PLAG is synthetic component which is composed of 1-palmitic acid-2-linoleic acid-3-acetylglycerol (Fig. 1), which can be separated from the chloroform extracts of the deer antler (Korean Patent No. 0283010), or alternatively can be prepared to small scale by the following 2 methods (Korean Patent Application No. 2000-0045168) and purified using several column purification steps. The first method includes the steps of obtaining reaction products by the reaction of glycerol and palmitic acid, separating 1-palmitoylglycerol by using a column chromatograpy from the reaction products, and successively carrying esterification reactions for the separated 1-palmitoylglycerol to obtain PLAG. The second method for preparing PLAG utilizes an acetolysis reaction of phosphatidyl choline. Recently, we developed the patented large scale synthetic process of PLAG without column purification. (Korean Patent No KR 10-1278874; PCT/KR2012/007644).

The subjects included those who 1) have body weight of ≥45 kg with BMI of 18.5~30.0 kg/m², 2) are in subhealthy state with WBC count of 2,000~5,000 during screening, 3) can visit the hospital on schedule and make their blood taken, 4) have agreed to birth control during and after the clinical test at least for 2 month including those who have themselves sterilized, and 5) have volunteered for the research and submitted written consent to follow the clinical protocols. Those subjects were excluded from this study who 1) have troubles or histories in liver, kidney, digestive, respiratory, musculoskeletal, secretory, neurological, and cardiovascular diseases, 2) have gastrointestinal diseases (e.g. Cohn's disease and ulcer) or experienced gastrointestinal operations (excluding appendectomy and herniotomia), 3) are sensitive to medications especially those extracted from deer antlers or exhibited clinical symptoms, 4) have a history of drug addiction, 5) have unusual diet which can affect the abruption, distribution, metabolism, and excretion of the medicine within 30 days, 6) have medication for other clinical drug(s) within 60 days, 7) have donated whole blood within 60 days, or transfused within 30 days, 8) have taken a medication for the inducer or suppressor of drug metabolizing enzyme including barbitals, 9) are heavy drinkers (>21 units/week, 1 unit matches 10 ml of pure alcohol), and 10) have failed to meet the criteria of clinicians from other issues. Seventy-five subjects were enrolled from 19 November 2012 to 10 December 2012. After a sufficient explanation of the research purposes and methods, participants gave written informed consent. The study received approval from the Institutional Review Board of Myongji Hospital (IRB No. 12-083). Of the 75 subjects enrolled in the study, 24 participants were determined to be unsuitable and were excluded from the data analysis. Two subjects (one in the control group and one in the PLAG group) did not participate after 4 weeks of intervention and were excluded from the data analysis. One subject refused blood sampling, and the other travelled abroad during the study period. Baseline and post-intervention characteristics are presented for 49 subjects (22 in the control group and 27 in the PLAG group at the completion of study) (Fig. 2).

The study was designed as a 4-week double-blind, randomized, controlled trial to investigate the effect of PLAG supplements on immune function in healthy people. Subjects were divided into two groups, and doses were administered before a meal twice a day. The supplement capsule contained 500 mg PLAG and the placebo capsule contained 500 mg soybean oil (all from ENZYCHEM Lifesciences, Daejeon, Korea). The soft capsules for the PLAG supplement and the placebo were the same in color, size, and shape. Blood sampling was performed during patient fasting to measure subject's immune function prior to administering PLAG or placebo. According to the clinical protocol, subjects returned to the clinic one month after the start of administration. Doctors confirmed that the subjects had taken their soft capsules regularly and asked whether there were any side effects. On the second visit, after 4 weeks from the first visit, subjects were instructed to fast during the visit and peripheral blood samples were taken to evaluate immune function after the administration of PLAG or placebo (Fig. 2).

Peripheral blood samples were collected on two occasions: before administration and 4 weeks after administration of PLAG. All blood samples were taken after subjects had fasted for 8 hours or more. The whole blood counts including white blood cell (WBC) and red blood cell counts; leukocyte differential counts including percent neutrophils, percent lymphocytes, and percent monocytes; and hemoglobin, hematocrit, and platelet counts were surveyed with a reference automated hematology analyzer from each manufacturer. Serum biochemical values including organ function profiles such as blood urea nitrogen, creatinine, and total bilirubin; and blood metabolic profile such as blood glucose and serum enzyme profile including aspartate amino-transferase, alanine amino-transferase, and r-glutamyl transpeptidase were obtained using the span diagnostic test kit with semiautoanalyzer (CECIL Instruments Limited, Cambridge, UK) with standard techniques. Commercial kits of radial immunodiffusion plate (Biomagherb, Tunisia) were used to determine the concentrations of IgA, IgG, and IgM, and C3 and C4 complement components for the subjects. Antibodies for immunostaining flow cytometry analysis were used to measure the lymphocyte subsets, including antihuman anti-CD3-FITC (BD Biosciences, San Jose, CA, USA), anti-CD4-PE (BD Biosciences), anti-CD8-PE (BD Biosciences), and CD56-PE (BD Biosciences). In addition, 15 ml of blood samples were taken into a vacutainer tube containing heparin for ex vivo study. As such, total 15 ml of blood were diluted with the same volume of phosphate buffered saline (PBS), and then placed carefully on Ficoll-Hypaque to form layers. After centrifugation at 400 g for 30 minutes at room temperature, the buffy coat was separated and harvested and then washed twice in complete medium.

Cultured peripheral monocytes were stimulated with Concanavalin A (Con A) to secrete cytokines, followed by the centrifugal separation of supernatants. Extracted supernatants were stored at -80℃ until cytokine measurements were taken. The isolated lymphocytes were incubated with the Con A to measure IL-2 over 24 hours and to measure IFN-γ, IL-4, and IL-6 over 48 hours. The Cytokines IL-2 (Human IL-2 Quantikine ELISA kit, R&D Systems, Minneapolis, MN, USA), IL-4 (Human IL-4 Quantikine ELISA kit, R&D Systems), IL-6 (Human IL-6 Quantikine ELISA kit, R&D Systems), and IFN-γ (Human IFN-γ Quantikine ELISA kit, R&D Systems) were examined at a wavelength of 450~570 nm using an automatic microplate reader (Molecular Devices, CA, USA).

The isolated lymphocytes were tested for a proliferative response to a 48-hour challenge of the mitogens Con A and LPS. The isolated lymphocytes were plated cells in a 96-well flat-bottomed microplate after adjusting the concentration of cells to 5×10⁴/well and incubated with the 10 µg/ml Con A or 20 µg/ml LPS to be tested at +37℃ in a humidified 5% CO₂ for 48 hours. Cultured peripheral monocytes' (T cell and B cell) proliferative response to mitogens was measured with Bromodeoxyuridine (BrdU, Cell Proliferation ELISA, colorimetric). The 96-well microplate wells were labeled cells by adding BrdU and incubating the cells for 2~4 hours at 37℃ in a humidified 5% CO₂. Genomic DNAs were denatured by adding Fixing Solution to each well and then added anti-BrdU antibody with peroxidase. Absorbance was measured at a wave-length of 450 nm using TMB substrate and an automatic microplate reader (Molecular Devices).

NK cell activity was measured with NKTEST® (Orpegen Pharma, Heidelberg, Germany) using fluorescence-labeled K562 target cells. Effector cells were resuspended in 1 ml of complete medium and adjusted cell concentration to 5×10⁶ cells/ml. Fluorescently labeled K562 cells were rapidly thawed, suspended in 50 ml of prewarmed complete medium and centrifuged. Harvested cells were resuspended in 1 ml of complete medium and adjusted cell concentration to 1×10⁵ cells/ml. Effector cells (E) were mixed with K562 target cells (T) at the desired E:T ratios ranging from 25:1 and incubated tubes for 2 to 4 hours in a humidified CO₂. At the end of the incubation, tubes were placed on ice until flow cytometric analysis. Cells were analyzed with flow cytometry using the blue-green excitation light (FACSCalibur system, Becton Dickinson Biosciences, CA, USA). After the incubation period in the cytotoxicity assay, dead target cells were identified by a DNA-stain, which penetrates the dead cells and specifically stains their nuclei. This method allows the percentage of target cells killed by effector NK cells to be determined.

The phagocytic activity of leukocytes was measured using the PHAGOTEST™ (Glycotope Biotechnology GmbH, Heidelberg, Germany), which facilitates the quantitative determination of fluorescein-labelled opsonized Escherichia coli bacteria. Heparinized whole blood was incubated with the fluorescein-labelled E. coli bacteria at 37℃ and a negative control sample remains on ice. After incubation, the phagocytosis was stopped by placing the samples on ice and adding cold quenching solution. This solution enabled the discrimination between attachment and internalization of bacteria by quenching the fluorescence of surface-bound bacteria, leaving the fluorescence of internalized particles unaltered. After two washing steps with washing solution, erythrocytes are removed by the addition of Lysing solution. Cells were spun down and the supernatant discarded. After washing the samples, the DNA-staining solution was added, mixed, and put on ice prior to flow cytometric analysis, excluding aggregation artifacts of bacteria or platelets. Cells were analyzed by flow cytometry using the FACSCalibur system (Becton Dickinson Biosciences, CA, USA).

Data are expressed as the mean±SD or percentage. Sociological characteristics of the two groups were compared using an independent t-test and the Chi-square test. The paired t-test was used to compare the changes in immune function on ex vivo and peripheral blood before and after the administration of PLAG or placebo, respectively. The Mann-Whitney U test was used to calculate and compare percent change of the immune function after 4 weeks of intervention between the two groups. SPSS for Windows (Version 15.0; SPSS Inc.; Chicago, IL, USA) was used to analyze these statistics; statistical significance was defined as a p-value of less than 0.05.

Pure 1-palmitoyl-3-acetyl-rac-glycerol is a key intermediate for the preparation of pure PLAG and was synthesized using 1-palmitoyl-rac-glycerol and acetylchloride as starting materials. And resulting crude 1-palmitoyl-3-acetyl-rac-glycerol was purified in hexane to give pure 1-palmitoyl-3-acetyl-rac-glycerol. To synthesize PLAG, carboxylic acid moiety of linoleic acid was activated by pivaloylchloride in the presence of triethylamine in hexane to give corresponding mixed anhydride, and then the resulting mixed anhydride was reacted with 1-palmitoyl-3-acetyl-rac-glycerol and catalytic amount of 4-dimethylaminopyridine (DMAP) as a catalyst to give PLAG.

The two groups did not differ statistically in body mass and sex ratio of men and women (Table I). The average age of subjects in the PLAG group was 39.6 years while the average age in the placebo group was 48.8 years; incidentally, there were more older subjects in the PLAG group than in the control group (Table I). The basal status such as smoking status, alcohol intake, regular exercise, hypertension, and dyslipidemia were checked for each subject in the two groups; there was no statistical difference between the control group and the PLAG group (Table I). We checked out that there is no adverse effects on kidney or liver function in the healthy adults taking EC-18 for 4 weeks (IRB No. 12-083). Normal levels of creatinine in the blood are approximately 0.6 to 1.2 mg per deciliter (dl) in adult males and 0.5 to 1.1 mg per dl in adult females (Data not shown).

There were no statistically significant differences (p>0.05) of WBC counts before and after supplementation in both groups (Table II). Among total T cells, there was no change of CD8⁺ (%) T cells before and after the 4-week intervention in both the PLAG group and the control group. In the PLAG group, CD3⁺ (%) T cells increased from 69.8±8.4 to 72.2±7.4 and CD4⁺ (%) cells increased from 39.2±9.3 to 41.8±8.4. Proportion of CD56⁺ (NK cell, %) cells decreased from 18.5±7.9 to 16.2±7.9. In the control group, there were no differences in CD3⁺ (%), CD4⁺ (%) and CD56⁺ (NK cells, %) cells after the 4-week intervention. Among immunoglobulins, there was no change of IgA before or after the 4-week intervention in both the PLAG group and the control group (Table III). In the PLAG group, IgG (mg/dl) decreased from 1,327.0±170.6 to 1,240.3±184.5, and IgM (mg/dl) decreased from 114.6± 56.3 to 106.5±51.9. In the control group, IgG (mg/dl) decreased from 1,312.0±203.6 to 1,238.5±166.0, and IgM (mg/dl) decreased from 118.0±50.8 to 109.9±44.9. Among complements, C3 (mg/dl) decreased from 109.5±13.0 to 99.7±12.4 in the PLAG group and it was unchanged in the compared group. C4 (mg/dl) remained unchanged in both PLAG and control group.

NK cell activity increased from 17.1±6.5 to 22.0±4.4 in the PLAG group whereas it also increased from 15.2±6.5 to 22.2±5.2 in the control group (Table IV). B cell activity decreased from 0.27±0.48 to 0.07±0.06 in the PLAG group only (Table IV). Con A-stimulated IL-4 production (pg/ml) was well regulated from 262.1±203.1 to 98.0±56.4 in the PLAG group and from 204.8±137.8 to 116.5±65.1 in the control group (Table V). IL-6 production (pg/ml) increased from 10,427.6±7,455.3 to 5,017.1±3,692.7 in the PLAG group and from 13,862.4±15,241.8 to 7,154.8±5,847.6 in the control group (Table V). Con A-stimulated IFN-γ production was not statistically changed in both PLAG and control groups (p>0.05) (Table V).

There was no difference in percent change of phagocyte, NK cell activity, and T cell proliferation before and after 4 week intervention between PLAG and control groups (Table VI). But B cell proliferation pattern was different with statistical significance between two groups; decreased by 53.1% in PLAG group vs. by 0.2% in control group (Table VI). Among cytokine production, there was no difference in percent change of IL-2, IL-6, IFN-γ, IFN-γ/IL-4 before and after 4 week intervention between PLAG and control groups (Table VI). But IL-4 production pattern was different with statistical significance between two groups; decreased by 36.0% in control group vs. decreased by 56.1% in PLAG group (Table VI). IL-2/IL-4 production pattern was also different with statistical significance between two groups; increased by 55.1% in control group vs. increased by 173.3% in PLAG group (Table VI). C3 downregulation pattern was intend to significantly different between two groups (p=0.064, Table VI). Therefore, when we statistically re-analyzed C3 pattern after the outlier-data exclusion using the 2 Standard Deviation (SD), C3 decrease in PLAG group was statistically significant compared to the control group (p<0.05, Table VII). There was no difference in percent change of WBC, T cell, NK cell, immunoglobulins, and complements before and after 4 week intervention between PLAG and control groups (Table VI). From these data, it could be suggested that PLAG has a modulating effect on Th2 cytokine overexpression and a possible role for improvement of hypersensitive diseases (e.g., allergies, asthma, and autoimmune diseases) by controlling Th1/Th2 balance.