Research On The Extraction,Purification And In Vitro Anticoagulant Activity Of Polysaccharides From Gentiana Scabra Bunge Roots

The genus Gentiana belonging to the family Gentianaceae is a perennial herb. It is endowed with a variety of pharmacological activities for hundreds of years. Polysaccharides are active constituents of Gentiana scabra Bunge roots. Gentiana scabra Bunge roots were as raw material to study preliminary structural characterization, physicochemical properties and in vitro anticoagulant activities of polysaccharides from Gentiana scabra Bunge roots (GSP) in the study. The main contents and results obtained in the research were as follows:1. Crude GSP were obtained using hot water extraction. On the basis of the single factor test, factors of the response surface design were determined. The results showed that optimization of process were as follows:extraction temperature 99.7℃, extraction time 160 min, 25 mL/g liquid material ratio, polysaccharides yield of 18.48%.2. The effects of NaCl, CaCl2, TCA and polyamide adsorption on the removing protein were evaluated by the deproteinization efficiency and the recovery of polysaccharide. The results showed that the deproteinization efficiency of four deproteinization methods was 63.4%, 92.4%,80.6%,73.6%, respectively. The recovery of polysaccharide was 65.0%,62.9%,64.3%,90.4%, separately. Polyamide adsorption was the optimal method for removing protein. The results indicated that the static polyamide method determined by UV spectra had the better effect on the removal of protein.3. The effects of H2O2, AB-8 macroporous absorption resin and active carbon on the removing the pigments were evaluated by the decolorization efficiency and the recovery of polysaccharide. The results showed that the decolorization efficiency of three decolorization methods was 59.5%,63.9%, and 61.1%, respectively; the recovery of polysaccharide was 59.3%,68.2%, and 62.9%, respectively. AB-8 macroporous absorption resin showed a beneficial decolorization effect. It was worth noting that the protein content could not be detected by the Bradford’s method when we employed the best method for decoloration.4. GSP was obtained by removing proteins and pigments. GSP was isolated by using an anion exchange chromatography column, resulting in three independent elution peaks of GSP-1, GSP-2 and GSP-3 with yields of 5%,35% and 23%, respectively. Among them, GSP-1 was the neutral polysaccharide, which was obtained by eluting with distilled water. GSP-2 and GSP-3 were two acidic components, which were obtained by eluting with a gradient concentration of NaCl solution (0.1,0.2 M). Taking yield and purity determination into consideration, the main fraction GSP-2 was further purified by a Sepharose CL-6B chromatograph column and the main peak fraction was collected.5. Preliminary structure of GSP and its fractions were determined by UV spectra, FT-IR spectra, high performance gel permeation chromatography (HPGPC) and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The results of UV spectra showed that no absorption at 280 or 260 nm were observed for GSP-1, GSP-2 and GSP-3, revealing the absence of protein and nucleic acid. The FT-IR spectra for GSP and purified polysaccharide fractions were very similar and typical for carbohydrate polysaccharides. The weight-average molecular weights of GSP-2 and GSP-3, as determined by HPGPC, were 28849 Da and 58203 Da, respectively. HPAEC-PAD analysis revealed that rhamnose, arabinose, galactose, glucose and galacturonic acid were main monosaccharides of GSP, GSP-1 and GSP-3. However, the component monosaccharides of GSP-3 were rhamnose, arabinose, galactose and galacturonic acid, of which the contents were 9.0%,40.6%,39.2% and 11.1%, correspondingly.6. We subsequently evaluated the anticoagulant activities of GSP and its fractions in vitro, using three indexes to evaluate the properties, including activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT). The results demonstrated that compared with the negative control group (saline), GSP and its fractions (GSP-1, GSP-2 and GSP-3) could prolong APTT and TT, but not PT. And GSP, GSP-1 and GSP-3 at 1.5 mg/mL showed a significant difference compared with that of saline both in APTT and in TT assays (P< 0.01 or P< 0.05). Furthermore, the blood clotting time of GSP-3 at 1.5 mg/mL in APTT assay was approximately 1.3-fold longer than that of the saline. Compared with the negative control group, the TT increased 1.1-fold with the addition of GSP-3 at 1.5 mg/mL. In addition, GSP-3 possessed the potent anticoagulant activity and might have potential for application in clinical or the food industry.The innovation points were as follows:1. The weight-average molecular weights of purified polysaccharides (named GSP-2 and GSP-3) were analyzed by HPGPC.2. The anticoagulant activity of purified fractions were reported for the first time.