US20110123562A1 - Anti-cancer active substance from antrodia camphorata, method for preparing the same and use thereof - Google Patents

Anti-cancer active substance from antrodia camphorata, method for preparing the same and use thereof Download PDF

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US20110123562A1
US20110123562A1 US12/954,112 US95411210A US2011123562A1 US 20110123562 A1 US20110123562 A1 US 20110123562A1 US 95411210 A US95411210 A US 95411210A US 2011123562 A1 US2011123562 A1 US 2011123562A1
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ethyl acetate
hexane
cancer
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fraction
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Been-Huang CHIANG
Yu-Wei Lin
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National Taiwan University NTU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a novel anti-cancer compound identified in mycelium of Antrodia camphorata , the methods of cultivation, and applications of the said compound.
  • the extracted compound is a bioactive anti-cancer compound that can inhibit the proliferation of hepatoma liver cancer cells and provide protection against liver cancer.
  • Antrodia camphorata is a medicinal fungus growing inside the Cinnamomum kanchirai Hayata, Lauraceae trunk, a native species tree found in Taiwan. Antrodia camphorata has been reported to have many medicinal properties including anti-oxidant, anti-inflammation, and anti-cancer properties (Chang and Chou, 2004; Mau, Huang; Huang and Chen, 2004; Song and Yen; 2003; Hseu, Chang, Hseu, Lee, Yech, and Chen, 2002; Shen, Chou, Wang, Chen, Chou, and Lu, 2004; Ao et al., 2009). This particular type of fungus was widely used as a treatment drug for liver-associated diseases in traditional medicine. Recently, the anti-cancer activity of Antrodia camphorata , particularly the anti-liver cancer activity, has attracted great interests.
  • Antrodia camphorata Many compounds identified in Antrodia camphorata were demonstrated to exhibit anti-cancer activities. Lien et al. have purified 4,7-dimethoxy-5-methyl-1,3-benzodioxole from dry fruiting body of Antrodia camphorata and discovered that this compound can inhibit the proliferation of human colon epithelial cells (Lien et al., 2009). In addition, 24-methylenelanosta-7,9-(11)-diene-3 ⁇ ,15 ⁇ -diol-21-oic (MMH01), another compound identified in Antrodia camphorata mycelium, was shown to inhibit the growth of human leukemia cancer cells (U937) and pancreatic cancer cells (B ⁇ PC3) (Chen, Chou, and Chang, 2009).
  • MMH01 24-methylenelanosta-7,9-(11)-diene-3 ⁇ ,15 ⁇ -diol-21-oic
  • B ⁇ PC3 pancreatic cancer cells
  • Antrodia camphorata Aside from their anti-cancer activities, some compounds isolated from Antrodia camphorata have also displayed anti-inflammatory activities. Yang et al. (2009) purified 5 different compounds from Antrodia camphorata which include antroquinonol B, 4-acetyl-antroquinonol B, 2,3-(methylenedioxy)-6-methyl benzene-1,4-diol, 2,4-dimethoxy-6-methyl-benzene-1,3-diol and antrodin D, and found that they can efficiently inhibit NO production and exhibit certain anti-inflammatory effects.
  • Antrodia camphorata Due to the high medicinal value and slow growth rate of Antrodia camphorata , the fruiting body of Antrodia camphorata is on high demand nowadays. In order to meet with the market demand of this rare fungus, many approaches have been explored and industrial-level production of Antrodia camphorata mycelium by using liquid culture medium has been developed (Shin, Pan, and Hsieh et al. 2006). While the medicinal effects of Antrodia camphorata have drawn much attention, limited studies are available in terms of identification and characterization of these bioactive anti-cancer compounds found in mycelium. Nakamura et al.
  • VEGF receptor vascular endothelial growth factor receptor
  • Hepatoma is the leading lethal cause of malignant cancers in China, Taiwan, Korea and south of the Sahara in Africa (Seow, Liang, Leow and Chung, 2001; Kern, Breuhahn and Schirmacher, 2002).
  • Previous studies have shown that mycelium of Antrodia camphorata can protect the liver from damage caused by alcohol, CCl4 and lipopolysaccharides (Dai et al., 2003; Lu et al., 2007; Hsiao et al., 2003; Song and Yen, 2003; Hattori and Sheu, 2006; Ao et al., 2009). Guo et al.
  • the present invention provides a novel bioactive, anti-cancer compound extracted from Antrodia camphorata mycelium, and said compound can inhibit the proliferation of hepatoma liver cancer cells and can protect against liver cancer.
  • a bioactive, anti-cancer compound which can be used as a drug to treat cancer(s) is provided.
  • the method used to produce the bioactive anti-cancer compound from Antrodia camphorata comprises the following steps:
  • Step 1 Cultivation of the Antrodia camphorata by liquid fermentation
  • Step 2 Extraction of the pellet of mycelium with ethanol so as to prepare the ethanol extract
  • Step 3 Dissolve the ethanol extract from step 2 in water, and extract with equal volume of ethyl acetate so as to obtain the ethyl acetate extract;
  • Step 4 Purification of ethyl acetate extract obtained from step 3 by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase (hexane/ethyl acetate gradient elution, ratios of hexane/ethyl acetate were from 100:0 to 0:100). At the end, 100% methanol was used to elute out final residues. Fractions collected (700 mL per fraction) were then further purified by thin-layer chromatography.
  • Fractions f and g were eluted with 80% hexane/20% ethyl acetate to 70% hexane/30% ethyl acetate and 70% hexane/30% ethyl acetate to 60% hexane/40% ethyl acetate, respectively.
  • Step 5 Purification of the fraction g obtained from Step 4 by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase (hexane/ethyl acetate gradient elution, ratios of hexane/ethyl acetate were from 80:20 to 50:50) and finally elute with 100% ethyl acetate. Collect the eluates (50 mL per fraction) which contain the fractions E, F, G, H and I.
  • Fraction E was eluted with 80% hexane/20% ethyl acetate to 75% hexane/25% ethyl acetate
  • fractions F and G were in the eluate of 75% hexane/25% ethyl acetate
  • fraction H and I were eluted with 75% hexane/25% ethyl acetate to 70% hexane/30% ethyl acetate, and 70% hexane/30% ethyl acetate, respectively.
  • Step 6 Further purification of the eluates containing fractions E, F, G, H, and I obtained from step 5 with appropriate purification methods and 4-acetylantroquinonol B was then collected.
  • the purification methods include, but are not limited to silica gel column chromatography.
  • the crude ethanol extract, ethyl acetate extract, eluted fraction f, eluted fraction g, eluted fractions E, F, G, H, 1 and 4-acetylantroquinonol B are the bioactive anti-cancer compounds.
  • These purified compounds can be used 1) to treat cancer directly, 2) as one of the cocktail drugs for cancer therapy, or 3) as constituents for other appropriate applications.
  • bioactive anti-cancer compounds described in present invention refer to the crude ethanol extract, ethyl acetate extract, hexane/ethyl acetate extract, fraction f, fraction g, fractions E, F, G, H, 1 and 4-acetylantroquinonol B thereof that are prepared by the method in present invention.
  • the Antrodia camphorata (BCRC35716) were obtained from Biosources Collection and Research Center (BCRC) in the Food industry Research and Development Institute (Hsinchu, Taiwan).
  • preparation of the bioactive anti-cancer compounds is not limited to this particular species.
  • the liquid fermentation refers to culturing mycelium of Antrodia camphorata in fermentation medium containing 2% glucose and 2% malt extract with pH adjusted to 4.5 ⁇ 5.0, cultured at 20-25° C., 20-100 rpm agitation and an aeration of 0.5-1 vvm in a 5-ton fermenter for 4 weeks.
  • steps for ethanol extraction are mixing the dry mycelium with 90 ⁇ 99% ethanol (the ratios of mycelium to ethanol is 1:10 to 1:50 (g/mL)) and homogenized with a polytron for 12 ⁇ 48 hrs.
  • the size of the silica gel column used in step 4 is 750-75 mm, 230-400 mesh.
  • the size of the silica gel column used in step 5 is 420-25 mm, 230-400 mesh.
  • the silica gel column used in step 6 is Luna 5u Silica (2) 100A column (4.6 ⁇ 250 mm) and mobile phase is the mixture of hexane/ethyl acetate in the ratio of 80 to 20.
  • bioactive anti-cancer compound which includes the effective dose of the compound and adequate diluents, excipients or carriers.
  • such compound mixtures can inhibit the proliferation of cancer cells.
  • the present invention further identified an anti-cancer drug mixture which consists of effective dose of 4-acetylantroquinonol B and a carrier.
  • the present invention also provides a novel application for the above 4-acetylantroquinonol B in which the compound can be used for anti-cancer drug(s) development and such drug(s) can inhibit the proliferation of cancer cells.
  • FIG. 1 shows anti-proliferative activities of fractions partitioned by various solvents. Means with different letters (a ⁇ d) within 72 and 96 hrs treatments are significantly different (P ⁇ 0.05).
  • FIGS. 2A-2E show the HPLC profiles of E, F, G, H, and I fractions after the second silica gel column chromatography.
  • 2 A, 2 B, 2 C, 2 D and 2 E represent fraction E, F, G, H, and I, respectively. These fractions all contain one major peak at the same retention time as indicated by the arrows.
  • FIG. 3 shows the inhibitory and cytotoxic activity of 4-acetylantroquinonol B on the proliferation of HepG2cells.
  • FIG. 4 shows the structure of the bioactive anti-cancer compound, 4-acetylantroquinonol B.
  • Antrodia camphorata used in present invention was obtained from the Biosources Collection and Research Center (BCRC) in the Food industry Research and Development Institute (Hsinchu, Taiwan). However, extraction of the bioactive anti-cancer compounds described in present invention is not limited to this particular strain.
  • the fermentation broth was centrifuged to separate mycelium from broth filtrate.
  • the mycelium pellet was washed twice with distilled water to remove the trace of broth and then freeze dry and store at 4° C.
  • dry mycelium pellet was mixed with 95% ethanol in the ratio of 1 g to 20 mL and homogenized with high-speed polytron and agitated for 24 hrs to extract the ethanol-soluble compounds.
  • the extract was further concentrated with a rotary evaporator and stored at ⁇ 80° C. for further studies.
  • the fraction containing the most potent inhibitory activity was then purified by silica gel chromatography (750-75 mm, 230-400 mesh), and eluted with hexane/ethyl acetate gradient solutions (the ratios of hexane/ethyl acetate vary from 100:0 to 0:100). Finally, 100% Methanol was used to remove the trace of residues.
  • Each fraction (700 mL) was collected and analyzed its composition pattern by thin-layer chromatography (TLC, silica gel 60 F 254 , Merck Co., Darmstadt, Germany) using ethyl acetate/hexane (50/50; v/v) for development. UV 254 nm-illuminating yellow fluorescence was used to group fractions with similar skeleton. According to the analyzed results from thin-layer chromatography, 13 fractions were collected from the eluates and tested for their anti-cancer activity.
  • the fraction f was obtained from hexane/ethyl acetate wash (with the ratios of hexane to ethyl acetate vary from 80/20 to 70/30) and collection tubes are at number 36 to 42 with the final volume of 4.9 L.
  • the fraction g was obtained from hexane/ethyl acetate wash (with the ratios of hexane to ethyl acetate vary from 70/30-60/40) and collection tubes are at number 43 to 55 with the final volume of 9.1 L.
  • the most effective anti-cancer extract collected was further purified using another silica gel column (750-75 mm, 230-400 mesh) and washed with hexane/ethyl acetate buffers (the ratios of hexane to ethyl acetate vary from 80/20 to 50/50). Pure ethyl acetate was then used for final elution and 12 fractions were collected. The eluates were collected every 50 mL and fraction E was obtained from 80/20-75/25 hexane/ethyl acetate wash and collection tubes are 44 to 56 with the final of 650 mL.
  • Fractions F and G were obtained from the wash of 75/25 hexane/ethyl acetate, collection tubes are 57 to 61 and 62 to 69 and collected volume are 250 mL and 400 mL, respectively.
  • Fraction H was obtained from 75/25 to 70/30 hexane/ethyl acetate wash, collection tubes are 70 to 73 and collected final volume is 200 mL.
  • Fraction I was obtained from 70/30 hexane/ethyl acetate wash, collection tubes are 74 to 84 and collected total volume is 550 mL. All these fractions were tested for their anti-cancer activity and the most effective fraction was then further purified.
  • collected fractions were further purified by using a high-performance liquid chromatography (HPLC) system equipped with a tunable absorbance detector (model 1100 series, Agilent, U.S.A.). Elution was carried out at flow rate of 1 mL/min with a column temperature at 25° C. and UV wavelength of 254 nm.
  • the superlative fraction was separated by a silica column (4.6 ⁇ 250 mm, Luna 5u Silica (2) 100A column) with hexane/ethyl acetate (80:20, v/v) as solvent system so as to obtain the anti-cancer bioactive compound.
  • the structure of the purified compound was further identified by nuclear magnetic resonance (NMR, Bruker AMX-400).
  • HepG2 cells were obtained from American Type Culture Collection, ATCC, Rockville, Md., U.S.A. and were maintained in Williams medium E (WME), containing 10 mM/L HEPES, 5 ⁇ g/mL insulin, 2 ⁇ g/mL, glucagon, 0.05 ⁇ g/mL hydrocortisone, and 5% fetal bovine serum (Gibco Life Technologies, Grand Island, N.Y., USA).
  • Colon cancer cells C26, BCRC 60443
  • prostate cancer cells LNCaP, BCRC 60088
  • breast cancer cells MDA-MB-231, BCRC 60425) were obtained from the Biosources Collection and Research Center (BCRC) in the Food industry Research and Development Institute (Hsinchu, Taiwan).
  • HepG2 cells in WME were cultured on a 96-well cell culture plate (2.5 ⁇ 104cells per well) and incubate at 37° C. with 5% CO2. After 4 hours incubation, replace the medium with different concentrations of Antrodia camphorata sample extracts.
  • the Antrodia camphorata extracts were dissolved in 1% dimethyl sulfoxide (DMSO) and mixed with WME medium so as to prepare extracts at different concentrations and the final concentration of DMSO was maintained below 1%.
  • Control cultures contained only extraction solvents, and blank wells contained 100 ⁇ L growth medium with no cells.
  • cell proliferation was determined by MTS assay (MTS-based cell titer 96 non-radioactivity cell proliferation assay, Promega, Madison, Wis., U.S.A.) so as to examine the anti-hepatoma activity.
  • MTS is a colorimetric method utilizing a tetrazolium reagent.
  • Cell proliferation was determined at 72 and 96 h from the MTS absorbance reading at 490 nm for each concentration compared with the control. The CC50 value was determined as the cytotoxic concentration of a sample that reduced the cell viability to 50% of control in 24 h.
  • the EC50 of a sample is defined as the median effective dose of a sample (the concentration of the sample which results in 50% of cell viability) after treating the cancer cells for a certain period of time. At least three replications of each sample were tested to confirm cell proliferation and cytotoxicity. The selective effect on cancer cells was expressed as SI (selective index) value. The SI value was determined as the ratio of CC50 versus EC50 for a tested sample.
  • the weights of ethyl acetate fraction, n-butanol fraction, and water fraction from the ethanol extract of mycelium were 574 g, 196 g, and 87 g, respectively. Their anti-proliferative activities are shown in FIG. 1 .
  • the EC50 of crude extract, ethyl acetate fraction, n-butanol fraction and water fraction for anti-proliferation of HepG2 cells for 72 h treatment were 5.59 ⁇ 0.16, 2.83 ⁇ 0.06, 18.26 ⁇ 2.72, and >100 ⁇ g/mL, respectively.
  • EC50 of these fractions for 96 h treatment were 2.76 ⁇ 0.01, 1.94 ⁇ 0.13, 5.3 ⁇ 0.00, and 9.35 ⁇ 0.32 ⁇ g/mL, respectively. It was found that the ethyl acetate fraction showed the best anti-proliferative activity among all fractions; thus this fraction was further purified by the open silica gel column.
  • the EC50 of g fraction toward HepG2 cells were 1.33 ⁇ g/mL and 0.82 ⁇ g/mL for 72 h and 96 h treatments, respectively.
  • the SI value of g fraction was 86 for 96 h treatment. Due to the contaminated compounds found in g fraction based on the result of TLC, it was necessary to use an open silica gel column to further purify the bioactive compound.
  • fractions F, G, and H Twelve fractions were isolated from the g fraction and the EC50 of these 12 fractions were shown in FIG. 4 .
  • the CC50 of all fractions on HepG2 cells was >50 ⁇ g/mL.
  • the SI values of these fractions ranged from 111 to 119 suggesting that these fractions were well-selective from hepatoma cells.
  • FIG. 3 shows the anti-proliferative activity of this compound.
  • the EC50 of this compound were 0.1 and 0.08 ⁇ g/mL for 72 and 96 h treatments, respectively. This compound showed the best anti-proliferative activity among the fractions obtained throughout the entire separation process. Compared to crude extract, this compound displayed 50 times the anti-proliferative activity.
  • the CC50 of this compound was >10 ⁇ g/mL.
  • present invention also tested the anti-proliferative activity of 4-acetylantroquinonol B against other cancer cells.
  • CT26 colon cancer
  • LNCaP prostate cancer
  • MDA-MB-231 breast cancer cells
  • the bioactive compound was elucidated by spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and mass spectral analyses.
  • the structure is shown in FIG. 4 .
  • this bioactive anti-cancer compound characterized in present invention is identical to one of the five anti-inflammatory compounds, 4-acetylantroquinonol B, formerly reported by Yang et al. in 2009. However, their investigation did not expose the effects of such compound in inhibition of cancer cells growth and its applications for treating liver, colon, prostate and breast cancers (Yang et al., 2009).
  • the present invention not only provides several bioactive anti-cancer compounds (different extracted fractions), but also novel applications of 4-acetylantroquinonol B for anti-cancer therapy. Therefore, compared with previous findings, the present invention offers advantageous methods of isolation and preparation of the bioactive compounds, and novel applications for anti-cancer therapy.
  • the anti-cancer compound was purified to definite constituent and was shown to have inhibitory effects on cancer cell proliferation at very low concentrations (e.g. 0.1 ⁇ g/mL for 72 hours or 0.08 ⁇ g/mL for 96 hours).
  • Such compound not only can be purified from Antrodia camphorata mycelium by liquid fermentation, but also can be produced through chemical synthesis.
  • This novel process can significantly reduce the cost of preparation and solve the issue of high demand for scarce Antrodia camphorata .
  • compounds obtained previously by others are crude extract and their constitutions were not determined. Such crude extract can only be obtained by extraction of fermentation broth, and that process cannot be simplified. Subsequently, the cost for preparation is expensive.
  • the present invention provides a novel application of 4-acetylantroquinonol B which is to be used as an anti-cancer drug through its anti-proliferative activity on cancer cells.
  • the present invention presents an original approach for extraction of bioactive anti-cancer compounds and further identified their multifunctional properties in terms of anti-proliferative activities on cancer cells.

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