CN105693789A - Preparation method of phenylpropanoid compound - Google Patents
Preparation method of phenylpropanoid compound Download PDFInfo
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- CN105693789A CN105693789A CN201610153508.4A CN201610153508A CN105693789A CN 105693789 A CN105693789 A CN 105693789A CN 201610153508 A CN201610153508 A CN 201610153508A CN 105693789 A CN105693789 A CN 105693789A
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Abstract
The invention discloses a preparation method of a phenylpropanoid compound, and relates to the technical field of medicine, in particular to a phenylpropanoid compound separated from dried roots of flemingia macrophylla. The compound is prepared through the steps of solvent extraction, column chromatography separation, liquid phase preparation, and separation and purification. The compound obtained through the method is reported for the first time and is high in purity. Experiments prove that the obtained phenylpropanoid compound can restrain the content of the inflammatory cytokine NO and then achieve anti-inflammatory activity by restraining NO approaches, is beneficial to treatment of various inflammatory diseases such as cervicitis, endometritis, pelvic inflammation, mastitis, sphagitis and/or arthritis, and can be developed into new medicine. The structural formula of the phenylpropanoid compound and medically-acceptable salt thereof is the formula (I) in the description.
Description
Technical field
The present invention relates to pharmaceutical technology field, more particularly, to the preparation method of a kind of phenylpropanoids。
Background technology
From natural drug, extract that to separate the constituent structure that obtains various, active significantly, it is easily separated purification, structural modification, transformation and complete synthesis, is always up a main thought of new drug development。
TNF-α: be a kind of can direct killing tumor cell and to the normal cell cytokine without overt toxicity, be one of bioactie agent that the direct killing function of tumor found up to now is the strongest, but its toxic and side effects be also very serious。
IL-1 β: collaborative APC and the T cell activation of stimulating, promotion B cell proliferation and secretory antibody when local low concentration, carries out immunomodulating。There is endocrine effect during a large amount of generation: induced liver acute phase protein synthesizes, cause heating and cachexia。
IL-6: mankind's IL-6 gene is positioned on No. 7 chromosome；IL-6 molecular weight is between 21～30KD。Main by mononuclear phagocyte, Th2 cell, vascular endothelial cell, fibroblast generation。Activating B cell can be stimulated to breed, secretory antibody；Stimulate T cell propagation and CTL activation；Cell cultured supernatant synthesized acute phase albumen, participates in inflammatory reaction；Promote blood cell development。
IL-6 can be synthesized by various kinds of cell, including T cell and B cell, monocytes/macrophages, endotheliocyte, epithelial cell and the fibroblast etc. of activation。The target cell of IL-6 effect is a lot, including macrophage, hepatocyte, static T cell, the B cell of activation and plasma cell etc.；Its biological effect is also sufficiently complex。
Macrophage can produce inflammation medium and participate in inflammatory reaction, and wherein, NO is the important cellular inflammation factor, and NO participates in multiple pathological processes, and excessive NO can promote generation and the development of diseases associated with inflammation, and can also induce other inflammatory factors。
Therefore, seek a kind of new preparation method, from natural plants, separate the compound made new advances, to suppress the content of cellular inflammation factor NO, be applied to the treatment of diseases associated with inflammation, be necessary。
Radix Flemingiae Philippinensis medical material is the dry root of pulse family (Leguminosae) Moghania (Flemingl.Roxb. or Moghania) plant Flemingia macrophylla (Moghaniamacrophylla (Willd.) O.Kuntze), is distributed mainly on region of Southeast in China。This plant is in Chinese Plants will (1995,41:313), Flora of Taiwan (1977,3:258), Hainan flora (1965,2:311), " Chinese Higher plant illustrated handbook " (1972,2:510), China main plant figure says (1955, and Guangzhou flora (1956, pp361) is all included pp707)。Radix Flemingiae Philippinensis medical material is the genuine medicinal materials of Guangxi province, and history is loaded in " Zhiwu Mingshi Tukao ", has the basis of medication among the people widely。Its nature and flavor are sweet, micro-puckery, flat, have the effects such as removing damp-heat, are mainly used in the gynaecopathias such as treatment rheumatic ostalgia, traumatic injury, chronic nephritis, dysmenorrhea and leucorrhea be many。This medical material has recorded at present into version " Chinese Pharmacopoeia " annex in 2005。
The composition that Flemingia macrophylla has been reported mainly has flavonoid, steroid class, terpenoid, Anthraquinones, volatile oil composition, is respectively provided with certain pharmacologically active。
Flemingia macrophylla pharmacologically active is various, reports the more neuroprotective that has, and antiinflammatory, antioxidation, to the anthelmintic action of pathogenic microorganism, parahormone effect, cytotoxicity, antibacterial action and immunological enhancement, antifatigue effect。
Radix Flemingiae Philippinensis is now widely used for the Chinese patent medicine of the type such as gynecological, rheumatic arthralgia and produces, such as FUKE QIANJIN PIAN, FUKE QIANJIN JIAONANG, JINJI CHONGJI, JINJI JIAONANG etc., this type of Chinese patent medicine be mainly used in gynaecopathia (dysmenorrhea, cold uterus be infertile, uterine prolapse, pelvic inflammatory disease, mastitis, leucorrhea are many, puerperal blood deficiency, arthralgia, puerperal waist and knee, hypogalactia and breast ulcer etc.), weak anemia (woman anemia, deficient qi and blood and the after being ill deficiency of vital energy etc.)。In recent years, what clinical report was more is FUKE QIANJIN PIAN effect in treatment gynecological inflammation。
At present, in the document of Radix Flemingiae Philippinensis, the document of report phenylpropanoids is few, find a kind of new preparation method, from natural plants, isolate effective Phenylpropanoid Glycosides class noval chemical compound, it is easily separated purification, structural modification and synthesis, developing new drug, it is applied to the treatment of diseases associated with inflammation, significant。
Summary of the invention
The preparation method that it is an object of the invention to provide a kind of a kind of phenylpropanoids separating from the dry root of Flemingia macrophylla and obtaining, separate, by the extraction of this preparation method, the compound obtained and can suppress the content of cellular inflammation factor NO, and then by suppressing NO approach to realize anti-inflammatory activity, it is of value to the treatment of various diseases associated with inflammation, it is possible to this compound is developed into new drug。
Specifically, the preparation method of phenylpropanoids of the present invention comprises the steps:
S1. the root taking Flemingia macrophylla is raw material, dry, and stripping and slicing is extracted through alcoholic solution, is merged by extracting solution, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in water by gained extractum in step S1, adopt macroporous adsorptive resins that it is carried out eluting, eluant is ethanol-water system, collects the eluent of front 3 column volumes, and called after MM-1 is standby；
S3. the flow point MM-1 reversed material ODS column chromatography collected in step S2 is carried out eluting, eluant is methanol-water system, 18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collect 6 flow points in order, it is respectively designated as MM-11, MM-12, MM-13, MM-14, MM-15, MM-16, standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, mobile phase is methanol-water-acetic acid system, collects eluent by peak sequence, collects 7 flow points altogether, it is respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. being prepared by the flow point MM-124 collected in step S4 liquid phase purification, mobile phase is methanol-water-acetic acid system, collects eluent, obtains described phenylpropanoids after recrystallization。Described phenylpropanoids purity is 99.31%。The structural formula of described phenylpropanoids is such as shown in formula I:
Preferably, in step S1, the concentration of ethanol is 50～80 volume %, and more preferably concentration of alcohol is 60 volume %。
Preferably, in step S1, the extraction time of ethanol is 2～4 times, extracts 1～3 hour every time, and more preferably the extraction time of ethanol is 3 times, each 2 hours。
Preferably, in step S2, macroporous adsorbent resin adopts D101 macroporous adsorbent resin。
Preferably, in step S2, the volume ratio of ethanol and water is 0:100～15:85。
Preferably, in step S3, the volume ratio of methanol and water is 20:80～30:70, more preferably 25:75。
Preferably, in step S4, the volume ratio of methanol-water-acetic acid is 10:90:0.01～35:65:0.01, more preferably 15:85:0.01。
Preferably, in step S4, preparative liquid chromatography post is YMC, 20mm*250mm, and flow rate of mobile phase is 5～10mL/min, it is preferred to flow velocity 5mL/min。
Preferably, in step S5, the volume ratio of methanol-water-acetic acid is 15:85:0.01。
Preferably, in step S5, preparative liquid chromatography post is YMC, 20mm*250mm, and flow rate of mobile phase is 5mL/min。
The present invention provides the phenylpropanoids that described preparation method prepares。
And with phenylpropanoids salt that described phenylpropanoids prepares for raw material through chemical synthesis process。
Preferably, the inorganic acid salt that described phenylpropanoids salt is the compound shown in formula I to be formed with mineral acid, or the sulfonate formed with sulfonic acid, or the alkali metal salt formed with alkali metal hydroxide, or the ammonium salt formed with ammonium。
Preferably, described inorganic acid salt is hydrochlorate, hydrobromate, hydrofluoride, hydriodate, sulfate, nitrate, carboxylate, phosphate or lactate；Described alkali metal salt is potassium salt, sodium salt, calcium salt, magnesium salt or lithium salts。
Beneficial effects of the present invention:
The present invention is new phenylpropanoids and pharmaceutically acceptable salt provides a kind of preparation method, a kind of new phenylpropanoids obtained is separated first from the dry root of Flemingia macrophylla, the compound prepared can suppress the content of cellular inflammation factor NO, and then by suppressing NO approach to realize anti-inflammatory activity, it is possible to it is used as such as the medicine of the diseases associated with inflammation such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis and/or arthritis。
It is an object of the invention to from traditional prescriptions of Chinese medicine, by in the prescription from FUKE QIANJIN PIAN and FUKE QIANJIN JIAONANG, a kind of noval chemical compound is prepared by solvent extraction, column chromatography for separation, preparation liquid phase separation, purification, and experiments prove that, it can apply to diseases associated with inflammation, such as the treatment of the diseases such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis and/or arthritis。
Specifically, inventor by from FUKE QIANJIN PIAN, FUKE QIANJIN JIAONANG prescription, science chooses the dry root of Flemingia macrophylla, by solvent extraction, column chromatography for separation, preparation liquid phase separation, purification, obtain phenylpropanoids of the present invention, then this compound is carried out test cell line, measure its suppression degree to cellular inflammation factor NO, experiments show that, the NO content that LPS is caused by this compound in concentration (4.87 14.62 μ g/mL) scope raises obvious inhibitory action, and shows obvious dose-dependence。
Accompanying drawing explanation
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram of phenylpropanoids of the present invention。
Fig. 2 is the carbon-13 nmr spectra figure of phenylpropanoids of the present invention。
Fig. 3 is that cytoactive is affected figure by phenylpropanoids of the present invention。
Fig. 4 is the phenylpropanoids of the present invention inhibitory action figure to NO。
Fig. 5 is the phenylpropanoids of the present invention inhibitory action figure to TNF-α。
Fig. 6 is the phenylpropanoids of the present invention inhibitory action figure to IL-1 β。
Fig. 7 is the phenylpropanoids of the present invention inhibitory action figure to IL-6。
Fig. 8 is the phenylpropanoids of the present invention inhibitory action figure to OH。
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but the present invention is not limited in any form by embodiment。Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus。Unless stated otherwise, raw material used by the present embodiment and equipment are the conventional commercial raw material of the art and equipment。
The compound of the present invention is the phenylpropanoids shown in described formula I and this compound pharmaceutically acceptable salt。This compound can adopt the method extracted for raw material with Flemingia macrophylla provided by the invention to prepare, it is also possible to prepares according to methods such as the chemosynthesis of structural formula provided by the invention combination employing this area。
As the salt of phenylpropanoids of the present invention, as long as pharmaceutically acceptable salt, the inorganic acid salt formed with the mineral acid such as hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid can be enumerated as；Sulfonate with sulfonic acid formation；The alkali metal salt formed with the alkali-metal hydroxide such as potassium, sodium, calcium, magnesium, lithium, the ammonium salt etc. formed with ammonium。
Phenylpropanoids of the present invention can be used as such as the medicine of the diseases associated with inflammation such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis and/or arthritis。
The compounds of this invention can be used as pharmaceutical composition together with the adjuvant pharmaceutically allowed and/or carrier, can also being used as pharmaceutical composition when adding the adjuvant and/or carrier that pharmaceutically allow with the combination of one or more Chinese crude drugs in Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, Radix Codonopsis or extract, the compounds of this invention can also be used as pharmaceutical composition together with other pharmaceutically acceptable active ingredient。
As pharmaceutical composition, it is possible to be tablet, capsule, powder, granule, pill, solution, suspensoid, syrup, injection, ointment, suppository, spray etc.。
Further, tablet can be make when adding the adjuvant that pharmaceutically allows and/or carrier coated tablet, Film coated tablets, enteric coatings ply or two-ply, multilayer tablet。
Adjuvant and/or the carrier of the present invention can be such that
Make solid preparation, additive can be used, for instance sucrose, lactose, cellulose sugar, maltose alcohol, glucose, starch based, agar, alginates, chitin, chitosan class, pectin class, Radix Acaciae senegalis class, gelatin class, collagen class, casein, albumin, calcium phosphate, Sorbitol, glycine, glycerol, Polyethylene Glycol, sodium bicarbonate, Talcum etc.。
Make semi-solid preparation, it is possible to use of animal or plant nature oils and fats (olive oil, Semen Maydis oil, Semen Ricini wet goods), mineral oils and fats (vaseline, white vaseline, solid paraffin etc.), wax class (Jojoba oil, Brazil wax, Cera Flava etc.), partial synthesis or complete synthesis fatty acid glyceride (lauric acid, myristic acid, Palmic acid etc.) etc.。
Make liquid preparation, additive can be used, for instance sodium chloride, glucose, Sorbitol, glycerol, olive oil, propylene glycol, ethanol etc.。When especially making injection, it is possible to use aseptic aqueous solution, for instance normal saline, isotonic solution, oiliness liquid, such as Oleum Sesami, soybean oil。Furthermore it is also possible to as required, and with suitable suspending agent, such as sodium carboxymethyl cellulose, nonionic surfactant, cosolvent, such as benzyl benzoate, benzyl alcohol etc.。
0.01～80 weight % that amount is preparation of the effective ingredient of these preparations, is suitably 1～50 weight %, and dosage changes according to differences such as the symptom of patient, body weight, ages。
The preparation of embodiment 1 phenylpropanoids
The present embodiment provides a kind of preparation method of phenylpropanoids shown in formula I, comprises the steps:
S1. Flemingia macrophylla 50kg is taken, with root for raw material, dry, it is cut into small pieces。Through the alcohol reflux 3 times of 8 times amount 60%, each 2 hours, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in 10L water by the extractum after concentration in step S1, adopt D101 macroporous adsorptive resins that it is carried out eluting, eluant is water, 3 column volumes of eluting, collection eluent, and called after MM-1 is standby；
S3. the flow point MM-1 collected in step S2 is carried out eluting with anti-phase ODS column chromatography, eluant is methanol-water system, and its volume ratio is 25:75,18 column volumes of eluting, the eluent of a flow point is collected by every 3 column volumes, collect 6 flow points in order, be respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 25:75:0.01, collect eluent by peak sequence, collect 7 flow points altogether, be respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. the flow point MM-124 collected in step S4 is prepared liquid phase purification, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 15:85:0.01, collects eluent, obtains described phenylpropanoids after recrystallization。
The preparation of embodiment 2 phenylpropanoids
The present embodiment provides a kind of preparation method of phenylpropanoids shown in formula I, comprises the steps:
S1. Flemingia macrophylla 40kg is taken, with root for raw material, dry, it is cut into small pieces。Through the alcohol reflux 2 times of 6 times amount 50%, each 1 hour, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in 5L water by the extractum after concentration in step S1, adopt D101 macroporous adsorptive resins that it is carried out eluting, the volume ratio that eluant is ethanol and water is 15:85,3 column volumes of eluting, collection eluent, and called after MM-1 is standby；
S3. the flow point MM-1 collected in step S2 is carried out eluting with anti-phase ODS column chromatography, eluant is methanol-water system, and its volume ratio is 20:80,18 column volumes of eluting, the eluent of a flow point is collected by every 3 column volumes, collect 6 flow points in order, be respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 10ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 35:65:0.01, collect eluent by peak sequence, collect 7 flow points altogether, be respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. the flow point MM-124 collected in step S4 is prepared liquid phase purification, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 15:85:0.01, collects eluent, obtains described phenylpropanoids after recrystallization。
The preparation of embodiment 3 phenylpropanoids
The present embodiment provides a kind of preparation method of phenylpropanoids shown in formula I, comprises the steps:
S1. Flemingia macrophylla 60kg is taken, with root for raw material, dry, it is cut into small pieces。The alcohol reflux of 7 times amount 70% 4 times, each 3 hours, merges extracting solution, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in 8L water by the extractum after concentration in step S1, adopt D101 macroporous adsorptive resins that it is carried out eluting, the volume ratio that eluant is ethanol and water is 10:90,3 column volumes of eluting, collection eluent, and called after MM-1 is standby；
S3. the flow point MM-1 collected in step S2 is carried out eluting with anti-phase ODS column chromatography, eluant is methanol-water system, and its volume ratio is 30:70,18 column volumes of eluting, the eluent of a flow point is collected by every 3 column volumes, collect 6 flow points in order, be respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 10ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 30:70:0.01, collect eluent by peak sequence, collect 7 flow points altogether, be respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. the flow point MM-124 collected in step S4 is prepared liquid phase purification, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 15:85:0.01, collects eluent, obtains described phenylpropanoids after recrystallization。
The preparation of embodiment 4 phenylpropanoids
The present embodiment provides a kind of preparation method of phenylpropanoids shown in formula I, comprises the steps:
S1. Flemingia macrophylla 50kg is taken, with root for raw material, dry, it is cut into small pieces。The alcohol reflux of 8 times amount 60% 2 times, each 1.5 hours, merges extracting solution, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in 6L water by the extractum after concentration in step S1, adopt D101 macroporous adsorptive resins that it is carried out eluting, the volume ratio that eluant is ethanol and water is 5:95,3 column volumes of eluting, collection eluent, and called after MM-1 is standby；
S3. the flow point MM-1 collected in step S2 is carried out eluting with anti-phase ODS column chromatography, eluant is methanol-water system, and its volume ratio is 25:75,18 column volumes of eluting, the eluent of a flow point is collected by every 3 column volumes, collect 6 flow points in order, be respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 10ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 25:75:0.01, collect eluent by peak sequence, collect 7 flow points altogether, be respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. the flow point MM-124 collected in step S4 is prepared liquid phase purification, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 15:85:0.01, collects eluent, obtains described phenylpropanoids after recrystallization。
The preparation of embodiment 5 phenylpropanoids
The present embodiment provides a kind of preparation method of phenylpropanoids shown in formula I, comprises the steps:
S1. Flemingia macrophylla 50kg is taken, with root for raw material, dry, it is cut into small pieces。The alcohol reflux of 8 times amount 80% 2 times, each 1.5 hours, merges extracting solution, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in 6L water by the extractum after concentration in step S1, adopt D101 macroporous adsorptive resins that it is carried out eluting, the volume ratio that eluant is ethanol and water is 10:90,3 column volumes of eluting, collection eluent, and called after MM-1 is standby；
S3. the flow point MM-1 collected in step S2 is carried out eluting with anti-phase ODS column chromatography, eluant is methanol-water system, and its volume ratio is 28:72,18 column volumes of eluting, the eluent of a flow point is collected by every 3 column volumes, collect 6 flow points in order, be respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 10ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 10:90:0.01, collect eluent by peak sequence, collect 7 flow points altogether, be respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. the flow point MM-124 collected in step S4 is prepared liquid phase purification, preparative liquid chromatography post is: YMC, 20mm*250mm, flow velocity: 5ml/min, mobile phase is methanol-water-acetic acid system, methanol: water: the volume ratio of acetic acid is 15:85:0.01, collects eluent, obtains described phenylpropanoids after recrystallization。
Compound embodiment 1 to embodiment 5 prepared carries out the detection of mass spectrum, proton nmr spectra, carbon-13 nmr spectra, and result proves that gained compound is: and 4 '-glucosyl group (6 "-glucosyl group)-3 '-methoxyphenyl 3-hydroxy-4-methyl methyl valerate。Its structural formula is such as shown in formula I:
Its mass spectrum, proton nmr spectra, carbon-13 nmr spectra spectral data as follows:
HR-ESIMS shows [M+Na]+for m/z601.2243, and in conjunction with nuclear-magnetism feature, can obtain molecular formula is C25H38O15, degree of unsaturation is 7。
1HNMR (400MHz, MeOD) δ 6.77 (d, J=2.3Hz, 1H), 6.70 (d, J=8.6Hz, 1H), 6.57 (d, J=8.6Hz, 2.3Hz, 1H), 5.47 (s, 1H), 4.85 (s, 1H), 4.67 (t, 1H), 4.26 (m, 2H), 3.20-4.00 (glc-H), 2.81 (s, 1H), 1.43,1.14 (s, 6H)。
13C-NMR(150MHz,CD3OD):176.5(C-1),151.4(C-4'),147.9(C-1'),141.5(C-3'),114.7(C-2'),112.7(C-6'),109.1(C-5'),102.3(C”),101.1(C”'),88.4(C-3),60.5(C-2),55.1(-OCH3),37.7(C-4),24.8(C-5),20.2(-CH3),61.5-77.8(glc-C)。
The preparation of embodiment 6 phenylpropanoids salt
The preparation of phenylpropanoids hydrochlorate:
This compound methanol solution will drip saturated hydrochloric acid to pH value 2～3 under stirring, drip acetonitrile, sucking filtration under stirring, dry and obtain white powder solid, be the hydrochlorate of compound。
The preparation of phenylpropanoids sulfonate:
Reaction system containing this phenylpropanoids, solvent, sulfonic acid, neutral oil and accelerator adds alkali-metal hydroxide, add solvent, lower alcohol and kicker, pass into carbon dioxide, separate and obtain white powder solid, be the sulfonate of compound。
The preparation of phenylpropanoids potassium salt or sodium salt:
KOH or NaOH being dissolved in ethanol is added in this compound, the lower heating reflux reaction of stirring, cold room temperature processed, under stirring, drip acetonitrile, sucking filtration, dry to obtain white solid, be potassium salt or the sodium salt of this compound。
The preparation of phenylpropanoids ammonium salt:
This compound methanol solution will drip saturated ammonia to pH value 9～11 under stirring, drip acetonitrile, sucking filtration under stirring, dry and obtain white solid, be the ammonium salt of compound。
The spectral data of above-claimed cpd salt:
Phenylpropanoids hydrochlorate: ESIMS shows m/z614.38, nuclear-magnetism feature1H-NMR(600MHz,CD3OD):1HNMR (400MHz, MeOD) δ 6.74 (d, J=2.3Hz, 1H), 6.67 (d, J=8.6Hz, 1H), 6.44 (d, J=8.6Hz, 2.3Hz, 1H), 5.41 (s, 1H), 4.80 (s, 1H), 4.60 (t, 1H), 4.21 (m, 2H), 3.20-4.00 (glc-H), 2.77 (s, 1H), 1.43,1.14 (s, 6H)。
Phenylpropanoids sulfonate: ESIMS is shown as m/z642.77, nuclear-magnetism feature1H-NMR(600 MHz,CD3OD):1HNMR (400MHz, MeOD) δ 6.78 (d, J=2.3Hz, 1H), 6.72 (d, J=8.6Hz, 1H), 6.59 (d, J=8.6Hz, 2.3Hz, 1H), 5.48 (s, 1H), 4.89 (s, 1H), 4.69 (t, 1H), 4.28 (m, 2H), 3.20-4.00 (glc-H), 2.83 (s, 1H), 1.43,1.14 (s, 6H)。
Phenylpropanoids potassium salt or sodium salt:
Potassium salt ESIMS shows m/z616.17, nuclear-magnetism feature1H-NMR(600MHz,CD3OD):1HNMR (400MHz, MeOD) δ 6.71 (d, J=2.3Hz, 1H), 6.66 (d, J=8.6Hz, 1H), 6.54 (d, J=8.6Hz, 2.3Hz, 1H), 5.44 (s, 1H), 4.81 (s, 1H), 4.61 (t, 1H), 4.26 (m, 2H), 3.20-4.00 (glc-H), 2.80 (s, 1H), 1.43,1.14 (s, 6H)。
Sodium salt ESIMS shows m/z600.67,1HNMR (400MHz, MeOD) δ 6.79 (d, J=2.3Hz, 1H), 6.63 (d, J=8.6Hz, 1H), 6.47 (d, J=8.6Hz, 2.3Hz, 1H), 5.34 (s, 1H), 4.84 (s, 1H), 4.67 (t, 1H), 4.20 (m, 2H), 3.20-4.00 (glc-H), 2.77 (s, 1H), 1.43,1.14 (s, 6H)。
Phenylpropanoids ammonium salt: ESIMS shows m/z593.49, nuclear-magnetism feature1H-NMR(600MHz,CD3OD):1HNMR (400MHz, MeOD) δ 6.70 (d, J=2.3Hz, 1H), 6.69 (d, J=8.6Hz, 1H), 6.49 (d, J=8.6Hz, 2.3Hz, 1H), 5.41 (s, 1H), 4.76 (s, 1H), 4.61 (t, 1H), 4.23 (m, 2H), 3.20-4.00 (glc-H), 2.77 (s, 1H), 1.43,1.14 (s, 6H)。
The partial structural formula of above-mentioned phenylpropanoids salt is such as shown in formula II～formula VI。
Wherein, formula II is the phenylpropanoids hydrochlorate prepared, formula III is the one of which sulfonate of the phenylpropanoids prepared, formula IV is the one of which potassium salt of the phenylpropanoids prepared, formula (V) is the one of which sodium salt of the phenylpropanoids prepared, and formula VI is the one of which ammonium salt of the phenylpropanoids prepared。
Experimental example 7 application test
The RAW264.7 oxidative macrophage of compound of the present invention and salt pair LPS induction stress with the impact of inflammation。(in order in experimentation, record is convenient, being numbered by phenylpropanoids of the present invention below: medicine MM-124, namely namely heretofore described medicine MM-124 refers to phenylpropanoids shown in formula I of the present invention or its pharmaceutically acceptable salt。)
1 materials and methods
1.1 medicines and instrument
Lipopolysaccharide (lipopolysaccharide, LPS), MTT available from Sigma；Mouse macrophage Raw264.7 is purchased from the refined cell bank in Hunan；PBS；DMEM high glucose medium, hyclone, penicillin and streptomycin；Full-automatic microplate reader；Constant temperature CO2 incubator。
Mice IL-1β (IL-1-β) ELISA detection kit, lot number: 2014/06 (96T)；Little mIL6 (IL-6) ELISA detection kit, lot number: 2014/06 (96T)；Murine tumor necrosis factor-α (TNF-α) ELISA detection kit, lot number: 2014/06 (96T)；Mouse nitrous oxide (NO) ELISA detection kit, lot number: 2014/10 (96T)；Mice hydroxyl radical free radical (OH) ELISA detection kit, lot number: 2014/10 (96T)。
Prepared by 1.2 medicines
First dissolve with a small amount of DMSO, be then diluted to certain concentration with DMEM, make in final concentration DMSO content less than 1 ‰。
1.3 cells are cultivated
Mouse macrophage Raw264.7 is incubated in the DMEM culture medium inactivateing the hyclone (FBS) of (56 DEG C, 30min), 10U/mL penicillin sodium, 100 μ g/mL streptomycins containing 10% heat, 37 DEG C, 5%CO2Constant incubator is hatched growth。
1.4 cell viabilities measure
Cell viability is measured by mtt assay。Cell is made cell suspension inoculation and hatches 24h in 96 orifice plates (1 × 104/hole), resynchronization 24h, then the medicine of variable concentrations is acted on cell 2h, then adding LPS (30 μ g/mL) and stimulate 24h, inhale and abandon former culture medium, every hole adds the MTT (0.5mg/mL) of 100 μ L and continues to hatch 4h, culture medium is abandoned in suction, every hole adds the DMSO of 150 μ L, shaking table jolting 10min, measures absorbance at 490nm place。
1.5NO assay
Raw264.7 cell is inoculated in 96 orifice plate 24h, resynchronization 24h, then the medicine of variable concentrations is acted on cell 2h, then add LPS (30 μ g/mL) and stimulate 24h, finally collect supernatant, and in the centrifugal 5min of 10000rpm, subpackage supernatant is placed in 80 DEG C and saves backup。By mice NO kit measurement NO content。
1.6 inflammatory factor TNF-α, IL-1 β, IL-6 measure
Sample takes 1.5 samples prepared for subsequent inflammation factor determination。Cell produces TNF-α, and the amount of IL-1 β, IL-6 is by mice TNF-α, and IL-1 β, IL-6 test kit measures。
1.7OH assay
Sample takes 1.5 samples prepared for OH factor determination。By OH kit measurement content。
1.8 statistical analysiss
Adopting SPSS17.0 software, experimental data is so that (x ± s represents；The data obtained is by using one factor analysis of variance, and homogeneity of variance LSD checks, and heterogeneity of variance DunnettT3 checks。
2 experimental results
2.1 cell viabilities
The impact of cell viability is evaluated by medicine by mtt assay。As it is shown on figure 3, Raw264.7 cell viability is had no significant effect by medicine MM-124 in (1.62-14.62 μ g/mL) concentration range；Therefore the drug level under this range of concentrations is suitable for subsequent experimental。
The generation of 2.2 Drug inhibition NO
As shown in Figure 4, stimulating Raw264.7 cell by LPS, it produces NO (65.81 ± 2.93IU/mL) content significantly raised compared with normal group NO (33.61 ± 2.19IU/mL) (p < 0.01)。The NO content that LPS is caused by medicine MM-124 in concentration (4.87-14.62 μ g/mL) scope raises obvious inhibitory action, and shows obvious dose-dependence。
2.3 Drug inhibition TNF-α, the generation of IL-1 β, IL-6
As shown in Figures 5 to 7, Raw264.7 cell is stimulated by LPS, Raw264.7 cellular inflammation factor TNF-α (132.16 ± 5.28pg/mL), IL-1 β (358.80 ± 24.64pg/mL), IL-6 (198.39 ± 5.97pg/mL) content and normal group TNF-α (65.41 ± 6.29pg/mL), IL-1 β (172.67 ± 10.06pg/mL), IL-6 (103.34 ± 2.88pg/mL) compare content significantly raised (p < 0.01)；Illustrate that LPS can stimulate Raw264.7 cell to produce a large amount of inflammatory factors。
LPS is stimulated the Raw264.7 cellular inflammation factor TNF-α caused by medicine MM-124 at various concentrations, and IL-1 β, IL-6 content is all without obvious inhibitory action (p > 0.05)。
The generation of 2.4 Drug inhibition OH
As shown in Figure 8, stimulating Raw264.7 cell by LPS, it produces OH (113.58 ± 6.03ng/mL) content significantly raised compared with normal group OH (63.40 ± 1.19ng/mL) (p < 0.01)。
Medicine MM-124 at various concentrations to OH all without obvious inhibitory action。
This experiment, through In vitro culture, have studied medicine MM-124 to mouse macrophage NO, TNF-α, the impact that IL-1 β, IL-6, OH generate。
Factor NO is had stronger inhibition by medicine MM-124, and to cellular inflammation factor TNF-α, IL-1 β, IL-6 content all has no significant effect, and illustrates that its anti-inflammatory activity is mainly through suppressing NO approach to realize；It, to the basic unrestraint effect of OH, illustrates that its antioxidant activity is inconspicuous。
Embodiment 8
The preparation of tablet: first prepare the phenylpropanoids shown in formula I by embodiment 1 method, and utilize the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, excipient, pelletizing press sheet is added with excipient weight than the ratio for 1:10 in this compound or its any one salt。
Embodiment 9
The preparation of powder: first prepare the phenylpropanoids shown in formula I by embodiment 1 method, and utilizing the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, powder method for making makes powder routinely。
Embodiment 10
The preparation of capsule or granule: first prepare the phenylpropanoids shown in formula I by embodiment 1 method, and utilize the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, add excipient with excipient weight than the ratio for 1:10 in this compound or its any one salt, make capsule or granule。
Embodiment 11
The preparation of injection: first prepare the phenylpropanoids shown in formula I by embodiment 1 method, and utilize the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, water for injection routinely, fine straining, injection is made in embedding sterilizing。
Embodiment 12
A kind of pharmaceutical composition, the phenylpropanoids shown in formula I is prepared containing embodiment 1 method, and utilize the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, and the powder that Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, Radix Codonopsis are made, and adjuvant。
Embodiment 13
A kind of pharmaceutical composition, prepares the phenylpropanoids shown in formula I containing embodiment 1 method, and the powder that Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, Radix Codonopsis are made, and adjuvant。
Embodiment 14
A kind of pharmaceutical composition, prepares the phenylpropanoids shown in formula I containing embodiment 1 method, and Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, Radix Codonopsis extract, and adjuvant。Extract is that in any one by patent announcement CN1078079C, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C, CN1296073C or several patent documents, extracting method prepares。
Embodiment 15
A kind of pharmaceutical composition, the phenylpropanoids shown in formula I is prepared containing embodiment 1 method, and utilize the salt that this compound and mineral acid (example hydrochloric acid, hydrobromic acid, Fluohydric acid., hydroiodic acid, sulphuric acid, nitric acid, carboxylic acid, phosphoric acid, lactic acid) or sulfonic acid or alkali-metal hydroxide (such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, Lithium hydrate) or ammonium make, and Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, Radix Codonopsis extract, and adjuvant。Extract is that in any one by patent announcement CN1078079C, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C, CN1296073C or several patent documents, extracting method prepares。
The ultimate principle of the present invention and the advantage of principal character and the present invention have more than been shown and described。Skilled person will appreciate that; the present invention is not restricted to the described embodiments; described in above-described embodiment and description is that principles of the invention is described; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications; this will be apparent to those skilled in the art, and these changes and improvements both fall within the claimed scope of the invention。Claimed scope is defined by appending claims and equivalent thereof。
Claims (10)
1. the preparation method of a phenylpropanoids, it is characterised in that the structural formula of described phenylpropanoids is such as shown in formula I,
(I)；
Described preparation method comprises the steps:
S1. the root taking Flemingia macrophylla is raw material, dry, and stripping and slicing is extracted through alcoholic solution, is merged by extracting solution, is concentrated into without alcohol taste, obtains extractum standby；
S2. being dissolved in water by gained extractum in step S1, adopt macroporous adsorptive resins that it is carried out eluting, eluant is ethanol-water system, collects the eluent of front 3 column volumes, and called after MM-1 is standby；
S3. the flow point MM-1 reversed material ODS column chromatography collected in step S2 is carried out eluting, eluant is methanol-water system, 18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collect 6 flow points in order, it is respectively designated as MM-11, MM-12, MM-13, MM-14, MM-15, MM-16, standby;
S4. being prepared by the flow point MM-12 collected in step S3 liquid phase separation, mobile phase is methanol-water-acetic acid system, collects eluent by peak sequence, collects 7 flow points altogether, it is respectively designated as MM-121, MM-122, MM-123, MM-124, MM-125, MM-126, MM-127, standby；
S5. being prepared by the flow point MM-124 collected in step S4 liquid phase purification, mobile phase is methanol-water-acetic acid system, collects eluent, obtains described phenylpropanoids after recrystallization。
2. preparation method according to claim 1, it is characterised in that: in step S1, the concentration of alcoholic solution is 50 ~ 80 volume %, it is preferred to the concentration of alcoholic solution is 60 volume %。
3. preparation method according to claim 1, it is characterised in that: in step S1, the extraction time of alcoholic solution is 2 ~ 4 times, extracts 1 ~ 3 hour every time, it is preferred to the extraction time of alcoholic solution is 3 times, each 2 hours。
4. preparation method according to claim 1, it is characterised in that: in step S2, macroporous adsorbent resin adopts D101 macroporous adsorbent resin。
5. preparation method according to claim 1, it is characterised in that: in step S2, the volume ratio of eluant ethanol and water is 0:100 ~ 15:85。
6. preparation method according to claim 1, it is characterised in that: in step S3, the volume ratio of eluant methanol and water is 20:80 ~ 30:70, it is preferred to 25:75。
7. preparation method according to claim 1, it is characterised in that: in step S4, the volume ratio of methanol-water-acetic acid is 10:90:0.01 ~ 35:65:0.01, it is preferred to 15:85:0.01。
8. preparation method according to claim 1, it is characterised in that: in step S4, the chromatographic column of preparation liquid phase is YMC, and 20mm*250mm, flow rate of mobile phase is 5 ~ 10mL/min, it is preferred to flow velocity 5mL/min。
9. preparation method according to claim 1, it is characterised in that in step S5, the volume ratio of methanol-water-acetic acid is 15:85:0.01。
10. preparation method according to claim 1, it is characterised in that: in step S5, the chromatographic column of preparation liquid phase is YMC, and 20mm*250mm, flow rate of mobile phase is 5mL/min。
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| CN102274341A (en) * | 2010-06-10 | 2011-12-14 | 上海中医药大学 | Extracting and refining process for medicinal components of figwort root |
| CN102631390A (en) * | 2012-04-12 | 2012-08-15 | 贵州师范大学 | Preparation method of periploca forrestii schltr extract as well as product and application of periploca forrestii schltr extract |
| CN104529984A (en) * | 2014-12-25 | 2015-04-22 | 株洲千金药业股份有限公司 | Method for extracting genistin from largeleaf flemingia |
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| CN102274341A (en) * | 2010-06-10 | 2011-12-14 | 上海中医药大学 | Extracting and refining process for medicinal components of figwort root |
| CN102631390A (en) * | 2012-04-12 | 2012-08-15 | 贵州师范大学 | Preparation method of periploca forrestii schltr extract as well as product and application of periploca forrestii schltr extract |
| CN104529984A (en) * | 2014-12-25 | 2015-04-22 | 株洲千金药业股份有限公司 | Method for extracting genistin from largeleaf flemingia |
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