Isolation And Structural Characterization Of A Polysaccharide DDP-1-D From The Stems Of Dendrobium Densiflorum

Dendrobium is a commonly used precious traditional Chinese medicine,"Shen Nong’s Herbal Classic" records:it is one of the best Chinese medicines. There are more than76species of Dendrobium plants found in China, in which more than40species are used as Herba Dendrobii in China. Herba Dendrobii has been used in traditional Chinese medicine as a therapeutic agent for curing cataract, throat inflammation, fever and chronic superficial gastritis or as a tonic for promoting the production of body fluid. Modern pharmacological researches indicate that Herba Dendrobii plays an important role in the treatment of cataracts, gastrointestinal disorders, and hypoglycemic. The main active ingredients of Herba Dendrobii are polysaccharides and alkaloids, especially the polysaccharides which have antioxidant, immune enhancement and anti-tumor activities and become research focus. At present, Herba Dendrobii is mainly from Dendrobium nobile, Dendrobium officinale, Dendrobium moniliforme and Dendrobium chryseum. Medicinal plants of Dendrobium resources are in a short supply situation, which is adverse to the research and development of Dendrobium polysaccharides. Dendrobium densiflorum widely distributes in Guangdong province, Hainan province, Guangxi province and other places, and the yield is relatively high. And therefore it has become one of the main substitutes for Herba Dendrobii. Guizhou Province Chinese Materia Medica Standards (1988version) and Jiangsu Province Chinese Materia Medica Standards (1998version) recorded Dendrobium densiflorum as Herba Dendrobii. In this paper, Dendrobium densiflorum was selected as raw materials, and the extraction technology of polysaccharides from Dendrobium densiflorum was optimaized by the orthogonal design. Then the total polysaccharides were purified by DEAE-52cellulose column and gel column chromatography, to obtain Dendrobium densiflorum homogeneous polysaccharides. Physical and chemical properties, relative molecular mass, monosaccharide composition and other structural characterization of the Dendrobium densiflorum homogeneous polysaccharides, were investigated. The aim of this paper is to lay the material foundation to further demonstrate the mechanism of the pharmacological effects of Dendrobium densiflorum polysaccharide, and also provide a scientific basis for the development and utilization of Dendrobium densiflorum polysaccharide in the field of drugs.1. Optimization of Extraction technology of polysaccharides from Dendrobium densiflorum by Orthogonal Design.Using the content of polysaccharide from Dendrobium densiflorum as an evaluation index, choosing extraction temperature, dosage of water, extraction time and extraction times as the observation factors, the influencing factors of the extraction technology of polysaccharide from Dendrobium densiflorum were studied by L9(34) orthogonal test. The dried stem of D. densiflorum was defatted with acetone and subsequently with methanol. The extracts were discarded and the residue was dried. Subsequently, the residue was extracted according to the orthogonal design. The combined extracts were concentrated and centrifuged and the supernatant was concentrated and precipitated by95%EtOH. The precipitate was dissolved in deionized water and deproteinized4times using the Sevag reagent. The resulting aqueous fraction was extensively dialyzed against deionized water. After centrifugation, the supernatant was lyophilized to yield the crude polysaccharide (DDP), which was accurately weighed. The content of polysaccharide from Dedrobium densiflorum was analyzed with phenol-H2SO4method. The degree of influence of various factors in extraction process of the Dendrobium densiflorum polysaccharide was as follows:extraction temperature> extraction times> extraction time> add water multiples. The optimum extraction technology of polysaccharide from Dendrobium densiflorum was as follows:extracting for three times-using ten times amount of water, with every extraction time of2h at90℃. The result of verification test showed that the method has good reproducibility (RSD=0.027). The extraction rate of polysaccharides was3.15%in the optimum extraction process.2. The extraction and purification of Dendrobium densiflorum polysaccharide and UV, FTIR analysis.The dried stem of D. densiflorum was defatted. The extracts were discarded and the residue was dried. Subsequently, the residue was extracted three times with10volumes of hot deionized water (90℃). The combined extracts were concentrated and centrifuged at a speed of10000rpm for lOmin and the supernatant was concentrated and precipitated by adding4times of volumes of95%EtOH. After centrifugation, the precipitate was dissolved in H2O. This process was repeated two times. The precipitate was dissolved in deionized water and deproteinized4times using the Sevag reagent. The resulting aqueous fraction was extensively dialyzed against deionized water for48h. After centrifugation, the supernatant was lyophilized to yield the crude polysaccharide (DDP). The crude polysaccharides portion was further fractionated on a DEAE-cellulose-52column with water and different concentrations of stepwise NaCl solution elution (0.05,0.1,0.2,0.3,0.5and1.0M NaCl). DDP-1was obtained by water elution, leading to a neutral polysaccharide DDP-1and five acidic polysaccharides DDP-2, DDP-3, DDP-4, DDP-5and DDP-6. DDP-1was separated on a Sephacryl S-200column, giving five fractions:DDP-1-A, DDP-1-B, DDP-1-C, DDP-1-D and DDP-1-E. The ultraviolet-visible spectra of DDP-1-C, DDP-1-D and DDP-1-E revealed no peak at260and280nm, respectively, indicating the absence of nucleic acid and protein. The infrared spectra of the DDP-1-C, the DDP-1-D and DDP-1-E were recorded on a FT-IR spectrometer, respectively. All samples exhibited a broad stretching intense characteristic peak at around3500-3300cm-1for the hydroxyl group. The band at1653-1639cm-1was due to the bound water. Each particular polysaccharide has a specific band in the1200-1000cm-1region, this region was dominated by ring vibrations overlapped with stretching vibrations of (C-OH) side groups and the (C-O-C) glycosidic band vibration. The938cm-1absorption was the characteristic peak of a-D-Glc;897cm-1was the absorption peak for the (3-side basis to heterogeneous C-H angle vibration.869and816cm-1were the characteristic peaks of D-Man. The DDP-1-C and DDP-1-D and DDP-1-E were presumed to be mannoglucan with pyranose.3. Structural characterization of a neutral polysaccharide DDP-1-D from the stems of Dendrobium Densiflorum.DDP-1-D was isolated from Dendrobium densiflorum for the first time. Its average molar mass was estimated to be9440Da by gel-permeation chromatography, using dextrans of known molecular weight as standards. The optical rotation of DDP-1-D was[α]D20+119.19°. DDP-1-D had been investigated by chromatographies (TLC and GC-MS), spectroscopes (UV, IR,1H-NMR,13C-NMR, COSY, TOCSY, HSQC and HMBC) and periodate oxidation-Smith degradation. The results indicate that DDP-1-D was composed of glucose and mannose in the molar ratios of3.01:1. A total of0.860mol NaIO4was consumed per mole of sugar residues. The production of formic acid was0.324moles per mole of sugar residues. It was thus deduced that the nonreducing terminal residues or (1→6)-linked glycosyl bonds amounted to32.4%;(1→2)-/(l→4)-linked and (1→3)-linked glycosyl bonds amounted to21.2%and less than46.4%, respectively. The oxidized product was reduced and hydrolyzed. GC-MS analysis identified the presence of monosaccharide (mannose and glucose), erythritol, and glycerol in the ratio of1:17.4:3.8, indicating that (1→4)-linked glycosyl residues, the nonreducing terminal, and (1→3)-linked mannose and glucose exist in DDP-l-D. The results also indicate that the (1→4)-linked glycosyl residues and (→3)-linked glycosyl were in the molar ratio of17.4:1. It consists of→4)-α-D-Glcp-(l→,→6)-α-D-Glcp-(→,→2)-a-D-Manp-(l→and→4)-β-D-Manp-(l→. The structure of the repeating unit of DDP-l-D is:→6)-α-D-Glcp-(1→2)-α-D-Manp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→and [→4-α-D-Glcp-(1→]nIn summary, we optimized extraction technology of polysaccharides from Dendrobium densiflorum by orthogonal design. Also, DDP-l-D was isolated from Dendrobium densiflorum for the first time, and its structure was characterized. We lay to the material foundation to further demonstrate the mechanism of the pharmacological effects of Dendrobium densiflorum polysaccharides, and also provide a scientific basis for the development and utilization of Dendrobium densiflorum polysaccharides in the field of drugs.