| Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma, which belongs to Liliaceae family, is indigenous to Hubei province, China. The tuberous root, recorded as Radix Liriopes in Pharmacopoeia of the People's Republic of China, is frequently used as "maidong" in prescriptions of traditional Chinese medicine for the treatment of diabetes mellitus (DM). Recently, some scholars have discovered that Radix Ophiopogonis (Ophiopogon japonicus (Thunb.) Ker-Gawl), which is another type of traditional Chinese medicine "maidong", showed considerable hypoglycemic effects and the water extract as well as polysaccharides were the main active components. Thus, the tuberous root of Liriope spicata var. prolifera might also have potential anti-diabetic activities. However, anti-diabetic effects of the tuberous root of Liriope spicata var. prolifera and the polysaccharides from it have not been reported. Therefore, supported by the Key Technologies R & D Program and the National Natural Science Foundation of China, the study on screening of active substance from the tuberous root of Liriope spicata var. prolifera on streptozotocin (STZ)-induced type II diabetic mice was carried out, as well as the preparation, physical-chemical properties and structural characteristics of the active substances (polysaccharides).My work was composed by four parts. The first part was about the anti-diabetic activity of Liriope spicata var. prolifera, as well as the mechanisms of action. The second part was about the preparation of a polysaccharide faction (TLSP) from Liriope spicata var. prolifera. The third part was about physical-chemical properties and structural characteristics of TLSP and two new polysaccharides (LSP1 and LSP2) separated from it. The last part was a review on natural compounds with anti-diabetic potential reported from 2005 to 2010. Part OneTo investigate whether the tuberous root of Liriope spicata var. prolifera possesses anti-diabetic (type 2 diabetic) activities and find out the active substances, this study firstly screened the activities of 95% ethanol extract (EE) and water extract (WE) form the tuberous root on a type 2 diabetic mice model induced by diets high in fat and fructose and intraperitoneal injection of STZ at a low dose (40 mg/kg). The results indicated that only the water extract (WE) showed significant anti-diabetic effects (a marked decrease of fasting blood glucose (FBG) and a significant improvement on glucose tolerance).After that, based on the separation and purification, the crude polysaccharides (CP), a polysaccharide faction (TLSP) and a non-polysaccharide faction (NP, contained no polysaccharides) were obtained from WE. Activity screening of WE, CP, TLSP and NP was also carried out on STZ-induced type 2 diabetic mice. The results proved that WE, CP and TLSP all showed significant anti-diabetic effects (a marked decrease of FBG and a significant improvement on glucose tolerance). Compared the activities, WE<CP<TLSP, but NP did not show any anti-diabetic effect. TLSP with the highest polysaccharide purity showed the highest activity, which indicated that polysaccharides were the main anti-diabetic substances of Liriope spicata var. prolifera.In order to further investigate the anti-diabetic material foundation of polysaccharides from Liriope spicata var. prolifera, separation was performed on TLSP to obtain two polysaccharides both with a single molecular weight, LSP1 and LSP2. Activity screening of TLSP, LSP1 and LSP2 was also carried out on STZ-induced type 2 diabetic mice. The results proved that they all caused a marked decrease of FBG and a significant improvement on glucose tolerance and insulin resistance (HOMA-IR), and all the above effects were the same marked as rosiglitazone. In addition, while lowering total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) cholesterol levels, TLSP, LSP1 and LSP2 elevated the relative high-density lipoprotein (HDL) cholesterol level (HDL/TC) in serum, and the activities of them were more marked than rosiglitazone. All the above results reflected polysaccharides from Liriope spicata var. prolifera could therapy type 2 diabetes and release symptoms in many ways. Compared with the synthetic activities, there was no difference among TLSP, LSP1 and LSP2 (TLSP?LSP1?LSP2,100 mg/kg?200 mg/kg). Because the preparation of TLSP was easier, shorter (time) and cost-lower than LSP1 and LSP2, our group chose TLSP as a candidate drug for further exploitation.During the pharmacodynamics research of polysaccharides from Liriope spicata var. prolifera, we found that TLSP, LSP1 and LSP2 could improve insulin resistance (HOMA-IR). So, following the idea of insulin resistance, our group studied the effects of TLSP, LSP1 and LSP2 on insulin signaling transduction and glucose metabolism to explain the possible anti-diabetic mechanisms. The results indicated that after oral administration of TLSP, LSP1 and LSP2, the protein expression levels of insulin receptor-?(InsR-?), insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3K) in renal tissues of the diabetic mice were significantly increased. In addition, the glycogen content and glucokinase (GK) activity in liver were significantly increased, yet the hepatic glucose-6-phosphatase (G6Pase) activity was decreased. So, the possible anti-diabetic mechanisms of TLSP, LSP1 and LSP2 may be due to the improvement of insulin signaling transduction and glucose metabolism.Part TwoWhen TLSP was chosen as a candidate drug for further exploitation, orthogonal experiments or/and single-factor designs were investigated to get the best preparation conditions of TLSP from the tuberous root of Liriope spicata var. prolifera, including the extraction by hot water, deproteinization by the Papain enzymolysis, dialysis by a regenerated cellulose membrane tube, repeated precipitation by ethanol, decolorization by a diethylaminoethyl cellulose 52 (DEAE-cellulose 52) column. By repeated verification, we got a stabile preparation of TLSP. The best preparation conditions were as follows:Powdered tuberous root was boiled in distilled water three times,0.5 h each time, and the ratios of liquid to solid were 4?1 (v/w),4?1,2?1, respectively. Each extract was then filtered and combined. The water extract was deproteinated by an enzymolysis method with Papain. Deproteinization conditions were pH 5.91 (phosphate buffer), ratio of Papain (12?/mg) to raw material was 0.3%, and kept in water-bath (45?) for 2 h. The deproteinated extract was then boiled for 5 min, stored overnight at 4?and fitered, the filtrate dialyzed using the regenerated cellulose membrane tube (Mw cut-off 1000) against tap water for 1 days and distilled water for another day. The retentate portion (nondialysate) was concentrated to a volume which was 1.5 times of the powdered material weight, and then precipitated by addition of ethanol to a final concentration of 80% (v/v); after 24 h, the precipitates were collected, the supernatant was concentrated to a volume which was 1.2 times of the powdered material weight, and then precipitated again by addition of ethanol to a final concentration of 80%(v/v); after 24 h, the precipitates were collected. The precipitates obtained by twice ethanol precipitation were combined, vacuum dried to obtain the crude polysaccharides. The crude polysaccharides were dissolved in water in a concentration of 0.2 g/mL, applied to a DEAE-cellulose 52 column. The sample volume was 5 mL/g column filler, the flow was 2.5 column body volumes (BV) per hour, the amount of water used for elution was 3 BV, and the collection amount of elution was 6 BV (from the 2nd BV to 7th BV of the elution). The fraction eluted with water was further vcuum concentrated, vacuum dried or lyophilized to obtain TLSP.Part ThreeIn order to make the quality of TLSP be easy controlled, this study investigated the physical-chemical properties of TLSP, LSP1 and LSP2, as well as the primary structural characteristics of LSP1 and LSP2.The main results as follows:(1) Two new polysaccharides, LSP1 and LSP2, were separated from TLSP. Their yields were 25.4% and 64.2%, respectively.(2) On the basis of several chemical and physical methods, the stable physical-chemical parameters of TLSP, LSP1 and LSP2 were finally determined.(3) The primary structural characteristics of LSP1 and LSP2 were investigated by HPGPC, FT-IR, UV, NMR spectroscopy, periodate oxidation and Smith degradation, methylation analysis and GC-MS. Based on the data obtained, LSP1 and LSP2 were two fructans with the molecular weights 3.20 and 4.29 kDa, respectively. They both have a backbone structure with six repeating units; each repeating unit in LSP1 comprises (1, 2?)-linked, (1,2,6?)-linked, and (2?)-linked fructosyl residues in the ratio of 1?1?1 and in LSP2 in the ratio of 2?1?1; a glucosyl residue and a fructosyl residue linked with the two terminals of the backbone of both LSP1 and LSP2, respectively.Part FourThe last part was a review on natural compounds with anti-diabetic potential reported in the literature from January 2005 to December 2010, in order to identify the research needs in this area. it focuses on some new and known chemical compounds isolated mainly from medicinal plants possessing anti-diabetic properties, including saponins, flavonoids, alkaloids, anthraquinones, terpenes, coumarins, phenolics, polysaccharides, and some other compounds. |
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