul Center). For GC-MS analysis of FFAs, samples were saponified then methylated. A 5- mg GEF sample in a screw-capped glass tube was hydrolyzedwith 1 mL 1 M KOH in 70% ethanol at 90◦C for 1 h then cooled down.Four milliliters of 10% boron triflouride (BF3) in methanol was added and derivatized at 60◦C for 20 min for methylation.
The re- action mixture was allowed to cool to room temperature, then fattyacid methyl esters (FAMEs) were extracted with 2 mL hexane. For phase separation, 1 mL water was added, the sample was shaken well, and the upper layer of hexane was collected through sodium sulfate filtration [15]. The FAMEs in the GEF samples were deter- mined using a 7890B GC, equipped with a 5977A mass selective detector quadrupole mass spectrometer system (Agilent Technol- ogies, Palo Alto, CA, USA). The DB-5 MS capillary column (30 m × 0.25 mm i.d., 0.25 mm film thickness, 5% diphenyl-95% dimethylsiloxane phase; J&W Scientific (Folsom, CA, USA) was used for separating the FAMEs using helium gas as a carrier at a flowrate of 1 mL/min. The GC oven temperature was maintained at 50◦Cfor 1 min, and then ramped to 320◦C at 5◦C per min, thenmaintained for 10 min. Samples of 1 mL volume were injected with a 2:1 split ratio. The temperatures of the GC injection port and MS interface were set at 280◦C and 300◦C, respectively. The mass se- lective detector was run in electron impact mode, with an electronenergy at 70 eV. The mass spectrometer was operated in full scan mode between 50 and 700 amu. Individual FAMEs were identified by the processes of peak separation and consultation with the Wiley 7n electron impact mass spectral library and comparing their retention times with authentic standards (Supelco, 37 Component FAME Mix certified reference material).Stock solutions of LPLs and PLs were prepared and diluted in high- pressure liquid chromatography (HPLC)egrade methanol. All solu- tions were stored at 4◦C.
LPLs and PLs were quantified by LC-MS/MSusing an Agilent series 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) instrument, which consisted of a G1311A quart pump, G1313A autosampler, G1322A degasser, G1316A column oven, and an API 2000 LC-MS/MS system (Applied Biosystems, Foster City, CA, USA) [16]. Tandem mass spectrometric (MS/MS) analysis was carried out using an electrospray ion source in positive (LPC, PC) and negative (LPA, LPE, LPI, PA, PI) modes. Data acquisition was performed in multiple reaction monitoring (MRM) mode and SCIEX Analyst 1.4.2 software (ABI Inc., Foster City, CA, USA) was used for data manage- ment and control. Chromatographic separations of LPA, LPC, LPE, and LPI were performed on a XBridge C18 analytical column (2.0 × 100 mm, 3.5 mm particle size; Waters Corporation, Milford, MA, USA; Fig. 2). The lipids for PA, PC, PI were separated on a XBridge Hydrophilic Interaction analytical column (2.1 × 100 mm, 3.5 mm particle size; Waters Corporation; Fig. 3). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid and 10 mM ammonium formate inwater (B). The isocratic pump mode was run at a flow rate of 0.2e0.25 mL/min and 2-mL aliquots were injected intothe column. The column temperature was maintained at 25◦C. Theion spray voltage was set at —3.5 to —4.5 kV in negative ion mode and5.5 kV in positive ion mode. The capillary temperature was set at 400e450◦C. The operating conditions, optimized by a flow injection of all analytes, were as follows: nebulizing (GS1), auxiliary (GS2), andcurtain gas flows of 30.0 to 50.0, 70.0, and 10 to 15 PSI, respectively. The MRM transitions of the analytes, the collision energy, and declustering potential for lipids are summarized in Table 1.
Quanti- tation was performed via MRM of the precursor ions and the related product ions using an internal standard method with peak area ratios. A 100-mg GEF sample in a 50-mL centrifuge tube was dissolved with 10 mL water and vortex-mixed. One milliliter of the middle layer was transferred to a new centrifuge tube and diluted with methanol. The mixture was vortexed and then filtered through a 0.2-mm syringe filter (Millex-LG; Merck Millipore Corporation,Merck KGaA, Darmstadt, Germany). A volume of 2 mL was injectedfor LC-MS/MS analysis..5.Cell cultureRat beta cells INS-1 were kindly gifted from Prof. Yup Kang (Ajou University School of Medicine, Suwon, South Korea) and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 units/mL penicillin, and 100 mg/mL streptomycin.Insulin secretion from INS-1 cells was determined using the ELISA. Briefly, INS-1 cells were seeded at 3 × 105 cells per well into 24-well plates. After 24 h, cells were washed with Krebs-Ringerbicarbonate (KRB) buffer (24 mM NaHCO3, 1.2 mM MgCl2, 1 mM HEPES, 129 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2,0.2 mM glucose, pH 7.4), and then preincubated in KRB buffer for 1 h [17]. The cells were incubated with gintonin, ginsenosides, or LPA at indicated concentrations for 2 h. In some experiments, Ki16425 was also added before gintonin treatment. Supernatants were collected and insulin concentration of each sample was determined using rat insulin ELISA kit according to the manufac- turer’s instruction (Shibayagi).(R ≥ 0.95). The linearity of each calibration curve was determined by peak area analyte/peak area internal standard at six concen- tration levels of individual analytes. All values are presented as mean relative standard deviation (%) from three different sam- ples of white ginseng.
3.Results
The calibration curves were evaluated via weighted linear (1/x) and quadratic (1/x2) regression with their correlation coefficientsIn previous reports, we showed that GEF from ginseng contains about 20% lipids. We confirmed through qualitative analysis that gintonin and GEF contain three representative fatty acids:linoleic (C18:2), palmitic (C16:0), and oleic acids (C18:1), and other minors [6]. In the present study, we performed quantitative anal- ysis to determine the concentrations of major FFAs in GEF. As shown in Table 2, we found that GEF contains a large amount (7.53%) of linoleic acid (C18:2) compared with two other fatty acids, palmitic (C16:0; 2.82%) and oleic acids (C18:1; 1.46%). This quan- titative assay of fatty acids showed that GEF contains about 12% fatty acids and that the primary FFA in GEF is linoleic acid (C18:2).In previous reports, we showed that gintonin contains LPA C18:2, LPA C16:0, and LPA C18:1 as the representative functional components. LPAs in GEF are LPLs with simple structures (for re- view, see Ref. [18]). In biological systems, there are several addi- tional bioactive LPLs such as LPC, LPE, lysophosphatidylglycerol (LPG), LPI, lysophosphatidylserine (LPS), and lysophosphatidyl- threonine (LPT). Most of them also exhibit gintonin’s effects through activation of G protein-coupled receptors [18]. Next, we determined the concentrations of free LPLs, including LPAs, in GEF.
We found that GEF contains about 0.2% LPA C18:2 and lesser con- centrations of LPA C16:0 and LPA C18:1 (Table 2). Next, we found that GEF contains a small amount, 0.082%, of LPC. Interestingly, we found that GEF contains 0.093% LPI C18:2, 0.038% LPI C16:0, and 0.011% LPI C18:1, which is known as a kind of endocannabinoid GPR55 receptor ligand [19]. We also found that GEF contains 0.012% LPE C18:2 and 0.019% LPE C16:0 (Table 2). GEF did not contain LPG, LPS, or LPT (data not shown). These results confirmed that LPAs are the major LPLs in GEF, along with lesser amounts of other LPL li- gands, including LPC, LPE, and LPI. This quantitative assay of LPLs shows that GEF contains about 0.54% LPLs, including LPAs.Next, we examined the free PL contents of GEF and quantified PA, PC, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PI, phosphatidylserine (PS), and phosphatidylthreonine. Surpris- ingly, we found that GEF contains large amounts of PAs, in the orderof 1.0% PA 16:0-18:2 > 0.5% PA 18:2-18:2 > 0.15% PA 16:0-18:1. Inaddition, GEF also contains 0.05% PC and a trace amount of PI. However, GEF does not contain PE, PG, PS, or phosphatidyl- threonine. These results indicate that GEF contains about total 1.75% PLs and that PAs are the main PLs in GEF.
The absence of PE, PG, and PS in GEF might be associated with the trace amounts of LPG, LPS, and LPT in GEF.In previous reports, we showed that GEF exhibits cellular effects in neuronal cells via LPA receptor activation [20]. In the present study, we further examined whether GEF also stimulates insulin secretion through LPA receptors in non-neuronal INS-1 cell, which is known to secrete insulin and used for model cell for insulin secretion. As shown in Fig. 4A, GEF stimulated insulin secretion with concentration-dependent manner. Interestingly, GEF-induced insulin secretion was not blocked by LPA1/3 receptor antagonist Ki16425 and the effect of GEF on insulin secretion was much stronger than that of LPA (Fig. 4B). In addition, when we compared the effects of GEF on insulin secretion with ginsenosides such as Rb1, Rg1, and Rg3, we found that GEF was more effective than ginsenosides in insulin secretion. These results indicate that GEF- induced insulin secretion in INS-1 cell was not mediated via LPA receptors.
4.Discussion
In previous reports, we showed that gintonin mainly consists of carbohydrates and lipid and ginseng protein complexes. We further demonstrated that one representative lipid component of gintonin, LPA C18:2, is the main functional ligand for the activation of G protein-coupled LPA receptors. We also showed gintonin-mediated effects from in vitro hormone or neurotransmitter releases to in vivo cognition-enhancing effects via LPA receptors [20]. As shown in Fig. 1, LPA is simply composed of G3P, fatty acids, and phosphate, but exhibits diverse biological effects. Previous reports also showed a possibility that GEF might contain other bioactive PLs related to LPAs and other bioactive lipids [6,20]. In the present study, we focused on determining fatty acid, LPL, and PL contents in GEF, because previous reports showed that fatty acids, LPLs, and PL components could exhibit biological activities through membrane receptor signaling pathways (for review, see Ref. [18]). In this pre- sent study, we did not focus on simple glycolipids such as mono- acylglycerol, diacylglycerol, and triacylglycerols, because they act as lipid storage for energy supply. We found three main characteristics of GEF through lipid content analysis. First, GEF contains about 12% FFAs, including linoleic acid (C18:2), followed by palmitic (C16:0) and oleic (C18:1). These results are also consistent with a previous report that Panax ginseng and American ginseng also contain high concentration of linoleic acid (C18:2) compared with other fatty acids [21]. Second, GEF contains a high concentration of LPA C18:2 compared with other LPLs such as LPC, LPE, and LPI, and its total LPL contents are 0.54%. Third, GEF also contains about 1.8% PLs such as PA 16:0-18:2, PA 18:2-18:2, PA 16:0-18:1, and PCs. Thus, the total lipid content of GEF was about 14.1%. These results indicate that linoleic acid (C18:2), LPA C18:2, and PA 16:0-18:2 might be the representative lipid components of GEF (Table 2).
Plants usually keep low concentrations of PAs, which are syn- thesized from LPAs by transferring one fatty acid to LPAs (Fig. 1C), because they are intermediate metabolites for larger molecules and are rapidly further processed into glycolipids and PLs for plasma membrane compositions or lipid storage [22]. Interestingly, GEF contains a relatively high concentration of PAs (Table 2). In addition, we observed a novel specificity of fatty acid components in PAs of GEF. The most abundant PA in GEF is PA 16:0-18:2 (Table 2). The concentration of PA 16:0-18:2 is about two-fold higher than that of PA 18:2-18:2 in GEF. It is noteworthy that most plant-derived PAs, including those in soy, consist of PA 18:2-18:2 [23], whereas most PAs derived from animals consist of PA 16:0-18:1, but GEF contains a small amount of the PA 16:0-18:1 found in animal systems [23]. On the other hand, the LPA C18:2 content in GEF is also much higher than that in other foodstuffs and herbal medicines [9]. Thus, ginseng contains unique LPA C18:2 and PA 16:0-18:2, different from those of animals and other plants. In addition, we found that polyunsaturated linoleic acid (C18:2) is the main fatty acid component of LPA, other LPLs, and PAs in GEF (Table 2), raising the possibility that linoleic acid (C18:2) is the primary fatty acid component of LPA, LPL, and PA synthesis in ginseng. Thus, Panax ginseng LPAs are synthesized from G3P by transferring an acyl group, which could be linoleic acid (C18:2), by GPAT in GEF. LPAs are further acylated to form PAs by LPA acyl- transferase [24], of which the second acyl group could be linoleic (C18:2), palmitic (C16:0), or oleic acid (C18:1) in GEF (Fig. 1C).
Thus, it is probable that in ginseng LPA and PA syntheses, the main acyl group will be in the order of linoleic (C18:2) >> palmitic (C16:0) > oleic acid (C18:1). As shown in Fig. 1A, LPA C18:2 can be transformed into PA 16:0-18:2, if C16:0 is transferred into LPA C18:2. LPA C18:2 can be transformed into PA 18:2-18:2, if C18:2 is transferred into LPA C18:2. LPA C18:1 can be transformed PA 16:0-18:1, if C16:0 is transferred into LPA C18:1. Those PAs could be further processed to form other PLs or glycerolipids. Thus, LPAs and PAs are intermediate metabolites for synthesis of diverse glycer- olipids, LPLs, and PLs in ginseng. Interestingly, it was revealed that the amounts of LPAs and PAs in GEF were much higher than those in other foodstuffs or herbal medicines [25]. Considering physiological roles of fatty acids, linoleic acid (C18:2) is a polyunsaturated fatty acid. Linoleic acid (C18:2) acts as an endogenous ligand of GPR40, a fatty acid receptor found in various organs [26]. Linoleic acid (C18:2) is also an essential fatty acid, and, in animals, absorbed linoleic acid (C18:2) is usually further metabolized into arachidonic acid and further processed to other arachidonic acid-derived bioactive ingredients such as
prostaglandins [27]. Thus, there is a possibility that the linoleic acid (C18:2) derived from GEF also could be further transformed into other physiologically active lipid-derived agents in animals. On the other hand, in plant systems, PAs are components of cellular membranes.
PAs are also produced as a response to stresses, including osmotic stress, pathogens, and freezing/cold/wounding, and act as lipid-derived signaling molecules against stresses [28]. PAs also act as an intracellular second messenger signal molecule in animal systems [29]. A recent study showed that PAs are strong activators of mammalian target of rapamycin (mTOR), which functions as a nutrient/energy/redox sensor and controls protein synthesis and regulates cellular metabolism, growth, and prolifer- ation [30]. Interestingly, it has been reported that the functions of PA in mTOR activation is dependent on the structure of its fatty acid components. For example, two saturated fatty acids promote stor- age, whereas one saturated and one unsaturated fatty acids promote signaling for the activation of mTOR [31]. Joy et al [32] further demonstrated that PA isolated from eggs with high satu- rated fatty acids exhibited less activation of mTOR than did PA from soy with unsaturated fatty acids. These previous reports suggest that GEF-derived PAs could be a candidate for mTOR activation.
Besides LPA and PAs, several LPLs act as plasma membrane signaling molecules. GEF contains small amounts of LPLs besides LPA, and one of them, LPI, may act as a G protein-coupled receptor ligand such as GPR55 [18]. Interestingly, we found that although GEF contains 0.26% LPC, LPE, and LPI, GEF does not contain LPG, LPS, or LPT (data not shown). In future studies, it will be inter- esting to examine the roles of linoleic acid (C18:2), PAs, and LPI in GPR40, mTOR, and GPR55 regulation, respectively, although their amounts in GEF are different from each other to some extent. If it is true for their individual functions, it will provide a chance to expand GEF application beyond LPA receptor-mediated biological effects.
Previous reports on whole ginseng root lipid analyses showed that Panax ginseng contains a total of about 1.13e1.24% free lipids [33]. Ginseng lipid content is composed of FFAs, mainly in the order of linoleic (C18:2) >> palmitic (C16:0) > oleic acid (C18:1), glyco-
lipids in the order of sterol glucoside > monogalactosyl diglyceride > digalactosylglyceride, and PLs in the order of PE = PG > PC > PI > PA. Comparing total lipid contents of whole ginseng with that of GEF, GEF contains at least 10-fold higher lipid concentrations (Table 2), although we did not quantitate glycolipids in GEF. In other words, GEF could be enriched with lipids, especially with linoleic acid (C18:2), LPAs, and PAs, compared with other lipids. Currently, we cannot clearly explain why specific lipid components are enriched during GEF preparation from whole ginseng root. Further study will be required to explain this phe- nomenon. In addition, it should be noted that we observed some degree of variation in the concentrations of bioactive lipids in the GEF examined among white ginseng batches, although we kept 4- year old ginseng roots for the preparation of GEF. The variations in the contents of the analyzed bioactive lipids might be due to regional differences in ginseng cultivation areas, soil conditions, and ginseng harvest seasons.
It will be interesting to determine why GEF from ginseng con tains such high concentrations of polyunsaturated linoleic acid (C18:2) and PAs compared with saturated fatty acids and other PLs. Ginseng is a representative perennial herbal medicine that grows in
high mountains or high latitude areas with winter temperatures below 0◦C. Therefore, it might be due to the cold growth environ- ments of ginseng. It is known that linoleic acid (C18:2) and PA are required for tolerance to cold stress [34]. There is evidence that gene transcriptions related to fatty acid desaturation increase during cold stress and the increased unsaturated fatty acids and PA enhance membrane fluidization and protect against cell membrane destruction under freezing conditions [28,35]. Thus, ginseng might produce polyunsaturated fatty acids, linoleic acid (C18:2), and PAs to keep itself alive under cold stress. Interestingly, GEF-mediated increase of insulin secretion was not associated with LPA receptors (Fig. 4B), because LPA1/3 receptor antagonist did not block GEF action. In previous reports, we showed that the effects of gintonin and GEF on neuronal cells were atten- uated or blocked by LPA1/3 receptor antagonist [20]. Considering the lipid compositions of GEF found in the present study, it seems that GEF-mediated insulin secretion achieved via other bioactive lipid(s) rather than LPAs. Further study will be required to identify novel candidate related with insulin secretion in INS-1 cells. Taken together, the present study shows that GEF might contain diverse Ki16198 bioactive components differentiating from LPAs.
In conclusion, we quantified the concentrations of the main lipid contents of GEF. We found that GEF has a unique composition of polyunsaturated linoleic acid (C18:2), LPA C18:2, and PA 16:0-18:2. GEF-induced insulin secretion in INS-1 cell was not mediated via LPA receptors, showing that GEF contains other bioactive compo- nent(s) besides LPAs.