WO2017157260A1 - 一种苯丙素类化合物及其制备方法和应用 - Google Patents

一种苯丙素类化合物及其制备方法和应用 Download PDF

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WO2017157260A1
WO2017157260A1 PCT/CN2017/076455 CN2017076455W WO2017157260A1 WO 2017157260 A1 WO2017157260 A1 WO 2017157260A1 CN 2017076455 W CN2017076455 W CN 2017076455W WO 2017157260 A1 WO2017157260 A1 WO 2017157260A1
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acid
preparation
pharmaceutically acceptable
phenylpropanoid
water
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PCT/CN2017/076455
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English (en)
French (fr)
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张鹏
彭开锋
龚云
李伏君
刘逆夫
夏伯候
林丽美
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株洲千金药业股份有限公司
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Priority claimed from CN201610154676.5A external-priority patent/CN105708845B/zh
Priority claimed from CN201610154680.1A external-priority patent/CN105732736B/zh
Priority claimed from CN201610154851.0A external-priority patent/CN105777821B/zh
Application filed by 株洲千金药业股份有限公司 filed Critical 株洲千金药业股份有限公司
Publication of WO2017157260A1 publication Critical patent/WO2017157260A1/zh
Priority to US16/132,506 priority Critical patent/US10329316B2/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
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    • 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)
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    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/738—Rosa (rose)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/758—Zanthoxylum, e.g. pricklyash
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate

Definitions

  • the present invention relates to the field of medical technology, and more particularly to a phenylpropanoid compound and a preparation method and application thereof.
  • the components extracted and extracted from natural medicines have various structures and activities, and their separation, purification, structural modification, transformation and total synthesis have always been a main idea for the development of new drugs.
  • TNF- ⁇ is a cytokine that can directly kill tumor cells without significant toxicity to normal cells. It is one of the most active bioactive factors found to kill tumors so far, but its side effects are also very serious.
  • IL-1 ⁇ synergistically stimulates APC and T cell activation at local low concentrations, promotes B cell proliferation and secretion of antibodies, and performs immunomodulation. Endocrine effect occurs when produced in large quantities: induces acute protein synthesis in the liver, causing fever and cachexia.
  • IL-6 The human IL-6 gene is located on chromosome 7; the molecular weight of IL-6 is between 21 and 30 KD. It is mainly produced by mononuclear macrophages, Th2 cells, vascular endothelial cells, and fibroblasts. It can stimulate the proliferation of activated B cells, secrete antibodies; stimulate T cell proliferation and CTL activation; stimulate hepatocytes to synthesize acute phase proteins, participate in inflammatory reactions; promote blood cell development.
  • IL-6 can be synthesized by a variety of cells, including activated T cells and B cells, monocyte-macrophages, endothelial cells, epithelial cells, and fibroblasts. IL-6 acts on many target cells, including macrophages, hepatocytes, resting T cells, activated B cells, and plasma cells; its biological effects are also complex.
  • OH is the most active reactive oxygen species in biological systems and can cause oxidative damage to DNA, proteins and lipids in cells and organisms.
  • Macrophages can produce a variety of inflammatory mediators involved in the inflammatory response. Among them, NO is an important cellular inflammatory factor. NO participates in various physiological and pathological processes. Excessive NO promotes the development and development of inflammatory diseases, and can also induce other inflammatory factors. .
  • a new preparation method is sought to isolate new compounds from natural plants, inhibit the content of cellular inflammatory factor NO, inhibit the expression of cytokine TNF- ⁇ , and inhibit the activity of hydroxyl radical (-OH). It is very necessary to apply to the treatment of inflammatory diseases.
  • the dried root of the genus Leguminosae (Flemingl. Roxb. or Moghania) is a dry root of Moghania macrophylla (Willd.) O. Kuntze, which is mainly distributed in the southeastern part of China.
  • the plant is in the Chinese Flora (1995, 41: 313), Taiwan Flora (1977, 3: 258), Hainan Flora (1965, 2: 311), "Chinese Higher Plants Illustration” (1972, 2: 510), The main plant maps in China (1955, pp707) and Guangzhou Flora (1956, pp361) are included.
  • the medicinal materials are the authentic medicinal materials in Guangxi, and the history is contained in the "Phyto-physical map", which has a wide range of folk medicine. Its sweet, slightly sputum, flat, with heat and dehumidification, etc., mainly used for the treatment of rheumatic bone pain, bruises, chronic nephritis, dysmenorrhea and vaginal discharge and other gynecological diseases.
  • the medicinal material is currently included in the appendix of the 2005 edition of the Chinese Pharmacopoeia.
  • the ingredients reported by Daye Qianjin are mainly flavonoids, anthraquinones, anthraquinones, anthraquinones, volatile oils, all of which have certain pharmacological activities. Their pharmacological activities are diverse, and more reported neuroprotective effects. , anti-oxidation, deworming effect on pathogenic microorganisms, hormonal effects, cytotoxic effects, antibacterial effects, immune enhancement, anti-fatigue effects.
  • the technical problem to be solved by the present invention is to provide a novel phenylpropanoid compound and a pharmaceutically acceptable salt thereof.
  • Another technical problem to be solved by the present invention is to provide a pharmaceutical composition of the phenylpropanoid compound and/or a pharmaceutically acceptable salt.
  • Another technical problem to be solved by the present invention is to provide a preparation method of a phenylpropanoid compound isolated from a dried root of a large leaf, and the compound obtained by the preparation method can inhibit the inflammatory factor NO of the cell.
  • the content and the expression of the cytokine TNF- ⁇ , with the freedom of hydroxyl The inhibitory action of (-OH), which has anti-inflammatory and anti-oxidative activities, is beneficial to the treatment of various inflammatory diseases, and the compound can be developed into a new drug.
  • a phenylpropanoid compound having a structural formula represented by formula (I), and a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt has a structure such as formula (II) or Formula (III):
  • R is a mineral acid
  • R 1 or R 2 or R 3 is any one or any two or any three of a sulfonate group, an alkali metal ion or an ammonium group.
  • the inorganic acid is hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, nitric acid, carboxylic acid, phosphoric acid or lactic acid;
  • the sulfonate is a sulfonate having an aryl group;
  • the alkali metal ion It is potassium ion, sodium ion, calcium ion, magnesium ion or lithium ion.
  • the sulfonate having an aryl group is benzenesulfonate or p-toluenesulfonate.
  • the pharmaceutically acceptable salt is an ammonium salt.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the phenylpropanoid compound of the above formula (I) and/or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition contains the phenylpropanoid compound of the above formula (I) and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant and/or carrier.
  • the pharmaceutical composition contains the phenylpropanoid compound represented by the above formula (I) and/or a pharmaceutically acceptable salt thereof, and other medicinal components.
  • the pharmaceutical composition further comprises one or more of sakura root, single-sided needle, spatholobus, succulent wood, andrographis paniculata, angelica, and codonopsis.
  • the pharmaceutical composition further comprises an extract of one or more of sassafras, unilateral needle, spatholobus, sambucus, andrographis, angelica, and codonopsis.
  • the extract is prepared by the extraction method described in any one or several patent documents of Patent Publication Nos. CN1078079C, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C, CN1296073C.
  • the pharmaceutical composition may be in the form of a tablet, a capsule, a powder, a granule, a pill, a solution, a suspension, a syrup, an injection, an ointment, a suppository or a spray, and other prior art achievable Dosage form.
  • the phenylpropanoid compound and a pharmaceutically acceptable salt thereof have an inhibitory effect on the content of the cellular inflammatory factor NO and the cytoinflammatory factor TNF- ⁇ , and have an inhibitory effect on the (-OH) of the hydroxyl radical, and further have
  • the anti-inflammatory and anti-oxidative activities can be applied to the preparation of a medicament for treating an inflammatory disease including, but not limited to, cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the object of the present invention is to obtain a new compound from the prescription of traditional Chinese medicine by solvent extraction, column chromatography separation, preparative liquid phase separation and purification from the prescription of gynecological Qianjin tablets and gynecological Qianjin capsules, and through experiment It has been confirmed that it can be applied to the treatment of inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the inventors obtained the dry root of the large leaf extract from the prescription of the gynecological Qianjin tablets and the gynecological Qianjin capsule, and obtained the liquid phase separation and purification by solvent extraction, column chromatography separation, preparative liquid phase separation, and purification.
  • the phenylpropanoid compound is then subjected to a cell test to determine the degree of inhibition of the cellular inflammatory factors NO, TNF- ⁇ and hydroxyl radical (-OH).
  • NO cellular inflammatory factors
  • TNF- ⁇ hydroxyl radical
  • TNF- ⁇ was significantly inhibited in Raw 264.7 cells (p ⁇ 0.05) in a dose-dependent manner at concentrations (4.13–12.38 ⁇ g/mL).
  • the increase in OH content caused by LPS has a significant inhibitory effect and shows a dose-dependent relationship.
  • the preparation method of the phenylpropanoid compound of the present invention comprises the following steps:
  • the extract obtained in the step S1 is dissolved in water, and eluted by a macroporous adsorption resin column.
  • the eluent is an ethanol-water system, and the first three column volumes of the eluate are collected and named as MM-1. spare;
  • the fraction MM-1 collected in step S2 is eluted by reverse phase material ODS column chromatography.
  • the eluent is a methanol-water system, eluting 18 column volumes, and collecting one for every 3 column volumes.
  • the eluate of the fraction was collected in sequence for 6 fractions, which were named MM-11, MM-12, MM-13, MM-14, MM-15, MM-16, and spare;
  • step S4 The fraction MM-12 collected in step S3 is separated by a preparative liquid phase, and the mobile phase is a methanol-water-acetic acid system.
  • the eluate is collected in the order of the peaks, and a total of 7 fractions are collected and named as MM. -121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, spare;
  • the fraction MM-125 collected in the step S4 is purified by a preparative liquid phase, and the mobile phase is a methanol-water-acetic acid system, and the eluate is collected, and the phenylpropanoid compound is obtained after recrystallization.
  • the phenylpropanoid compound had a purity of 99.84%.
  • the structural formula of the phenylpropanoid compound is as shown in the formula (1):
  • the concentration of the ethanol solution is 50 to 80% by volume, and more preferably the concentration of the ethanol solution is 60% by volume.
  • step S1 the extraction times of the ethanol solution are 2 to 4 times, and each extraction is performed for 1 to 3 hours.
  • the number of extractions of the ethanol solution in one step is 3 times, 2 hours each time.
  • the macroporous adsorption resin is a D101 macroporous adsorption resin.
  • the volume ratio of ethanol to water is: 0:100 to 15:85.
  • the volume ratio of methanol to water is from 20:80 to 30:70, further preferably 25:75.
  • the volume ratio of methanol-water-acetic acid is 10:90:0.01 to 35:65:0.01, further preferably 15:85:0.01.
  • the liquid chromatography column is prepared as YMC, 20 mm*250 mm, and the mobile phase flow rate is 5-10 ml/min, preferably 5 ml/min.
  • step S5 the volume ratio of methanol-water-acetic acid is 15:85:0.01.
  • the liquid chromatography column is prepared as YMC, 20 mm*250 mm, and the mobile phase flow rate is 5 mL/min.
  • the present invention provides a phenylpropanoid compound prepared by the preparation method.
  • a phenylpropanoid compound and a pharmaceutically acceptable salt thereof are provided for use as a medicament for the preparation of a medicament for treating an inflammatory disease.
  • the phenylpropanoid compound and a pharmaceutically acceptable salt thereof are used in the preparation of a medicament for inhibiting the content of cellular inflammatory factor NO, or inhibiting the expression of the cellular inflammatory factor TNF- ⁇ , or inhibiting the activity of hydroxyl radicals.
  • the inflammatory disease is cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the drug contains pharmaceutically acceptable excipients and/or carriers.
  • the medicament also contains other medicinal ingredients.
  • the medicine further comprises one or more of sakura root, single-sided needle, spatholobus, succulent wood, andrographis paniculata, angelica, and codonopsis.
  • the medicament further comprises an extract of one or more of sakura root, unilateral needle, spatholobus, sambucus, andrographis, angelica, and codonopsis.
  • the pharmaceutical dosage form is a tablet, a capsule, a powder, a granule, a pill, a solution, a suspension, a syrup, an injection, an ointment, a suppository or a spray.
  • the present invention provides a novel phenylpropanoid compound, and at the same time provides a pharmaceutically acceptable salt of the phenylpropanoid compound, the phenylpropanoid compound or a pharmaceutically acceptable salt thereof inhibits cellular inflammatory factors
  • the content of NO and the expression of cytokine TNF- ⁇ have an inhibitory effect on (-OH) of hydroxyl radicals, and thus have anti-inflammatory and anti-oxidative activities, and can be well applied for the preparation of inflammatory diseases.
  • Therapeutic drugs including but not limited to inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis, provide a powerful technical basis for the development of anti-inflammatory drugs.
  • the present invention provides a preparation method for a novel phenylpropanoid compound and a pharmaceutically acceptable salt thereof, which is obtained by first extracting a novel phenylpropanoid compound from a dried root of a large leaf.
  • the compound can inhibit the expression of cellular inflammatory factor NO and the expression of cytokine TNF- ⁇ , has an inhibitory effect on (-OH) of hydroxyl radicals, and has anti-inflammatory and anti-oxidative activities, and can be used as cervicitis, for example.
  • a therapeutic drug for inflammatory diseases such as endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the object of the present invention is to prepare a novel phenylpropanoid compound from the prescription of traditional Chinese medicine by solvent extraction, column chromatography separation, liquid phase separation and purification from the prescription of gynecological Qianjin tablets and gynecological Qianjin capsules. And it has been confirmed by experiments that it can be applied to the treatment of inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the inventors obtained the benzene of the present invention by solvent extraction, column chromatography separation, preparative liquid phase separation and purification, from the prescriptions of gynecological Qianjin tablets and gynecological Qianjin capsules.
  • the compound is then subjected to a cell test to determine the degree of inhibition of cellular inflammatory factors NO, TNF- ⁇ and hydroxyl radical (-OH).
  • the experiment shows that the compound is at a concentration (6.88–12.38 ⁇ g/mL). Within the range, the LPS-induced increase in NO content was significantly inhibited and showed a significant dose-dependent relationship.
  • the inflammatory factor TNF- ⁇ was significantly inhibited in Raw 264.7 cells (p ⁇ 0.05) in a dose-dependent manner at concentrations (4.13–12.38 ⁇ g/mL). It has a significant inhibitory effect on the increase of OH content caused by LPS, and shows a dose-dependent relationship.
  • novel phenylpropanoid compounds provided by the invention have simple structure and high purity, and the extraction and separation method is simple and easy to synthesize, and can be adapted to the industrial application of the new drug.
  • Fig. 1 is a nuclear magnetic resonance spectrum of a phenylpropanoid compound of the present invention.
  • Fig. 2 is a nuclear magnetic resonance carbon spectrum of the phenylpropanoid compound of the present invention.
  • Figure 3 is a graph showing the effect of the phenylpropanoid compounds on cell viability of the present invention.
  • Fig. 4 is a graph showing the inhibition of NO by the phenylpropanoid compound of the present invention.
  • Fig. 5 is a graph showing the inhibitory effect of the phenylpropanoid compound on TNF- ⁇ of the present invention.
  • Fig. 6 is a graph showing the inhibitory effect of the phenylpropanoid compound on IL-1 ⁇ of the present invention.
  • Fig. 7 is a graph showing the inhibitory effect of the phenylpropanoid compound on IL-6 of the present invention.
  • Fig. 8 is a graph showing the inhibition of OH by the phenylpropanoid compounds of the present invention.
  • the compound of the present invention is a phenylpropanoid compound represented by the above formula (I) and a pharmaceutically acceptable salt of the compound represented by the formula (II) or the formula (III).
  • the compound can be prepared by the method of extracting from the raw material provided by the present invention, or can be prepared according to the structural formula provided by the present invention and by chemical synthesis in the art.
  • the salt of the phenylpropanoid compound of the present invention may be a pharmaceutically acceptable salt, and examples thereof include hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, nitric acid, carboxylic acid, and phosphoric acid.
  • An inorganic acid salt formed by an inorganic acid such as lactic acid; a sulfonic acid salt formed with a sulfonic acid; an alkali metal salt formed with a hydroxide of an alkali metal such as potassium, sodium, calcium, magnesium or lithium; an ammonium salt formed with ammonium; .
  • the phenylpropanoid compound of the present invention can be used as a therapeutic drug for inflammatory diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngitis and/or arthritis.
  • the compounds of the present invention can be used as a pharmaceutical composition together with a pharmaceutically acceptable adjuvant and/or carrier, or in the case of the addition of pharmaceutically acceptable excipients and/or carriers, with sakura root, single-sided needle, spatholobus, succulent wood
  • a pharmaceutically acceptable adjuvant and/or carrier or in the case of the addition of pharmaceutically acceptable excipients and/or carriers, with sakura root, single-sided needle, spatholobus, succulent wood
  • the combination of one or more Chinese herbal medicines or extracts of andrographis paniculata, angelica, and codonopsis is used as a pharmaceutical composition, and the compound of the present invention can also be used as a pharmaceutical composition together with other pharmaceutically acceptable medicinal ingredients.
  • the pharmaceutical composition may be a tablet, a capsule, a powder, a granule, a pill, a solution, a suspension, a syrup, an injection, an ointment, a suppository, a spray or the like.
  • the tablet may be a sugar-coated tablet, a film-coated tablet, an enteric coated tablet or a double-layer tablet, a multilayer tablet prepared with the addition of a pharmaceutically acceptable adjuvant and/or carrier.
  • excipients and/or carriers of the invention may be as follows:
  • additives such as sucrose, lactose, cellulose sugar, maltitol, glucose, starch, agar, alginates, chitin, chitosan, pectin, gum arabic, gelatin can be used.
  • animal and vegetable oils oil, corn oil, castor oil, etc.
  • mineral oils Vaseline, white petrolatum, solid paraffin, etc.
  • waxes jojoba oil, carnauba wax, beeswax
  • Etc. partially synthesized or fully synthesized glycerol fatty acid esters (lauric acid, myristic acid, palmitic acid, etc.).
  • an additive such as sodium chloride, glucose, sorbitol, glycerin, olive oil, propylene glycol, ethanol or the like can be used.
  • a sterile aqueous solution such as physiological saline, an isotonic solution, or an oily liquid such as sesame oil or soybean oil can be used.
  • a suitable suspending agent such as sodium carboxymethylcellulose, a nonionic surfactant, a co-solvent such as benzyl benzoate, benzyl alcohol or the like may be used as needed.
  • the amount of the active ingredient of these preparations is 0.01 to 80% by weight, preferably 1 to 50% by weight, based on the symptoms, body weight, age, and the like of the patient.
  • This embodiment provides a preparation method of the phenylpropanoid compound represented by the formula (I), comprising the following steps:
  • step S2 The concentrated extract in step S1 is dissolved in 10 L of water, and eluted with a D101 macroporous adsorption resin column.
  • the eluent is water, eluted with 3 column volumes, and the eluate is collected and named as MM. -1, spare;
  • step S3 The fraction MM-1 collected in step S2 is eluted by reverse phase ODS column chromatography, and the eluent is a methanol-water system, the volume ratio is 25:75, and the elution is 18 column volumes. Collect a fraction of eluate every 3 column volumes, and collect 6 fractions in order, named: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 ;
  • step S4 The collected fraction MM-12 in step S3 is separated by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, and the mobile phase is a methanol-water-acetic acid system.
  • the volume ratio of methanol:water:acetic acid was 25:75:0.01, and the eluate was collected in the order of peaks.
  • a total of 7 fractions were collected and named separately. For MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, spare;
  • step S5 The fraction MM-125 collected in step S4 is purified by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, mobile phase is methanol-water-acetic acid system, methanol The volume ratio of water:acetic acid was 15:85:0.01, and the eluate was collected and recrystallized to obtain the phenylpropanoid compound.
  • This embodiment provides a preparation method of the phenylpropanoid compound represented by the formula (I), comprising the following steps:
  • step S2 The concentrated extract in step S1 is dissolved in 5 L of water and eluted with a D101 macroporous adsorption resin column.
  • the eluent is a volume ratio of ethanol to water of 15:85, and elution of 3 column volumes. , collect the eluent, named MM-1, spare;
  • step S3 The fraction MM-1 collected in step S2 is eluted by reverse phase ODS column chromatography, and the eluent is a methanol-water system, the volume ratio is 20:80, and the elution is 18 column volumes. Collect a fraction of eluate every 3 column volumes, and collect 6 fractions in order, named: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 ;
  • step S4 The fraction MM-12 collected in step S3 is separated by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 10 ml/min, mobile phase is methanol-water-acetic acid system, methanol : Water: The volume ratio of acetic acid is 35:65:0.01, and the eluate is collected according to the peak order.
  • a total of 7 fractions are collected and named as MM-121, MM-122, MM-123, MM-124, MM. -125, MM-126, MM-127, spare;
  • step S5 The fraction MM-125 collected in step S4 is purified by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, mobile phase is methanol-water-acetic acid system, methanol The volume ratio of water:acetic acid was 15:85:0.01, and the eluate was collected and recrystallized to obtain the phenylpropanoid compound.
  • This embodiment provides a preparation method of the phenylpropanoid compound represented by the formula (I), comprising the following steps:
  • step S2 Dissolve the concentrated extract in step S1 in 8L of water, and use D101 macroporous adsorption resin column to advance it. Elution, the eluent is a volume ratio of ethanol to water of 10:90, eluting 3 column volumes, collecting the eluent, named MM-1, standby;
  • step S3 The fraction MM-1 collected in step S2 is eluted by reverse phase ODS column chromatography, and the eluent is a methanol-water system, the volume ratio is 30:70, and the elution is 18 column volumes. Collect a fraction of eluate every 3 column volumes, and collect 6 fractions in order, named: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 ;
  • step S4 The collected fraction MM-12 in step S3 is separated by a preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 10 ml/min, and the mobile phase is a methanol-water-acetic acid system.
  • the volume ratio of methanol:water:acetic acid was 30:70:0.01, and the eluent was collected according to the peak order.
  • a total of 7 fractions were collected and named as MM-121, MM-122, MM-123, MM-124, respectively.
  • step S5 The fraction MM-125 collected in step S4 is purified by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, mobile phase is methanol-water-acetic acid system, methanol The volume ratio of water:acetic acid was 15:85:0.01, and the eluate was collected and recrystallized to obtain the phenylpropanoid compound.
  • This embodiment provides a preparation method of the phenylpropanoid compound represented by the formula (I), comprising the following steps:
  • step S2 The concentrated extract in step S1 is dissolved in 6 L of water and eluted with a D101 macroporous adsorption resin column.
  • the eluent is a volume ratio of ethanol to water of 5:95, and elution of 3 column volumes. , collect the eluent, named MM-1, spare;
  • step S3 The fraction MM-1 collected in step S2 is eluted by reverse phase ODS column chromatography, and the eluent is a methanol-water system, the volume ratio is 25:75, and the elution is 18 column volumes. Collect a fraction of eluate every 3 column volumes, and collect 6 fractions in order, named: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 ;
  • step S4 The fraction MM-12 collected in step S3 is separated by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 10 ml/min, mobile phase is methanol-water-acetic acid system, methanol : Water: The volume ratio of acetic acid is 25:75:0.01, and the eluate is collected according to the peak order.
  • a total of 7 fractions are collected and named as MM-121, MM-122, MM-123, MM-124, MM. -125, MM-126, MM-127, prepared use;
  • step S5 The fraction MM-125 collected in step S4 is purified by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, mobile phase is methanol-water-acetic acid system, methanol The volume ratio of water:acetic acid was 15:85:0.01, and the eluate was collected and recrystallized to obtain the phenylpropanoid compound.
  • This embodiment provides a preparation method of the phenylpropanoid compound represented by the formula (I), comprising the following steps:
  • step S2 Dissolve the concentrated extract in step S1 in 6L of water, and elute it with a D101 macroporous adsorption resin column.
  • the eluent is a volume ratio of ethanol to water of 10:90, eluting 3 column volumes. , collect the eluent, named MM-1, spare;
  • step S3 The fraction MM-1 collected in step S2 is eluted by reverse phase ODS column chromatography, and the eluent is a methanol-water system, the volume ratio is 28:72, and the elution is 18 column volumes. Collect a fraction of eluate every 3 column volumes, and collect 6 fractions in order, named: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 ;
  • step S4 The collected fraction MM-12 in step S3 is separated by a preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 10 ml/min, and the mobile phase is a methanol-water-acetic acid system.
  • the volume ratio of methanol:water:acetic acid was 10:90:0.01.
  • the eluent was collected in the order of peaks.
  • a total of 7 fractions were collected and named as MM-121, MM-122, MM-123, MM-124. MM-125, MM-126, MM-127, spare;
  • step S5 The fraction MM-125 collected in step S4 is purified by preparative liquid phase to prepare a liquid chromatography column: YMC, 20 mm*250 mm, flow rate: 5 ml/min, mobile phase is methanol-water-acetic acid system, methanol The volume ratio of water:acetic acid was 15:85:0.01, and the eluate was collected and recrystallized to obtain the phenylpropanoid compound.
  • Phenylpropanoid hydrochloride ESIMS showed m/z 412.67, NMR characteristics 1 H-NMR (600 MHz, CD 3 OD): 1 H-NMR (600 MHz, CD 3 OD): 6.63 (d, 1H), 6.49 (d, 1H), 5.05 (s, 1H), 4.98 (m, 2H), 3.00-4.00 (glc-H), 2.74 (m, 2H).
  • Phenylpropanoid sulfonate ESIMS showed m/z 504.28, NMR characteristics 1 H-NMR (600 MHz, CD3OD): 1 H-NMR (600 MHz, CD 3 OD): 6.55 (d, 1H), 6.36 ( d, 1H), 4.90 (s, 1H), 4.91 (m, 2H), 3.00-4.00 (glc-H), 2.51 (m, 2H).
  • the potassium salt ESIMS shows m/z 452.19, NMR characteristics 1 H-NMR (600 MHz, CD 3 OD): 6.70 (d, 1H), 6.57 (d, 1H), 4.94 (s, 1H), 4.91 (m, 2H) , 3.00-4.00 (glc-H), 2.70 (m, 2H).
  • the sodium salt ESIMS shows m/z 420.19, NMR characteristics 1 H-NMR (600 MHz, CD 3 OD): 6.56 (d, 1H), 6.46 (d, 1H), 4.74 (s, 1H), 4.51 (m, 2H) , 3.00-4.00 (glc-H), 2.60 (m, 2H).
  • Ammonium salt of phenylpropanoid compound shows m/z 406.57, NMR characteristics 1 H-NMR (600 MHz, CD3OD): 1 H-NMR (600 MHz, CD 3 OD): 6.63 (d, 1H), 6.33 (d, 1H), 4.94 (s, 1H), 4.91 (m, 2H), 3.00-4.00 (glc-H), 2.77 (m, 2H).
  • the formula (IV) is the prepared phenylpropanoid hydrochloride
  • the formula (V) is one of the prepared sulfonate compounds
  • the formula (VI) is the prepared styrene
  • One of the potassium salts of the compound is one of the sodium salts of the prepared phenylpropanoid compound
  • the formula (VIII) is one of the ammonium salts of the prepared phenylpropanoid compound.
  • the phenylpropanoid compound of the present invention is labeled as follows: the drug MM-125, that is, the drug MM-125 described in the present invention means the formula (I) of the present invention. a phenylpropanoid compound or a pharmaceutically acceptable salt thereof.
  • Lipopolysaccharide (LPS), MTT was purchased from Sigma; mouse macrophage Raw264.7 was purchased from Xiangya cell bank; PBS; DMEM high glucose medium, fetal bovine serum, penicillin and streptomycin; Standard instrument; constant temperature CO 2 incubator.
  • Mouse IL-1 ⁇ (IL-1- ⁇ ) ELISA kit batch number: 2014/06 (96T); mouse interleukin 6 (IL-6) ELISA kit, batch number: 2014/06 (96T); Mouse tumor necrosis factor- ⁇ (TNF- ⁇ ) ELISA kit, batch number: 2014/06 (96T); mouse nitric oxide (NO) ELISA Test kit, batch number: 2014/10 (96T); mouse hydroxyl free radical (OH) ELISA test kit, batch number: 2014/10 (96T).
  • Mouse macrophage Raw 264.7 was cultured in DMEM medium containing 10% heat-inactivated (56 ° C, 30 min) fetal bovine serum (FBS), 10 U/mL penicillin sodium, 100 ⁇ g/mL streptomycin, 37 ° C, Incubate in a 5% CO 2 incubator.
  • FBS fetal bovine serum
  • penicillin sodium 100 ⁇ g/mL streptomycin
  • Cell viability was determined by the MTT method.
  • the cells were made into cell suspensions and seeded in 96-well plates (1 ⁇ 10 4 cells/well) for 24 hours, then synchronized for 24 hours. Then different concentrations of drugs were applied to the cells for 2 hours, then LPS (30 ⁇ g/mL) was added to stimulate 24 hours.
  • the original medium was aspirated, 100 ⁇ L of MTT (0.5 mg/mL) was added to each well for 4 h, the medium was aspirated, 150 ⁇ L of DMSO was added to each well, shaken for 10 min on a shaker, and the absorbance was measured at 490 nm.
  • Raw 264.7 cells were seeded in 96-well plates for 24 h, then synchronized for 24 h, then different concentrations of drugs were applied to the cells for 2 h, then LPS (30 ⁇ g/mL) was added for 24 h. Finally, the supernatant was collected and centrifuged at 10,000 rpm for 5 min. The supernatant was dispensed and stored at -80 ° C for later use. The NO content was determined by a mouse NO kit.
  • Samples were taken from 1.5 samples for subsequent inflammatory factor determination.
  • the amount of TNF- ⁇ , IL-1 ⁇ , and IL-6 produced by the cells was measured by a mouse TNF- ⁇ , IL-1 ⁇ , IL-6 kit.
  • Samples were taken from 1.5 samples for OH factor determination. The content was determined by an OH kit.
  • Raw 264.7 cells were stimulated by LPS, Raw 264.7 cell inflammatory factor TNF- ⁇ (127.98 ⁇ 4.65 pg/mL), IL-1 ⁇ (347.55 ⁇ 9.56 pg/mL), IL-6 (201.67).
  • the content of ⁇ 10.32pg/mL was significantly higher than that of normal group TNF- ⁇ (67.12 ⁇ 4.00pg/mL), IL-1 ⁇ (160.88 ⁇ 13.10pg/mL) and IL-6 (111.13 ⁇ 8.06pg/mL).
  • High p ⁇ 0.01); indicating that LPS can stimulate Raw264.7 cells to produce a large number of inflammatory factors.
  • Drug MM-125 significantly inhibited the production of inflammatory factor TNF- ⁇ in Raw 264.7 cells at a concentration (6.88-12.38 ⁇ g/mL) (p ⁇ 0.05), and showed a significant dose-dependent relationship; at each concentration, it could not significantly inhibit Raw 264.7 cells produced IL-1 ⁇ , IL-6 (p>0.05).
  • Drug MM-125 significantly inhibited the increase of OH content induced by LPS in the concentration range (4.13-12.38 ⁇ g/mL) and showed a dose-dependent relationship.
  • Drug MM-125 has a significant inhibitory effect on the production of cellular inflammatory factor NO, and also has a certain inhibitory effect on TNF- ⁇ , but it has no obvious inhibitory effect on IL-1 ⁇ and IL-6, indicating that it has certain anti-inflammatory activity. It has a significant inhibitory effect on the production of OH, indicating that it has better antioxidant activity.
  • the phenylpropanoid compound represented by the formula (I) is prepared by the method of the first embodiment, and the compound and the inorganic acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, a salt made of nitric acid, carboxylic acid, phosphoric acid, lactic acid or a sulfonic acid or an alkali metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide) or ammonium,
  • the compound or any one of the salts and the excipients are added to the excipient in a weight ratio of 1:10, and granulated and compressed.
  • the phenylpropanoid compound represented by formula (I) is prepared according to the method of Example 1, and the compound and inorganic acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, nitric acid) are utilized.
  • the method is made into a powder.
  • Capsules or Granules The phenylpropanoid compound represented by the formula (I) is prepared by the method of Example 1, and the compound and the inorganic acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid are utilized).
  • the compound and the inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid are utilized.
  • an alkali metal hydroxide such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide
  • ammonium is added in a ratio of the compound or any one of the salts to the excipient in a weight ratio of 1:10 to prepare a capsule or granule.
  • the phenylpropanoid compound represented by the formula (I) is obtained by the method of Example 1, and the compound and the inorganic acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, nitric acid) are utilized.
  • Injectables are prepared by water, fine filtration and potting.
  • a pharmaceutical composition comprising the method of Example 1 to obtain a phenylpropanoid compound of the formula (I), and the use of the compound with a mineral acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid) a salt made of a nitric acid, a carboxylic acid, a phosphoric acid, a lactic acid or a sulfonic acid or an alkali metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide) or ammonium, and Golden cherry root, single-sided needle, spatholobus, eucalyptus, andrographis, angelica, powder made from Codonopsis pilosula, and excipients.
  • a mineral acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid
  • a salt made of a nitric acid, a
  • a pharmaceutical composition comprising the phenylpropanoid compound represented by the formula (I), and the powder of the sakura root, the unilateral needle, the spatholobus, the mulberry, the andrographis, the angelica, the ginseng, And accessories.
  • a pharmaceutical composition comprising the phenylpropanoid compound represented by the formula (I), and the extract of the sakura root, the unilateral needle, the spatholobus, the mulberry, the andrographis, the angelica, the codonopsis, and the method of the method of the first embodiment, and Excipients.
  • the extract is prepared according to the extraction method of any one or several patent documents of Patent Publication No. CN1078079C, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C, CN1296073C.
  • a pharmaceutical composition comprising the method of Example 1 to obtain a phenylpropanoid compound of the formula (I), and the use of the compound with a mineral acid (such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid) a salt made of a nitric acid, a carboxylic acid, a phosphoric acid, a lactic acid or a sulfonic acid or an alkali metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide) or ammonium, and Golden cherry root, single-sided needle, spatholobus, succulent wood, andrographis, angelica, extract of Codonopsis pilosula, and accessories.
  • a mineral acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, sulfuric acid
  • a salt made of a nitric acid, a carboxylic acid, a phospho
  • the extract is prepared according to the extraction method of any one or several patent documents of Patent Publication No. CN1078079C, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C, CN1296073C.

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Abstract

提供一种苯丙素类化合物及其制备方法和应用。所述苯丙素类化合物结构如式(I)所示，所述药学上可接受的盐的结构如式(II)或式(III)所示。所述苯丙素类化合物及其药学上可接受的盐具有抑制细胞炎症因子NO的含量及细胞炎症因子TNF-α的表达作用，具有对羟基自由基的(-OH)的抑制作用，进而具有抗炎及抗氧化活性，在制备治疗与上述因子相关疾病，例如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等炎症性疾病的药物方面具有良好的应用前景。

Description

一种苯丙素类化合物及其制备方法和应用 技术领域
本发明涉及医药技术领域，更具体地，涉及一种苯丙素类化合物及其制备方法和应用。
背景技术
从天然药物中提取分离得到的成分结构多样、活性显著，对其进行分离纯化、结构修饰、改造和全合成，一直是新药研发的一个主要思路。
TNF-α：是一种能够直接杀伤肿瘤细胞而对正常细胞无明显毒性的细胞因子，是迄今为止所发现的直接杀伤肿瘤作用最强的生物活性因子之一，然而其毒副作用也非常严重。
IL-1β：在局部低浓度时协同刺激APC和T细胞活化，促进B细胞增殖和分泌抗体，进行免疫调节。大量产生时有内分泌效应：诱导肝脏急性期蛋白合成，引起发热和恶病质。
IL-6：人类IL-6基因位于第7号染色体上；IL-6分子量在21～30KD之间。主要由单核巨噬细胞、Th2细胞、血管内皮细胞、成纤维细胞产生。能够刺激活化B细胞增殖，分泌抗体；刺激T细胞增殖及CTL活化；刺激肝细胞合成急性期蛋白，参与炎症反应；促进血细胞发育。
IL-6可由多种细胞合成，包括活化的T细胞和B细胞、单核－巨噬细胞、内皮细胞、上皮细胞以及成纤维细胞等。IL-6作用的靶细胞很多，包括巨噬细胞、肝细胞、静止的T细胞、活化的B细胞和浆细胞等；其生物效应也十分复杂。
OH为生物系统中最具活性的活性氧物质，能导致细胞及生物体内DNA，蛋白质和脂质氧化损伤。
巨噬细胞能产生多种炎症介质参与炎症反应，其中，NO为重要的细胞炎症因子，NO参与多种生理病理过程，过量NO会促进炎症性疾病的发生与发展，并且还能诱导其他炎症因子。
因此，寻求一种新的制备方法，从天然植物中分离出新的化合物，以抑制细胞炎症因子NO的含量、抑制细胞炎症因子TNF-α的表达作用，抑制羟基自由基(-OH)的活性，应用于炎症性疾病的治疗，是非常有必要的。
千斤拔药材为豆科(Leguminosae)千斤拔属(Flemingl.Roxb.或Moghania)植物大叶千斤拔(Moghania macrophylla(Willd.)O.Kuntze)的干燥根，在我国主要分布于东南部地区。该植物在中国植物志(1995，41:313)、台湾植物志(1977，3:258)、海南植物志(1965，2:311)、《中国高等植物图鉴》(1972，2:510)、中国主要植物图说(1955，pp707)和广州植物志(1956，pp361)中均有收录。千斤拔药材为广西地区的道地药材，史载于《植物名实图考》，有着广泛的民间用药基础。其性味甘，微涩，平，具有清热除湿等功效，主要用于治疗风湿骨痛、跌打损伤、慢性肾炎、痛经和白带多等妇科疾病。该药材目前已收载入2005年版《中国药典》附录。
大叶千斤拔已报道的成分主要有黄酮类、甾类、萜类、蒽醌类、挥发油类成分，均具有一定的药理活性，其药理活性多样，报道较多的有神经保护作用，抗炎、抗氧化作用，对病原微生物的驱虫作用、类激素作用、细胞毒作用，抗菌作用以及免疫增强作用、抗疲劳作用。
千斤拔目前广泛应用于妇科、风湿痹痛等类型的中成药生产，如妇科千金片、妇科千金胶囊、金鸡冲剂、金鸡胶囊等，此类中成药主要用于妇科病(痛经、子宫寒冷不孕、子宫下垂、盆腔炎、乳腺炎、白带多、产后血虚、关节痛、产后腰膝痛、缺乳和乳疮等)，虚弱贫血(妇女贫血，气血虚弱和病后气虚等)。近年来，临床报道较多的是妇科千金片在治疗妇科炎症方面的作用。
目前，在千斤拔的文献中，报道苯丙素类化合物的文献极少，寻找有效的苯丙素类新化合物及其制备方法，对其进行分离纯化、结构修饰和合成，开发新药，应用于炎症性疾病的治疗，意义重大。
发明内容
本发明要解决的技术问题是提供一种新的苯丙素类化合物及其药学上可接受的盐。
本发明要解决的另一技术问题是提供所述苯丙素类化合物和/或药学上可接受的盐的药物组合物。
本发明要解决的另一技术问题是提供从大叶千斤拔的干燥根中分离得到的一种苯丙素类化合物的制备方法，通过该制备方法提取分离得到的化合物可以抑制细胞炎症因子NO的含量及细胞炎症因子TNF-α的表达作用，具有对羟基自由 基的(-OH)的抑制作用，进而具有抗炎及抗氧化活性，有益于各种炎症性疾病的治疗，可以将该化合物开发成新药。
本发明的目的是提供一种苯丙素类化合物的医药应用。
本发明的目的通过以下技术方案予以实现：
提供一种苯丙素类化合物及其药学上可接受的盐，所述苯丙素类化合物的结构式如式(Ⅰ)所示，所述药学上可接受的盐的结构如式(Ⅱ)或式(Ⅲ)所示：
Figure PCTCN2017076455-appb-000001
其中，R为无机酸，R1或R2或R3为磺酸根、碱金属离子或铵根中的任意一种或任意两种或任意三种。
优选地，所述无机酸为盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸或乳酸；所述磺酸根为具有芳基的磺酸根；所述碱金属离子为钾离子、钠离子、钙离子、镁离子或锂离子。
优选地，所述具有芳基的磺酸根为苯磺酸根或对甲苯磺酸根。
优选地，所述药学上可接受的盐为铵盐。
本发明同时提供一种药物组合物，所述药物组合物含有上述式(Ⅰ)所示苯丙素类化合物和/或其药学上可接受的盐。
优选地，所述药物组合物含有上述式(Ⅰ)所示苯丙素类化合物和/或其药学上可接受的盐、以及药学上允许的辅料和/或载体。
优选地，所述药物组合物含有上述式(Ⅰ)所示苯丙素类化合物和/或其药学上可接受的盐、以及其他药性成分。
优选地，所述药物组合物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种。
优选地，所述药物组合物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种的提取物。
所述提取物为按专利公告号CN1078079C、CN1170549C、CN1158087C、CN1330335C、CN1296071C、CN1321631C、CN1296072C、CN1296073C的任意一件或几件专利文件中所述的提取方法制备得到。
所述药物组合物的剂型可以为片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂、混悬剂、糖浆剂、注射剂、软膏剂、栓剂或喷雾剂，以及其他现有技术可实现的剂型。
所述苯丙素类化合物及其药学上可接受的盐具有抑制细胞炎症因子NO的含量及细胞炎症因子TNF-α的表达作用，具有对羟基自由基的(-OH)的抑制作用，进而具有抗炎及抗氧化活性，可应用于制备治疗炎症性疾病的药物，所述炎症性疾病包括但不限于宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等。
本发明的目的是从传统的中药处方中，通过从妇科千金片及妇科千金胶囊的处方中，通过溶剂提取、柱层析分离、制备液相分离、纯化制得一种新化合物，并通过实验证实，其可以应用于炎症性疾病，如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等疾病的治疗。
具体地，发明人通过从妇科千金片、妇科千金胶囊的处方中，选取大叶千斤拔的干燥根，通过溶剂提取、柱层析分离、制备液相分离、纯化，得到本发明所 述苯丙素类化合物，然后对该化合物进行细胞试验，测定其对细胞炎症因子NO，TNF-α及羟基自由基(-OH)的抑制程度，实验表明，该苯丙素类化合物在浓度(6.88–12.38μg/mL)范围内对LPS引起的NO含量升高有明显的抑制作用，并表现出明显的剂量依赖关系。在浓度(6.88–12.38μg/mL)范围内，能显著抑制Raw 264.7细胞产生TNF-α(p＜0.05)，并呈显著的剂量依赖关系，在浓度(4.13–12.38μg/mL)范围内对LPS引起的OH含量升高有明显的抑制作用，并表现出一定的剂量依赖关系。
本发明所述苯丙素类化合物的制备方法包括如下步骤：
S1.取大叶千斤拔的根部为原料，干燥，切块，经乙醇溶液提取，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中所得浸膏加水溶解，采用大孔吸附树脂柱对其进行洗脱，洗脱剂为乙醇-水系统，收集前3个柱体积的洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相材料ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为MM-11，MM-12，MM-13，MM-14，MM-15，MM-16，备用；
S4.将步骤S3中收集到的流分MM-12用制备液相分离，流动相为甲醇-水-乙酸系统，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，流动相为甲醇-水-乙酸系统，收集洗脱液，重结晶后得到所述苯丙素类化合物。所述苯丙素类化合物纯度为99.84％。所述苯丙素类化合物的结构式如式(1)所示：
Figure PCTCN2017076455-appb-000002
优选地，步骤S1中，乙醇溶液的浓度为50～80体积％，进一步优选为乙醇溶液浓度为60体积％。
优选地，步骤S1中，乙醇溶液的提取次数为2～4次，每次提取1～3小时，进 一步优选为乙醇溶液的提取次数为3次，每次2小时。
优选地，步骤S2中，大孔吸附树脂采用D101大孔吸附树脂。
优选地，步骤S2中，乙醇与水的体积比为：0:100～15:85。
优选地，步骤S3中，甲醇与水的体积比为20:80～30:70，进一步优选为25:75。
优选地，步骤S4中，甲醇-水-乙酸的体积比为10:90:0.01～35:65:0.01，进一步优选为15:85:0.01。
优选地，步骤S4中，制备液相色谱柱为YMC，20mm*250mm，流动相流速为：5～10ml/min，优选为流速5ml/min。
优选地，步骤S5中，甲醇-水-乙酸的体积比为15:85:0.01。
优选地，步骤S5中，制备液相色谱柱为YMC，20mm*250mm，流动相流速为5mL/min。
本发明提供所述制备方法制备得到的苯丙素类化合物。
提供一种苯丙素类化合物及其药学上可接受的盐作为制备治疗炎症性疾病的药物中的应用。
优选地，所述苯丙素类化合物及其药学上可接受的盐在制备抑制细胞炎症因子NO的含量，或抑制细胞炎症因子TNF-α的表达，或抑制羟基自由基的活性的药物中的应用，应用于治疗炎症性疾病的药物中。
优选地，所述炎症性疾病为宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎。
所述药物含有药学上允许的辅料和/或载体。
优选地，所述药物还含有其他药效成分。
优选地，所述药物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种。优选地，所述药物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种的提取物。
优选地，所述药物的剂型为片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂、混悬剂、糖浆剂、注射剂、软膏剂、栓剂或喷雾剂。
本发明的有益效果是：
本发明提供了一种新的苯丙素类化合物，同时提供了所述苯丙素类化合物药学上可接受的盐，该苯丙素类化合物或其药学上可接受的盐可以抑制细胞炎症因 子NO的含量及细胞炎症因子TNF-α的表达作用，具有对羟基自由基的(-OH)的抑制作用，进而具有抗炎及抗氧化活性，可很好地应用于制备治疗炎症性疾病方面的药物，包括但不限于如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等炎症性疾病的治疗药物，为抗炎药物的发展提供有力的技术基础。
本发明为新的苯丙素类化合物及其药学上可接受的盐提供了一种制备方法，首次从大叶千斤拔的干燥根中分离得到的一种新苯丙素类化合物，制得的化合物可以抑制细胞炎症因子NO的含量及细胞炎症因子TNF-α的表达作用，具有对羟基自由基的(-OH)的抑制作用，进而具有抗炎及抗氧化活性，可以用作如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等炎症性疾病的治疗药物。
本发明的目的是从传统的中药处方中，通过从妇科千金片及妇科千金胶囊的处方中，通过溶剂提取、柱层析分离、制备液相分离、纯化制得一种新苯丙素类化合物，并通过实验证实，其可以应用于炎症性疾病，如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等疾病的治疗。
具体地，发明人通过从妇科千金片、妇科千金胶囊的处方中，科学选取大叶千斤拔的干燥根，通过溶剂提取、柱层析分离、制备液相分离、纯化，得到本发明所述苯丙素类化合物，然后对该化合物进行细胞试验，测定其对细胞炎症因子NO，TNF-α及羟基自由基(-OH)的抑制程度，实验表明，该化合物在浓度(6.88–12.38μg/mL)范围内对LPS引起的NO含量升高有明显的抑制作用，并表现出明显的剂量依赖关系。在浓度(6.88–12.38μg/mL)范围内，能显著抑制Raw 264.7细胞产生炎症因子TNF-α(p＜0.05)，并呈显著的剂量依赖关系，在浓度(4.13–12.38μg/mL)范围内对LPS引起的OH含量升高有明显的抑制作用，并表现出一定的剂量依赖关系。
本发明提供的新的苯丙素类化合物结构简单、纯度高，且提取分离方法简便、易于合成，可适应新药的产业化应用。
附图说明
图1为本发明所述苯丙素类化合物的核磁共振氢谱图。
图2为本发明所述苯丙素类化合物的核磁共振碳谱图。
图3为本发明所述苯丙素类化合物对细胞活性的影响图。
图4为本发明所述苯丙素类化合物对NO的抑制作用图。
图5为本发明所述苯丙素类化合物对TNF-α的抑制作用图。
图6为本发明所述苯丙素类化合物对IL-1β的抑制作用图。
图7为本发明所述苯丙素类化合物对IL-6的抑制作用图。
图8为本发明所述苯丙素类化合物对OH的抑制作用图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，本实施例所用的原料和设备均为本技术领域常规市购的原料和设备。
本发明的化合物是所述式(Ⅰ)所示的苯丙素类化合物及式(Ⅱ)或式(Ⅲ)所示该化合物药学上可接受的盐。该化合物可以采用本发明提供的以大叶千斤拔为原料提取的方法制备得到，也可以根据本发明提供的结构式结合采用本领域的化学合成等方法制备得到。
作为本发明所述苯丙素类化合物的盐，只要是药学上可接受的盐即可，可列举为与盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸等无机酸形成的无机酸盐；与磺酸形成的磺酸盐；与钾、钠、钙、镁、锂等碱金属的氢氧化物形成的碱金属盐，与铵形成的铵盐等。
本发明苯丙素类化合物可用作如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎等炎症性疾病的治疗药物。
本发明化合物可以与药学上允许的辅料和/或载体一起用作药物组合物，也可以在加入药学上允许的辅料和/或载体的情况下与金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种中药材或提取物的组合用作药物组合物，本发明化合物还可以与其他药学上可接受的药效成分一起用作药物组合物。
作为药物组合物，可以是片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂、混悬剂、糖浆剂、注射剂、软膏剂、栓剂、喷雾剂等。
进一步地，片剂可以是在添加药学上允许的辅料和/或载体的情况下制成的糖衣片、薄膜衣片、肠溶性包衣片或双层片、多层片。
本发明的辅料和/或载体可以如下：
制成固体制剂，可以使用添加剂，例如蔗糖、乳糖、纤维素糖、麦芽糖醇、葡萄糖、淀粉类、琼脂、海藻酸盐类、甲壳素、壳聚糖类、果胶类、阿拉伯树胶类、明胶类、胶原类、酪蛋白、白蛋白、磷酸钙、山梨糖醇、甘氨酸、甘油、聚乙二醇、碳酸氢钠、滑石等。
制成半固体制剂，可以使用动植物性油脂(橄榄油、玉米油、蓖麻油等)、矿物性油脂(凡士林、白凡士林、固态石蜡等)、蜡类(霍霍巴油、巴西棕榈蜡、蜂蜡等)、部分合成或全合成的甘油脂肪酸酯(月桂酸、肉豆蔻酸、棕榈酸等)等。
制成液体制剂，可使用添加剂，例如氯化钠、葡萄糖、山梨糖醇、甘油、橄榄油、丙二醇、乙醇等。尤其制成注射剂的情况下，可以使用无菌水溶液，例如生理盐水、等渗液、油性液，如麻油、大豆油。另外，还可以根据需要，并用适当的助悬剂，如羧甲基纤维素钠，非离子表面活性剂、助溶剂，如苯甲酸苄酯、苯甲醇等。
这些制剂的有效成分的量为制剂的0.01～80重量％，适宜为1～50重量％，给药量根据患者的症状、体重、年龄等不同而变化。
实施例1苯丙素类化合物的制备
本实施例提供式(Ⅰ)所示苯丙素类化合物的一种制备方法，包括如下步骤：
S1.取大叶千斤拔50kg，以根部为原料，干燥，切成小块。经8倍量60％的乙醇回流提取3次，每次2小时，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中浓缩后的浸膏溶于10L水中，采用D101大孔吸附树脂柱对其进行洗脱，洗脱剂为水，洗脱3个柱体积，收集洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，其体积比为25:75，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为：MM-11，MM-12，MM-13，MM-14，MM-15，MM-16备用；
S4.将步骤S3中的收集到的流分MM-12用制备液相分离，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为25:75:0.01，按出峰顺序收集洗脱液，共收集7个流分，分别命名 为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为15:85:0.01，收集洗脱液，重结晶后得到所述苯丙素类化合物。
实施例2苯丙素类化合物的制备
本实施例提供式(Ⅰ)所示苯丙素类化合物的一种制备方法，包括如下步骤：
S1.取大叶千斤拔40kg，以根部为原料，干燥，切成小块。经6倍量50％的乙醇回流提取2次，每次1小时，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中浓缩后的浸膏溶于5L水中，采用D101大孔吸附树脂柱对其进行洗脱，洗脱剂为乙醇与水的体积比为15:85，洗脱3个柱体积，收集洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，其体积比为20:80，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为：MM-11，MM-12，MM-13，MM-14，MM-15，MM-16备用；
S4.将步骤S3中收集到的流分MM-12用制备液相分离，制备液相色谱柱为：YMC，20mm*250mm，流速：10ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为35:65:0.01，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为15:85:0.01，收集洗脱液，重结晶后得到所述苯丙素类化合物。
实施例3苯丙素类化合物的制备
本实施例提供式(Ⅰ)所示苯丙素类化合物的一种制备方法，包括如下步骤：
S1.取大叶千斤拔60kg，以根部为原料，干燥，切成小块。7倍量70％的乙醇回流提取4次，每次3小时，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中浓缩后的浸膏溶于8L水中，采用D101大孔吸附树脂柱对其进 行洗脱，洗脱剂为乙醇与水的体积比为10:90，洗脱3个柱体积，收集洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，其体积比为30:70，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为：MM-11，MM-12，MM-13，MM-14，MM-15，MM-16备用；
S4.将步骤S3中的收集到的流分MM-12用制备液相分离，制备液相色谱柱为：YMC，20mm*250mm，流速：10ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为30:70:0.01，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为15:85:0.01，收集洗脱液，重结晶后得到所述苯丙素类化合物。
实施例4苯丙素类化合物的制备
本实施例提供式(Ⅰ)所示苯丙素类化合物的一种制备方法，包括如下步骤：
S1.取大叶千斤拔50kg，以根部为原料，干燥，切成小块。8倍量60％的乙醇回流提取2次，每次1.5小时，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中浓缩后的浸膏溶于6L水中，采用D101大孔吸附树脂柱对其进行洗脱，洗脱剂为乙醇与水的体积比为5:95，洗脱3个柱体积，收集洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，其体积比为25:75，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为：MM-11，MM-12，MM-13，MM-14，MM-15，MM-16备用；
S4.将步骤S3中收集到的流分MM-12用制备液相分离，制备液相色谱柱为：YMC，20mm*250mm，流速：10ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为25:75:0.01，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备 用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为15:85:0.01，收集洗脱液，重结晶后得到所述苯丙素类化合物。
实施例5苯丙素类化合物的制备
本实施例提供式(Ⅰ)所示苯丙素类化合物的一种制备方法，包括如下步骤：
S1.取大叶千斤拔50kg，以根部为原料，干燥，切成小块。8倍量80％的乙醇回流提取2次，每次1.5小时，将提取液合并，浓缩至无醇味，得浸膏备用；
S2.将步骤S1中浓缩后的浸膏溶于6L水中，采用D101大孔吸附树脂柱对其进行洗脱，洗脱剂为乙醇与水的体积比为10:90，洗脱3个柱体积，收集洗脱液，命名为MM-1，备用；
S3.将步骤S2中收集到的流分MM-1用反相ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，其体积比为28:72，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为：MM-11，MM-12，MM-13，MM-14，MM-15，MM-16备用；
S4.将步骤S3中的收集到的流分MM-12用制备液相分离，制备液相色谱柱为：YMC，20mm*250mm，流速：10ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为10:90:0.01，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
S5.将步骤S4中收集到的流分MM-125用制备液相纯化，制备液相色谱柱为：YMC，20mm*250mm，流速：5ml/min，流动相为甲醇-水-乙酸系统，甲醇：水：乙酸的体积比为15:85:0.01，收集洗脱液，重结晶后得到所述苯丙素类化合物。
将实施例1至实施例5制备得到的化合物进行质谱、核磁共振氢谱、核磁共振碳谱的检测，结果证明所得化合物为：3’,5’-二羟基-4’-葡萄糖基-苯基-3-羟基丙酸甲酯。其结构式如式(Ⅰ)所示：
Figure PCTCN2017076455-appb-000003
Figure PCTCN2017076455-appb-000004
其质谱、核磁共振氢谱、核磁共振碳谱的谱图数据如下：
HR-ESIMS显示[M+Na]+为m/z 399.1889，结合核磁特征，可得分子式为C15H20O11，不饱和度为6。
1H-NMR(600MHz,CD3OD):6.60(d,1H),6.47(d,1H),4.95(s,1H),4.94(m,2H),3.00-4.00(glc-H),2.71(m,2H)。
13C-NMR(150MHz,CD3OD):170.5(C-1),150.7(C-1'),150.2(C-4'),135.5(C-3',5'),114.7(C-2',6'),101.2(C-1”),61.5-77.8(C2”-C6”),61.1(C-2),38.69(C-3)。
实施例6苯丙素类化合物盐的制备
苯丙素类化合物盐酸盐的制备：
搅拌下将该化合物甲醇溶液中滴加饱和盐酸至pH值2-3，搅拌下滴加乙腈，抽滤，干燥得到白色粉末固体，即为化合物的盐酸盐。
苯丙素类化合物磺酸盐的制备：
在含有该苯丙素类化合物、溶剂、磺酸、中性油和促进剂的反应体系中加入碱金属的氢氧化物，加入溶剂、低级醇和助促进剂，通入二氧化碳，分离得到白色粉末固体，即为化合物的磺酸盐。
苯丙素类化合物钾盐或钠盐的制备：
将溶于乙醇中的KOH或NaOH加入该化合物中，搅拌下加热回流反应，冷制室温，搅拌下滴加乙腈，抽滤，干燥得白色固体，即为该化合物的钾盐或钠盐。
苯丙素类化合物铵盐的制备：
搅拌下将该化合物甲醇溶液中滴加饱和氨水至pH值9-11，搅拌下滴加乙腈，抽滤，干燥得到白色固体，即为苯丙素类化合物的铵盐。
上述化合物盐的谱图数据：
苯丙素类化合物盐酸盐：ESIMS显示m/z 412.67，核磁特征1H-NMR(600MHz,CD3OD):1H-NMR(600MHz,CD3OD):6.63(d,1H),6.49(d,1H),5.05(s,1H),4.98(m,2H),3.00-4.00(glc-H),2.74(m,2H)。
苯丙素类化合物磺酸盐：ESIMS显示为m/z 504.28，核磁特征1H-NMR(600MHz,CD3OD):1H-NMR(600MHz,CD3OD):6.55(d,1H),6.36(d,1H),4.90(s,1H), 4.91(m,2H),3.00-4.00(glc-H),2.51(m,2H)。
苯丙素类化合物钾盐或钠盐：
钾盐ESIMS显示m/z 452.19，核磁特征1H-NMR(600MHz,CD3OD):6.70(d,1H),6.57(d,1H),4.94(s,1H),4.91(m,2H),3.00-4.00(glc-H),2.70(m,2H)。
钠盐ESIMS显示m/z 420.19，核磁特征1H-NMR(600MHz,CD3OD):6.56(d,1H),6.46(d,1H),4.74(s,1H),4.51(m,2H),3.00-4.00(glc-H),2.60(m,2H)。
苯丙素类化合物铵盐：ESIMS显示m/z 406.57，核磁特征1H-NMR(600MHz,CD3OD):1H-NMR(600MHz,CD3OD):6.63(d,1H),6.33(d,1H),4.94(s,1H),4.91(m,2H),3.00-4.00(glc-H),2.77(m,2H)。
上述苯丙素类化合物盐的结构式如式(Ⅳ)～式(Ⅷ)所示。
Figure PCTCN2017076455-appb-000005
Figure PCTCN2017076455-appb-000006
其中，式(Ⅳ)为制备得到的苯丙素类化合物盐酸盐，式(Ⅴ)为制备得到的苯丙素类化合物的其中一种磺酸盐，式(Ⅵ)为制备得到的苯丙素类化合物的其中一种钾盐，式(Ⅶ)为制备得到的苯丙素类化合物的其中一种钠盐，式(Ⅷ)为制备得到的苯丙素类化合物的其中一种铵盐。
实验例7应用试验
本发明所述化合物以及盐对LPS诱导的RAW 264.7巨噬细胞氧化应激与炎症的影响。(为了实验过程中记录方便，以下将本发明所述的苯丙素类化合物标号为：药物MM-125，即本发明中所述的药物MM-125即是指本发明式(Ⅰ)所示苯丙素类化合物或其药学上可接受的盐。)
1材料与方法
1.1药品及仪器
脂多糖(lipopolysaccharide,LPS)，MTT购自Sigma公司；小鼠巨噬细胞Raw264.7购自湘雅细胞库；PBS；DMEM高糖培养基、胎牛血清、青霉素与链霉素；全自动酶标仪；恒温CO2培养箱。
小鼠白介素1-β(IL-1-β)ELISA检测试剂盒，批号：2014/06(96T)；小鼠白介素6(IL-6)ELISA检测试剂盒，批号：2014/06(96T)；小鼠肿瘤坏死因子-α(TNF-α)ELISA检测试剂盒，批号：2014/06(96T)；小鼠一氧化氮(NO)ELISA 检测试剂盒，批号：2014/10(96T)；小鼠羟基自由基(OH)ELISA检测试剂盒，批号：2014/10(96T)。
1.2药物制备
首先用少量DMSO溶解，然后用DMEM稀释至一定的浓度，使终浓度中DMSO含量少于1‰。
1.3细胞培养
小鼠巨噬细胞Raw 264.7培养于含10％热灭活(56℃，30min)的胎牛血清(FBS)、10U/mL青霉素钠、100μg/mL链霉素的DMEM培养基中，37℃、5％CO2恒温培养箱中孵育生长。
1.4细胞活力测定
细胞活力通过MTT法来测定。将细胞制成细胞悬液接种于96孔板(1×104个/孔)孵育24h，再同步化24h，然后将不同浓度的药物作用于细胞2h，然后再加入LPS(30μg/mL)刺激24h，吸弃原培养基，每孔加入100μL的MTT(0.5mg/mL)继续孵育4h，吸弃培养基，每孔加入150μL的DMSO，摇床振摇10min，在490nm处测定吸光度。
1.5NO含量测定
Raw 264.7细胞接种于96孔板24h，再同步化24h，然后将不同浓度的药物作用于细胞2h，然后再加入LPS(30μg/mL)刺激24h，最后收集上清液，并于10000rpm离心5min，分装上清并置于-80℃保存备用。通过小鼠NO试剂盒测定NO含量。
1.6炎症因子TNF-α,IL-1β,IL-6测量
样品取1.5制备好的样品用于后续炎症因子测定。细胞产生TNF-α,IL-1β,IL-6的量通过小鼠TNF-α,IL-1β,IL-6试剂盒来测定。
1.7OH含量测定
样品取1.5制备好的样品用于OH因子测定。通过OH试剂盒测定含量。
1.8统计分析
采用SPSS17.0软件，实验数据以x±s表示；所得数据通过用单因素方差分析，方差齐性用LSD检验，方差不齐用Dunnett T3检验。
2实验结果
2.1细胞活力
药物对细胞活力的影响通过MTT法来评价。如图3所示，药物MM-125在1.37-12.37μg/mL浓度范围内对Raw 264.7细胞活力没有显著影响；因此在此范围浓度下的药物浓度对于后续实验是合适的。
2.2药物抑制NO的产生
如图4所示，通过LPS刺激Raw 264.7细胞，其产生NO(48.66±1.66IU/mL)含量与正常组NO(29.05±0.85IU/mL)相比明显升高(p＜0.01)。药物MM-125在浓度(6.88±12.38μg/mL)范围内对LPS引起的NO含量升高有明显的抑制作用，并表现出明显的剂量依赖关系。
2.3药物抑制TNF-α,IL-1β,IL-6的产生
如图5至图7所示，通过LPS刺激Raw 264.7细胞，Raw 264.7细胞炎症因子TNF-α(127.98±4.65pg/mL)，IL-1β(347.55±9.56pg/mL)，IL-6(201.67±10.32pg/mL)含量与正常组TNF-α(67.12±4.00pg/mL),IL-1β(160.88±13.10pg/mL),IL-6(111.13±8.06pg/mL)相比含量明显升高(p＜0.01)；说明LPS能够刺激Raw264.7细胞产生大量炎症因子。
药物MM-125在浓度(6.88-12.38μg/mL)范围内能够显著抑制Raw 264.7细胞产生炎症因子TNF-α(p＜0.05)，并呈显著的剂量依赖关系；在各浓度下，不能显著抑制Raw 264.7细胞产生IL-1β，IL-6(p＞0.05)。
2.4药物抑制OH的产生
如图8所示，通过LPS刺激Raw 264.7细胞，其产生OH(106.00±3.90ng/mL)含量与正常组OH(64.62±2.18ng/mL)相比明显升高(p＜0.01)。
药物MM-125在浓度(4.13-12.38μg/mL)范围内对LPS引起的OH含量升高有明显的抑制作用，并表现出一定的剂量依赖关系。
本实验经体外培养，研究了药物MM-125对小鼠巨噬细胞NO,TNF-α,IL-1β,IL-6,OH生成的影响。
药物MM-125对细胞炎症因子NO的产生有明显抑制作用，并且对TNF-α也有一定的抑制作用，但其对IL-1β,IL-6无明显抑制作用，说明其有一定的抗炎活性；其对OH的产生有明显的抑制效果，说明其有较好的抗氧化活性。
实施例8
片剂的制备：按实施例1方法先制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，按该化合物或其任意一种盐与赋形剂重量比为1:10的比例加入赋形剂，制粒压片。
实施例9
散剂的制备：按实施例1方法先制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，按常规散剂制法制成散剂。
实施例10
胶囊剂或颗粒剂的制备：按实施例1方法先制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，按该化合物或其任意一种盐与赋形剂重量比为1:10的比例加入赋形剂，制成胶囊剂或颗粒剂。
实施例11
注射剂的制备：按实施例1方法先制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，按常规注射用水，精滤，灌封灭菌制成注射剂。
实施例12
一种药物组合物，含有实施例1方法制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，以及金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参制成的粉末，和辅料。
实施例13
一种药物组合物，含有实施例1方法制得式(Ⅰ)所示的苯丙素类化合物，以及金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参制成的粉末，和辅料。
实施例14
一种药物组合物，含有实施例1方法制得式(Ⅰ)所示的苯丙素类化合物，以及金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参的提取物，和辅料。提取物是按专利公告号CN1078079C、CN1170549C、CN1158087C、CN1330335C、CN1296071C、CN1321631C、CN1296072C、CN1296073C的任意一件或几件专利文件中提取方法制备得到。
实施例15
一种药物组合物，含有实施例1方法制得式(Ⅰ)所示的苯丙素类化合物，以及利用该化合物与无机酸(如盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸、乳酸)或磺酸或碱金属的氢氧化物(如氢氧化钾、氢氧化钠、氢氧化钙、氢氧化镁、氢氧化锂)或铵制成的盐，以及金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参的提取物，和辅料。提取物是按专利公告号CN1078079C、CN1170549C、CN1158087C、CN1330335C、CN1296071C、CN1321631C、CN1296072C、CN1296073C的任意一件或几件专利文件中提取方法制备得到。
以上显示和描述了本发明的基本原理和主要特征和本发明的优势。本领域的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这对本领域技术人员而言是显而易见的，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (26)

  1. 一种苯丙素类化合物及其药学上可接受的盐，其特征在于，所述苯丙素类化合物的结构式如式(Ⅰ)所示，所述药学上可接受的盐的结构如式(Ⅱ)或式(Ⅲ)所示：
    Figure PCTCN2017076455-appb-100001
    其中，R为无机酸，R1或R2或R3为磺酸根、碱金属离子或铵根中的任意一种或任意两种或任意三种。
  2. 根据权利要求1所述苯丙素类化合物及其药学上可接受的盐，其特征在于，所述无机酸为盐酸、氢溴酸、氢氟酸、氢碘酸、硫酸、硝酸、羧酸、磷酸或乳酸；所述磺酸根为具有芳基的磺酸根；所述碱金属离子为钾离子、钠离子、钙离子、镁离子或锂离子。
  3. 根据权利要求1所述苯丙素类化合物及其药学上可接受的盐，其特征在 于，所述具有芳基的磺酸根为苯磺酸根或对甲苯磺酸根。
  4. 根据权利要求1所述苯丙素类化合物及其药学上可接受的盐，其特征在于，所述药学上可接受的盐为铵盐。
  5. 一种药物组合物，其特征在于，含有权利要求1至4任一项所述苯丙素类化合物和/或其药学上可接受的盐。
  6. 根据权利要求5所述药物组合物，其特征在于，所述药物组合物还含有药学上允许的辅料和/或载体。
  7. 根据权利要求5所述的药物组合物，其特征在于，还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种。
  8. 根据权利要求5所述的药物组合物，其特征在于，所述药物组合物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种的提取物。
  9. 根据权利要求5所述的药物组合物，其特征在于，所述药物组合物的剂型为片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂、混悬剂、糖浆剂、注射剂、软膏剂、栓剂或喷雾剂。
  10. 一种权利要求1至4任一项所述苯丙素类化合物的制备方法，其特征在于，所述制备方法包括如下步骤：
    S1.取大叶千斤拔的根部为原料，干燥，切块，经乙醇溶液提取，将提取液合并，浓缩至无醇味，得浸膏备用；
    S2.将步骤S1中所得浸膏加水溶解，采用大孔吸附树脂柱对其进行洗脱，洗脱剂为乙醇-水系统，收集前3个柱体积的洗脱液，命名为MM-1，备用；
    S3.将步骤S2中收集到的流分MM-1用反相材料ODS柱层析进行洗脱，洗脱剂为甲醇-水系统，洗脱18个柱体积，按每3个柱体积收集一个流分的洗脱液，按顺序收集6个流分，分别命名为MM-11，MM-12，MM-13，MM-14，MM-15，MM-16，备用；
    S4.将步骤S3中收集到的流分MM-12用制备液相分离，流动相为甲醇-水-乙酸系统，按出峰顺序收集洗脱液，共收集7个流分，分别命名为MM-121，MM-122，MM-123，MM-124，MM-125，MM-126，MM-127，备用；
    S5.将步骤S4中收集到的流分MM-125用制备液相纯化，流动相为甲醇- 水-乙酸系统，收集洗脱液，重结晶后得到所述苯丙素类化合物。
  11. 根据权利要求10所述的制备方法，其特征在于：步骤S1中，乙醇溶液的浓度为50～80体积％，优选为乙醇溶液浓度为60体积％。
  12. 根据权利要求10所述的制备方法，其特征在于：步骤S1中，乙醇的提取次数为2～4次，每次提取1～3小时，优选为乙醇的提取次数为3次，每次2小时。
  13. 根据权利要求10所述的制备方法，其特征在于：步骤S2中，大孔吸附树脂采用D101大孔吸附树脂。
  14. 根据权利要求10所述的制备方法，其特征在于：步骤S2中，乙醇与水的体积比为0:100～15:85。
  15. 根据权利要求10所述的制备方法，其特征在于：步骤S3中，洗脱剂甲醇与水的体积比为20:80～30:70，优选为25:75。
  16. 根据权利要求10所述的制备方法，其特征在于：步骤S4中，甲醇-水-乙酸的体积比为10:90:0.01～35:65:0.01，优选为15:85:0.01。
  17. 根据权利要求10所述的制备方法，其特征在于：步骤S4中，制备液相的色谱柱为YMC，20mm*250mm，流动相流速为5～10mL/min，优选为流速5mL/min。
  18. 根据权利要求10所述的制备方法，其特征在于，步骤S5中，甲醇-水-乙酸的体积比为15:85:0.01。
  19. 根据权利要求10所述的制备方法，其特征在于：步骤S5中，制备液相的色谱柱为YMC，20mm*250mm，流动相流速为5mL/min。
  20. 一种权利要求1至4任一项所述苯丙素类化合物及其药学上可接受的盐在制备治疗炎症性疾病的药物中的应用。
  21. 根据权利要求20所述应用，其特征在于，是在制备抑制细胞炎症因子NO的含量或抑制细胞炎症因子TNF-α的表达或抑制羟基自由基的活性的药物中的应用。
  22. 根据权利要求20所述应用，其特征在于，所述炎症性疾病为宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎和/或关节炎。
  23. 根据权利要求20至22任一项所述应用，其特征在于，所述药物含有 药学上允许的辅料和/或载体。
  24. 根据权利要求23所述应用，其特征在于，所述药物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种。
  25. 根据权利要求23所述应用，其特征在于，所述药物还含有金樱根、单面针、鸡血藤、功劳木、穿心莲、当归、党参中的一种或几种的提取物。
  26. 根据权利要求23所述应用，其特征在于，所述药物的剂型为片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂、混悬剂、糖浆剂、注射剂、软膏剂、栓剂或喷雾剂。
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