Study On Preparative Isolation Of Glycoglycerolipid And Its Bioactivity

Glycoglycerolipids have various bioactivities, such as antioxidation, antiviral, antibacterial, anti-tumor, anti-inflammation and anti-atherosclerosis. Glycoglycerolipids occur in nerve tissue of animals, plants and microorganisms. In this study, a method for isolation and purification of glycoglycerolipids from pumpkin and ginger, was successfully established by a combination of high-speed countercurrent chromatography and preparative high-performance liquid chromatography. Moreover, the hypoglycemic, anti-inflammatory, anti-tumor and antioxidant activity of the glycoglycerolipid monomers related to structure were investigated. The results could provide scientnfic evidence for the development and utilization of pumpkin and ginger.The results are as follows:1. The n-butanol extract of the fruits of pumpkin was eluted through D4020 macroporous adsorption resin with water,20,40,60,80 and 95% ethanolic solutions to fraction Fr60 riched on glycoglycerolipids. Fr60 was separated by HSCCC using a solvent system composed of dichloromethane-light petroleum-ethanol-water (6:2:4:4, v/v/v/v) resulting in three sub-fractions GCL1, GCL2 and GCL3 which were subjected to separation with preparative HPLC. GCL1,GCL2 and GCL3 were further separated into its molecular species by preparative HPLC. Reversed-phase HPLC seoaration of GCL1, GCL2 and GCL3 with an Prep-ODS-BP-A column (250 mm×30 mm,15μm) eluuted with acetonitrile/water (55:45, v/v) at a flow rate of 10 mL/min to yield four gly cogly cerolipid monomers,1-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[β-D-gal-actopyranosyl-(1→6)-O-β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl] glycerol, 1-O-(9Z,12Z-octadecadienoyl)-3-O-[β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl] glycerol,1-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl] glycerol and 1-O-(9Z,12Z-octadecadienoyl)-3-O-[β-D-galactopyranosyl-(1→6)-O-β-D-galactop-yranosyl-(16)-O-β-D-galactopyranosy l-(1→6)-O-β-D-galactopyranosy 1] glycerol. The structures of the four compounds were elucidated on the basis of spectroscopic analysis including 2D-NMR (TOCSY, HMQC, HMBC, and NOESY), and ESI-MS and chemical methods. The four glycoglycerolipids belong to new compounds.2. The n-butanol extract of the fruits of ginger was eluted through reversed-phase MCI GEL CHP 20P with water,30,60 and 90% methanolic solutions to give Fr30, Fr60 and Fr90. Fr90 was separated by HSCCC using a solvent system composed of light petroleum-ethyl acetate-methanol-water (2:1:1:1, v/v/v/v) to yield Soya-cerebroside I and FrA which was enriched-glycoglycerolipids. FrA were further separated into its molecular species by preparative HPLC. Reversed-phase HPLC of FrA with an Prep-ODS-BP-A column (250 mm×30 mm,15μm) eluted with acetonitrile/water (55:45, v/v) to yield two glycoglycerolipid monomers, 1-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[α-D-galactopyranosyl-(1→6)-O-β-D-gal-actopyranosyl] glycerol and 1-O-(9Z, 12Z-octadecadienoyl)-3-O-[α-D-galactopy-ranosyl-(1→6)-O-β-D-galactopyranosy 1] glycerol. In addition, Fr30 was separated by HSCCC using a solvent system composed of chloroform-methanol-water (5:4:3, v/v/v) to yield 1-(4-O-β-D-Glucopyranosyl-3-methoxyphenyl)-3,5-dihydroxyde-cane and apigenin-7-O-rutinoside. Fr60 was separated by HSCCC using a solvent system composed of TBME-methanol-water (2:1:1, v/v/v) to yield 5-hydroxybornyl-2-O-β-D-glucopyranoside, (R)-3,7-dimethyloct-2E-en-1,6,7-triol, p-epi-menthane-1,2,8-triol 2-O-β-D-glucopyranoside and p-menthane-1,2,8-triol 2-O-β-D-glucopyranoside. Their structures of the eight compounds were elucidated on the basis of spectroscopic analysis including 2D-NMR (TOCSY, HMQC, HMBC, and NOESY) and ESI-MS and chemical methods. p-epi-menthane-1,2,8-triol 2-O-β-D-glucopyranoside and p-menthane-1,2,8-triol 2-O-β-D-glucopyranoside belong to new compounds.3. The hypoglycemic activity (type II) of glycoglycerolipidswas tested by diabetic mouse model induced by long-term high-glucose and high-fat diet and streptozocin (STZ). The results showed that glycoglycerolipids, especially for QGMG containing linolenic-acid (18:3) group had hypoglycemic effect. Compared with the control group, hypoglycemic effect of QGMG reached a significant level after one week, and a highly significant level after two weeks. The effect of QGMG was better than that of the reference drug metformin (P<0.05).Structure-activity relationship study suggested that the hypoglycemic activity of glycoglycerolipids depended on glycerol-sugar backbone and the saturated degree of the fatty acid groups. The more the number of sugar groups and the double bonds, the stronger hypoglycemic effect The results indicated that sugar moiety and unsaturated acyl groups could significantly synergistic action to hypoglycemic activity.4. The anti-inflammatory potencies of the glycoglycerolipids were determined by croton-oil-induced ear oedema model. The results showed that glycoglycerolipids had anti-inflammatory effect. Structure-activity relationship suggested that the anti-inflammatory activity of glycoglycerolipids decreased as increase of galactosyl group number and double bond number.5. The cytotoxicity of glycoglycerolipids on tumor cell COLO205, B16-F10 and A549 was evaluated by MTT method. Glycoglycerolipids possess inhibitory effects on the growth of tumor cell lines COLO205, B16-F10 and A549 with dose-response relationships. The inhibition of glycoglycerolipids against tumor cells COLO205 and B16-F1 was obviously greater than that of tumor cell A549.The preliminary relationship of structure-activity revealed that the anti-tumor activity of glycoglycerolipids decreased as the increase of galactosyl group number. Furthermore, the type of glycosidic bond influenced the activity to some extent. DGMG (18:3) exhibited the same effect on lung adenocarcinoma as DGMG (18:2), andwhile DGMG (18:3) had stronger effect on human colon cancer cell COLO205 than DGMG (18:2), and DGMG (18:2) had stronger effect on mouse melanoma cell than DGMG (18:3). For the glycoglycerolipids with singleβ-galactose, linoleic acyl chain showed better effect than inolenic acyl chains.6. The antioxidant activity of the glycoglycerolipids was investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. The results showed that glycoglycerolipids have higher scavenging ability on DPPH at a concentration of 50-500μg/mL. The relationship of structure-activity indicated that the radical-scavenging activity of glycoglycerolipids increased as increase of galactosyl group number, and the type of glycosidic bond and saturated degree of the fatty acid groups influenced antioxidant activity. For the glycoglycerolipids with singleβ-galactose, linoleic acyl chain showed better effect than inolenic acyl chains.