Method for identifying the molecular configuration of ganoderic acid A from <i>Ganoderma lucidum</i>

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 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).

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, wherein, the Ganoderma lucidum is subjected to DNA extraction before extracting the ganoderic acid, the extracted DNA of the Ganoderma lucidum uses the primer ITS1F shown in SEQ ID NO:1 and the primer ITS4 shown in SEQ ID NO:2 undergoes polymerase chain reaction and then DNA sequencing is performed to ensure that it is Ganoderma lucidum.

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 Kα radiation at 295K.

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

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic view of Ganoderma lucidum.

(2) 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.

(3) FIG. 3 is an ORTEP diagram of ganoderic acid A of the present invention.

(4) FIG. 4 is a theoretical calculation diagram of ganoderic acid A of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(5) 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.

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

(7) (a) .sup.1H NMR (CDCl.sub.3) δ: 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);

(8) (b) .sup.13C-NMR (100 MHz, CDCl.sub.3) δ: 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);

(9) (c) at room temperature (295 K), unit cell dimensions: a=7.1982(5) Å, b=12.8985(9) Å, c=9.3138(7) Å; α=90°, β=120°, γ=90°; space group=P2(1); volume: 4332.2(5) Å.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.

(10) Specifically, the ganoderic acid A is isolated from fruit bodies of Ganoderma lucidum. The fruit bodies are evidenced by the phylogenetic tree constructed with ITS sequence. The molecular identification is as follows:

(11) 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 μl containing 25 μl 2×PCR Master mix, 0.2 μM of each primer and 5 μl 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.

(12) Ganoderic Acid a Extraction:

(13) 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.

(14) Production of Single Crystal of Ganoderic Acid A:

(15) 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 Kα radiation with 0.7107 Å λ at 295K.

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

(17) Calculation Methods and Input:

(18) 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.

(19) Result:

(20) The obtained geometric structures of ganoderic acid A is shown as follows:

(21) ##STR00001##

(22) 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.

(23) 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*.

(24) TABLE-US-00001 TABLE 1 Atomic bond lengths (Å) Crystallographic data B3LYP/6-31G* O1—C2 1.194(5) 1.219 O3—C11 1.214(4) 1.226 O5—C21 1.202(5) 1.218 O7—C25 1.305(5) 1.353 C1—C27 1.536(6) 1.544 C1—C6 1.572(5) 1.570 C3—C4 1.515(7) 1.537 C5—C10 1.523(5) 1.554 C5—C6 1.558(5) 1.566 C7—C8 1.524(5) 1.522 C9—C10 1.352(4) 1.365 C10—C11 1.513(5) 1.504 C12—C13 1.535(5) 1.530 C13—C17 1.554(5) 1.565 C14—C29 1.548(5) 1.455 C15—C16 1.538(5) 1.549 C17—C18 1.546(5) 1.548 C18—C19 1.528(6) 1.539 C21—C22 1.490(6) 1.523 C23—C25 1.510(5) 1.526 O2—C8 1.425(4) 1.425 O4—C15 1.430(4) 1.442 O6—C25 1.212(5) 1.213 C1—C2 1.524(6) 1.546 C1—C26 1.542(7) 1.552 C2—C3 1.434(7) 1.518 C4—C5 1.543(5) 1.555 C5—C28 1.545(5) 1.552 C6—C7 1.522(5) 1.528 C8—C9 1.531(5) 1.533 C9—C14 1.528(5) 1.539 C11—C12 1.487(6) 1.528 C13—C30 1.534(5) 1.402 C13—C14 1.564(4) 1.576 C14—C15 1.553(4) 1.549 C16—C17 1.549(5) 1.565 C18—C20 1.514(6) 1.403 C20—C21 1.505(5) 1.527 C22—C23 1.518(5) 1.529 C23—C24 1.525(6) 1.521

(25) 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.

(26) TABLE-US-00002 TABLE 2 Atomic torsion angle (°) Crystallographic data B3LYP/6-31G* C2—C1—C27 106.8(4) 107.8 C27—C1—C26 108.7(4) 107.5 C27—C1—C6 108.4(3) 109.4 O1—C2—C3 117.2(5) 120.9 C3—C2—C1 123.5(4) 117.6 C3—C4—C5 112.8(4) 112.6 C10—C5—C28 109.3(3) 107.1 C10—C5—C6 107.3(3) 107.9 C28—C5—C6 113.6(3) 115.0 C7—C6—C1 113.1(3) 114.2 C6—C7—C8 110.3(3) 109.5 O2—C8—C9 111.1(3) 112.8 C10—C9—C14 121.4(3) 120.4 C14—C9—C8 116.0(2) 117.0 C9—C10—C5 124.4(3) 123.5 O3—C11—C12 119.4(4) 118.1 C12—C11—C10 119.5(3) 119.6 C30—C13—C12 108.1(3) 108.2 C12—C13—C17 118.3(3) 117.8 C12—C13—C14 107.6(3) 107.1 C9—C14—C29 104.7(3) 104.4 C29—C14—C15 107.4(7) 107.5 C29—C14—C13 113.4(3) 113.7 O4—C15—C16 109.8(3) 112.9 C16—C15—C14 103.2(3) 104.0 C18—C17—C16 113.0(3) 112.7 C16—C17—C13 102.5(3) 102.3 C20—C18—C17 111.1(3) 110.1 C21—C20—C18 113.2(3) 112.1 O5—C21—C20 120.6(4) 120.9 C21—C22—C23 114.7(3) 113.3 C25—C23—C24 109.4(4) 109.6 O6—C25—O7 123.3(4) 122.4 O7—C25—C23 112.0(3) 120.0 C2—C1—C26 107.3(4) 107.2 C2—C1—C6 111.2(3) 110.2 C26—C1—C6 114.3(4) 114.5 O1—C2—C1 119.3(5) 121.4 C2—C3—C4 118.4(5) 114.1 C10—C5—C4 110.0(3) 110.7 C4—C5—C28 110.8(3) 110.0 C4—C5—C6 105.7(3) 106.1 C7—C6—C5 110.1(3) 109.7 C5—C6—C1 117.0(3) 118.3 O2—C8—C7 110.6(3) 107.1 C7—C8—C9 113.0(3) 113.0 C10—C9—C8 122.1(3) 122.0 C9—C10—C11 118.6(3) 119.4 C11—C10—C5 117.0(3) 116.9 O3—C11—C10 121.0(4) 122.3 C11—C12—C13 110.8(3) 111.0 C30—C13—C17 109.9(3) 109.6 C30—C13—C14 110.6(3) 111.7 C17—C13—C14 102.2(2) 102.3 C9—C14—C15 121.3(3) 120.6 C9—C14—C13 110.9(2) 111.8 C15—C14—C13  99.4(2) 99.2 O4—C15—C14 115.1(3) 111.9 C15—C16—C17 108.0(3) 107.4 C18—C17—C13 117.7(3) 119.0 C20—C18—C19 109.5(4) 108.9 C19—C18—C17 114.2(3) 114.0 O5—C21—C22 121.1(4) 121.6 C22—C21—C20 118.3(3) 117.6 C25—C23—C22 111.7(3) 109.7 C22—C23—C24 111.3(4) 112.2 O6—C25—C23 124.5(4) 125.8

(27) 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.

(28) 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) Å b = 12.8985(9) Å c = 9.3138(7) Å β(°) or γ(°) 120 volume (Å.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 θ 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 > 2σ(I)] R1 = 0.0797; WR2 = 0.1829 R indices(all data) R1 = 0.1070; WR2 = 0.2003 Largest diff. peak/hole[e Å.sup.−3] 0.417/−0.212

(29) 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.