WO2006112686A1 - Novel lignan compounds, their preparation methods and their uses - Google Patents

Novel lignan compounds, their preparation methods and their uses Download PDF

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WO2006112686A1
WO2006112686A1 PCT/KR2006/001524 KR2006001524W WO2006112686A1 WO 2006112686 A1 WO2006112686 A1 WO 2006112686A1 KR 2006001524 W KR2006001524 W KR 2006001524W WO 2006112686 A1 WO2006112686 A1 WO 2006112686A1
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lignan
present
chemical formula
acid
lignan compound
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PCT/KR2006/001524
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French (fr)
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Mee Ree Kim
Dai Eun Sok
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The Industry & Academic Cooperation In Chungnam National University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A61F7/0053—Cabins, rooms, chairs or units for treatment with a hot or cold circulating fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N2/00—Magnetotherapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A61F2007/0001—Body part
    • A61F2007/0018—Trunk or parts thereof
    • A61F2007/0022—Abdomen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A61F2007/0054—Heating or cooling appliances for medical or therapeutic treatment of the human body with a closed fluid circuit, e.g. hot water
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0659—Radiation therapy using light characterised by the wavelength of light used infrared
    • A61N2005/066—Radiation therapy using light characterised by the wavelength of light used infrared far infrared
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0004—Applications of ultrasound therapy
    • A61N2007/0008—Destruction of fat cells

Definitions

  • the present invention relates to a novel lignan compound having anti-oxidative and neuroprotective activity, more particularly to a novel lignan compound having anti- oxidative and neuroprotective activity that is isolated from an extract of Petasites aponicus and health foods and pharmaceutical compositions comprising the same.
  • the excitatory neurotransmitter such as L-kainic acid, L-glutamic acid and domoic acid
  • the L-kainic acid stimulates the incorporation of cation such as sodium and calcium through an ion channel of brain nerve cell and causes polarization between membranes of nerve cells in order to excite the nerve cell.
  • excito-toxicity caused by the L-kainic acid, makes endogenous glutamic acids or other excitatory neurotransmitters discharged in an excessive amount at a time so as to damage nerve tissues and affect neuropathgenesis (Choi, et al., Ann. Rev. Neurosci., 13, 171-182, 1990).
  • the L-kainic acid kills pyramidal neurons located in C1 and CA3 domains of hippocampus selectively so as to cause epilepsies following limbic motor seizure or convulsion (Ben-Ari, Y., Limbic seizure and brain damage produced by kainic acid:mechanisms and relevance to human temporal lobe epilepsy. Neuroscience, 14(2): 375-403, 1985).
  • the kainic acid has been used as a therapeutic agent to induce temporal lobe epilepsy in experimental animals. Further, in practice, it is demonstrated as a model system to develop a drug inhibiting neural toxicity selectively in the hippocampus.
  • Extracts from Petasites plants have been used for thousands of i years for therapeutic purposes in folk medicine.
  • the extract of Petasites hybridus a native European perennial, has been found to contain spasmolytic (Ziolo and Samochowiec, 1988) and analgesic (Grossman and Schmidramsl, 2001) compounds such as petasin or isopetasin.
  • Petasites formosanus an indigenous species of Petasites in Taiwan, has been used for the treatment of hypertension; one of hypotensive constituents was identified to be S-petasin, a sesquiterpene (Wang et al., 2001).
  • phenolic compounds such as phenylpropenoyl derivatives were isolated from the leaves of this species (Lin et ai, 2001 ; Lin et al., 2004).
  • Petasites japonicus Sieb. et Zucc.
  • Maxim. Compositae
  • petasinophenol Mozushina et al., 2002
  • flavonoid glycosides Mozushina et al., 2003
  • isolated from P. japonicus inhibited eukaryotic DNA polymerase rhamda and DNA polymerase alpha, respectively.
  • the butanol-soluble fraction was found to contain a remarkable antioxidative action in DPPH radical scavenging assay.
  • the present invention provides a lignan compound having the Chemical Formula 1 , or a pharmaceutically acceptable salt or hydrate thereof: [Chemical Formula 1]
  • the present invention provides a pharmaceutical composition having neuroprotective activity, which comprises a effective amount of the lignan compound according to the present invention as an active ingredient, together with a pharmaceutically acceptable carrier.
  • the present invention provides a health food comprising the lignan compound according to the present invention as an active ingredient.
  • the present invention provides a method for isolating a llignan compound comprising the following steps: 1) extracting Petasites japonicus with methanol to obtain a solid extract; 2) extracting the solid extract with butanol after suspending it in water; 3) eluting the butanol extract with a stepwise gradient of chloroform and methanol on a silica gel colum to separate multiple fractions;
  • the term "pharmaceutically acceptable salt” means alkali metal salts, alkaline earth metal salts, salts of inorganic acids, salts of organic acids, salts with amino acids, etc.
  • the concrete examples of the pharmaceutical salts include salts of inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; and salts of organic carboxylic acids, such as acetic acid, triflu- oroacetic acid, citric acid, formic acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid; and the like.
  • These acid addition salts can be prepared by known methods on the basis of the said Chemical
  • hydrate means a compound of the present invention or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound thereto by non-covalent intermolecular forces.
  • Other terms used herein can be interpreted as having their usual meanings in the art to which the present invention pertains, unless their definitions are explained in the description.
  • the lignan compounds of the present invention are novel Furofuran lignan compounds isolated from Petasites japonicus.
  • the lignan compounds have antioxidative and neurotoxity inhibitory activity and therefore can protect the brain against oxidative neurotoxicity that disrupt or degenerate neuronal cells.
  • the lignan compound of the present invention has strong antioxidative activity and suppresses oxidative activity of kainic acid that is an excitatory neurotransmitter, which can prevent failure of neuronal tissure, generation of neuronal disease, death of pyramidal neurons in hippocampus and therefore alleviate epilepsy by suppresing excessive secretion of the excitatory transmitter.
  • the lignan compounds according to the present invention have excellent antioxidant activity, relief of neurotoxicity and neuroprotective effect and thus, can be usefully used in the prevention and treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic- ischemic injury, and chronic spinal cord injury.
  • brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic- ischemic injury, and chronic spinal cord injury.
  • the BuOH-soluble fraction of the leaves of P. japonicus was subjected to repeated column chromatographies on normal and reverse phase silica gels and HPLC, and finally the lignan compound was obtained as an amorphous powder with a negative optical rotation, [ ⁇ ] D -10.0°.
  • the molecular formula of lignan compound of the present invention was found to be C2 6 H 32 O 12 , based on a quasi-molecular ion at m/z 535 [M - H] "
  • the active neuroprotective compound which was isolated as above, was identified by LC/MS and NMR to be the following Chemical Formula 2 and named as 2- (4'-hydroxy-3'-methoxyphenyl)-6-(4"-hydroxy-3"-methoxyphenyl)-8-hydroxy-3,7- dioxabicyclo[3.3.0]octane 4'-O-(D-glucopyranoside) according to IUPAC nomenclature.
  • Chemical Formula 2 2- (4'-hydroxy-3'-methoxyphenyl)-6-(4"-hydroxy-3"-methoxyphenyl)-8-hydroxy-3,7- dioxabicyclo[3.3.0]octane 4'-O-(D-glucopyranoside) according to IUPAC nomenclature.
  • the natural source of the lignan compound is not limited to P. japonicus but can be any natural plant which contains the lignan compound.
  • the method for extracting and isolating the lignan compound is not limited to the method specifically disclosed in the description, but any conventional methods which can extract and isolate the lignan compound can be used.
  • the compound of Chemical Formula 2 can be hydrolysed to obtain Aglycone of Chemical Formula 3 and D-glucose
  • Reaction Formula 1 is one example for preparing the compound of Chemical Formula 3.
  • any method can be used to obtain Aglycone from the lignan compound of Chemical Formula 2 which can hydrolyze the compound. Therefore, any enzyme can be used, which have the same activity as naringinase.
  • the Aglycone of Chemical Formula 3 has the same activity as the lignan compound of Chemical Formula 2 and therefore can be used in place of the lignan compound of Chemical Formula 2.
  • the lignan compound of the present invention and its pharmaceutically acceptable salts or hydrates are combined with pharmaceutically acceptable carriers and depending upon use, may be formulated into oral dosage forms, such as tablets, soft or hard capsules, chewing capsules, powder, liquid agents or suspension, and parenteral dosage forms such as injectable liquid agents or suspension.
  • binders such as Arabia gum, corn starch, minute crystallic cellulose or gelatin; excipients such as calcium diphosphate or lactose; disintegrants such as alginic acid, corn starch or potato starch; lubricants such as magnesium stearic acid; sweetening agents such as sucrose or saccharin; and flavoring agents such as peppermint, methyl salicylic acid or fruit flavor may be included.
  • liquid carriers such as polyethylene glycol or fat oil in addition to above- mentioned components may be included.
  • the injectable agent for parenteral dosage forms may be administered parenterally, for example, subcutaneously, intravenously, intramuscularly or peritoneally.
  • the injectable solution or suspension may be prepared by blending active ingredients of pharmaceutically acceptable carriers such as water, salt water, soluble dextrose, and other sucrose solution, non-volatile oil, ethanol, glycerin, polyethylene glycol and propylene glycol in effective amounts homogeneously.
  • active ingredients such as water, salt water, soluble dextrose, and other sucrose solution, non-volatile oil, ethanol, glycerin, polyethylene glycol and propylene glycol in effective amounts homogeneously.
  • other additives such as anti-bacterial agents, chelates, buffering agents may be included.
  • the pharmaceutically acceptable carriers can be any additives if they are pharmaceutically pure, substantially non-toxic and not affecting the action of the active components.
  • compositions comprising the lignan compound of the present invention in an effective amount can be administered orally in 25 to 100 mg/kg weight per day once or several times and preferably, administered separately three times to four times per day.
  • effective amount means an amount of active ingredients effective to alleviate, ameliorate or prevent symptoms of disease or decrease or delay the onset of clinical markers or symptoms of disease.
  • the individual patient with a particular body weight and life style may readily determine the proper dosage by starting out with the general dosage level set forth above and adjust the dosage as necessary to alleviate the disease.
  • the lignan compound of the present invention was extracted from petasites japonicus which have been used as a traditional food and therefore is edible without an adverse effect to the human body.
  • the lignan compound can be used as an active ingredient in health food, such as solid food or liquid drink.
  • the lignan compound of the present invention can be mixed with conventional sweetening agents, vitamins, amino acids, minerals organic acids, odorants, preservatives, etc. to formulate solution, suspension or any conventional formulation form, which may be a good health food that can prevent adult diseases such as neuronal disease due to its antioxidative activity. Therefore, the present invention can provide a new health food which comprises the lignan compound as an active ingredient, and, if required, additionally contains assitant components selected from other galenical extracts, vitamins, amino acids, sweetening agents, minerals, organic acids, odorant, fruit juices or preservatives, etc.
  • the vitamins used in the present invention can be vitamin A, vitamin Bi, B 2 , B 6 ,
  • Bi 2 vitamin C, vitamin D, vitamin E, vitamin F, vitamin G, vitamin H, vitamin K, or conventional vitamins required in the human body.
  • lipid-soluble vitamin it is preferable to add a sutable amount of emulsifier.
  • amino acids used in the present invention can be glycine, alanine, valine, glutamic acid, lycin, arginin, histidine, phenylalanine, tyrosine, tryptophan, proline, oxyproline, etc.
  • the organic acids used in the present invention can be citric acid, tartaric acid, malic acid, acetic acid, or other edible organic acid.
  • the odorant used in the present invention can be mint water, mint oil, orange oil, strawberry essence, or other conventional fruit odor or plant essence.
  • the fruit juice used in the present invention can be apple juice, strawberry juice, orange juice, lemon juice, peach juice, pineapple juice, banana juice, paraya juice, plum juice or other conventional fruit juice.
  • the above assitant components selected from other galenical extract, vitamins, amino acids, sweetening agent, minerals, organic acids, odorant, fruit juice or preservative can be added to the health food in a conventional amount.
  • Fig. 1 shows a schematic draw of extraction, fractionation, and isolation of a lignan compound of the present invention from Petasites japonicus
  • Fig. 2 shows a Mass spectrum of a lignan compound of the present invention
  • Fig. 3 is a graph showing the effect of a lignan compound of the present invention on TBARS in brain tissue of mice administered with kainic acid
  • Fig. 4 is a graph showing the effect of a lignan compound of the present invention on total glutathione level in the brain tissue of mice administered with kainic acid;
  • Fig. 5 is a graph showing the change of glutathione peroxidase activity in the brain tissue of mice administered with kainic acid
  • Fig. 6 is a graph showing the change of glutathione reductase activity in the brain tissue of mice administered with kainic acid
  • Fig. 7 is a graph showing the effect of a lignan compound of the present invention on Evans blue uptake into the brain of mice administerd with kainic acid.
  • the leaves of P. japonicus were extracted with MeOH at room temperature to obtain 220 g of the solid extract.
  • the MeOH extract was suspended in H 2 O and extracted successively with hexane (3 L), CHCI 3 (3 L), EtOAc (3 L), and BuOH (3 L) to give the hexane (8.1 g), CHCI 3 (7.6 g), EtOAc (8.9 g), and BuOH-soluble fractions (26.6 g), respectively.
  • the extraction was performed three times.
  • the BuOH- soluble fraction (78 g) was chromatographed on a silica gel column (5 x 60 cm) eluted with a stepwise gradient of CHCI 3 and MeOH to yield four fractions: 10% MeOH 3000 ml (A), 30% MeOH 2,500 ml (B), 50% MeOH 4000 ml (C) and 100% MeOH 3000 ml (D). The four fractions were dried into 2.2g(A), 5.3g(B), 7.Og (C) and 3.3g (D).
  • the fraction (B) was chromatographed on a RP C-18 column (3.5 cm x 60 cm) eluted with MeOH-H 2 O (1 : 4) to obtain four subfractions: 5000 ml (1), 1000 ml (2), 1000 ml (3) and 1000 ml (4).
  • the four subfraction were dried into B-1 : 70mg, B-2: 291.8mg, B- 3:220.7mg and B-4: 238.5mg.
  • the fraction B-2 was precipitated from MeOH at 4 0 C to yield 88 mg of amorphous powder.
  • the amorphous powder was identified by LC/MS and NMR as Example 3(1) to be 2-(4-hydroxy-3-methoxyphenyl)-6-(4-hydroxy-3- methoxyphenyl)-8-hydroxy-3,7-dioxabicyclo[3.3.0]octane-4-O-(-D-glucopyranoside).
  • the schematic draw of extraction, fractionation, and isolation of lignan compound of the present invention from Petasites japonicus is depicted in Fig. 1.
  • Naringinase (100 mg, from Penicillium decumbens) was added to a suspension of lignan compound of Example 1(1) (8 mg) in 50 mM acetate buffer (pH 5.5) and the mixture was stirred at 37 °C for 5 h.
  • the reaction mixture was extracted with EtOAc (10 mL x 3).
  • the extract was isolated by silica gel TLC (EtOAc-MeOH-H 2 O-AcOH, 65 : 20 : 15 : 15).
  • the spot on the TLC plate was visualized by an anisaldehyde-H 2 SO 4 reagent.
  • the stereochemistry of glucose was determined by GC method as described in Example 3(2).
  • D-glucose (Min et a/., 2000).
  • the configuration of the glycosidic linkage for the glucopyranoside unit was determined to be ⁇ form, based on the J 1 2 value of the anomeric proton at 7.2 Hz (£ 4.89).
  • the linkage of D-glucose moiety was determined, based on the HMBC correlation between at 4H 4.89 (H-V”) and S 0 145.9 (C-4').
  • the stereochemistry of glucose seperated from aglycone was determined by GC method.
  • the sugar derivative thus obtained showed a retention time of 21.30 min, identical to that of authentic D-glucose. Therefore, it is noted that the enzyme can hydrolyze D- glucose, which is bound to the present lignan compound as a glucoside, to give Aglycone.
  • mice To examine the neuroprotective activity of the present lignan compounds suppressing the neurotoxicity of kainic acid, an oxidative-stress generator, in brain tissue, the effect of the present lignan compounds on suppression of seizure and mortality caused by kainic acid was analyzed using mice.
  • ICR Mice were administered orally with the compounds of Example 1(1) and (2) (50 mg/kg and 100 mg/kg), suspended in saline, using an esophagus needle for 4 days consecutively before kainic acid injection. Then, the mice were injected intraperitoneally (50 mg/kg) with kainic acid, dissolved in saline, and control mice were administered with the same volume of saline. Following the challenge with kainic acid, the onset time of seizure and the viability (mortality) of the animals were monitored for 60 min.
  • the oral administration of the present lignan compounds delayed the onset time of seizure more than 2 X and increased the viability more than 2 X compared with the control group.
  • quercetin despite its antioxidant action, showed no significant effect on the seizure and mortality caused by kainic acid.
  • the present lignan compounds have examine the neuroprotective activity suppressing the neurotoxicity of kainic acid.
  • Brain tissue rinsed with 0.15 M KCI solution containing 2 mM EDTA, was homogenized in 9 volumes of 10 mM phosphate buffer (pH 7.4) using a tissue homogenizer with a Teflon pestle.
  • To the brain homogenate (1.0 ml) was added 1.0 ml of 8.1 % SDS, 2 ml of 20% acetic acid and 1 ml of 0.75% thiobarbituric acid (TBA).
  • the mixture was boiled for 30 min and then centrifuged (14,000 rpm, 10 min), and then the absorbance of the supernatant was measured at 533 nm.
  • Example 1(1) and 1(2) compounds were related to the prevention against oxidative stress in the brain tissue of mice intoxicated with kainic acid, we examined the effect of the compounds on the level of
  • TBARS a biochemical marker of oxidative stress
  • Figure 3 shows that TBARS value was increased to 168 % of control value (P ⁇ 0.05) in the homogenate of whole brain of mice treated with kainic acid.
  • Example 1(1)(40 mg/kg) reduced the TBARS value, which was enhanced by kainic acid challenge, to the level of control group (P ⁇ 0.05).
  • Example 1(2) compound(40 mg/kg) suggest that the neuroprotective action of lignan compounds of the present invention may be in part due to its preventive action against oxidative stress in the brain.
  • Brain tissue (about 0.27-0.30 g wet wt) was pulverized in a cooled ceramic percussion mortar with 6 % metaphosphoric acid, and the mixture was centrifuged (27,00Og, 20 min) at 4 0 C .
  • Total GSH was determined enzymatically according to a published procedure with a slight modification.
  • To 0.02 ml of supernatant was added 0.39 mL of 100 mM phosphate buffer (pH 7.4) containing 5 mM EDTA, 0.025 ml of 10 mM DTNB and 0.08 ml of 5 mM NADPH. After 3 min equilibration at 25 ° C , the reaction was started by adding 1 unit of GSH reductase.
  • the formation of 2-nitro-5-thiobenzoic acid was continuously recorded at 412 nm with a UV/VIS spectrophotometer.
  • the total amount of GSH in the samples was determined from a standard curve obtained by plotting the known
  • Example 1(1) and 1(2) compounds were examined the effect of Example 1(1) and 1(2) compounds on the level of total glutathione, another biomarker of oxidative stress, in the brain of mice administered with kainic acid (50 mg/kg).
  • the administration of kainic acid (50 mg/kg) reduced the level of total glutathione in the cytosol fraction of brain homogenate to approximately 73 % of control level.
  • the administration of Example 1(1) and 1(2) (40 mg/kg) elevated the level of total GSH in the brain cytosol from 73 % in kainic acid- treated group to 97 % of control group, further confirming the notion that the neuroprotective effect of lignan compounds of the present invention may be related to its prevention against oxidative stress in the brain.
  • a mixture containing 0.1 M phosphate buffer (pH 7.0), 3 mM EDTA, 1 mM GSH, 0.1 mM NADPH, 2 units of GSH reductase and 0.05 ml of supernatant was incubated for 3 min, and 0.01 ml of 10 mM cumene hydroperoxide was then added to the reaction mixture.
  • GSH reductase activity was measured in a mixture containing 0.1 M phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM GSSG, 0.1 mM NADPH, and 0.05 ml of the supernatant.
  • Example 1(1) and 1(2) the change in glutathione-related enzymes such as GSH peroxidase and GSH reductase in lignan compound/kainic acid-treated group was examined.
  • Fig. 5 indicates that the treatment with Example 1(1) and 1(2) appeared to restore the loss of the GSH peroxidase activity, which was caused by kainic acid.
  • Fig. 6 there was no significant change of GSH-reductase activity among vehicle- treated group, kainic acid-treated group, and lignan compound/kainic acid-treated group (Fig. 6), suggesting that under the experimental condition used, the neurotoxicity of kainic acid may not be sufficient to alter the level of GSH-reductase activity.
  • the brain tissue was homogenized with 50% trichloroacetic acid in fourfold, and the homogenate was centrifuged at 30,000 rpm for 20 min and the absorbance was measured at 615 nm. Values were calculated as Evans Blue mg/g tissue.
  • Example 1 (1) and 1(2) the protective effect of Example 1 (1) and 1(2) on damage of blood brain barrier caused by kainic acid (50 mg/kg) was examined.
  • Evans blue (3 mL/kg, 2% in saline) was iv administered to mice, either the lignan compound- treated group or the vehicle-treated group, 30 min before KA administration, and the brain tissue was analyzed for Evans blue retaining.
  • the tissue was homogenized, and the absorbance of the supernatant of the brain homogenate was measured at 615 nm (Figure 7), the amount of Evans blue retained in the brain following KA intoxication was significantly higher as compared to control.
  • Example 1(1) and 1(2) significantly reduced the Evans blue level in the KA-treated group compared to the control level.
  • the lignan compounds of the present invention is assumed to prevent against oxidative damage of the blood-brain barrier in KA-treated mice.
  • the syrup containing 2% (m/v) of lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. An acid-added salt of the present lignan compounds, saccharin and sugar were dissolved in 8Og of warm water. The solution was cooled and mixed with a solution comprising glycerin, saccharin, flavor, ethanol, sorbic acid and distilled water. The mixture was added by water into 100 ml. The acid-added salt may be replaced with another pharmaceutically acceptable salt.
  • the tablet containing lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. Hydrochloride of the present lignan compounds 250 g was mixed with lactose 175.9 g, potato starch 180 g and colloidal silicic acid 32 g. The mixture was added with 10% gelatin solution, pulverized and passed through 14 mesh sieve. The resultant was dried and added with potato 160 g, talc 50 g and Mg. sterarate 5 g. The final mixture was compressed into tablets by common techniques.
  • the injection containing lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. Hydrochloride of the present lignan compounds 1 g, NaCI 0.6 g and ascorbic acid 0.1 g were dissolved in distilled water into 100 ml_. The solution was inputted in a vial and sterilized by heating at 200 0 C in 30 min.
  • the lignan compounds or their pharmaceutically acceptable salt or hydrate according to the present invention have excellent antioxidant activity, relief of neurotoxicity and neuroprotective effect and thus, can be usefully used in the prevention and treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic- ischemic injury and chronic spinal cord injury.

Abstract

The present invention relates to novel lignan compounds having antioxidative and neuroprotective activity, more particularly, to a lignan compound having Chemical Formula 1 or a pharmaceutically acceptable salt or hydrate thereof and health food and pharmaceutical compositions comprising the same. The lignan compounds according to the present invention have excellent antioxidant activity, relief of neurotoxicity and neuroprotective effect and thus, can be usefully used in the prevention and treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic-ischemic injury and chronic spinal cord injury.

Description

NOVEL LIGNAN COMPOUNDS, THEIR PREPARATION METHODS AND THEIR
USES
Technical Field
The present invention relates to a novel lignan compound having anti-oxidative and neuroprotective activity, more particularly to a novel lignan compound having anti- oxidative and neuroprotective activity that is isolated from an extract of Petasites aponicus and health foods and pharmaceutical compositions comprising the same.
Background Art
Generally, the excitatory neurotransmitter, such as L-kainic acid, L-glutamic acid and domoic acid, is an excitatory transmitter playing an important role in brain nerves. Especially, it is disclosed that the L-kainic acid stimulates the incorporation of cation such as sodium and calcium through an ion channel of brain nerve cell and causes polarization between membranes of nerve cells in order to excite the nerve cell. In addition, it is reported that excito-toxicity, caused by the L-kainic acid, makes endogenous glutamic acids or other excitatory neurotransmitters discharged in an excessive amount at a time so as to damage nerve tissues and affect neuropathgenesis (Choi, et al., Ann. Rev. Neurosci., 13, 171-182, 1990).
Besides, it is illustrated that if administered to the whole body, the L-kainic acid kills pyramidal neurons located in C1 and CA3 domains of hippocampus selectively so as to cause epilepsies following limbic motor seizure or convulsion (Ben-Ari, Y., Limbic seizure and brain damage produced by kainic acid:mechanisms and relevance to human temporal lobe epilepsy. Neuroscience, 14(2): 375-403, 1985).
For this reason, the kainic acid has been used as a therapeutic agent to induce temporal lobe epilepsy in experimental animals. Further, in practice, it is demonstrated as a model system to develop a drug inhibiting neural toxicity selectively in the hippocampus.
Extracts from Petasites plants (Compositae) have been used for thousands of i years for therapeutic purposes in folk medicine. The extract of Petasites hybridus, a native European perennial, has been found to contain spasmolytic (Ziolo and Samochowiec, 1988) and analgesic (Grossman and Schmidramsl, 2001) compounds such as petasin or isopetasin. Petasites formosanus, an indigenous species of Petasites in Taiwan, has been used for the treatment of hypertension; one of hypotensive constituents was identified to be S-petasin, a sesquiterpene (Wang et al., 2001). Additionally, phenolic compounds such as phenylpropenoyl derivatives were isolated from the leaves of this species (Lin et ai, 2001 ; Lin et al., 2004). Separately, the leaves of Petasites japonicus (Sieb. et Zucc.) Maxim. (Compositae), a perennial plant widely grown in Japan and Korea, is used as an edible vegetable. Recent studies showed that petasinophenol (Mizushina et al., 2002) and flavonoid glycosides (Mizushina et al., 2003), isolated from P. japonicus, inhibited eukaryotic DNA polymerase rhamda and DNA polymerase alpha, respectively. In the course of screening antioxidant compounds from the extract of leaves of P. japonicus, the butanol-soluble fraction was found to contain a remarkable antioxidative action in DPPH radical scavenging assay.
In the present invention, we isolated a new lignan compound having neuroprotective activity against oxidative damage in the brain of mice treated with kainic acid, from the butanol-soluble fraction of leaves of P. japonicus and completed the present invention.
Disclosure Of Invention
Therefore, it is an object of the present invention to provide novel lignan compounds having neuroprotective activity.
It is another object of the present invention to provide a health food comprising the lignan compounds as an active ingredient.
It is another object of the present invention to provide a pharmaceutical composition having neuroprotective activity comprising the lignan compounds as an active ingredient.
It is another object of the present invention to provide a method for isolating the lignan compounds from Petasites japonicus.
Hereinafter, the present invention will be described more clearly as follows.
In order to achieve the above object, the present invention provides a lignan compound having the Chemical Formula 1 , or a pharmaceutically acceptable salt or hydrate thereof: [Chemical Formula 1]
Figure imgf000004_0001
wherein, R is hydrogen or In order to achieve the above object, the present invention provides a pharmaceutical composition having neuroprotective activity, which comprises a effective amount of the lignan compound according to the present invention as an active ingredient, together with a pharmaceutically acceptable carrier.
In order to achieve the above object, the present invention provides a health food comprising the lignan compound according to the present invention as an active ingredient.
In order to achieve the above object, the present invention provides a method for isolating a llignan compound comprising the following steps: 1) extracting Petasites japonicus with methanol to obtain a solid extract; 2) extracting the solid extract with butanol after suspending it in water; 3) eluting the butanol extract with a stepwise gradient of chloroform and methanol on a silica gel colum to separate multiple fractions;
4) eluting the separated fractions with methanol to separate multiple fractions; and, 5) obtaining a lignan compound of the present invention from the separated fractions. Now, the present invention will be described in detail.
As used herein, the term "pharmaceutically acceptable salt" means alkali metal salts, alkaline earth metal salts, salts of inorganic acids, salts of organic acids, salts with amino acids, etc. The concrete examples of the pharmaceutical salts include salts of inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; and salts of organic carboxylic acids, such as acetic acid, triflu- oroacetic acid, citric acid, formic acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid; and the like. These acid addition salts can be prepared by known methods on the basis of the said Chemical
Formula 1.
As used herein, the term "hydrate" means a compound of the present invention or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound thereto by non-covalent intermolecular forces. Other terms used herein can be interpreted as having their usual meanings in the art to which the present invention pertains, unless their definitions are explained in the description.
The lignan compounds of the present invention are novel Furofuran lignan compounds isolated from Petasites japonicus. The lignan compounds have antioxidative and neurotoxity inhibitory activity and therefore can protect the brain against oxidative neurotoxicity that disrupt or degenerate neuronal cells.
Besides, the lignan compound of the present invention has strong antioxidative activity and suppresses oxidative activity of kainic acid that is an excitatory neurotransmitter, which can prevent failure of neuronal tissure, generation of neuronal disease, death of pyramidal neurons in hippocampus and therefore alleviate epilepsy by suppresing excessive secretion of the excitatory transmitter.
Therefore, the lignan compounds according to the present invention have excellent antioxidant activity, relief of neurotoxicity and neuroprotective effect and thus, can be usefully used in the prevention and treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic- ischemic injury, and chronic spinal cord injury. According to the present invention, the BuOH-soluble fraction of the leaves of P. japonicus was subjected to repeated column chromatographies on normal and reverse phase silica gels and HPLC, and finally the lignan compound was obtained as an amorphous powder with a negative optical rotation, [α]D -10.0°. The molecular formula of lignan compound of the present invention was found to be C26H32O12, based on a quasi-molecular ion at m/z 535 [M - H]" in the negative API-MS.
The active neuroprotective compound, which was isolated as above, was identified by LC/MS and NMR to be the following Chemical Formula 2 and named as 2- (4'-hydroxy-3'-methoxyphenyl)-6-(4"-hydroxy-3"-methoxyphenyl)-8-hydroxy-3,7- dioxabicyclo[3.3.0]octane 4'-O-(D-glucopyranoside) according to IUPAC nomenclature. [Chemical Formula 2]
Figure imgf000006_0001
However, the natural source of the lignan compound is not limited to P. japonicus but can be any natural plant which contains the lignan compound. Further, the method for extracting and isolating the lignan compound is not limited to the method specifically disclosed in the description, but any conventional methods which can extract and isolate the lignan compound can be used.
According to the following Reaction Formula 1 , the compound of Chemical Formula 2 can be hydrolysed to obtain Aglycone of Chemical Formula 3 and D-glucose
(βtype D-glucose) of Chemical Formula 4. Reaction Formula 1 is one example for preparing the compound of Chemical Formula 3.
[Reaction Formula 1] J
Figure imgf000007_0001
However, except for the above enzymatic hydrolysis method, any method can be used to obtain Aglycone from the lignan compound of Chemical Formula 2 which can hydrolyze the compound. Therefore, any enzyme can be used, which have the same activity as naringinase.
Further, the Aglycone of Chemical Formula 3 has the same activity as the lignan compound of Chemical Formula 2 and therefore can be used in place of the lignan compound of Chemical Formula 2.
When formulating a pharmaceutical composition of the present invention, the lignan compound of the present invention and its pharmaceutically acceptable salts or hydrates are combined with pharmaceutically acceptable carriers and depending upon use, may be formulated into oral dosage forms, such as tablets, soft or hard capsules, chewing capsules, powder, liquid agents or suspension, and parenteral dosage forms such as injectable liquid agents or suspension. When formulating the lignan compound of the present invention into oral dosage forms such as tablets, soft or hard capsules, chewing capsules, powder, liquid agents or suspension, binders such as Arabia gum, corn starch, minute crystallic cellulose or gelatin; excipients such as calcium diphosphate or lactose; disintegrants such as alginic acid, corn starch or potato starch; lubricants such as magnesium stearic acid; sweetening agents such as sucrose or saccharin; and flavoring agents such as peppermint, methyl salicylic acid or fruit flavor may be included. When formulating unit dosage form, liquid carriers such as polyethylene glycol or fat oil in addition to above- mentioned components may be included. In addition, the injectable agent for parenteral dosage forms, such as solution or suspension, may be administered parenterally, for example, subcutaneously, intravenously, intramuscularly or peritoneally. In general, the injectable solution or suspension may be prepared by blending active ingredients of pharmaceutically acceptable carriers such as water, salt water, soluble dextrose, and other sucrose solution, non-volatile oil, ethanol, glycerin, polyethylene glycol and propylene glycol in effective amounts homogeneously. Besides depending upon requirements, other additives such as anti-bacterial agents, chelates, buffering agents may be included.
Furthermore, the pharmaceutically acceptable carriers can be any additives if they are pharmaceutically pure, substantially non-toxic and not affecting the action of the active components.
The pharmaceutical compositions comprising the lignan compound of the present invention in an effective amount can be administered orally in 25 to 100 mg/kg weight per day once or several times and preferably, administered separately three times to four times per day. The term "effective amount" means an amount of active ingredients effective to alleviate, ameliorate or prevent symptoms of disease or decrease or delay the onset of clinical markers or symptoms of disease. The individual patient with a particular body weight and life style may readily determine the proper dosage by starting out with the general dosage level set forth above and adjust the dosage as necessary to alleviate the disease.
Further, the lignan compound of the present invention was extracted from petasites japonicus which have been used as a traditional food and therefore is edible without an adverse effect to the human body. The lignan compound can be used as an active ingredient in health food, such as solid food or liquid drink.
For example, the lignan compound of the present invention can be mixed with conventional sweetening agents, vitamins, amino acids, minerals organic acids, odorants, preservatives, etc. to formulate solution, suspension or any conventional formulation form, which may be a good health food that can prevent adult diseases such as neuronal disease due to its antioxidative activity. Therefore, the present invention can provide a new health food which comprises the lignan compound as an active ingredient, and, if required, additionally contains assitant components selected from other galenical extracts, vitamins, amino acids, sweetening agents, minerals, organic acids, odorant, fruit juices or preservatives, etc.
The vitamins used in the present invention can be vitamin A, vitamin Bi, B2, B6,
Bi2, vitamin C, vitamin D, vitamin E, vitamin F, vitamin G, vitamin H, vitamin K, or conventional vitamins required in the human body. In the case of lipid-soluble vitamin, it is preferable to add a sutable amount of emulsifier.
The amino acids used in the present invention can be glycine, alanine, valine, glutamic acid, lycin, arginin, histidine, phenylalanine, tyrosine, tryptophan, proline, oxyproline, etc.
The organic acids used in the present invention can be citric acid, tartaric acid, malic acid, acetic acid, or other edible organic acid.
The odorant used in the present invention can be mint water, mint oil, orange oil, strawberry essence, or other conventional fruit odor or plant essence.
The fruit juice used in the present invention can be apple juice, strawberry juice, orange juice, lemon juice, peach juice, pineapple juice, banana juice, paraya juice, plum juice or other conventional fruit juice.
The above assitant components selected from other galenical extract, vitamins, amino acids, sweetening agent, minerals, organic acids, odorant, fruit juice or preservative can be added to the health food in a conventional amount.
Brief Description of the Drawings The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;
Fig. 1 shows a schematic draw of extraction, fractionation, and isolation of a lignan compound of the present invention from Petasites japonicus;
Fig. 2 shows a Mass spectrum of a lignan compound of the present invention; Fig. 3 is a graph showing the effect of a lignan compound of the present invention on TBARS in brain tissue of mice administered with kainic acid; Fig. 4 is a graph showing the effect of a lignan compound of the present invention on total glutathione level in the brain tissue of mice administered with kainic acid;
Fig. 5 is a graph showing the change of glutathione peroxidase activity in the brain tissue of mice administered with kainic acid; Fig. 6 is a graph showing the change of glutathione reductase activity in the brain tissue of mice administered with kainic acid; and
Fig. 7 is a graph showing the effect of a lignan compound of the present invention on Evans blue uptake into the brain of mice administerd with kainic acid.
Best Mode for Carrying Out the Invention
Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
<Example 1> Materials
The leaves of Petasites japonicus were collected from agricultural fields in
Kongju, Korea in May, 2004, and verified by Dr. Young Jin Choi, Herbarium of Wild Vegetable Experiment Station, Kangwon-Do, Korea.. A voucher specimen was deposited in the Herbarium of Wild Vegetable Experiment Station, Kangwon-Do, Korea. Kainic acid, glutathione reductase (Type III from bakers yeast), reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5'-dithio-2-nitrobenzoic acid (DTNB), NADP, NADPH, thiobarbituric acid (TBA) and 1 ,1-Diphenyl-2-picrylhydrazyl (DPPH) were products of Sigma Chemical Co. (St. Louis, MO).
<Example 2> Preparation of lignan compounds of the present invention
(1) Preparation of 2-(4-hydroxy-3-methoxyphenyl)-6-(4-hydroxy-3- methoxyphenyl)-8-hydroxy-3,7-dioxabicyclo[3.3.0]octane-4-O-(-D-glucopyranoside)
The leaves of P. japonicus (1.5 kg) were extracted with MeOH at room temperature to obtain 220 g of the solid extract. The MeOH extract was suspended in H2O and extracted successively with hexane (3 L), CHCI3 (3 L), EtOAc (3 L), and BuOH (3 L) to give the hexane (8.1 g), CHCI3 (7.6 g), EtOAc (8.9 g), and BuOH-soluble fractions (26.6 g), respectively. The extraction was performed three times. The BuOH- soluble fraction (78 g) was chromatographed on a silica gel column (5 x 60 cm) eluted with a stepwise gradient of CHCI3 and MeOH to yield four fractions: 10% MeOH 3000 ml (A), 30% MeOH 2,500 ml (B), 50% MeOH 4000 ml (C) and 100% MeOH 3000 ml (D). The four fractions were dried into 2.2g(A), 5.3g(B), 7.Og (C) and 3.3g (D). The fraction (B) was chromatographed on a RP C-18 column (3.5 cm x 60 cm) eluted with MeOH-H2O (1 : 4) to obtain four subfractions: 5000 ml (1), 1000 ml (2), 1000 ml (3) and 1000 ml (4). The four subfraction were dried into B-1 : 70mg, B-2: 291.8mg, B- 3:220.7mg and B-4: 238.5mg. The fraction B-2 was precipitated from MeOH at 4 0C to yield 88 mg of amorphous powder. The amorphous powder was identified by LC/MS and NMR as Example 3(1) to be 2-(4-hydroxy-3-methoxyphenyl)-6-(4-hydroxy-3- methoxyphenyl)-8-hydroxy-3,7-dioxabicyclo[3.3.0]octane-4-O-(-D-glucopyranoside). The schematic draw of extraction, fractionation, and isolation of lignan compound of the present invention from Petasites japonicus is depicted in Fig. 1.
(2) Preparation of Aglicone
Naringinase (100 mg, from Penicillium decumbens) was added to a suspension of lignan compound of Example 1(1) (8 mg) in 50 mM acetate buffer (pH 5.5) and the mixture was stirred at 37 °C for 5 h. The reaction mixture was extracted with EtOAc (10 mL x 3). The extract was isolated by silica gel TLC (EtOAc-MeOH-H2O-AcOH, 65 : 20 : 15 : 15). The spot on the TLC plate was visualized by an anisaldehyde-H2SO4 reagent. The stereochemistry of glucose was determined by GC method as described in Example 3(2).
<Example 3> Analysis of physical properties of lignan compounds of the present invention
Optical rotation was measured with a JASCO DIP-370 digital polarimeter in MeOH. UV spectrum was recorded on a UV-2450 spectrometer. 1H and 13C NMR spectra were recorded on Varian Unity Inova 400 spectrometer with the tetramethylsilane as an internal standard. Chemical shifts were presented as ppm. API- MS was measured with a Perkin-Elmer SCIEX API III Biomolecular Mass Analyzer. The molecular formula of the present lignan compound was found to be C26H32O12, based on quasi-molecular ion at m/z 535 [M - H]" in the negative API-MS. The Mass spectrum of lignan compound of the present invention is shown in Fig. 2..
(1) lignan compound of the present invention
Molecular formula: C26H32Oi2API-MS m/z: 535 [M - H]", 373 [M - GIc - Hf Optical rotation: [α]D: -10.0°(c 0.23, MeOH); UV (MeOH) λmaχ (log ε): 204 (4.73), 229 (4.19), 279 (3.75) nm
1H-NMR (400 MHz, pyridine-cfe) & 7.10 (1 H, d, J = 2 Hz, H-2", 7.06 (1 H, d, J = 8.4 Hz, H-51, 6.95 (1 H, d, J = 2 Hz, H-21, 6.84 (2H, d, J = 2 Hz, H6'6", 6.71 (1 H1 d, J = 8.4 Hz1 H-5", 5.41 (1 H, d, J = 4 Hz, H-8), 4.89 (1 H, d, J = 7.2 Hz, H-1 '"), 4.76 (2H, d, J = 7.2 Hz, H-2,6), 4.12 (1H, dd, J = 8.8, 5.6 Hz, Hax-4), 3.91 (1 H, dd, J = 8.8, 2.6 Hz, HeqUa- 4), 3.77 (3H, s, OCH3-31, 3.76 (3H, s, OCH3-3"), 3.67 (1 H, dd, J = 11.6, 4.8 Hz, H-6111), 3.44 (1 H, dd, J = 11.6, 5.6 Hz, H-6"1), 3.30 (1 H, m, H-5"1), 3.26 (2H, m, H-2"1^111), 3.16 (1 H, m, H-4"1), 3.03 (1 H, m, H-5), 2.73 (1 H, d, J = 7.2 Hz, H-1).
13C-NMR (100 MHz, pyridine-d5) δ: 149.0 (C-31, 147.4 (C-3", 145.9 (C-41, 145.7 (C-4"), 135.9 (C-11), 134.0 (C-1", 118.8 (C-61), 118.0 (C-6"), 115.3 (C-51), 114.9 (C-5"), 110.7 (C-2"), 110.3 (C-21), 100.9 (C-8), 100.1 (C-1111), 86.1 (C-6), 82.5 (C-2), 77.0 (C-3111),
76.8 (C-5111), 73.2 (C-2111), 71.4 (C-4), 69.6 (C-4111), 62.0 (C-1), 60.6 (C-6111), 55.7 (OCH3- 3'), 55.5 (OCH3-3"), 53.3 (C-5).
(2) Aglycone
The enzymatic hydrolysis of the present lignan compound with naringinase yielded a monosaccharide unit, which was identified by co-TLC, as described in
Example 2(2). Its absolute configuration was determined by gas chromatography to be
D-glucose (Min et a/., 2000). The configuration of the glycosidic linkage for the glucopyranoside unit was determined to be β form, based on the J1 2 value of the anomeric proton at 7.2 Hz (£ 4.89). The linkage of D-glucose moiety was determined, based on the HMBC correlation between at 4H 4.89 (H-V") and S0 145.9 (C-4'). The stereochemistry of glucose seperated from aglycone was determined by GC method.
The sugar derivative thus obtained showed a retention time of 21.30 min, identical to that of authentic D-glucose. Therefore, it is noted that the enzyme can hydrolyze D- glucose, which is bound to the present lignan compound as a glucoside, to give Aglycone.
<Example 4> Effect of lignan compounds of the present invention on suppression of kainic acid
To examine the neuroprotective activity of the present lignan compounds suppressing the neurotoxicity of kainic acid, an oxidative-stress generator, in brain tissue, the effect of the present lignan compounds on suppression of seizure and mortality caused by kainic acid was analyzed using mice. ICR Mice were administered orally with the compounds of Example 1(1) and (2) (50 mg/kg and 100 mg/kg), suspended in saline, using an esophagus needle for 4 days consecutively before kainic acid injection. Then, the mice were injected intraperitoneally (50 mg/kg) with kainic acid, dissolved in saline, and control mice were administered with the same volume of saline. Following the challenge with kainic acid, the onset time of seizure and the viability (mortality) of the animals were monitored for 60 min.
[Table 1]
Effect of the compounds of the present invention on seizure in mice injected with kainic acid
Figure imgf000014_0001
[Table 2]
Effect of the compounds of the present invention on viability in mice injected with kainic acid
Figure imgf000014_0002
As a result, it was found that the oral administration of the present lignan compounds delayed the onset time of seizure more than 2 X and increased the viability more than 2 X compared with the control group. Under the same condition, quercetin, despite its antioxidant action, showed no significant effect on the seizure and mortality caused by kainic acid. Based on these observations, it is suggested that the present lignan compounds have examine the neuroprotective activity suppressing the neurotoxicity of kainic acid.
<Example 5> DPPH radical scavenging assay
To examinine the antioxidant effect of the present lignan compounds, scavenging activity of the present lignan compounds on DPPH radicals was measured as described previously (Sok D.-E., Oh S. H., Kim, Y.-B., Kim M. R., J. Agri. Food Chem., 51 , 4570- 4575, 2003; Blois M. S., Nature, 181 , 1199-2000, 1958). 0.135 ml of DPPH radical solution (0.15 mM) in methanol and 0.015 ml of various concentrations of the lignan compounds of Example 1(1) and (2) in DMSO were mixed in a microplate reader well. The mixture was kept at room temperature for 30 min, and the absorbance at 535 nm was measured. IC5O value was expressed as the concentration of compound to show 50 % inhibition of DPPH radicals.
[Table 3]
DPPH free radical scavenging activity of compounds
Figure imgf000015_0001
As a result, it was found that DPPH scavenging activity is proportional to the concentration of the present lignan compounds and the lignan compound of Example
1(2), which is emzymatically deglycosylated, has more than 4 X of DPPH scavenging activity than the liganan compound of Example 1(1), which is almost equal with that of alpha-tocopherol.
<Exarnple 6> Measurement of lipid peroxidation in Brain Tissue
Brain tissue, rinsed with 0.15 M KCI solution containing 2 mM EDTA, was homogenized in 9 volumes of 10 mM phosphate buffer (pH 7.4) using a tissue homogenizer with a Teflon pestle. To the brain homogenate (1.0 ml) was added 1.0 ml of 8.1 % SDS, 2 ml of 20% acetic acid and 1 ml of 0.75% thiobarbituric acid (TBA).
The mixture was boiled for 30 min and then centrifuged (14,000 rpm, 10 min), and then the absorbance of the supernatant was measured at 533 nm.
To see whether the neuroprotective action of Example 1(1) and 1(2) compounds were related to the prevention against oxidative stress in the brain tissue of mice intoxicated with kainic acid, we examined the effect of the compounds on the level of
TBARS, a biochemical marker of oxidative stress, in the brain of mice administered with kainic acid (50 mg/kg). Figure 3 shows that TBARS value was increased to 168 % of control value (P < 0.05) in the homogenate of whole brain of mice treated with kainic acid. Meanwhile, Example 1(1)(40 mg/kg) reduced the TBARS value, which was enhanced by kainic acid challenge, to the level of control group (P <0.05). Also, a similar results was observed with Example 1(2) compound(40 mg/kg). These results suggest that the neuroprotective action of lignan compounds of the present invention may be in part due to its preventive action against oxidative stress in the brain.
<Example 7> Determination of total GSH
Brain tissue (about 0.27-0.30 g wet wt) was pulverized in a cooled ceramic percussion mortar with 6 % metaphosphoric acid, and the mixture was centrifuged (27,00Og, 20 min) at 4 0C . Total GSH was determined enzymatically according to a published procedure with a slight modification. To 0.02 ml of supernatant was added 0.39 mL of 100 mM phosphate buffer (pH 7.4) containing 5 mM EDTA, 0.025 ml of 10 mM DTNB and 0.08 ml of 5 mM NADPH. After 3 min equilibration at 25 °C , the reaction was started by adding 1 unit of GSH reductase. The formation of 2-nitro-5-thiobenzoic acid was continuously recorded at 412 nm with a UV/VIS spectrophotometer. The total amount of GSH in the samples was determined from a standard curve obtained by plotting the known amount of GSH versus.
We examined the effect of Example 1(1) and 1(2) compounds on the level of total glutathione, another biomarker of oxidative stress, in the brain of mice administered with kainic acid (50 mg/kg). As shown in Fig. 4, the administration of kainic acid (50 mg/kg) reduced the level of total glutathione in the cytosol fraction of brain homogenate to approximately 73 % of control level. Then, the administration of Example 1(1) and 1(2) (40 mg/kg) elevated the level of total GSH in the brain cytosol from 73 % in kainic acid- treated group to 97 % of control group, further confirming the notion that the neuroprotective effect of lignan compounds of the present invention may be related to its prevention against oxidative stress in the brain.
<Example 8> Assay of GSH peroxidase and reductase
Brain tissue was homogenized in 9 volumes of 20 mM phosphate buffer containing 0.1 M KCI, 1 mM EDTA and 0.5% Triton X-100. The homogenate was centrifuged (15,00Og, 15 min), and the supernatant was re-centrifuged (105,00Og, 30 min). The last supernatant was retained for enzymatic assays. The assay of GSH peroxidase activity was carried out as described previously. A mixture containing 0.1 M phosphate buffer (pH 7.0), 3 mM EDTA, 1 mM GSH, 0.1 mM NADPH, 2 units of GSH reductase and 0.05 ml of supernatant was incubated for 3 min, and 0.01 ml of 10 mM cumene hydroperoxide was then added to the reaction mixture. GSH reductase activity was measured in a mixture containing 0.1 M phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM GSSG, 0.1 mM NADPH, and 0.05 ml of the supernatant.
In order to elucidate the mechanism responsible for the restoration of GSH level by Example 1(1) and 1(2) (40 mg/kg), the change in glutathione-related enzymes such as GSH peroxidase and GSH reductase in lignan compound/kainic acid-treated group was examined. Fig. 5 indicates that the treatment with Example 1(1) and 1(2) appeared to restore the loss of the GSH peroxidase activity, which was caused by kainic acid. However, there was no significant change of GSH-reductase activity among vehicle- treated group, kainic acid-treated group, and lignan compound/kainic acid-treated group (Fig. 6), suggesting that under the experimental condition used, the neurotoxicity of kainic acid may not be sufficient to alter the level of GSH-reductase activity.
<Example 9> Blood brain barrier breakdown during kainic acid-induced seizures
Animals were immobilized in a loose-fitting plaster cast, and then 3ml/kg of 2 % Evan-blue in saline solution was injected from tail vein as a visual indicator. Thirty min later, seizures were intraperitoneally induced by 50 mg/kg kainic acid administration. At the end of experiments, 60 min after the injection of kainic acid, mice were killed and the brains perfused transaortically with a saline solution and removed. Thereafter, the brain section was selected and cut. Sections were viewed under the video microscope. Separately, the brain tissue was homogenized with 50% trichloroacetic acid in fourfold, and the homogenate was centrifuged at 30,000 rpm for 20 min and the absorbance was measured at 615 nm. Values were calculated as Evans Blue mg/g tissue.
Separately, the protective effect of Example 1 (1) and 1(2) on damage of blood brain barrier caused by kainic acid (50 mg/kg) was examined. For the purpose, Evans blue (3 mL/kg, 2% in saline) was iv administered to mice, either the lignan compound- treated group or the vehicle-treated group, 30 min before KA administration, and the brain tissue was analyzed for Evans blue retaining. When the tissue was homogenized, and the absorbance of the supernatant of the brain homogenate was measured at 615 nm (Figure 7), the amount of Evans blue retained in the brain following KA intoxication was significantly higher as compared to control. Meanwhile, pretreatment with Example 1(1) and 1(2) significantly reduced the Evans blue level in the KA-treated group compared to the control level. Thus, the lignan compounds of the present invention is assumed to prevent against oxidative damage of the blood-brain barrier in KA-treated mice.
<Example 10> Preparion of formulations containing lignan compounds of the present invention
1. Preparation of Syrup
The syrup containing 2% (m/v) of lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. An acid-added salt of the present lignan compounds, saccharin and sugar were dissolved in 8Og of warm water. The solution was cooled and mixed with a solution comprising glycerin, saccharin, flavor, ethanol, sorbic acid and distilled water. The mixture was added by water into 100 ml. The acid-added salt may be replaced with another pharmaceutically acceptable salt.
[Talbe 4] Composition of the Syrup Hydrochloride of the present lignan compounds 2 g
Saccharin 0.8 g
Glycerin 8.O g
Flavor 0.04 g
Ethanol 4.0 g Sorbic acid 0.4 g
Distilled water Balance
2. Preparation of Tablet
The tablet containing lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. Hydrochloride of the present lignan compounds 250 g was mixed with lactose 175.9 g, potato starch 180 g and colloidal silicic acid 32 g. The mixture was added with 10% gelatin solution, pulverized and passed through 14 mesh sieve. The resultant was dried and added with potato 160 g, talc 50 g and Mg. sterarate 5 g. The final mixture was compressed into tablets by common techniques.
[Table 5] Composition of the Tablet
Hydrochloride of the present lignan compounds 250 g
Lactose 175.9 g
Potato starch 18O g Colloidal silicic acid 32 g
10% Gelatin solution
Potato starch 16O g
Talc 5O g
Mg. Stearate 5 g
3. Preparation of Injection
The injection containing lignan compounds of the present invention or its pharmaceutically acceptable salt was prepared as follows. Hydrochloride of the present lignan compounds 1 g, NaCI 0.6 g and ascorbic acid 0.1 g were dissolved in distilled water into 100 ml_. The solution was inputted in a vial and sterilized by heating at 2000C in 30 min.
[Table 6] Composition of the Injection
Hydrochloride of the present lignan compounds 1 g NaCI 0.6 g
Ascorbic Acid 0.1 g
Distilled water Balance
Industrial Applicability As illustrated and confirmed above, the lignan compounds or their pharmaceutically acceptable salt or hydrate according to the present invention have excellent antioxidant activity, relief of neurotoxicity and neuroprotective effect and thus, can be usefully used in the prevention and treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic- ischemic injury and chronic spinal cord injury.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention.
Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A lignan compound having the Chemical Formula 1 , or a pharmaceutically acceptable salt or hydrate thereof: [Chemical Formula 1]
Figure imgf000021_0001
wherein, R is hydrogen or
2. A health food comprising the lignan compound according to claim 1 as an active ingredient.
3. The health food according to claim 2, which additionally comprises one or more of assistant foods selected from the group consisting of galenical extracts, vitamins, amino acids, sweetening agents, minerals, organic acids, odorants and fruit juices and one or more of assitant agents selected from the group consisting of excipient, diluent and antioxidant, and is formulated into liguid drug, soft capsule, tablet, granule or capsule.
4. A pharmaceutical composition having neuroprotective activity, which comprises an effective amount of the lignan compound according to claim 1 as an active ingredient, together with a pharmaceutically acceptable carrier.
5. The pharmaceutical composition according to claim 4, wherein the neuroprotective activity is performed by suppressing neurotoxicity of excitatory neurotransmitter.
6. The pharmaceutical composition according to claim 5, wherein the excitatory neurotransmitter is kainic acid.
7. A method for isolating a lignan compound comprising the following steps: 1) extracting Petasites japonicus with methanol to obtain a solid extract; 2) extracting the solid extract with butanol after suspending it in water;
3) eluting the butanol extract with a stepwise gradient of chloroform and methanol on a silica gel colum to separate multiple fractions;
4) eluting the separated fractions with methanol to separate multiple fractions; and,
5) obtaining a lignan compound having the Chemical Formula 2 from the separated fractions.
[Chemical Formula 2]
Figure imgf000022_0001
8. The method for isolating a llignan compound according to claim 7, which has an additional step for leaving out β-D-glucose from the lignan compound having the Chemical Formula by naringinase or β-D-glucosidase to obtain a lignan compound having the Chemical Formula 3. [Chemical Formula 3]
Figure imgf000023_0001
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US9101160B2 (en) 2005-11-23 2015-08-11 The Coca-Cola Company Condiments with high-potency sweetener
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WO2008057963A3 (en) * 2006-11-02 2008-09-04 Coca Cola Co High-potency sweetener composition with phytoestrogen and compositions sweetened therewith
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US9101161B2 (en) 2006-11-02 2015-08-11 The Coca-Cola Company High-potency sweetener composition with phytoestrogen and compositions sweetened therewith
WO2024027845A1 (en) * 2022-08-05 2024-02-08 中国科学院上海药物研究所 USE OF BIS-TETRAHYDROFURAN COMPOUND AS α-SYNUCLEIN AGGREGATION INHIBITOR, AND PHARMACEUTICAL COMPOSITION COMPRISING SAME

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