Patent Application
20120178945
The application claims the benefit of Taiwan Patent Application No. 100100874, filed on Jan. 10, 2011, in the Taiwan Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.
The present invention relates to an extract of the fruiting body of Antrodia cinnamomea (abbreviated as A. cinnamomea or AC). In particular, the present invention relates to the benzenoid compounds of the fruiting body of A. cinnamomea, the preparation method and the analysis method thereof.
Antrodia cinnamomea (AC), by name niu-chang-chih or jang-jy is an endemic fungus in Taiwan and grows in the internal heartwood (or the dark/humid wood surface) of the particular Cinnamomum kanehirai in 400 to 2000 meters altitude. Therefore, it is uneasily to find out the wide fruiting body of AC or identify the morphological appearance of this Aphyllophorales fungus. In addition, the price of AC is still high due to their biologically active components having potential pharmaceutical value.
Since the fruiting body of AC cannot be easily found and be artificially cultured, mycelia products of AC are popular in the market and announce to own anticancer activity, reduced treatment-related symptoms and other side effects. In addition, mycelia products of AC have recently been reported to have anti-oxidant, antihypersensitive and immunostimulatory effects (Liu et al., 2007). It has been claimed of these mycelia products that they contain active components similar to the wild fruiting bodies with cytotoxic triterpenes, steroids, as well as immunostimulatory polysaccharides reported previously (Chen et al., 1995; Yang et al., 1996).
Traditionally AC has been used as health food to prevent inflammation, hypertension, itchy skin and liver cancer. Therefore, extracts of mycelia and fruiting body of AC are deemed as a potential chemotherapeutic agent against hepatoma, as well as prostate, bladder, lung cancer cells and so on (Chen et al., 2007; Hsu et al., 2007; Peng et al., 2007; Song et al., 2005; Wu et al., 2006). However, the chemical distribution and pharmacological research of niu-chang-chih products are not clarified up to now.
In addition, Taiwan Patent No. 1299665 discloses the extract of AC and the preparation thereof, in which the mycelia of AC is extracted with ethanol to obtain polysaccharides for inhibiting matrix metalloproteinase activities. However, the extract is not extracted with the fruiting body of AC, and the mycelia product thereof cannot inhibit cancer cell growth. Taiwan Patent No. 1279439 discloses that the mycelia of AC is cultured to obtain the cultured products by adjusting pH value of medium. However, there is no extraction method disclosed. Taiwan Patent No. 591110 discloses that γ-aminobutyric acid is extracted from the lyophilized mycelia of AC with water or organic solvents. However, the above-mentioned inventions did not disclose any product of the fruiting body of AC extracted with water or organic solvent, and there is no targeted second metabolites contained in the AC being identified.
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.
In the present invention, the extracts which are located in the lower polarity layer but still have activities are extracted from the fruiting body of AC, and the novel benzenoid compounds included in AC are identified. By the extraction method, the present invention can be applicable in detecting the types and amounts of benzenoid compounds in niu-chang-chih healthcare food and medicines and the fruiting body of AC, and can be used in industries.
The present invention provides a method for preparing an n-hexane extract of the fruiting body of A. cinnamomea, including steps of: providing the fruiting body of AC; extracting the fruiting body with an ethanol solution to obtain an ethanol extract; and extracting the ethanol extract with an n-hexane solution to obtain the n-hexane extract including at least one benzenoid compound.
The at least one benzenoid compound includes 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and/or antrocamphine A. The preparing method further includes a step of grinding the fruiting body as fine powder.
The present invention further provides the n-hexane extract of the fruiting body of AC, which includes at least one benzenoid compound. The n-hexane extract includes characteristics that a single hydrogen signal on an aromatic ring is ranged at δ 6.4 to 6.6, a methylenedioxy signal is ranged at δ 5.8 to 6.1, a double bond signal is ranged at δ 5.4 to 5.6, a methoxy signal is ranged at δ 3.7 to 4.1 and an aromatic methyl signal is ranged at δ 2.1 to 2.6 on a 1H nuclear magnetic resonance (1H NMR) spectrum when the n-hexane extract is solved in pyridine-D5 (C5D5N).
The n-hexane extract is obtained by sequentially extracting the fruiting body of AC with the ethanol solution and the n-hexane solution. The types of the contained at least one benzenoid compound is the same as aforementioned.
The present invention further provides an n-hexane extract of the fruiting body of AC, which includes at least one benzenoid compound. The n-hexane extract includes characteristics of having a single hydrogen signal ranged at δ 6.2 to 6.4 on an aromatic ring, a methylenedioxy signal ranged at δ 5.8 to 6.0, a double bond signal ranged at δ 5.2 to 5.5, a methoxy signal ranged at δ 3.6 to 4.1 and an aromatic methyl signal ranged at δ 2.1 to 2.4 on the 1H NMR spectrum when the n-hexane extract is solved in deuterium chloroform (CDCl3).
The present invention further provides a method for detecting an amount of at least one benzenoid compound in the fruiting body of AC, including steps of: providing the n-hexane extract extracted from the fruiting body; detecting whether the at least one benzenoid compound is present in the n-hexane extract with the 1H NMR; and detecting the amount by using a high performance liquid chromatography (HPLC) when the at least one benzenoid compound is present in the n-hexane extract.
The aforementioned detecting method further including a step of detecting a signal of the at least one benzenoid compound with the 1H NMR, wherein the signal is one selected from a group consisting of aromatic signals, a double bond signal, a methoxy signal, a methyl signal and a combination thereof.
When the n-hexane extract is solved in C5D5N, the chemical shifts of the present signals on the 1H NMR spectrum are described as above. Similarly, when solving in CDCl3, the chemical shifts of the present signals on the 1H NMR spectrum are also described as above.
Preferably, the HPLC used in the experiment includes a detector, and the detector is one selected from a group consisting of a full wavelength detector, a single wavelength detector and/or an electrospray ionization mass spectrometer. The full wavelength detector is configured to detect wavelengths at 254 nm and 270 nm.
The present invention further provides a method for detecting at least one benzenoid compound in the n-hexane extract of the fruiting body of AC with 1H NMR based on an internal standard corresponding to the n-hexane extract, the method including steps of: detecting whether a methoxy signal ranged at δ 3.9 to 4.0 exists in the n-hexane extract; detecting whether a first methyl signal ranged at δ 2.1 to 2.2 exists in the n-hexane extract; and detecting whether a second methyl signal ranged at δ 2.3 to 2.4 exists in the n-hexane extract.
The methoxy signal, the first methyl signal and the second methyl signal are respectively present to indicate that the n-hexane extract includes 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and an antrocamphine A.
Additionally, the first intensity of the methoxy signal is calculated based on the internal standard (pyrazine) when the methoxy signal exists. Similarly, the second intensity of the first methyl signal and/or the third intensity of the second methyl signal are calculated based on pyrazine when the first and/or the second methoxy signal exist. Furthermore, the amounts of 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and antrocamphine A in the n-hexane extract are sequentially determined by the first, the second and the third intensities.
The present invention further provides benzenoid compounds, extracted from the fruiting body of AC, the chemical formulas of benzenoid compounds are described as the preceding paragraph.
The present invention further provides a detecting method including a step of simultaneously detecting amounts of 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and antrocamphine A with HPLC.
The present invention further provides a detecting method including a step of detecting an amount of at least one of 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and antrocamphine A with 1H NMR.
The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings.
a) to 5(d) respectively illustrate the 1H NMR spectra of (a) the n-hexane extract, (b) compound 1, (c) compound 2 and (d) compound 3 solved in CDCl3 at 400 MHz.
a) to 6(d) respectively illustrate (a) the 1H NMR spectra of the n-hexane extract and internal standard (piperazine) solved in the CDCl3 at 400 MHz, (b) the magnification diagram of target characteristics of compound 1 in NMR method 1, (c) the magnification diagram of target characteristics of compounds 2 and 3 in NMR method 1 and (d) the magnification diagram of target characteristics of compounds 1, 2 and 3 in NMR method 2.
a) and 7(b) respectively illustrate the HPLC spectra of (a) the n-hexane extract and (b) compounds 1 to 3 at 254 nm in HPLC method 1.
a) and 8(b) respectively illustrate the HPLC spectra of (a) n-hexane extract and (b) compounds 1 to 3 at 270 nm in HPLC method 1.
a) and 9(b) respectively illustrate the HPLC spectra of n-hexane extract at (a) 254 nm and (b) 270 nm in HPLC method 2.
The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
Experiment 1: Preparation of the n-hexane Extract of the Fruiting Body of Antrodia cinnamomea (AC)
Please refer to preparation method 10 in
For exploring novel compounds in the n-hexane extract and for proving the novel compounds only existed therein rather than other extracts of the fruiting body of AC, the debris was sequentially extracted with ethyl acetate and ethanol according to the method disclosed in Taiwan Publication No. 201029658 to obtain the ethyl acetate extract and the second ethanol extract, and the n-hexane extract of the present invention was compared with the aforementioned extracts.
Experiment 2: Analysis of the NMR Spectra of the n-hexane Extract
The n-hexane extract was solved in pyridine-D5 (C5D5N) as the concentration of 11.6 mg/0.75 ml, and 1H NMR spectrum experiment was performed at a resolution of 400 MHz. Please refer to
Experiment 3: Separation of the Components of the n-hexane Extract
Compounds 1 to 3 were extracted in the present invention, and the corresponding structural formulas (Formulas 1 to 3) of compounds 1 to 3 were detailedly listed as follows.
For distinguishing two hydrogen atoms linked to C-4′ in formulas 1 and 3, both hydrogen atoms were nominated as “Ha” and “Hb”, respectively.
For identifying the components of the benzenoid-concentrating layer, the n-hexane extract was separated with the column chromatography. The n-Hexane extract (897.7 mg) was chromatographed with silica gel 60 (Merck, 230 to 400 mesh) and n-hexane-ethyl acetate (EA) (1:0, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1 sequentially) to afford 12 fractions. Each fraction product was solved in CDCl3 solution, and was analyzed with 1H NMR spectrum at a resolution of 200 MHz. The major characteristic signals of the aromatic components of fractions 1 and 3 were shown on the spectra and were detailedly described as follows.
Fraction 1 (245.4 mg) was chromatographed with Sephadex LH-20 resin and EA-dichloromethane (CH2Cl2) (1:1) to separate as five subfractions. Subfraction 1-4 (55.01 mg) was chromatographed with prepared-thin layer chromatography (pre-TLC) and n-hexane-EA (10:1) to afford a subfraction (47.5 mg). This subfraction (47.5 mg) then was purified with ODS high performance reverse chromatography column (250×10 mm, acetonitrile-H2O (80:20), flow rate: 2 ml/min) to give 3.3 mg of compound 2, i.e. 4,7-dimethoxy-5-methyl-1,3-benzodioxole, at retention time of 9.94 minutes, and give 3.3 mg of compound 1, i.e. 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole, at retention time of 17.25 minutes.
Fraction 3 (27.7 mg) was separated with Sephadex LH-20 resin and EA-CH2Cl2 (1:1) to afford two subfractions. Subfraction 3-2 (11.8 mg) was chromatographed with pre-TLC and separated with CH2Cl2 to give 10.0 mg of compound 3, i.e. antrocamphine A, or named 1,2,5-trimethoxy-3-methyl-4-(3-methylbut-3-en-1-ynyl)benzene.
Experiment 4: Structural Characterization of the Major Components of the n-hexane Extract
Compound 1 is a white amorphous solid, and the molecular formula is C15H26O4 using electrospray ionization mass spectrometry (ESIMS, m/z 261 [M+H]+, 283 [M+Na]+) and NMR spectrum. Please refer to Table 1, 1H NMR spectrum of compound 1 showed two methyl signals at δH 2.01 (3H, s) and 2.27 (3H, s), two methoxy signals at δH 3.87 (3H, s) and 3.98 (3H, s), two termial olefinic methylene protons (δH 5.26 and 5.37) and one methylenedioxy signal at δH 5.94 (2H, s). By the assistance of quantum cohenrence (QC) and heteronuclear multiple-bond cohenrence (HMBC), it could be determined the signals of 13C NMR spectrum corresponding to those of 1H NMR spectrum (Table 1), and it showed an aromatic methyl signal at δC 13.9 (6-CH3), a set of 3-methylbut-3-en-1-ynyl signals at δC 83.5 (C-1′), 97.5 (C-2′), 127.2 (C-3′), 121.0 (CH2-4′) and 23.6 (3′-CH3), two methoxy signals at δC 60.4 (4-OCH3) and 60.0 (7-OCH3) and a set of benzodioxole signals at δC 139.5 (C-1), 101.4 (CH2-2) and 136.2 (C-3).
1H and 13C NMR data of compound 1 (600 and 150 MHz of CDCl3, δ:
Please refer to
Please refer to
Compound 2 is a white amorphous solid, and the molecular formula is C10H12O4 using ESIMS (m/z 197 [M+H]−) and NMR spectrum. The characteristic signals of 1H NMR spectrum (400 MHz of CDCl3) of compound 2 showed that an aromatic methyl signal at δH 2.18 (3H, d, J=0.6 Hz), two methoxy signals at δH 3.84 (7-OCH3, s) and 3.88 (4-OCH3, s), a methylenedioxy signal at δH 5.93 (2H, s) and an aromatic single hydrogen signal at δH 6.30 (1H, d, J=0.6 Hz). The characteristic signals of 13C NMR (100 MHz of CDCl3) showed an aromatic methyl signal at δC 15.9, two methoxy signals at δC 56.9 and 59.9, a methylenedioxy signal at δC 101.4. According to the analytic data of the NOESY spectrum (δH 3.88 (4-OCH3)/2.18 (5-CH3), δH 2.18 (5-CH3)/6.30 (6-H) and 6.30 (6-H)/3.84 (7-OCH3)), the substitutions on the benzene ring for each functional groups in compound 2 was determined. It could be identified six aromatic carbon signals at δC 134.7 (C-1), 138.6 (C-3), 136.5 (C-4), 123.7 (C-5), 108.8 (CH-6) and 138.8 (C-7) by the help of HMBC. The abovementioned structure was determined.
Compound 3 is a yellow oil, and the molecular formula is C15H18O3 using ESIMS (m/z 247 [M+H]+) and NMR spectrum. The characteristic signals of 1H NMR (400 MHz of CDCl3) of compound 3 were two methyl signals at δH 2.01 (3′-CH3, t, J=1.6 Hz) and 2.36 (3-CH3, s), three methoxy signals at δH 3.72 (2-OCH3, s), 3.86 (1-OCH3, s) and 3.88 (5-OCH3, s), two termial olefinic methylene protons (δH 5.25 and 5.37) and one methylenedioxy signal at δH 6.33 (1H, s). The characteristic signals of 13C NMR spectrum (100 MHz of CDCl3) showed an aromatic methyl signal at δC 14.1 (3-CH3), a set of 3-methylbut-3-en-1-ynyl signal at δC 83.5 (C-1′), 97.5 (C-2′), 127.3 (C-3′), 120.7 (CH2-4′) and 23.7 (3′-CH3), three methoxy signals at δC 56.3 (1-OCH3), 60.4 (2-OCH3) and 55.8 (5-OCH3), and six aromatic carbon signals at δC 157.2 (C-1), 141.1 (C-2), 135.3 (C-3), 104.8 (C-4), 153.4 (C-5) and 94.4 (CH-6). The aforementioned chemical structure of compound 3 could be determined by the assistance of NOSEY and HMBC.
Experiment 5: Comparison of NMR of the n-hexane Extract with its Major Components
The n-hexane extract and compounds 1 to 3 were solved in CDCl3 solution, and their 1H NMR spectra were compared. Please refer to
Experiment 6: Detection of Amounts of Benzenoid Compounds with NMR Spectrum
The detection procedures were described as follows. An adequate internal standard was first chosen. This standard must has high purity and high stability, and its characteristic signals in the NMR spectrum are not interfered by the characteristic signals of the analyzed sample. Next, an specific amount of internal standard was added in the sample, an adequate deuterium solvent was selected to perform the NMR spectrum analysis, integral area ratios of characteristic signals of the compound to those of the internal standard were calculated, and the absolute amount of each compound was obtained by introducing the ratio to the absolute amount formula.
NMR method 1. The quantitative analysis of the major compounds 1 to 3 in the n-hexane extract was performed using NMR spectrum analysis in the present invention. The experimental conditions were listed as follows. The n-hexane extract (10.0 mg) was added in the internal standard, pyrazine (0.132 mg), which was solved in CDCl3 solution (0.6 mL) to be the test solvent for the NMR spectrum analysis. The NMR spectrometer was Varian UNITY plus 400 MHz spectrometer, the scanning number was 10 (7 minutes), the width of spectrum was 6002.4 Hz, and the width of intensity impulse was 6.3 micro-second (μs). Furthermore, the start point and end point of the targeted characteristic proton absorption signal of each compound were manually selected to calculate the integral area of peak to be the basis of this quantitative analysis. The whole experiment was made in triplicate, and its relative standard deviation (RSD %) was determined.
By the 1H NMR spectrum signals of three major compounds obtained in Experiment 4, the characteristic proton signals, methoxy signal at δH 3.98 (3H, s) at C-4 of compound 1, methyl signal at δH 2.18 (3H, d, J=0.6 Hz) at C-5 of compound 2 and methyl signal at δH 2.36 (3H, s) at C-3 of compound 3, of the respective compounds were chosen to be the targeted characteristic signals. Please refer to
The integral area ratio was introduced to the following quantitative formula 1 to determine the amount of compounds 1 to 3 in the n-hexane extract.
Eighty (80) is referred to the molecular weight of the internal standard (pyrazine). A is indicated to the integral area ratio of targeted characteristic signals of compounds 1 to 3 to characteristic signals of internal standard. B is referred to the number of proton of internal standard (the number of proton of pyrazine is 4). H is referred to the number of proton in the characteristic signal of compounds 1 to 3 (the targeted characteristic signal of compound 1 is 4-OCH3 and H value is 3; that of compound 2 is 5-CH3 and H value is 3; and that of compound 3 is 3-CH3 and H value is 3). MW is referred to molecular weight of each compound (molecular weights of compounds 1, 2 and 3 were 260, 196 and 246 respectively).
The absolute amount and the RSD % of compounds 1 to 3 of the n-hexane extract in the experiment were obtained from the above-mentioned detection method. Please refer to Table 3, the RSD value in triplicate were at the acceptable range, and it could be known that compounds 1 to 3 were the major components in the n-hexane extract and also were the major components of the benzenoid compounds of the fruiting body of AC.
NMR method 2. By the 1H NMR spectrum signals of three major compounds obtained in Experiment 4, the characteristic proton signals, methoxy signal at δH 3.98 (3H, s) at C-4 of compound 1, methoxy signal at δH 3.84 (3H, s) at C-7 of compound 2 and methoxy signal at δH 3.72 (3H, s) at C-2 of compound 3, of the respective compounds were chosen to be the targeted characteristic signals. Please refer to
The integral area ratio was introduced to the following quantitative formula 1 to determine the amount of compounds 1 to 3 in the n-hexane extract.
Eighty (80) is referred to the molecular weight of the internal standard (pyrazine). A is indicated to the integral area ratio of targeted characteristic signals of compounds 1 to 3 to characteristic signals of internal standard. B is referred to the number of proton of internal standard (the number of proton of pyrazine is 4). H is referred to the number of proton in the characteristic signal of compounds 1 to 3 (the targeted characteristic signal of compound 1 is 4-OCH3 and H value is 3; that of compound 2 is 7-OCH3 and H value is 3; and that of compound 3 is 2-OCH3 and H value is 3). MW is referred to molecular weight of each compound (molecular weights of compounds 1, 2 and 3 were 260, 196 and 246 respectively).
The absolute amount and the RSD % of compounds 1 to 3 of the n-hexane extract in the experiment were obtained from the above-mentioned detection method. Please refer to Table 5, the RSD value in triplicate were at the acceptable range, and it could be known that compounds 1 to 3 were the major components in the n-hexane extract and also were the major components of the benzenoid compounds of the fruiting body of AC.
Experiment 7: Amount Detection of the Benzenoid Compounds with HPLC
HPLC method 1. The relative amount analysis of the major components in the n-hexane extract was performed using HPLC, and the HPLC spectra of three obtained major compounds 1 to 3 were compared with that of the n-hexane extract. The conditions for HPLC were listed as follows. HPLC was Shimadzu LC-10AT, detector was Shimadzu SPD-M10A photodiode array detector, the auto sampler was Shimadzu SIL-20A prominence auto sampler; the HPLC column was Cosmosil 5C-18-MS (250×4.6 mm, 5 μm); solvents A and B in the mobile phase respectively were acetonitrile and water, flow rate was 1 ml/min, the temperature of column was room temperature, and the detection wavelength was 254 nm and 270 nm. The conditions of the solvent system were listed as follows. The mobile phase included solvents A and B, the linear gradient was 30% A to 100% A within 0 to 60 minutes, and the flow rate and the temperature of column were described as above.
Please refer to
Please refer to
HPLC method 2. The relative amount analysis of the major components in the n-hexane extract was performed using HPLC, and the HPLC spectra of three obtained major compounds 1 to 3 were compared with that of the n-hexane extract. The conditions for HPLC were listed as follows. HPLC was Shimadzu LC-10AT, detector was Shimadzu SPD-M10A photodiode array detector, the auto sampler was Shimadzu SIL-20A prominence auto sampler; the HPLC column was Agilent Poroshell 120 SB-C18 (150×4.6 mm, 2.7 μm); solvents A and B in the mobile phase respectively were acetonitrile and water (contain 0.1 formic acid), flow rate was 1.2 ml/min, the temperature of column was room temperature, and the detection wavelength was 254 nm and 270 nm. The conditions of the solvent system were listed as follows. The mobile phase included solvents A and B, the gradient program was used as follows: the initial elution condition was A-B (47:53, v/v), linearly changed to A-B (55:45, v/v) at 10.5 min and held for 4.5 min, then linearly changed to A-B (85:15, v/v) at 20 min, A-B (100:0, v/v) from 20 min to 23 min. Over the next 7 min, the percentage of mobile phase A kept in 100%., and the flow rate and the temperature of column were described as above.
Please refer to
Please refer to
Additionally, the molecular weights of the major components, i.e. compounds 1 to 3 (standard sample), of the n-hexane extract were determined using high performance liquid chromatography electrospray ionization tandem mass chromatography (HPLC-ESI-MS) with the positive ion mode. The conditions of HPLC were listed as follows. The HPLC meter was Agilent 1100 series, the HPLC column was Cosmosil 5C-18-MS 250×4.6 mm, the solvents A and B in the mobile phase respectively were acetonitrile and 0.1% formic acid H2O, flow rate was 1 ml/min, the temperature of column was room temperature, and the detection wavelengths were 254 nm and 270 nm. The conditions of the solvent system were listed as follows. The mobile phase included solvents A and B, the linear gradient was 30% A to 100% A within 0 to 60 minutes, and flow rate and column temperature were described as above. The mass spectrometer was Thermo Finnigan LCQ DECA XPplus. The retention time of compound 1 was 38.22 minutes, its major ion peak was at m/z 197 [M+H]+, and the determined molecular weight (MW) of compound 1 was 260. The retention time of compound 2 was 22.39 minutes, its major ion peak was at m/z 247 [M+H]+, and the determined MW of compound 3 was 246.
It could be known from the results of the above NMR analysis and HPLC-ESI-MS analysis that 4,7-dimethoxy-5-(3-methylbut-3-en-1-ynyl)-6-methyl-1,3-benzodioxole (compound 1), 4,7-dimethoxy-5-methyl-1,3-benzodioxole (compound 2) and 1,2,5-trimethoxy-3-methyl-4-(3-methylbut-3-en-1-ynyl)benzene (compound 3) were the major components in the n-hexane extract of the fruiting body of Antrodia cinnamomea. The forementioned experimental methods could be the good tool for detecting the benzenoid compound in the fruiting body of AC in the industries.
While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100100874 | Jan 2011 | TW | national |
