Method for identifying the molecular configuration of ganoderic acid A from Ganoderma lucidum

Abstract

Disclosed is a method for identifying the molecular configuration of ganoderic acid A which comprises extracting ganoderic acid A from fruit bodies of Ganoderma lucidum, producing crystals of the Ganoderic Acid, analyzing the crystals of the ganoderic acid A by X-ray structural analysis to obtain values of the three-dimensional coordinate of the crystals of the ganoderic acid A and using the values of the three-dimensional coordinate of the crystals of the ganoderic acid A obtained from the X-ray structural analysis as initial coordinates in the input to the calculation program of the B3LYP method included in the Gaussian 03 package software together with the 6-31G* basis set function of the density functional theory (DFT).

Claims

1. A method for identifying the molecular configuration of ganoderic acid A, comprising: extracting ganoderic acid A from Ganoderma lucidum; producing crystals of the ganoderic acid A; analyzing the crystals of the ganoderic acid A by X-ray structural analysis to obtain values of the three-dimensional coordinate of the crystals of the ganoderic acid A; and using the values of the three-dimensional coordinate of the crystals of the ganoderic acid A obtained from the X-ray structural analysis as initial coordinates in the input to the calculation program of the B3LYP method included in the Gaussian 03 package software together with the 6-31G* basis set function of the density functional theory (DFT) to determine the molecular configuration of the ganoderic acid A.

2. The method of claim 1, wherein the crystals of the ganoderic acid A are obtained by recrystallization followed by a crystal-growing process.

3. The method of claim 1, wherein the crystals of the ganoderic acid A are analyzed by X-ray structural analysis on a SMART CCD diffract meter with Mo K radiation at 295K.

4. The method of claim 3, wherein is 0.7107 on the SMART CCD diffract meter with Mo K radiation.

5. The method of claim 1, wherein the ganoderic acid A is extracted from fruit bodies of Ganoderma lucidum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows a schematic view of Ganoderma lucidum.

[0013] FIG. 2 is a maximal likelihood tree showing inferred phylogenetic relationships of fungal ITS gene sequences from cultured Ganoderma lucidum voucher TJ1 and NCBI Genbank.

[0014] FIG. 3 is an ORTEP diagram of ganoderic acid A of the present invention.

[0015] FIG. 4 is a theoretical calculation diagram of ganoderic acid A of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The preferred embodiments of the present invention are described below with reference to FIGS. 1-4. The description is only the explanation of the preferred embodiments, and is not the limitation of the implementation of the present invention.

[0017] The ganoderic acid A of the present invention is extracted from Ganoderma lucidum as shown in FIG. 1 having the following physico-chemical properties:

[0018] (a) 1H NMR (CDCl.sub.3) : 0.98 (3H, d, J=6.4 Hz), 1.09 (3H, s), 1.13 (3H, s), 1.16 (3H, s), 1.35 (3H, d), 1.43 (3H, s), 1.52 (3H, s), 1.60 (2H, t), 1.85 (1H, dd, J=13.2 Hz), 1.95 (1H, m), 1.97 (1H, m), 1.98 (1H, m), 2.16 (1H, m), 2.2 (1H, m), 2.25 (1H, m), 2.34 (1H, dd, J=16 Hz), 2.56 (t, 2H), 2.59 (1H, dd), 2.60 (1H, dd, J=9.6 Hz), 2.67 (1H, d), 2.94 (1H, d), 3.11 (1H, dd), 3.30 (1H, qt), 4.95 (1H, dd, J=10 Hz), 5.25 (1H, dd, J=9.2 Hz);

[0019] (b).sup.13C-NMR (100 MHz, CDCl.sub.3) : 36.0 (C-1), 34.6 (C-2), 216.1 (C-3), 46.7 (C-4), 48.9 (C-5), 29.6 (C-6), 68.7 (C-7), 161.5 (C-8), 139.9 (C-9), 38.3 (C-10), 199.7 (C-11), 52.4 (C-12), 47.0 (C-13), 54.7 (C-14), 72.2 (C-15), 36.9 (C-16), 48.6 (C-17), 17.5 (C-18), 19.6 (C-19), 33.1 (C-20), 19.7 (C-21), 49.9 (C-22), 209.0 (C-23), 47.1 (C-24), 35.6 (C-25), 178.2 (C-26), 17.6 (C-27), 20.3 (C-28), 27.2 (C-29), 20.8 (C-30);

[0020] (c) at room temperature (295 K), unit cell dimensions: a=7.1982(5) , b=12.8985(9) , c=9.3138(7) ; =90, =120, =90; space group=P2(1); volume: 4332.2(5) .sup.3; Z=6; and Dcalc=1.188 Mgm.sup.3, wherein the physico-chemical properties are determined by X-ray crystallography, NMR spectroscopy, and density functional theory calculations.

[0021] Specifically, the ganoderic acid A is isolated from fruit bodies of Ganoderma lucidum. The fruit bodies are evidenced by the phylogenetic tree contructed with ITS sequence. The molecular identification is as follows:

[0022] Dried fruit bodies are cut into small pieces and ground with liquid nitrogen. Approximately 0.1 g ground sample is collected in a sterile 1.5 ml Eppendorf tube and followed by DNA extraction using the Qiagen plant Dneasy mini kit. Fungal DNAs extracted from the dried fruit bodies are amplified using primers ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS4 (5-TCCTCCGCTTATTGATA TGC-3). The PCR reaction is carried out in a total volume of 50 l containing 25 l 2PCR Master mix, 0.2 M of each primer and 5 l template DNA. The PCR program includes an initial denaturation at 95 C. and 72 C. for 3 minutes, with cooling at 4 C. Therefore, PCR products are checked by 1% agarose gel electrophoresis and sequencing. Taxonomic identification of the fungal sequence is blasted against National Center for Biotechnology information database. As shown in FIG. 2, maximal likelihood tree is constructed with MEGA6. The sequence of Ganoderma lucidum voucher TJ1 and reference sequences deposited in the GenBank database are aligned by ClustalW. The robustness of inferred topologies is tested by bootstrap analysis and 1000 resamplings of trees.

[0023] Ganoderic Acid a Extraction:

[0024] Dried fruit bodies of Ganoderma lucidum are crushed and extracted with MeOH. The MeOH extracts residue is placed on a silica gel column and eluted with EtOAc gradually enriched with MeOH to afford 10 fractions. Ganoderic acid A (78.0 mg) is isolated by silica gel column from fraction 3 with CHCl.sub.3/MeOH 40:1 as elute solvent.

[0025] Production of Single Crystal of Ganoderic Acid A:

[0026] Single crystals of ganoderic acid A is obtained by recrystallization followed by a crystal-growing process. The recrystallization involves heating ganoderic acid A together with proper amount of methanol. Methanol vapor is allowed to slowly diffuse into the crystal-growing bottle until a perfect crystal is produced. The structure of the resulting single crystals is then analyzed by X-ray crystallography. Suitable crystals are selected, and then mounted on thin glass fibers using viscous oil. All measurements are made on a SMART CCD diffract meter with Mo K radiation with 0.7107 at 295K.

[0027] As shown in FIG. 3, through structure analysis using X-ray crystallography, the ORTEP diagrams of ganoderic acid A is identified.

[0028] Calculation Methods and Input:

[0029] All computations are carried out using the B3LYP method included in the Gaussian 03 package software together with the 6-31G* basis set function of the density (DFT) to understand structure features of the ganoderic acid A. The DFT theoretical calculation diagram of ganoderic acid A is shown in FIG. 4.

[0030] Result:

[0031] The obtained geometric structures of ganoderic acid A is shown as follows:

##STR00001##

[0032] The values of the three-dimensional coordinate obtained from the X-ray structural analysis are used as initial coordinates in the input to the calculation program to compare the reliabilities and reasonableness of the theoretical methods used in the research.

[0033] Table 1 shows the data of crystals of ganoderic acid A and the atomic bond lengths of the best structure obtained by using B3LYP/6-31G*.

TABLE-US-00001 TABLE 1 Atomic bond lengths () Crystallographic data B3LYP/6-31G* O1C2 1.194(5) 1.219 O3C11 1.214(4) 1.226 O5C21 1.202(5) 1.218 O7C25 1.305(5) 1.353 C1C27 1.536(6) 1.544 C1C6 1.572(5) 1.570 C3C4 1.515(7) 1.537 C5C10 1.523(5) 1.554 C5C6 1.558(5) 1.566 C7C8 1.524(5) 1.522 C9C10 1.352(4) 1.365 C10C11 1.513(5) 1.504 C12C13 1.535(5) 1.530 C13C17 1.554(5) 1.565 C14C29 1.548(5) 1.455 C15C16 1.538(5) 1.549 C17C18 1.546(5) 1.548 C18C19 1.528(6) 1.539 C21C22 1.490(6) 1.523 C23C25 1.510(5) 1.526 O2C8 1.425(4) 1.425 O4C15 1.430(4) 1.442 O6C25 1.212(5) 1.213 C1C2 1.524(6) 1.546 C1C26 1.542(7) 1.552 C2C3 1.434(7) 1.518 C4C5 1.543(5) 1.555 C5C28 1.545(5) 1.552 C6C7 1.522(5) 1.528 C8C9 1.531(5) 1.533 C9C14 1.528(5) 1.539 C11C12 1.487(6) 1.528 C13C30 1.534(5) 1.402 C13C14 1.564(4) 1.576 C14C15 1.553(4) 1.549 C16C17 1.549(5) 1.565 C18C20 1.514(6) 1.403 C20C21 1.505(5) 1.527 C22C23 1.518(5) 1.529 C23C24 1.525(6) 1.521

[0034] The calculations of ganoderic acid A are in closest agreement with the experiment and are also molecule predicted by DFT calculations. The atomic torsion angle of ganoderic acid A obtained by X-ray crystallography structural analysis and theoretical calculations is shown in Table 2.

TABLE-US-00002 TABLE 2 Atomic torsion angle () Crystallographic data B3LYP/6-31G* C2C1C27 106.8(4) 107.8 C27C1C26 108.7(4) 107.5 C27C1C6 108.4(3) 109.4 O1C2C3 117.2(5) 120.9 C3C2C1 123.5(4) 117.6 C3C4C5 112.8(4) 112.6 C10C5C28 109.3(3) 107.1 C10C5C6 107.3(3) 107.9 C28C5C6 113.6(3) 115.0 C7C6C1 113.1(3) 114.2 C6C7C8 110.3(3) 109.5 O2C8C9 111.1(3) 112.8 C10C9C14 121.4(3) 120.4 C14C9C8 116.0(2) 117.0 C9C10C5 124.4(3) 123.5 O3C11C12 119.4(4) 118.1 C12C11C10 119.5(3) 119.6 C30C13C12 108.1(3) 108.2 C12C13C17 118.3(3) 117.8 C12C13C14 107.6(3) 107.1 C9C14C29 104.7(3) 104.4 C29C14C15 107.4(7) 107.5 C29C14C13 113.4(3) 113.7 O4C15C16 109.8(3) 112.9 C16C15C14 103.2(3) 104.0 C18C17C16 113.0(3) 112.7 C16C17C13 102.5(3) 102.3 C20C18C17 111.1(3) 110.1 C21C20C18 113.2(3) 112.1 O5C21C20 120.6(4) 120.9 C21C22C23 114.7(3) 113.3 C25C23C24 109.4(4) 109.6 O6C25O7 123.3(4) 122.4 O7C25C23 112.0(3) 120.0 C2C1C26 107.3(4) 107.2 C2C1C6 111.2(3) 110.2 C26C1C6 114.3(4) 114.5 O1C2C1 119.3(5) 121.4 C2C3C4 118.4(5) 114.1 C10C5C4 110.0(3) 110.7 C4C5C28 110.8(3) 110.0 C4C5C6 105.7(3) 106.1 C7C6C5 110.1(3) 109.7 C5C6C1 117.0(3) 118.3 O2C8C7 110.6(3) 107.1 C7C8C9 113.0(3) 113.0 C10C9C8 122.1(3) 122.0 C9C10C11 118.6(3) 119.4 C11C10C5 117.0(3) 116.9 O3C11C10 121.0(4) 122.3 C11C12C13 110.8(3) 111.0 C30C13C17 109.9(3) 109.6 C30C13C14 110.6(3) 111.7 C17C13C14 102.2(2) 102.3 C9C14C15 121.3(3) 120.6 C9C14C13 110.9(2) 111.8 C15C14C13 99.4(2) 99.2 O4C15C14 115.1(3) 111.9 C15C16C17 108.0(3) 107.4 C18C17C13 117.7(3) 119.0 C20C18C19 109.5(4) 108.9 C19C18C17 114.2(3) 114.0 O5C21C22 121.1(4) 121.6 C22C21C20 118.3(3) 117.6 C25C23C22 111.7(3) 109.7 C22C23C24 111.3(4) 112.2 O6C25C23 124.5(4) 125.8

[0035] Please refer to Table 3, the overall B3LYP/6-31G* calculation of the molecular structures of ganoderic acid A is in excellent agreement with experimental data.

TABLE-US-00003 TABLE 3 Ganoderic acid A Empirical formula C.sub.30H.sub.44O.sub.7 Formula weight 516.65 Crystal system hexagonal Space group P2(1) Unit cell dimensions a = 7.1982(5) b = 12.8985(9) c = 9.3138(7) () or () 120 volume (.sup.3) 4332.2(5) Z (atoms/unit) 6 D.sub.calc/Mgm.sup.3 1.188 T(K) 295(2) Absorption coefficient 0.083 F(000) 1680 range (deg) 2.09~27.50 Crystal size 0.50 0.50 0.35 mm.sup.3 Index range h(14~8) k(4~14) 1(50~51) Reflection collection 19223 Independent reflection 6564(R(int) = 0.0436 Absorption correction Semi-empirical Max. and min. transmission 0.9715 and 0.9596 Data/restraints/parameters 6564/1/314 GOF on F.sup.2 1.064 Final R indices [I > 2(I)] R1 = 0.0797; WR2 = 0.1829 R indices(all data) R1 = 0.1070; WR2 = 0.2003 Largest diff. peak/hole[e .sup.3] 0.417/0.212

[0036] The above description is only the explanation of the preferred embodiments of the present invention. However, a person with ordinary skill in the art may make various modifications to the present invention. Those modifications shall still fall within the spirit and the scope defined by the appended claims.