Study on polysaccharide (1,3)-B-glucan from Ganoderma lucidum

Journal of Chemistry, Vol. 43 (2), P. 258 - 262, 2005<br /> <br /> <br /> study on polysaccharide (1,3)- -glucan<br /> from Ganoderma lucidum<br /> Received 17th-Sept.-2004<br /> TRAN DINH TOAI1,VU NGOC BAN2, LE CAM TU2, NGUYEN THANH HANH1<br /> 1<br /> Institute of Chemistry, Vietnamese Academy of Science and Technology<br /> 2<br /> Faculty of Chemistry, Hanoi University of Science<br /> <br /> SUMMARY<br /> In this paper, a biologically active substance of Lingzhi mushroom was studied. The optimal<br /> process seperating (1,3)- -D-glucan from Ganoderma lucidum has been formed and the<br /> molecular weight of the preparation is estimated. The structure of (1,3)- -D-glucan unit and its<br /> properties are characterized by using HyperChem 7.02 software.<br /> <br /> I - INTRODUCTION<br /> <br /> Vietnam is rich in highly valuable gene<br /> sources in plants and animals which are specific<br /> to tropical climate. One of them is Lingzhi<br /> mushroom Ganoderma lucidum. Ganoderma<br /> lucidum is a precious drug of the Orient<br /> medicine, which has been used for thousands of<br /> year. Lingzhi mushroom is believed to have<br /> extraordinary effects on tumors and cancers. Figure 1: The structure of (1,3)- -D-glucan<br /> However, the nature of marvellous drug-lingzhi Modern research has defined that the<br /> has never been explained before. Nowadays, the biologically active substance of Lingzhi<br /> characterization of the structure of biologically mushroom which can restrain the development<br /> active substances has been developed strongly. of cancer cells is (1,3)- -D-glucan. Thus, a<br /> The modern methods such as NMR, mass<br /> chemotheurapy based on (1,3)- -D-glucan to<br /> spectroscopy, HPLC, ..., help us determine<br /> treat cancers is a scientific challange now.<br /> correctly the structure of biologically active<br /> substances of lingzhi mushroom’s constitutions<br /> II - MATERIALS AND PRINCIPLES<br /> such as alcoloid, terpenoid, steroid, ergosterol,<br /> glycol, lectin, several types of polysaccharides<br /> 1. Materials<br /> (glucose, mantose, galactose) and some metals<br /> (Germanium). One of the most important Lingzhi mushrooms were planted on<br /> organic compounds of lingzhi mushroom is mushroom farm of Enzyme Department in<br /> polysaccharide. Besides, modern medical Institute of Chemistry, Vietnamese Academy of<br /> research has defined that the biologically active Science and Technology.<br /> substance in Linzghi mushroom which can 2. Principles<br /> restrain the development of cancer cells is (1,3)-<br /> -D-glucan. (1,3)- -D-glucan is separated with many<br /> 258<br /> different solvents: C2H5OH 80%, H2O, collected and dissolved by 15% NaOH solution.<br /> (NH4)2C2O4 2%, NaOH 15%, CH3COOH. The solution (IV) was incubated at 20h, 30oC<br /> The viscosity method was used in order to and adjusted to pH 9 by using acetic acid 10%.<br /> estimate the molecular weight of the obtained Then it was centrifuged to retain the residue and<br /> polysaccharide. This is a simply experimental dried. This product was stored and used in<br /> method which allows us to value molecular futher experiment.<br /> weight in expanse (M~104-106). Mak-Euvin Each step was repeated 3 times and mixed to<br /> expression was used to show the dependence of get homologous solution to use in the next step.<br /> viscosity on molecular weight: [ ] = KM or The extraction scheme was shown in chart 1.<br /> lg[ ] = lgK + lgM [7] where K and are<br /> constant which depend on temperature and the<br /> nature of solvent. By looking up K, and 10 g of ground fruit-body<br /> determining by experiment, the molecular<br /> Ethanol 80%, 3h, 100oC<br /> weight of the polysaccharide can be valued.<br /> 13<br /> C-NMR spectrum is used to define the Sediment I<br /> structure of (1,3)- -glucan. 20-30 mg of sample<br /> H2O, 5h, 100oC<br /> was dissolved in D2O and 1-5% DMSO to the<br /> concentration of 2-3% at 80oC. The spectrum Sediment II<br /> was defined on Bruker Avent 500 equipment in<br /> Institute of Chemistry, Vietnam Academy of (NH4)2C2O4 2%, 3h, 100oC<br /> Science and Technology.<br /> HyperChem 7.02 software was used to build Sediment III<br /> and optimize the geometric structure of (1,3)- - NaOH 15%, 20h, 30oC<br /> glucan. Concretely, we use MM+ force field,<br /> Polak Ribierre conjugate gradient technic, RMS Solution IV<br /> gradient 0.001 and maximum cycles 1000. The<br /> charges of atoms in one structural unit of (1,3)- Adjust pH = 9 by CH3COOH<br /> -glucan molecule was also estimated by 10%<br /> HyperChem. Besides we valued the chemical Solution V<br /> shifts of 13C and built 13C-NMR spectrum of<br /> Centrifuge and dry<br /> (1,3)- -glucan.<br /> Preparation<br /> III - RESULTS AND DISCUSSION<br /> <br /> 1. Separation of Polysaccharide Chart 1: The putative sheme of separating<br /> a) Separation of polysaccharide from ground polysaccharide from Lingzhi fruit-body powder<br /> Lingzhi fruit-body To optimize the process, a lot of<br /> experiments were performed. The table below is<br /> To separate polysaccharide from ground<br /> to find out the optimal volume of ethanol.<br /> Lingzhi mushroom fruit-body, first the ground<br /> fruit-body was suspended in 80% ethanol From 10 g of ground fruit-body, 3.6 g of dry<br /> solution. This suspension was left at 100oC for 3 preparation was obtained. The recovery of the<br /> hours. The sediment (I) of this stage was process is 36%. The dry preparation is soluble in<br /> collected and suspended with distilled water at diluted, warm alkali and unsoluble in water.<br /> 100oC. This suspension was left at 100oC for 5 b) Separation of polysaccharide from Lingzhi<br /> hours. Then the sediment (II) was retained and mass powder<br /> suspended in 2% (NH4)2C2O4 at 100oC for 3<br /> hours. After that the sediment (III) was Polysaccharide was separated from Lingzhi<br /> 259<br /> mass by the same way as from ground fruit- 2). Due to the chart, the molecular weight of<br /> body above (chart 2). Instead of 10g of ground polysaccharide of Ganoderma lucidum was<br /> fruit body, 20g of mass powder was used. estimated about 250,000 Da.<br /> From 20 g of Lingzhi mass powder, 3.0 g of 4,5<br /> dry preparation was obtained. The recovery of 4,0<br /> the process is 15%. The dry preparation has the 3,5<br /> same properties with that from ground fruit-<br /> <br /> <br /> <br /> <br /> Reduced viscosity<br /> 3,0<br /> body.<br /> 2,5<br /> 2. Properties of obtained polysaccharide 2,0<br /> a) Estimation of polysaccharide’s molecular 1,5<br /> weight 1,0<br /> The viscosity of polysaccharide aqueous 0,5<br /> solution depends on the its concentration. The 0<br /> results are shown in table 2. 0 0,2 0,4 0,6 0,8 1<br /> Basing on the data in table 2, the curve Concentration, g/ml<br /> showing the relationship between the viscosity Chart 2: The dependence of viscosity on<br /> and the concentration was built (shown in chart concentration of polysaccharide solution<br /> <br /> Table 1: The dependence of preparation’s weight on ethanol volume<br /> VC2H5OH, ml 100 120 140 160 180 200 220 240<br /> mpreparation, g 2.0 2.6 2.8 3.0 3.2 2.9 2.6 2.2<br /> <br /> Table 2: The viscosity of polysaccharide solution<br /> Concentration, g/100 ml 0% 0.2% 0.4% 0.6% 0.8%<br /> Time flowing through viscometer, s 28.54 44.24 64.22 87.33 117.01<br /> relative = t/to 1.55 2.25 3.06 4.10<br /> specific = relative -1 0.55 1.25 2.06 3.10<br /> educed = specific C 2.75 3.13 3.43 3.88<br /> <br /> b) Characterization of the structure and<br /> properties of (1,3)- -glucan<br /> Molecular structure of -glucan can be built<br /> by HyperChem. But this structure is still crude.<br /> In order to have minimum energy, similar to<br /> reality, it must be optimized. The optimal model<br /> of one (1,3)- -D-glucan structural unit is shown<br /> in figure 2.<br /> Besides, the charge of atoms in a (1,3)- -D-<br /> glucan structural unit were valuated by<br /> HyperChem 7.02. The results are shown in<br /> figure 3. The result helps us to find out which Figure 2: The optimal model of (1,3)- -D-<br /> atoms are the most active in reactions in bodies. glucan structural unit<br /> 260<br />  Figure 3: The charge of atoms in one (1,3)- -D-glucan structural unit<br /> <br /> The chemical shifts of 13C determined by HyperChem are shown in table 3. This result is<br /> useful to determine whether the obtained polysaccharide was pure.<br /> <br /> Table 3: The chemical shifts of 13C in (1,3)- -D-glucan structural unit<br /> Glucose Chemical shifts of 13C, ppm<br /> round C1 C2 C3 C4 C5 C6<br /> 1 101.85 73.8 75.60 71.25 74.95 62.1<br /> 2 95.18 73.1 83.72 70.10 74.57 62.1<br /> <br /> <br /> <br /> <br /> Figure 4: The ACD 13C-NMR spectrum of (1,3)- -D-glucan<br /> 261<br />  Comparing the ACD 13C-NMR spectrum of polysaccharide from Lingzhi mushroom was<br /> (1,3)- -D-glucan (figure 4) with the experimental defined by the viscosity determining method.<br /> 13<br /> C-NMR spectrum of (1,3)- -D-glucan (figure 5), The geometric structure of one (1,3)- -D-<br /> we can find out the similarity between those. That glucan unit was optimized by using HyperChem<br /> means the polysaccharide separated from Lingzhi 7.02 software.<br /> mushroom is (1,3)- -D-glucan. The charges of atoms were estimated. The<br /> chemical shifts of 13C were valuated to compare<br /> with the experimental 13C-NMR spectrum.<br /> <br /> References<br /> <br /> 1. Dong Thi Anh Dao. Separ. dry subs. and<br /> Saponin from Lingzhi mushroom<br /> (Ganoderma lucidum) to process drinking<br /> water. National Chem. Confer. (2003).<br /> 2. F. Yang, Y. Ke, S. Kuo. Enzyme Microb<br /> Technol 200 Aug. 1, 27 (3-5), P. 295 - 301.<br /> 3. S. B. Lin, C. H. Li, S. S. Lee, L. S. Kan. Life<br /> Sci. Apr. 11; 72, No. 21, P. 2381 - 90 (2003)<br /> Figure 5: The experimental 13C-NMR spectrum 4. B. Lakshmi, T. A. Ajith, N. Sheena, N.<br /> of (1,3)- -D-glucan from Ganoderma lucidum Gunapalan, K. K. Janardhanan. Mutagen, 23<br /> S.1, P. 85 - 97 (2003).<br /> IV - CONCLUSIONS 5. H. S. Kim, S. Kacew, B. M. Lee. Carcino-<br /> genesis, Aug., 20, No. 8, P. 1637 - 1640<br /> (1,3)- -D-glucan separating process from (1999).<br /> Lingzhi mushroom Ganoderma lucidum fruit 6. S. K. Eo, Y. S. Kim, C. K. Lee, S. S. Han. J.<br /> body and mass was obtained and optimized. Ethnopharmacol, Dec. 15, 68 (1 - 3), P. 175<br /> Besides the structure and properties of this - 81 (1999).<br /> polysaccharide was characterized: 7. Vu Ngoc Ban. Physical chemistry practices.<br /> The 250,000 Da molecular weight of Hanoi National University (1989).<br /> <br /> <br /> <br /> <br /> 262<br />