WO2017063150A1 - 酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔的用途，以及包含酪梨萃取物的保健食品 - Google Patents

酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔的用途，以及包含酪梨萃取物的保健食品 Download PDF

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WO2017063150A1
WO2017063150A1 PCT/CN2015/091904 CN2015091904W WO2017063150A1 WO 2017063150 A1 WO2017063150 A1 WO 2017063150A1 CN 2015091904 W CN2015091904 W CN 2015091904W WO 2017063150 A1 WO2017063150 A1 WO 2017063150A1
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virus
avocado extract
yne
cells
avocado
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PCT/CN2015/091904
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English (en)
French (fr)
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李景钦
陈益昇
张训硕
曾敬凯
林俊光
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高雄医学大学
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    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to an avocado extract, and in particular to its use for the preparation of a health food for the prevention of the Flaviviridae family virus.
  • the infection control of the Flaviviridae family virus is one of the important topics of public health in various countries.
  • the virus of the yellow fever virus family is mainly found in arthropods, and the infected object is mainly mammals.
  • the genetic material is single-stranded RNA, which is about 9.6 to 12.3 kb in length and has a viral envelope. structure.
  • the Flaviviridae family includes a variety of viruses, for example, Dengue virus, Yellow fever virus, West Nile virus, Japanese encephalitis virus. Hepatitis C virus or Bovine viral diarrhea virus.
  • the virus of the yellow fever virus family can cause encephalitis, encephalomyelitis, hemorrhagic diseases or other systemic infections in infected individuals.
  • Dengue virus belonging to the genus Flaviviridae Genus mainly consists of three structural proteins, capsid protein C, membrane protein M, and envelope protein E ( Envelope protein E) and 7 nonstructural proteins (NS). It is known that some non-structural proteins play an important role in the mechanism of dengue virus infection, for the symptoms caused by viral infections, such as dengue fever, dengue shock syndrome and dengue There is also a close relationship between dengue hemorrhagic fever.
  • dengue viruses can be divided into four types, serotypes, respectively.
  • DENV-1, DENV-2, DENV-3, and DENV-4 all have the ability to cause disease.
  • the dengue virus is mainly transmitted to humans by means of mosquitoes.
  • the diseases caused by dengue virus infection are mainly caused by the distribution of Aedes aegypyi and Aedes albopictus in tropical and subtropical regions.
  • dengue fever has begun to spread to countries and has gradually become a global public health problem.
  • An embodiment of the present invention provides a use of an extract of Acer (Persea americana) for the preparation of a health food for preventing viral infection by the Flaviviridae family.
  • Acer Persea americana
  • One embodiment of the present invention provides a use of an extract of Persea americana for the preparation of a food additive for preventing viral infection by the Flaviviridae family.
  • Yet another embodiment of the present invention provides a use of avocadenol B for the preparation of a medicament for treating or preventing a viral infection of the Flaviviridae family.
  • a (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne ((2R,4R)-1,2,4-trihydroxyheptadec-16-yne) is provided.
  • a further embodiment of the present invention provides a health food for inhibiting viral replication activity or viral inflammatory reaction of the Flaviviridae family, comprising an effective amount of aperiti (Persea americana) extract as an active ingredient and pharmaceutically acceptable Accepted carrier.
  • aperiti Persea americana
  • 2A-2C are relative RNAs of dengue virus in dengue virus-infected Huh-7 cells treated with different concentrations of avocado extract, avoB and THHY by RT-qPCR according to some embodiments of the present disclosure.
  • a histogram of the amount (*p ⁇ 0.05; **p ⁇ 0.01).
  • 3A-3B are serotypes of DENV-1, DENV-2, DENV-3, and DENV-4 serotypes treated with different concentrations of avocado extract, avoB, and THHY, according to some embodiments of the present disclosure. Histogram of relative RNA amount of dengue virus in dengue virus-infected Huh-7 cells (*p ⁇ 0.05; **p ⁇ 0.01).
  • 4A-4B, 5A-5B, and 6A-6B are diagrams of dengue virus-infected Huh- under different concentrations of avocado extract, avoB, and THHY by RT-qPCR according to some embodiments of the present disclosure.
  • OAS-1 was detected by dengue virus-infected Huh-7 cells under different concentrations of avocado extract, avoB and THHY by RT-qPCR.
  • dengue virus-infected Huh-7 cells were detected by RT-qPCR under different concentrations of avocado extract, avoB, and THHY treatment.
  • a histogram of relative RNA levels of TNF- ⁇ , IL-1 ⁇ , and IL-6 (*p ⁇ 0.05; **p ⁇ 0.01).
  • 13A-13C are line graphs of Huh-7 cell viability under different concentrations of avocado extract, avoB, and THHY treatment, in accordance with certain embodiments of the present disclosure.
  • Figure 14A is a bar graph of the relative RNA amount of Japanese encephalitis virus in BHK cells infected with Japanese encephalitis virus under different concentrations of avocado extract (*p ⁇ 0.05; **p ⁇ 0.01).
  • Figure 14B is a bar graph of the relative RNA amount of Japanese encephalitis virus in BHK cells infected with Japanese encephalitis virus at different concentrations of avoB (*p ⁇ 0.05; **p ⁇ 0.01).
  • Figure 15A is a bar graph of the relative RNA amount of hepatitis C virus in hepatitis B virus-infected BHK cells treated with different concentrations of avocado extract (*p ⁇ 0.05; **p ⁇ 0.01).
  • Figure 15B is a bar graph of the relative RNA amount of hepatitis C virus in hepatitis B virus-infected BHK cells treated with different concentrations of THHY (*p ⁇ 0.05; **p ⁇ 0.01).
  • Figure 15C is a bar graph of the relative RNA amount of hepatitis C virus in hepatitis B virus-infected BHK cells treated with different concentrations of avoB (*p ⁇ 0.05; **p ⁇ 0.01).
  • the extract of Acer Persea americana
  • the avocado extract contains avocadenol B or (2R, 4R)-1,2,4-trihydroxyheptadeca-16-yne as the main active ingredient.
  • the "avocado extract” as used in the present disclosure refers to an extract extracted from a plant body of Acer (Persea americana).
  • Acer Persea americana
  • avocado is native to Central America and Mexico and belongs to the angiosperm of the family Lauraceae. It contains a variety of vitamins, minerals and beneficial phytochemicals. It is rich in biological activity and antioxidant function and does not contain cholesterol. The world record is listed as the most nutritious fruit. It should be noted that the avocados used in the present disclosure may be derived from any origin or improved variety.
  • the avocado extract of the present disclosure is selected from avocado fruit, and after being sliced and dried, an organic solvent is used. Perform extraction.
  • the drying temperature may be from 20 ° C to 80 ° C, from 40 ° C to 60 ° C, for example, 50 ° C.
  • the above organic solvent may be a C1 to C12 alcohol, for example, methanol, ethanol, propanol, isopropanol, butanol, 2-butanol, pentane pure, hexanol, heptanol, octanol, decyl alcohol, decyl alcohol , undecyl alcohol, dodecanol or a combination thereof, but is not limited thereto.
  • the organic solvent may also be an aromatic hydrocarbon such as benzene, toluene or xylene, but is not limited thereto.
  • methanol is used as the extraction solution, and the concentration of methanol may be 80% to 100%, for example, 99.5%.
  • the temperature and time of the extraction can be determined depending on conditions such as the solvent characteristics to be used, and are not particularly limited.
  • the extraction temperature may be from 5 ° C to 50 ° C or from 10 ° C to 30 ° C, for example, 25 ° C.
  • the above extraction step may be repeated a plurality of times to obtain a higher purity extract, for example, three times of extraction may be repeated.
  • the avocado extract obtained in the above extraction step can be further subjected to a purification step to further increase the purity thereof.
  • the purification step can be column chromatography, thin layer chromatography, gas chromatography, high performance liquid chromatography, ion exchange chromatography (ion exchange). Chromatography or a combination thereof, for example, column chromatography using silica filling.
  • Avocadenol B the chemical formula is C 17 H 30 O 3 , the full name is (2R, 4R, 6E)-1,2,4-trihydroxyheptadeca-6-ene-16-yne ((2R, 4R, 6E)-1,2,4-trihydroxyheptadec-6-en-16-yne).
  • the inventors conducted a cell test on the above avocado extract and avocadenol B and (2R, 4R)-1,2,4-trihydroxyheptadeca-16-yne obtained by purifying the avocado extract.
  • avocado extract, avocaden B and (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne were found to have the effect of inhibiting the Flaviviridae family virus.
  • the "Yellowviridae” virus described in the present disclosure may include Dengue virus, Yellow fever virus, West Nile virus, Japanese encephalitis. Virus) or Hepatitis C virus, etc., may include all viruses belonging to the yellow fever virus family.
  • avocado extract, avocadenol B, and (2R,4R)-1,2,4-trihydroxyl can inhibit the protein and RNA production of dengue virus, that is, inhibit the replication activity of the virus.
  • the avocado extract, avocaden B, and (2R,4R)-1,2,4-trihydroxyheptadeca-16-alkyne inhibit the inflammatory response induced by dengue virus.
  • avocado extract, avocaden B, and (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne can induce interferon production by cells infected with dengue virus ( Interferon, IFN).
  • the present disclosure also provides an avocado extract, avocadenol B or (2R, 4R)-1,2,4-trihydroxyheptadeca-16-yne for the preparation of a virus against the infection of the yellow fever virus.
  • Use of health food or food supplements Furthermore, the present disclosure also provides an avocado extract, avocadenol B or (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne for the preparation or treatment of a yellow fever virus Use of an infected drug (eg, dengue virus).
  • an infected drug eg, dengue virus
  • it can also be used for the preparation of dietary supplements, nutritional products or medical foods.
  • the above-described health food, food supplement or medicament for the preparation of a virus against the infection of the Flaviviridae may further comprise a pharmaceutically acceptable carrier or salt.
  • the pharmaceutically acceptable carrier or salt may comprise from 0.5 to 99% by weight of the health food, food additive or drug, preferably from 5 to 95% by weight.
  • the above pharmaceutically acceptable carriers may include additives, excipients, preservatives, flavoring agents and the like which are generally used in the manufacture of foods or pharmaceuticals.
  • additives for example, starch, corn starch, lactose, dextrin, cyclodextrin, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, gelatin, gum, acacia, gum (guar) ), pectin, gum arabic, tragacanth, carrageenan, or similar additives.
  • the pharmaceutically acceptable carrier can also be a solvent, a dispersion medium, a coating, an antibacterial or antifungal agent, and the like.
  • the pharmaceutically acceptable salt may be an inorganic cation, for example, an alkali metal salt.
  • Classes such as sodium, potassium or amine salts, alkaline earth metal salts such as magnesium, calcium salts, salts containing divalent or tetravalent cations such as zinc, aluminum or zirconium salts.
  • the pharmaceutically acceptable salts may also be organic salts such as dicyclohexylamine salts, methyl-D-glucosamine, amine acid salts such as arginine, lysine, tissue Amine acid or glutamic acid amide.
  • the aforementioned drugs may be appropriately designed according to the administration route, and may be, for example, a tablet, a capsule, a film-coated tablet, a powder, a granule, a syrup, a suspension, an emulsion, an injection, a suppository or a patch.
  • the administration route may be, for example, oral administration, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, anal administration, inhalation administration, or topical administration.
  • the dosage of the drug can be appropriately formulated according to the patient's weight, age, symptoms of the affected part, physiological condition, and route of administration according to the condition of the patient or the deacon.
  • the "effective amount" as used in the present disclosure means having a dose which inhibits viral activity, kills a virus, reduces the number of viruses, or completely destroys a virus.
  • This effective amount is usually supplied to the patient depending on the surface area of the patient, the weight of the patient, and the condition of the patient.
  • the effective dose will also vary with the following conditions, including: the route of administration of the drug, the dosage form of the drug, or whether other treatments are used in combination.
  • the inventors of the present invention found that the avocado extract has the effect of inhibiting the Flaviviridae family virus.
  • the avocado extract inhibits dengue virus replication activity and viral inflammatory response, and can also induce dengue virus-infected cells to produce interferon (IFN) to fight the virus.
  • IFN interferon
  • the avocado extract contains avocadenol B or (2R, 4R)-1,2,4-trihydroxyheptadeca-16-yne as a main component for inhibiting the activity of dengue virus.
  • the immature avocado fruit (about 11.9 kg) can be sliced by the method of Y.-C. Lu et al. Second metabolites from the unripe of Persea americana and their antimycobacterial activities. Food Chemistry 135 (2012) 2904-0929. It was dried in an oven at 50 ° C to obtain a dried avocado sample (about 2.3 kg, 19.3% of the original weight). The dried avocado sample was extracted with methanol at a concentration greater than 99.5% at room temperature and the extraction step was repeated three times.
  • the ethyl acetate (ethyl acetate, EtOAc) aqueous solution EtOAc: H 2 O 1:1
  • EtOAc EtOAc: H 2 O 1:1
  • the fraction which was soluble in ethyl acetate was about 280 g
  • the fraction which was soluble in water was about 283 g
  • the portion soluble in ethyl acetate was the avocado extract used in the subsequent experiments.
  • ethyl acetate-soluble fraction (about 100 g) was added to a column packed with silica colloid (70-230, Merck) for chromatography and purification. After elution with a concentration gradient of elution n-hexane-ethyl acetate (n-hexane-EtOAc), 12 fractions (A-1 to A-12) were obtained. Next, 10.5 g of the A-12 separation liquid was recrystallized with n-hexane to obtain crystals (A-12-C) and a mother liquid (A-12-M).
  • A-12-M was chromatographed on a column packed with silica colloid (230-400 mesh, Merck), and eluted with a concentration gradient elution solution of n-hexane-ethyl acetate to obtain 7 Divided into paintings (A-12-M-1 to A-12-M-7).
  • Huh-7 cells of human hepatoma cell line were infected with dengue virus strain 16681 (DENV-2 serotype), in which Huh-7 cells were cultured in 24-well plates, and the density of Huh-7 cells was 5 ⁇ 10 4 cells/well.
  • the MOI (multiplicity of infection) of the infection was 0.2.
  • Huh-7 cells were treated with different concentrations of avocado extract, avoB and THHY, respectively, and cultured for 3 days. It should be noted that, except for special instructions, the experimental conditions for dengue virus-infected Huh-7 cells in the following experiments were the same. Next, the above cells were dissolved in RIPA lysis buffer, and cell lysate was centrifuged to collect total protein of Huh-7 cells.
  • the virus protein NS2B of dengue virus was used as a target, and its specific antibody (rabbit polyclonal anti-NS2B antibody, GeneTex, CA, USA) was used for detection, and GAPDH with stable expression in cells was used as an internal control group (internal). Control). Furthermore, signal detection was performed using an ECL detection kit (PerkinElmer, CT).
  • 1A to 1C respectively show the protein content of dengue virus in dengue virus-infected Huh-7 cells treated with different concentrations of avocado extract, avoB and THHY by Western blotting. 0.1% DMSO. 1A to 1C show that when the concentration of avocado extract, avoB, and THHY-treated Huh-7 cells was increased, the amount of dengue virus protein production in Huh-7 cells was decreased.
  • avocado extract, avoB and THHY all inhibited the protein production of dengue virus, and the inhibitory effect was significant and concentration-dependent. From this, it can be seen that the avocado extract, avoB, and THHY have the effect of inhibiting the protein produced by the dengue virus.
  • Dengue virus strain 16681 (DENV-2 serotype) or four different serotypes of dengue virus strains DENV-1, DENV-2, DENV-3 and DENV-4 (DENV-1 to DENV-4 strains, respectively) It is a biological material obtained from the Disease Control Agency, DENV-1 number 8700828A; DENV-2 number 454009A; DENV-3 number 8700829A; DENV-4 number S9201818) Huh- infected human liver cancer cell line 7 cells. Next, total cellular RNA of Huh-7 cells was purified using Trizol reagent (Invitrogen, Carlsbad, CA).
  • the immediate reverse transcription real-time quantitative polymerase chain reaction was carried out under a reaction volume of 10 ⁇ l, wherein the reaction solution contained 200 ng of cDNA, 5 ⁇ l of Power SYBER Green PCR Master, and a 0.4 ⁇ M primer pair.
  • the temperature condition of the PCR reaction was set to: react at 95 ° C for 10 minutes ⁇ [95 ° C reaction for 15 seconds ⁇ 60 ° C for 1 minute] cycle 40 times ⁇ 95 ° C reaction for 15 seconds ⁇ 60 ° C reaction for 1 minute ⁇ 95 ° C reaction 15 second.
  • the primer used has specific recognition for the virus protein NS2 of dengue virus, and the primer pair shown by the sequence identification numbers 1 and 2 was used in the experiment to detect the dengue virus RNA.
  • GAPDH as a control group
  • the primer pairs shown in SEQ ID Nos. 3 and 4 in the experiment were detected using the primer pairs shown in SEQ ID Nos. 3 and 4 in the experiment.
  • 2A to 2C show the relative RNA amount of dengue virus in Huh-7 cells infected with 16681 dengue virus by different concentrations of avocado extract, avoB and THHY, respectively, by RT-qPCR.
  • the control group in the figure was 0.1% DMSO.
  • the data in the figure are all quantified by normalization of GAPDH.
  • 2A-2C show that dengue virus RNA was significantly reduced in Huh-7 cells when the concentration of avocado extract, avoB and THHY in Huh-7 cells was increased (t-test, p ⁇ 0.05;p ⁇ 0.01).
  • the interpolation effect of avocado extract, avoB and THHY inhibited the generation of dengue virus RNA by effective concentration 50 (EC 50 ) of 36 ⁇ 3.4 ⁇ g / ml, 7.6 ⁇ 1.3 ⁇ M and 2.9, respectively. ⁇ 2.6 ⁇ M.
  • Figures 3A-3B show four different serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) by RT-qPCR at different concentrations of avocado extract, avoB, and THHY.
  • a histogram of the relative RNA amount of dengue virus in dengue virus-infected Huh-7 cells, and the data in the figure are all quantified by GAPDH normalization.
  • the concentration of the avocado extract of Huh-7 cells was increased, the RNA of dengue virus was significantly reduced in Huh-7 cells (t-test, p ⁇ 0.05; p ⁇ 0.01), and The situation was the same in both serotypes of dengue virus (DENV-1 to DENV-4).
  • the avocado extract can effectively inhibit the RNA synthesis of dengue viruses of DENV-1, DENV-2, DENV-3 and DENV-4.
  • the half effect concentration of the extract of avocado extract inhibiting DENV-1, DENV-2, DENV-3 and DENV-4 dengue virus RNA by interpolation was 65 ⁇ 5.1 ⁇ g/ml and 42 ⁇ 6.1, respectively. Gg/ml, 33 ⁇ 4.8 ⁇ g/ml and 74 ⁇ 3.4 ⁇ g/ml.
  • avoB and THHY can also effectively inhibit RNA synthesis of dengue viruses of DENV-1, DENV-2, DENV-3 and DENV-4 types.
  • the half effect concentrations of avoB inhibited DENV-1, DENV-2, DENV-3 and DENV-4 dengue virus RNA generation by interpolation were 14.4 ⁇ 2.1 ⁇ M, 8.4 ⁇ 1.8 ⁇ M, 13.4 ⁇ 2.2, respectively. ⁇ M and 15.2 ⁇ 4.1 ⁇ M.
  • the half effect concentrations of THHY inhibition of DENV-1, DENV-2, DENV-3 and DENV-4 dengue virus RNA production were 16.3 ⁇ 3.4 ⁇ M, 3.4 ⁇ 1.1 ⁇ M, 13.7 ⁇ 4.1 ⁇ M and 14.7 ⁇ 2.3 ⁇ M, respectively.
  • Interferon is a cytokine secreted by a virus after infection by a virus. Interferon acts on related receptors on surrounding uninfected cells to induce uninfected cells to synthesize antiviral proteins to prevent infection. Thus has an anti-viral effect.
  • many viruses have the ability to fight interferons, for example, dengue viruses can disrupt the signaling pathway of interferon production in host cells.
  • the experimental method was also carried out by the aforementioned real-time quantitative reverse transcription PCR, and the target of detection was changed to RNA of the interferon gene in the host cell.
  • primer pairs having specificity for recognition of interferon genes IFN- ⁇ 2 and IFN- ⁇ 17
  • primer pairs shown in SEQ ID Nos. 5 and 6 are used to detect RNA of IFN- ⁇ 2
  • the primer pairs shown in SEQ ID Nos. 7 and 8 were used to detect RNA of IFN- ⁇ 17.
  • 4A to 4B, 5A to 5B, and 6A to 6B respectively show IFN- ⁇ 2 in Huh-7 cells infected with 16681 dengue virus under different concentrations of avocado extract, avoB and THHY by RT-qPCR.
  • a histogram of the relative RNA amount of IFN- ⁇ 17, which is 0.1% DMSO in the figure, and the data in the figure are all quantified by GAPDH normalization.
  • the inventors also performed correlation detection on the downstream signaling molecules OAS-1, OAS-2, OAS-3 and PKR of interferon, further confirming the interferon signaling of avocado extract, avoB and THHY to infected cells.
  • the impact of the path Furthermore, the RNA of OAS-1 was detected using the primer set shown in SEQ ID Nos. 9 and 10; the RNA of OAS-2 was detected using the primer pairs shown by SEQ ID Nos. 11 and 12; the sequence identification numbers 13 and 14 were used.
  • the primer pair detects the RNA of OAS-3; and the primer pair shown in SEQ ID NOs. 15 and 16 detects the RNA of PKR.
  • Figures 7A to 7D show the detection of OAS-1, OAS-2, OAS-3 and PKR in Huh-7 cells infected with 16681 dengue virus by different concentrations of avocado extract, avoB and THHY by RT-qPCR.
  • Figures 8-9 show bar graphs of relative RNA levels of OAS-1, OAS-2, and OAS-3 in Huh-7 cells infected with 16681 dengue virus.
  • the inventors also conducted the following experiments to test avocado extract, avoB, and The effect of THHY on the inflammatory response caused by viral infection.
  • the experimental method is also carried out by the aforementioned instant reverse transcription instant quantitative polymerase chain reaction, and the target of detection is RNA of a factor or cytokine associated with an inflammatory reaction in a host cell.
  • a primer pair having specificity for tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-1 ⁇ (IL-1 ⁇ ), and cytokine-6 (IL-6) is used herein.
  • TNF- ⁇ RNA is detected using the primer pair shown in the sequence identification numbers 17 and 18
  • the IL-1 ⁇ RNA is detected using the primer pair shown in the sequence identification numbers 19 and 20
  • the sequence identification numbers 21 and 22 are used.
  • the primer pair shown is for detecting IL-6 RNA.
  • 10A to 10C, 11A to 11C, and 12A to 12C respectively show TNF- ⁇ in Huh-7 cells infected with 16681 dengue virus under different concentrations of avocado extract, avoB, and THHY by RT-qPCR.
  • TNF- ⁇ , IL-1 ⁇ and IL in Huh-7 cells were increased when the concentration of avocado extract, avoB and THHY in Huh-7 cells was increased.
  • the amount of RNA production of -6 was significantly reduced (t-test, p ⁇ 0.05; p ⁇ 0.01). From this, it can be seen that the avocado extract, avoB, and THHY are effective in inhibiting the inflammatory reaction caused by dengue virus infection.
  • the half effect concentrations of the avocado extracts inhibiting the production of TNF- ⁇ , IL-1 ⁇ and IL-6 RNA by interpolation were 56.7 ⁇ 2.1 ⁇ g/ml, 67.4 ⁇ 4.9 ⁇ g/ml and 80.6 ⁇ 1.1 ⁇ g, respectively. /ml.
  • the half-effect concentrations of avoB inhibiting TNF- ⁇ , IL-1 ⁇ and IL-6 RNA production were 11.7 ⁇ 3.4 ⁇ M, 8.7 ⁇ 3.1 ⁇ M and 22.4 ⁇ 5.7 ⁇ M, respectively.
  • the half effect concentrations of THHY inhibiting TNF- ⁇ , IL-1 ⁇ and IL-6 RNA production were 7.8 ⁇ 2.7 ⁇ M, 4.1 ⁇ 1.4 ⁇ M and 48.6 ⁇ 4.2 ⁇ M, respectively.
  • the inventors also conducted the following experiments to test the effects of the concentration of avocado extract, avoB, and THHY on Huh-7 cell viability.
  • Huh-7 cells were treated with different concentrations of avocado extract, avoB and THHY, respectively. After 3 days of culture, the cells were tested with a cytotoxicity test kit (MTS assay kit, CellTilter 96Aqueous One Solution Cell proliferation assay system, Promega, WI, USA) Determination of the effect of avocado extract, avoB and THHY on Huh-7 cell viability (cytotoxicity). The experiment was carried out according to the operation manual attached to the kit, and the absorbance at a wavelength of 490 nm was measured using a 550 BioRad plate-reader (Bio-Rad, Hertfordshire, UK).
  • MTS assay kit CellTilter 96Aqueous One Solution Cell proliferation assay system, Promega, WI, USA
  • avocado extract, avocadenol B and (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne can effectively inhibit the production of RNA and protein of dengue virus.
  • the efficacy of dengue virus replication activity can effectively inhibit the production of RNA and protein of dengue virus.
  • avocado extract, avocadenol B, and (2R,4R)-1,2,4-trihydroxyheptadeca-16-yne can induce dengue virus-infected cells to produce interferon to fight the virus.
  • they are also effective in inhibiting the inflammatory response induced by dengue virus.
  • RNA of Japanese encephalitis virus (standardized by GAPDH) was quantified after treatment with different concentrations of avocado extract (50-200 ⁇ g/ml) and AvoB (1-20 ⁇ M).
  • the RNA content of BHK cells (also infected with Japanese encephalitis virus) treated with 0.1% DMSO was used as a control group, and the results were as shown in Figs. 14A and 14B, respectively, and it was confirmed that the avocado extract and the avoB of the present invention have the same. It inhibits the activity of Japanese encephalitis virus replication.
  • Huh-7 cells infected with hepatitis C virus were selected and treated with different concentrations of avocado extract (20-50 ⁇ g/ml), THHY (1-20 ⁇ M) and avoB (1-20 ⁇ M).
  • the RNA of hepatitis C virus (standardized by GAPDH) and the RNA content of Huh-7 cells (also infected with hepatitis C virus) treated with 0.1% DMSO were used as a control group, and the results are shown in Figures 15A, 15B, and 15C, respectively. It was confirmed that the avocado extract, THHY, and avoB of the present invention have an activity of inhibiting the replication of hepatitis C virus.

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Abstract

本发明公开了酪梨萃取物用于制备预防黄热病毒科病毒感染的保健食品或食物添加物的用途。本发明也提供了avocadenol B和(2R,4R)-1,2,4-三羟基十七碳-16-炔用于制备治疗或预防黄热病毒科病毒感染的药物的用途。本发明更提供一种抑制黄热病毒科病毒复制活性或病毒性发炎反应的保健食品，包括有效量的酪梨萃取物为活性成分以及医药学上可接受的载剂。

Description

酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔的用途，以及包含酪梨萃取物的保健食品 技术领域
本发明是有关于一种酪梨萃取物，特别是关于其用于制备预防黄热病毒科(Flaviviridae family)病毒的保健食品的用途。
背景技术
黄热病毒科(Flaviviridae family)病毒之感染防治为各国公共卫生的重要课题之一。黄热病毒科的病毒主要发现于节肢动物(arthropods)中，而感染对象主要为哺乳类动物，其遗传物质为单股的RNA，长度约为9.6～12.3kb，具有病毒封套(viral envelope)的结构。
黄热病毒科(Flaviviridae family)包括多种病毒，例如，登革病毒(Dengue virus)、黄热病毒(Yellow fever virus)、西尼罗河病毒(West Nile virus)、日本脑炎病毒(Japanese encephalitis virus)、C型肝炎病毒(Hepatitis C virus)或牛科病毒性腹泻病毒(Bovine viral diarrhea virus)等。黄热病毒科的病毒会使遭受感染的个体产生脑炎、脑脊髓炎、出血性疾病(hemorrhagic diseases)或其他全身性感染的疾病。
其中属于黄热病毒属(Flaviviridae Genus)的登革病毒(Dengue virus，DENV)，主要由3个结构蛋白、鞘蛋白C(capsid protein C)、膜蛋白M(membrane protein M)、封套蛋白E(envelope protein E)以及7个非结构蛋白(nonstructural protein，简称NS)所构成。目前研究已知，部份非结构蛋白在登革病毒感染的机制中扮演重要角色，对于病毒感染所引起的症状，例如，登革热(dengue fever)、登革休克症候群(dengue shock syndrome)及登革出血热(dengue hemorrhagic fever)亦有密切关联。
再者，依其抗原性的不同，登革病毒可分为四种，血清型，分别为 DENV-1、DENV-2、DENV-3及DENV-4，皆具有感染致病的能力。登革病毒主要借助蚊子作为媒介传染给人类，登革病毒感染造成的疾病主要好发于热带、亚热带等有埃及斑蚊(Aedes aegypyi)及白线斑蚊(Aedes albopictus)分布的国家。但随着各国之间的交流及往返趋于频繁，自1980年代后，登革热亦开始有向各国蔓延的趋势，逐渐成为全球性的公共卫生问题。
然而，目前并没有特定用以治疗登革热的药物，而疫苗的研发可能是控制登革病毒感染的最好方法。近年来，许多研究尝试研发可同时对四种血清型登革病毒产生免疫的疫苗，但在临床上仍遭遇了许多困难，像是无法产生具有长期免疫效果的疫苗等。因此，如何有效地预防登革病毒的感染为相当重要的公共卫生议题。
发明内容
本发明一实施例提供一种酪梨(Persea americana)萃取物用于制备预防黄热病毒科(Flaviviridae family)病毒感染的保健食品的用途。
本发明一实施例提供一种酪梨(Persea americana)萃取物用于制备预防黄热病毒科(Flaviviridae family)病毒感染的食品添加物的用途。
本发明又一实施例提供一种avocadenol B用于制备治疗或预防黄热病毒科(Flaviviridae family)病毒感染的药物的用途。
本发明再一实施例提供一种(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)用于制备治疗或预防黄热病毒科(Flaviviridae family)病毒感染的药物的用途。
本发明更一实施例提供一种抑制黄热病毒科(Flaviviridae family)病毒复制活性或病毒性发炎反应的保健食品，包括有效量的酪梨(Persea americana)萃取物为活性成分以及医药学上可接受的载剂。
附图说明
图1A～1C是根据本揭露一些实施例中，以西方墨点法检测在不同浓度的酪梨萃取物、avoB及THHY处理下，经登革病毒感染的Huh-7 细胞中登革病毒的蛋白质含量。
图2A～2C是根据本揭露一些实施例中，以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经登革病毒感染的Huh-7细胞中登革病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图3A～3B根据本揭露一些实施例中，以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经DENV-1、DENV-2、DENV-3及DENV-4血清型之登革病毒感染的Huh-7细胞中登革病毒的相对RNA量之柱状图(*p<0.05；**p<0.01)。
第4A～4B、5A～5B及6A～6B图系根据本揭露一些实施例中，以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经登革病毒感染的Huh-7细胞中IFN-α2及IFN-α17的相对RNA量的柱状图。
图7A～7D及8～9根据本揭露一些实施例中，以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经登革病毒感染的Huh-7细胞中OAS-1、OAS-2、OAS-3及PKR的相对RNA量的柱状图。
图10A～10C、11A～11C及12A～12C根据本揭露一些实施例中，以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经登革病毒感染的Huh-7细胞中TNF-α、IL-1β及IL-6的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图13A～13C根据本揭露一些实施例中，在不同浓度的酪梨萃取物、avoB以及THHY处理下，Huh-7细胞存活率的折线图。
图14A以不同浓度的酪梨萃取物处理下，经日本脑炎病毒感染的BHK细胞中日本脑炎病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图14B以不同浓度的avoB处理下，经日本脑炎病毒感染的BHK细胞中日本脑炎病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图15A以不同浓度的酪梨萃取物处理下，经C型肝炎病毒感染的BHK细胞中C型肝炎病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图15B以不同浓度的THHY处理下，经C型肝炎病毒感染的BHK细胞中C型肝炎病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
图15C以不同浓度的avoB处理下，经C型肝炎病毒感染的BHK细胞中C型肝炎病毒的相对RNA量的柱状图(*p<0.05；**p<0.01)。
具体实施方式
以下公开许多不同的实施方法或是例子来实行本发明的不同特征，以下描述具体的元件及其排列的例子以阐述本发明。当然这些仅是例子且不该以此限定本发明的范围。
本案发明人发现酪梨(Persea americana)萃取物具有抑制登革病毒复制活性及病毒性发炎反应的效果，且亦具有诱导受登革病毒感染的细胞产生干扰素(interferon，IFN)的能力。特别地，酪梨萃取物包含avocadenol B或(2R,4R)-1,2,4-三羟基十七碳-16-炔作为主要的活性成分。
本揭露所述的「酪梨萃取物」是指萃取自酪梨(Persea americana)植物体的萃取物。酪梨原产于中美洲及墨西哥，属于被子植物门的樟科(Lauraceae)，其含有多种维生素、矿物质以及有益的植化素，富含生物活性及抗氧化功能且不具胆固醇，被金氏世界纪录列为最营养的水果。应注意的是，本揭露使用的酪梨可源自任何产地或为经改良的品种。
本揭露的酪梨萃取物选用酪梨果实，经切片烘干后，以有机溶剂 进行萃取。烘干温度可为20℃～80℃、40℃～60℃，例如可为50℃。而上述有机溶剂可为C1至C12醇类，例如，甲醇、乙醇、丙醇、异丙醇、丁醇、2-丁醇、戊纯、己醇、庚醇、辛醇、壬醇、癸醇、十一醇、十二醇或其组合，但不限于此。有机溶剂亦可为芳香烃类，例如，苯、甲苯或二甲苯，但不限于此。在一实施例中，是采用甲醇作为萃取溶液，甲醇的浓度可为80％～100％，例如可为99.5％。
萃取的温度及时间可视使用的溶剂特性等条件决定，没有特别限定。萃取温度可为5℃～50℃或10℃～30℃，例如可为25℃。再者，可重复多次上述萃取步骤以获得纯度较高的萃取物，例如，可重复进行3次萃取。
接着，上述萃取步骤得到的酪梨萃取物可再经纯化步骤，以更进一步的提升其纯度。纯化步骤可为管柱层析(column chromatography)、薄层层析(thin layer chromatography)、气相层析(gas chromatography)、高效液相层析(high performance liquid chromatography)、离子交换层析(ion exchange chromatography)或其组合，例如，可使用以二氧化硅填充之管柱层析。
经上述萃取及纯化步骤，以核磁共振(nuclear magnetic resonance，NMR)分析及比对，可获得avocadenol B(如下式(I)所示)以及(2R,4R)-1,2,4-三羟基十七碳-16-炔(如下式(II)所示)。
Figure PCTCN2015091904-appb-000001
Figure PCTCN2015091904-appb-000002
avocadenol B，化学简式为C17H30O3，全名为(2R,4R,6E)-1,2,4-三羟基十七碳-6-烯-16-炔((2R,4R,6E)-1,2,4-trihydroxyheptadec-6-en-16-yne)。已有研究证实avocadenol B具有抗分枝杆菌(antimycobacterial)的活性(Y.-C.Lu et al.Secondary metabolites from the unripe of Persea americana and their antimycobacterial activities.Food Chemistry 135(2012)2904-0929)。
(2R,4R)-1,2,4-三羟基十七碳-16-炔(2R,4R)-1,2,4-trihydroxyheptadec-16-yne)，化学简式为C17H32O3，尚无研究指出与其相关的生物活性。
发明人将上述酪梨萃取物及由该酪梨萃取物纯化取得的avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔进行细胞试验。发现酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔皆具有抑制黄热病毒科(Flaviviridae family)病毒的功效。
值得注意的是，至今从未有文献指出酪梨萃取物、avocadenol B或(2R,4R)-1,2,4-三羟基十七碳-16-炔与黄热病毒科(Flaviviridae family)病毒的感染的预防或治疗有相关性。
承上述，本揭露所述的「黄热病毒科」病毒可包括登革病毒(Dengue virus)、黄热病毒(Yellow fever virus)、西尼罗河病毒(West Nile virus)、日本脑炎病毒(Japanese encephalitis virus)或C型肝炎病毒(Hepatitis C virus)等，可包含所有属于黄热病毒科的病毒。
在一些实施例中，酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟 基十七碳-16-炔可抑制登革病毒的蛋白质及RNA生成，即，抑制病毒的复制活性。在一些实施例中，酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔可抑制登革病毒所诱导的发炎反应。又，在一些实施例中，酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔可诱导受到登革病毒感染的细胞产生干扰素(interferon，IFN)。
因此，本揭露也提供一种将上述酪梨萃取物、avocadenol B或(2R,4R)-1,2,4-三羟基十七碳-16-炔用于制备预防黄热病毒科病毒感染的保健食品或食物添加物的用途。再者，本揭露也提供一种将酪梨萃取物、avocadenol B或(2R,4R)-1,2,4-三羟基十七碳-16-炔用于制备治疗或预防黄热病毒科病毒(例如：登革病毒)的感染的药物的用途。除此之外，也亦可将其用于制备膳食增补剂、营养产品或医学食品等用途。
在一实施例中，上述用于制备预防黄热病毒科病毒感染的保健食品、食物添加物或药物可更包括医药学上可接受的载剂(carrier)或盐类。医药学上可接受的载剂或盐类可占保健食品、食物添加物或药物的0.5～99wt％，较佳为5～95wt％。
而上述医药学上可接受的载剂可包括一般在食品或药物的制造上可使用的添加剂、赋形剂、防腐剂、矫味剂等。例如，淀粉、玉米淀粉、乳糖、糊精、环糊精、甲基纤维素、羧甲基纤维素、羧甲基纤维素钠、明胶、树胶(gum)、洋菜胶、古阿树胶(guar)、果胶、阿拉伯胶、西黄耆胶(tragacanth)、鹿角胶(carrageenan)、或类似的添加剂。再者，医药学上可接受的载剂亦可为溶剂、分散媒(dispersion medium)、套膜(coating)、抗菌或抗真菌试剂等。
再者，医药学上可接受的盐类可为无机阳离子，例如，碱金属盐 类，如钠、钾或胺盐，碱土金族盐类，如镁、钙盐，含二价或四价阳离子的盐类，如锌、铝或锆盐。此外，医药学上可接受的盐类亦可为有机盐类，如二环己胺盐类、甲基-D-葡糖胺，胺基酸盐类，如精胺酸、离胺酸、组织胺酸或麸胺酸酰胺等。
前述药物可根据投予路径适当设计剂型，例如可为锭剂、胶囊剂、膜衣锭剂、散剂、颗粒剂、糖浆、悬浮剂(suspensions)、乳剂(emulsions)、注射剂、栓剂或贴剂等。投予路径可为，例如口服、皮下注射、腹腔内注射、静脉内注射、肌肉注射、肛门投予、吸入性投药或局部投药等。药物的使用剂量可依医师或执事人员根据患者体重、年龄、患部症状、生理状况、投药路径等条件，适当调配。
此外，本揭露所述的「有效量」是指具有可以抑制病毒活性、杀死病毒、减少病毒数目、或者完全消灭病毒的剂量。此有效量通常是根据病患体表面积，病患的重量以及病患情况的不同来供应给病患。如此技术人士所知，有效剂量亦会随着以下条件的不同而变化，包括：药物的投予路径，药物的剂型或是否并用其他治疗法等。
综上所述，本案发明人发现酪梨萃取物具有抑制黄热病毒科(Flaviviridae family)病毒的功效。特别地，酪梨萃取物可抑制登革病毒的复制活性及病毒性发炎反应，且也可诱导受登革病毒感染的细胞产生干扰素(interferon，IFN)以对抗病毒。再者，发明人亦发现酪梨萃取物包含avocadenol B或(2R,4R)-1,2,4-三羟基十七碳-16-炔作为抑制登革病毒活性的主要成分。
以下进一步以实施例及比较例具体说明本发明，然其并非用以限定本揭露的内容。
实施例
酪梨的萃取及纯化
可参照Y.-C.Lu et al.Secondary metabolites from the unripe of Persea americana and their antimycobacterial activities.Food Chemistry 135(2012)2904-0929的方法，将未成熟的酪梨果实(约11.9kg)切片后，置于50℃的烘箱干燥以取得干燥的酪梨样本(约2.3kg，占原重量的19.3％)。在室温下，利用浓度大于99.5％甲醇萃取干燥的酪梨样本，并且重复萃取步骤三次。接着，加入乙酸乙酯(ethyl acetate，EtOAc)水溶液(EtOAc：H2O为1：1)使甲醇萃取物分离为可溶于乙酸乙酯的部分(EtOAc-soluble fraction)及可溶于水之部分(H2O-soluble fraction)。得到可溶于乙酸乙酯的部分约为280g，而可溶于水的部分约为283g，其中可溶于乙酸乙酯的部分即为用于后续实验中的酪梨萃取物。
接着，将一部分可溶于乙酸乙酯的部分(约100g)加入填充二氧化硅胶体的管柱(70～230，Merck)进行层析及纯化。利用浓度梯度的洗提液(elution)正己烷-乙酸乙酯(n-hexane-EtOAc)洗提后，得到12个分画(fraction)(A-1～A-12)。接着将10.5g的A-12分离液利用正己烷重新结晶化，以获取结晶(A-12-C)以及母液(A-12-M)。
接着，将10g的A-12-M以填充二氧化硅胶体之管柱(230～400mesh，Merck)进行层析，利用浓度梯度的洗提液正己烷-乙酸乙酯洗提后，得到7个分画(A-12-M-1～A-12-M-7)。
再者，将7.3g的A-12-M-4以RP-C18管柱(spherical C18 100A reversed-phase silica gel(RP-18)，20～40μM，Silicycle)进行层析，并且利用丙酮-水(1：1)进行洗提，洗提后得到25.2mg的avocadenol B以及113mg的(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)。为方便说明，接续的叙述中将分别以avoB及THHY简称avocadenol B以及(2R,4R)-1,2,4-三羟基 十七碳-16-炔。
检测Huh-7细胞中生成的病毒蛋白质
使用登革病毒株16681(DENV-2血清型)感染人类肝癌细胞株的Huh-7细胞，其中Huh-7细胞培养于24孔盘，Huh-7细胞的密度为5x104cells/well，而病毒感染的MOI(multiplicity of infection)为0.2。接着分别以不同浓度的酪梨萃取物、avoB以及THHY处理Huh-7细胞，培养3天。应注意的是，除特别说明外，下述实验中登革病毒感染Huh-7细胞的实验条件皆与此相同。接着，将上述细胞溶解于RIPA lysis buffer中，并将细胞溶解液(cell lysate)离心以收集Huh-7细胞的总蛋白质(total protein)。
接着，利用西方墨点法(western blot)检测Huh-7细胞中登革病毒的蛋白质生成量。以登革病毒的病毒蛋白NS2B作为标靶，利用其专一性抗体(rabbit polyclonal anti-NS2B antibody，GeneTex,CA，USA)进行检测，并以细胞中表现量稳定的GAPDH作为内部对照组(internal control)。再者，使用ECL检测套组(PerkinElmer，CT)进行信号检测。
图1A～1C分别显示以西方墨点法检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经登革病毒感染的Huh-7细胞中登革病毒的蛋白质含量，图中对照组为0.1％DMSO。图1A～1C显示，当酪梨萃取物、avoB以及THHY处理Huh-7细胞浓度增加时，Huh-7细胞中登革病毒的蛋白质生成量下降。酪梨萃取物、avoB以及THHY皆抑制了登革病毒的蛋白质生成，且抑制效果有显著性及浓度依赖性。由此可知，酪梨萃取物、avoB以及THHY具有抑制登革病毒制造蛋白质的功效。
检测Huh-7细胞中生成的病毒RNA
此外，发明人亦检测Huh-7细胞中病毒的RNA生成量，以进一步确认上述蛋白质部分的实验结果。分别使用登革病毒株16681(DENV-2血清型)或四种不同血清型的登革病毒株DENV-1、DENV-2、DENV-3及DENV-4(DENV-1～DENV-4病毒株是从疾病管制署取得的生物材料，DENV-1的编号为8700828A；DENV-2的编号为454009A；DENV-3的编号为8700829A；DENV-4的编号为S9201818)感染人类肝癌细胞株的Huh-7细胞。接着，使用Trizol reagent(Invitrogen，Carlsbad，CA)纯化Huh-7细胞的总细胞RNA(total cellular RNA)。
接着，以即时反转录即时定量聚合酶连锁反应(real-time quantitative reverse transcription PCR)检测经登革病毒感染的Huh-7细胞中的病毒RNA生成量。即时反转录即时定量聚合酶连锁反应在反应体积为10μl的条件下进行，其中反应溶液含有200ng cDNA、5μl Power SYBER Green PCR Master及0.4μM的引子对(primer pair)。而PCR反应的温度条件设定为：在95℃反应10分钟→﹝95℃反应15秒→60℃反应1分钟﹞循环40次→95℃反应15秒→60℃反应1分钟→95℃反应15秒。其中，使用的引子对对于登革病毒的病毒蛋白NS2具有专一辨识性，实验中使用序列辨识号1及2所示的引子对以检测登革病毒的RNA。此外，实验中使用序列辨识号3及4所示的引子对检测寄主细胞的GAPDH(作为对照组)。
图2A～2C分别显示以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经16681登革病毒感染的Huh-7细胞中登革病毒的相对RNA量(relative RNA)的柱状图，图中对照组为0.1％DMSO，再者，图中的数据皆为经GAPDH标准化(normalization)的量化结果。
图2A～2C显示，当处理Huh-7细胞的酪梨萃取物、avoB以及 THHY的浓度增加时，Huh-7细胞中登革病毒的RNA显著地减少(t-test，p<0.05；p<0.01)。此外，利用内插法计算可得酪梨萃取物、avoB以及THHY抑制登革病毒RNA生成的半效应浓度(effective concentration 50，EC50)分别为36±3.4μg/ml、7.6±1.3μM以及2.9±2.6μM。
此外，图3A～3B显示以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经四种不同血清型(DENV-1、DENV-2、DENV-3及DENV-4)登革病毒感染的Huh-7细胞中登革病毒的相对RNA量的柱状图，图中数据皆为经GAPDH标准化(normalization)的量化结果。
如图3A所示，当处理Huh-7细胞的酪梨萃取物的浓度增加时，Huh-7细胞中登革病毒的RNA显著地减少(t-test，p<0.05；p<0.01)，且在四种血清型的登革病毒中情形皆相同(DENV-1～DENV-4)。易言之，酪梨萃取物能够有效抑制DENV-1、DENV-2、DENV-3及DENV-4型的登革病毒的RNA合成。另外，利用内插法计算可得酪梨萃取物抑制DENV-1、DENV-2、DENV-3及DENV-4登革病毒RNA生成的半效应浓度分别为65±5.1μg/ml、42±6.1μg/ml、33±4.8μg/ml以及74±3.4μg/ml。
再者，如图3B所示，当avoB以及THHY处理Huh-7细胞的浓度增加时，Huh-7细胞中登革病毒的RNA显著地减少(t-test，p<0.05；p<0.01)，且在四种血清型的登革病毒中皆相同(DENV-1～DENV-4)。由图可知，avoB以及THHY也能够有效地抑制DENV-1、DENV-2、DENV-3及DENV-4型的登革病毒的RNA合成。另外，利用内插法计算可得avoB抑制DENV-1、DENV-2、DENV-3及DENV-4登革病毒RNA生成的半效应浓度分别为14.4±2.1μM、8.4±1.8μM、13.4±2.2 μM以及15.2±4.1μM。而THHY抑制DENV-1、DENV-2、DENV-3及DENV-4登革病毒RNA生成的半效应浓度则分别为16.3±3.4μM、3.4±1.1μM、13.7±4.1μM以及14.7±2.3μM。
检测Huh-7细胞中生成的干扰素
干扰素(interferon，IFN)为细胞受到病毒感染后所分泌的细胞因子，干扰素借助与周围未感染的细胞上的相关受体作用，促使未感染的细胞合成抗病毒蛋白以防制感染扩大，因而具有抗病毒的作用。然而，许多病毒具有对抗干扰素的能力，例如，登革病毒可破坏宿主细胞内干扰素生成的信号传递路径。
承上述，发明人进行接续实验以探讨酪梨萃取物、avoB以及THHY是否会影响经登革病毒感染的细胞内的干扰素信号传递路径。
实验方法同样以前述的即时反转录即时定量聚合酶连锁反应(real-time quantitative reverse transcription PCR)进行，而检测的目标则改为宿主细胞中干扰素基因的RNA。在此使用对干扰素基因(IFN-α2及IFN-α17)具有识别专一性的引子对，详细而言，使用序列辨识号5及6所示的引子对以检测IFN-α2的RNA，而使用序列辨识号7及8所示的引子对以检测IFN-α17的RNA。
图4A～4B、5A～5B及6A～6B分别显示以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经16681登革病毒感染的Huh-7细胞中IFN-α2及IFN-α17的相对RNA量的柱状图，图中对照组为0.1％DMSO，再者，图中的数据皆为经GAPDH标准化(normalization)的量化结果。
如图4A～4B、5A～5B及6A～6B所示，当处理Huh-7细胞的酪梨萃取物、avoB以及THHY的浓度增加时，Huh-7细胞中IFN-α2及IFN-α17的RNA生成量皆明显地增加。由上述结果可知，酪梨萃取物、 avoB以及THHY皆能诱导受登革病毒感染的细胞产生干扰素以对抗登革病毒。
此外，发明人也针对干扰素的下游信息传递分子OAS-1、OAS-2、OAS-3及PKR进行相关检测，更进一步地确认酪梨萃取物、avoB以及THHY对于感染细胞的干扰素信号传递路径的影响。再者，使用序列辨识号9及10所示的引子对检测OAS-1的RNA；使用序列辨识号11及12所示的引子对检测OAS-2的RNA；使用序列辨识号13及14所示的引子对检测OAS-3的RNA；及使用序列辨识号15及16所示的引子对检测PKR的RNA。
图7A～7D显示以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经16681登革病毒感染的Huh-7细胞中OAS-1、OAS-2、OAS-3及PKR的相对RNA量的柱状图，图中对照组为0.1％DMSO，再者，图中的数据皆为经GAPDH标准化(normalization)的量化结果。相似地，图8～9显示经16681登革病毒感染的Huh-7细胞中OAS-1、OAS-2及OAS-3的相对RNA量的柱状图。
如图7A～7D及8～9所示，当处理Huh-7细胞的酪梨萃取物、avoB以及THHY的浓度增加时，Huh-7细胞中OAS-1、OAS-2、OAS-3及PKR的RNA生成量皆明显地增加。由此可知，酪梨萃取物、avoB以及THHY不仅能诱导受登革病毒感染的细胞产生干扰素，它们亦促使干扰素的下游信息传递分子进行合成。因此，酪梨萃取物、avoB以及THHY抑制登革病毒的机制的确与干扰素密切相关。再者，可进一步地推断酪梨萃取物、avoB以及THHY抑制登革病毒的机制，可能是借助恢复或增强抗病毒的干扰素的生成而达成。
检测Huh-7细胞中病毒诱导的发炎反应
此外，发明人也进行下述实验，以测试酪梨萃取物、avoB以及 THHY对于病毒感染所引起的发炎反应(inflammatory response)的影响。
实验方法亦以前述的即时反转录即时定量聚合酶连锁反应进行，而检测的目标则为宿主细胞中与发炎反应相关的因子或细胞激素的RNA。在此使用对肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、细胞激素-6(IL-6)具有识别专一性的引子对。详细而言，使用序列辨识号17及18所示的引子对检测TNF-α的RNA；使用序列辨识号19及20所示的引子对检测IL-1β的RNA；及使用序列辨识号21及22所示的引子对检测IL-6的RNA。
图10A～10C、11A～11C及12A～12C分别显示以RT-qPCR检测在不同浓度的酪梨萃取物、avoB以及THHY处理下，经16681登革病毒感染的Huh-7细胞中TNF-α、IL-1β及IL-6的相对RNA量的柱状图，图中对照组为0.1％DMSO，再者，图中的数据皆为经GAPDH标准化(normalization)的量化结果。
如图10A～10C、11A～11C及12A～12C所示，当处理Huh-7细胞的酪梨萃取物、avoB以及THHY的浓度增加时，Huh-7细胞中TNF-α、IL-1β及IL-6的RNA生成量皆显著地减少(t-test，p<0.05；p<0.01)。由此可知，酪梨萃取物、avoB以及THHY能够有效地抑制登革病毒感染所造成的发炎反应。此外，利用内插法计算可得酪梨萃取物抑制TNF-α、IL-1β及IL-6RNA生成的半效应浓度分别为56.7±2.1μg/ml、67.4±4.9μg/ml以及80.6±1.1μg/ml。而avoB抑制TNF-α、IL-1β及IL-6RNA生成的半效应浓度分别为11.7±3.4μM、8.7±3.1μM以及22.4±5.7μM。THHY抑制TNF-α、IL-1β及IL-6RNA生成的半效应浓度则分别为7.8±2.7μM、4.1±1.4μM以及48.6±4.2μM。
细胞毒性分析
此外，发明人也进行以下实验，以测试酪梨萃取物、avoB以及THHY的浓度对于Huh-7细胞存活率(cell viability)的影响。
分别以不同浓度的酪梨萃取物、avoB以及THHY处理Huh-7细胞，细胞经3天培养后，以细胞毒性测试套组(MTS assay kit，CellTilter 96Aqueous One Solution Cell proliferation assay system，Promega，WI，USA)测定酪梨萃取物、avoB以及THHY对于Huh-7细胞存活率的影响(细胞毒性)。根据套组所附的操作手册进行实验，使用550BioRad plate-reader(Bio-Rad,Hertfordshire，UK)，测定波长490nm下的吸光值。
实验结果经换算后，可得到不同浓度的酪梨萃取物、avoB以及THHY相对于Huh-7细胞存活率，结果如图13A～13C所示，图中对照组为0.1％DMSO，且图中数据为三次重复实验所得之结果。应注意的是，对Huh-7细胞而言，酪梨萃取物、avoB以及THHY的CC50(cytotoxic concentration 50)分别为960±5.8μg/ml、103±6.2μM以及142±4.7μM。
前述实验中处理Huh-7细胞的酪梨萃取物、avoB以及THHY的最高浓度分别不超过80μg/ml、20μM以及20μM，皆远小于酪梨萃取物、avoB以及THHY的CC50。由此可知，前述处理Huh-7细胞的酪梨萃取物、avoB及THHY浓度对于Huh-7细胞是没有毒性的。
综上所述，酪梨萃取物、avocadenol B以及(2R,4R)-1,2,4-三羟基十七碳-16-炔可有效地抑制登革病毒的RNA及蛋白质的生成，具有抑制登革病毒的复制活性的功效。再者，酪梨萃取物、avocadenol B以及(2R,4R)-1,2,4-三羟基十七碳-16-炔可诱导受登革病毒感染的细胞产生干扰素以对抗病毒。此外，它们亦可有效地抑制登革病毒所诱导的发炎反应。
值得注意的是，酪梨萃取物、avoB以及THHY抑制登革病毒的机制，很可能是借助恢复或增强经病毒感染的细胞中用以对抗病毒的干扰素而达成。
对日本脑炎病毒的抑制测试
本试验选用经日本脑炎病毒感染的BHK细胞，以不同浓度的酪梨萃取物(50～200μg/ml)、AvoB(1～20μM)处理后，定量日本脑炎病毒的RNA(经GAPDH标准化)，并以处理0.1％DMSO的BHK细胞(同样经日本脑炎病毒感染)的RNA含量作为对照组，其结果分别如图14A、14B所示，证实本发明的酪梨萃取物、avoB均具有可以抑制日本脑炎病毒复制的活性。
对C型肝炎病毒的抑制测试
本试验则是选用经C型肝炎病毒感染的Huh-7细胞，以不同浓度的酪梨萃取物(20～50μg/ml)、THHY(1～20μM)、avoB(1～20μM)处理后，定量C型肝炎病毒的RNA(经GAPDH标准化)，并以处理0.1％DMSO的Huh-7细胞(同样经C型肝炎病毒感染)的RNA含量作为对照组，其结果分别如图15A、15B、15C所示，证实本发明的酪梨萃取物、THHY、avoB均具有可以抑制C型肝炎病毒复制的活性。
虽然本发明已以数个较佳实施例揭露如上，然其并非用以限定本发明，任何所属技术领域中具有通常知识者，在不脱离本发明的精神和范围内，当可作任意的更动与润饰，因此本发明的保护范围当申请专利范围所界定者为准。

Claims (17)

  1. 一种酪梨萃取物(Persea americana)用于制备预防黄热病毒科(Flaviviridae family)病毒感染的保健食品的用途。
  2. 如权利要求1所述的用途，其中黄热病毒科(Flaviviridae family)病毒包括：登革病毒(Dengue virus)、黄热病毒(Yellow fever virus)、西尼罗河病毒(West Nile virus)、日本脑炎病毒(Japanese encephalitis virus)或C型肝炎病毒(Hepatitis C virus)。
  3. 如权利要求1所述的用途，其中该酪梨萃取物包括avocadenol B作为活性成分。
  4. 如权利要求1所述的用途，其中该酪梨萃取物包括(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)作为活性成分。
  5. 如权利要求1至4中任一项所述的用途，其中该酪梨萃取物具有抑制登革病毒(Dengue virus)的蛋白质及RNA生成的能力。
  6. 如权利要求1至4中任一项所述的用途，其中该酪梨萃取物具有抑制登革病毒(Dengue virus)所诱导的发炎反应的能力。
  7. 如权利要求1至4中任一项所述的用途，其中该酪梨萃取物具有诱导受到登革病毒(Dengue virus)感染的细胞产生干扰素的能力。
  8. 一种酪梨萃取物(Persea americana)用于制备预防黄热病毒科(Flaviviridae family)病毒感染的食品添加物的用途。
  9. 如权利要求8所述的用途，其中黄热病毒科(Flaviviridae family)病毒包括：登革病毒(Dengue virus)、黄热病毒(Yellow fever virus)、西尼罗河病毒(West Nile virus)、日本脑炎病毒(Japanese encephalitis virus)或C型肝炎病毒(Hepatitis C virus)。
  10. 如权利要求8所述的用途，其中该酪梨萃取物包括avocadenol B作为活性成分。
  11. 如权利要求8所述的用途，其中该酪梨萃取物包括(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)作为活性成分。
  12. 一种avocadenol B用于制备治疗或预防黄热病毒科(Flaviviridae family)病毒感染的药物的用途。
  13. 一种(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)用于制备治疗或预防黄热病毒科(Flaviviridae family)病毒感染的药物的用途。
  14. 一种抑制黄热病毒科(Flaviviridae family)病毒复制活性或病毒性发炎反应的保健食品，包括一有效量的酪梨(Persea americana)萃取物为活性成分以及一医药学上可接受的载剂或盐类。
  15. 如权利要求14所述的保健食品，其中黄热病毒科 (Flaviviridae family)病毒包括：登革病毒(Dengue virus)、黄热病毒(Yellow fever virus)、西尼罗河病毒(West Nile virus)、日本脑炎病毒(Japanese encephalitis virus)或C型肝炎病毒(Hepatitis C virus)。
  16. 如权利要求14所述的保健食品，其中该酪梨萃取物包括avocadenol B。
  17. 如权利要求14所述的保健食品，其中该酪梨萃取物包括(2R,4R)-1,2,4-三羟基十七碳-16-炔((2R,4R)-1,2,4-trihydroxyheptadec-16-yne)。
PCT/CN2015/091904 2015-10-14 2015-10-14 酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔的用途，以及包含酪梨萃取物的保健食品 WO2017063150A1 (zh)

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PCT/CN2015/091904 WO2017063150A1 (zh) 2015-10-14 2015-10-14 酪梨萃取物、avocadenol B及(2R,4R)-1,2,4-三羟基十七碳-16-炔的用途，以及包含酪梨萃取物的保健食品

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CN1156404A (zh) * 1994-05-20 1997-08-06 诺瓦瓦克斯有限公司 抗微生物水包油乳液
WO2004014298A2 (en) * 2002-07-31 2004-02-19 Access Business Group International Llc Composition and method for lowering cholesterol
WO2010026595A2 (en) * 2008-09-08 2010-03-11 Polyol Biotech Ltd. Cosmetic compositions comprising polyhydroxyltate fatty alcohols and derivatives and uses thereof

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