CN106046073A - Preparation method for phenylpropanoid compound - Google Patents
Preparation method for phenylpropanoid compound Download PDFInfo
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- CN106046073A CN106046073A CN201610153536.6A CN201610153536A CN106046073A CN 106046073 A CN106046073 A CN 106046073A CN 201610153536 A CN201610153536 A CN 201610153536A CN 106046073 A CN106046073 A CN 106046073A
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Abstract
The invention discloses a preparation method for a phenylpropanoid compound and specifically relates to a phenylpropanoid compound separated from the dried root of Flemingia macrophylla, belonging to the field of medical technology. The phenylpropanoid compound is prepared through solvent extraction, column chromatographic separation, and separation and purification of a preparative liquid phase. The phenylpropanoid compound prepared by using the method is reported for the first time and has high purity. Experiment results show that the novel phenylpropanoid compound is capable of inhibiting the expression of the inflammation cytokines TNF-alpha, IL-1beta and IL-6, so the phenylpropanoid compound has anti-inflammatory and anti-oxidation activity and is beneficial for treatment of inflammatory diseases like cervicitis, endometritis, pelvic inflammation, mastitis, pharyngolaryngitis and/or arthritis. A novel drug can be developed from the compound. The structural formula of the phenylpropanoid compound is as shown in a formula (I) which is described in the specification.
Description
Technical field
The present invention relates to pharmaceutical technology field, more particularly, to the preparation method of a kind of phenylpropanoids.
Background technology
The constituent structure extracting isolated from natural drug is various, active significantly, and it is carried out isolated and purified, structure
Modify, transform and complete synthesis, an always main thought of new drug development.
TNF-α: be a kind of can direct killing tumor cell and to normal cell without the cytokine of overt toxicity, be so far
One of bioactie agent that the direct killing function of tumor that found till the present is the strongest, but its toxic and side effects is the tightest
Weight.
IL-1 β: collaborative APC and the T cell activation of stimulating, promotion B cell proliferation and secretory antibody when local low concentration, enter
Row 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 list
Core macrophage, Th2 cell, vascular endothelial cell, fibroblast produce.Activating B cell can be stimulated to breed, and secretion is anti-
Body；Stimulate T cell propagation and CTL activation；Cell cultured supernatant synthesized acute phase albumen, participates in inflammatory reaction；Promote that hemocyte is sent out
Educate.
IL-6 can be synthesized by various kinds of cell, including activation T cell and B cell, monocytes/macrophages, endotheliocyte,
Epithelial cell and fibroblast etc..The target cell of IL-6 effect is a lot, thin including macrophage, hepatocyte, static T
Born of the same parents, the B cell of activation and plasma cell etc.；Its biological effect is the most sufficiently complex.
Therefore, seek a kind of new preparation method, from natural plants, separate the compound made new advances, to suppress cellular inflammation
Factor TNF-α, the expression of IL-1 β, IL-6, it is applied to the treatment of diseases associated with inflammation, is necessary.
Radix Flemingiae Philippinensis medical material is that pulse family (Leguminosae) Moghania (Flemingl.Roxb. or Moghania) is planted
Thing Flemingia macrophylla (Moghania macrophylla(Willd.) O.Kuntze) dry root, be mainly distributed in China
In region of Southeast.This plant is in Chinese Plants will (1995,41:313), TaiWan, China flora (1977,3:258), sea
South flora (1965,2:311), " Chinese Higher plant illustrated handbook " (1972,2:510), China main plant figure say (1955,
Pp707) and Guangzhou flora (1956, pp361) is all included.Radix Flemingiae Philippinensis medical material is the genuine medicinal materials of Guangxi province, history
It is loaded in " Zhiwu Mingshi Tukao ", has medication the most among the people basis.Its nature and flavor are sweet, micro-puckery, flat, have the merits such as removing damp-heat
Effect, is mainly used in treating rheumatic ostalgia, traumatic injury, chronic nephritis, dysmenorrhea and the gynaecopathia such as leucorrhea is many.This medical material is the most
Record into version " Chinese Pharmacopoeia " annex in 2005.
The composition that Flemingia macrophylla has been reported mainly has flavonoid, steroid, terpenoid, Anthraquinones, volatile oil composition, all has
There is certain pharmacologically active.
Flemingia macrophylla pharmacologically active is various, reports the more neuroprotective that has, and antiinflammatory, antioxidation, to disease
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, gynecological
A thousand pieces of gold capsule, JINJI CHONGJI, JINJI JIAONANG etc., this type of Chinese patent medicine is mainly used in gynaecopathia, and (dysmenorrhea, cold uterus be infertile, uterus
Sagging, pelvic inflammatory disease, mastitis, leucorrhea are many, blood deficiency in puerperal, arthralgia, waist and knee in puerperal, 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 that FUKE QIANJIN PIAN is scorching in treatment gynecological
Effect in terms of disease.
At present, in the document of Radix Flemingiae Philippinensis, the document of report phenylpropanoids is few, finds a kind of new preparation side
Method, isolates effective Phenylpropanoid Glycosides class noval chemical compound from natural plants, it is carried out isolated and purified, structural modification and synthesis,
Developing new drug, is applied to the treatment of diseases associated with inflammation, significant.
Summary of the invention
It is an object of the invention to provide a kind of a kind of Phenylpropanoid Glycosides class of isolated from the dry root of Flemingia macrophylla
The preparation method of compound, the compound being extracted isolated by this preparation method can suppress cellular inflammation factor TNF-α,
The expressional function of IL-1 β, IL-6, and then there is anti-inflammatory activity, it is of value to the treatment of various diseases associated with inflammation, can be by this chemical combination
Thing develops 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, is dried, stripping and slicing, extracts through ethanol solution, is merged by extracting solution, is concentrated into
Taste without alcohol, obtains extractum standby；
S2. being dissolved in water by gained extractum in step S1, use macroporous adsorptive resins that it is carried out eluting, eluant is second
Alcohol-water system, collects the eluent of front 3 column volumes, and named MM-1 is standby；
S3. the flow point MM-1 reversed material ODS column chromatography collected in step S2 being carried out eluting, eluant is methanol-water
System, 18 column volumes of eluting, collect the eluent of a flow point by every 3 column volumes, collect 6 flow points in order, respectively
Named MM-11, MM-12, MM-13, MM-14, MM-15, MM-16, standby;
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, flowing is methanol-water-acetic acid system mutually, presses
Peak sequence collects eluent, collects 7 flow points altogether, is respectively designated as MM-121, MM-122, MM-123, MM-124, MM-
125, MM-126, MM-127, standby；
S5. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, flowing is methanol-water-acetic acid system mutually, receives
Collection eluent, obtains described phenylpropanoids after recrystallization.The purity of described phenylpropanoids is 99.87%.Change
The structural formula of compound is as shown in formula I:
(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, more preferably second every time
The extraction time of alcohol is 3 times, each 2 hours.
Preferably, in step S2, macroporous adsorbent resin uses D101 macroporous adsorbent resin.
Preferably, in step S2, ethanol is 0:100 ~ 15:85 with the volume ratio of water.
Preferably, in step S3, methanol is 20:80 ~ 30:70, more preferably 25:75 with the volume ratio of water.
Preferably, in step S4, the volume ratio of methanol-water-acetic acid is 10:90:0.01 ~ 35:65:0.01, the most excellent
Elect 15:85:0.01 as.
Preferably, in step S4, preparative liquid chromatography post is YMC, 20mm*250mm, and flow rate of mobile phase is 5 ~ 10mL/
Min, more preferably 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.
Beneficial effects of the present invention:
The phenylpropanoids that the present invention is new provides a kind of preparation method, divides first from the dry root of Flemingia macrophylla
From a kind of new phenylpropanoids obtained, prepared compound can suppress cellular inflammation factor TNF-α, IL-1 β, IL-6
Expressional function, and then there is anti-inflammatory activity, can serve as such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis
And/or the medicine of the diseases associated with inflammation such as arthritis.
It is an object of the invention to from traditional prescriptions of Chinese medicine, by the prescription from FUKE QIANJIN PIAN and FUKE QIANJIN JIAONANG
In, prepare a kind of noval chemical compound 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 cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis and/or arthritis etc.
The treatment of disease.
Specifically, inventor by from FUKE QIANJIN PIAN, FUKE QIANJIN JIAONANG prescription, science chooses Flemingia macrophylla
Dry root, by solvent extraction, column chromatography for separation, preparation liquid phase separation, purification, obtain Phenylpropanoid Glycosides class chemical combination of the present invention
Thing, then carries out test cell line to this compound, measure its to cellular inflammation factor TNF-α, the suppression degree of IL-1 β, IL-6,
Experiment shows, LPS is stimulated Raw 264.7 cell caused scorching in the range of concentration (7.50 13.50 μ g/mL) by this compound
Inflammation factor TNF-α content has obvious inhibitory action (p ＜ 0.05), and shows obvious dose-dependence；In concentration
Under 13.50 μ g/mL, inflammatory factor IL-1 β content is had obvious inhibitory action (p ＜ 0.05)；At concentration (10.50 13.50 μ
G/mL) in the range of, inflammatory factor IL-6 content is all had obvious inhibitory action (p ＜ 0.05), shows obvious dose-dependant
Relation.
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 phenylpropanoids of the present invention affects figure to cytoactive.
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 embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are the examination of the art routine
Agent, method and apparatus.Unless stated otherwise, to be the art conventional commercial former for raw material used by the present embodiment and equipment
Material and equipment.
The compound of the present invention is the phenylpropanoids shown in described formula I.This compound can use the present invention
The method extracted for raw material with Flemingia macrophylla provided prepares, it is also possible to adopts according to the structural formula combination that the present invention provides
Prepare by methods such as the chemosynthesis of this area.
The compounds of this invention, can be used as such as cervicitis, endometritis, pelvic inflammatory disease, mastitis, pharyngolaryngitis and/or joint
The medicine of the diseases associated with inflammation such as inflammation.
The compounds of this invention can be used as pharmaceutical composition together with the adjuvant pharmaceutically allowed and/or carrier, it is also possible to
In the case of adding the adjuvant that pharmaceutically allows and/or carrier with Radix Rosae Laevigatae, Fructus Zanthoxyli Dissiti, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis,
One or more Chinese crude drugs or the combination of extract in Radix Angelicae Sinensis, Radix Codonopsis are used as pharmaceutical composition, and the compounds of this invention is all right
Pharmaceutical composition it is used as together with other pharmaceutically acceptable active ingredient.
As pharmaceutical composition, can be tablet, capsule, powder, granule, pill, solution, suspensoid, syrup
Agent, injection, ointment, suppository, spray etc..
Further, tablet can be the sugar-coat made in the case of adding the adjuvant and/or carrier pharmaceutically allowed
Sheet, 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, it is possible to use additive, such as sucrose, lactose, cellulose sugar, maltose alcohol, glucose, starch
Class, 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, Oleum Ricini etc.), mineral oil
Fat (vaseline, white vaseline, solid paraffin etc.), wax class (Jojoba oil, Brazil wax, Cera Flava etc.), partial synthesis or complete
The fatty acid glyceride (lauric acid, myristic acid, Palmic acid etc.) etc. of synthesis.
Make liquid preparation, can use additive, such as sodium chloride, glucose, Sorbitol, glycerol, olive oil, the third two
Alcohol, ethanol etc..In the case of especially making injection, it is possible to use aseptic aqueous solution, such as 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..
The amount of the effective ingredient of these preparations is 0.01 ~ 80 weight % of preparation, is suitably 1 ~ 50 weight %, dosage according to
The symptom of patient, body weight, age etc. are different and change.
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. take Flemingia macrophylla 50kg, with root as raw material, be dried, be cut into small pieces.Alcohol reflux 3 through 8 times amount 60%
Secondary, each 2 hours, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. the extractum after concentrating in step S1 is dissolved in 10L water, uses D101 macroporous adsorptive resins that it is carried out eluting,
Eluant is water, 3 column volumes of eluting, collection eluent, and named MM-1 is standby；
S3. with anti-phase ODS column chromatography, the flow point MM-1 collected in step S2 being carried out eluting, eluant is methanol-water system,
Its volume ratio is 25:75,18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collects 6 in order
Individual flow point, is respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, 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. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, methanol: water: the volume ratio of acetic acid is 15:85:0.01,
Collect eluent, after recrystallization, obtain described phenylpropanoids.
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. take Flemingia macrophylla 40kg, with root as raw material, be dried, be cut into small pieces.Alcohol reflux 2 through 6 times amount 50%
Secondary, each 1 hour, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. the extractum after concentrating in step S1 is dissolved in 5L water, uses D101 macroporous adsorptive resins that it is carried out eluting, washes
De-agent is ethanol and the volume ratio of water is 15:85,3 column volumes of eluting, collects eluent, and named MM-1 is standby；
S3. with anti-phase ODS column chromatography, the flow point MM-1 collected in step S2 being carried out eluting, eluant is methanol-water system,
Its volume ratio is 20:80,18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collects 6 in order
Individual flow point, is respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 10ml/min, flowing is methanol-water-acetic acid system mutually, 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. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, methanol: water: the volume ratio of acetic acid is 15:85:0.01,
Collect eluent, after recrystallization, obtain described phenylpropanoids.
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. take Flemingia macrophylla 60kg, with root as raw material, be dried, be cut into small pieces.The alcohol reflux of 7 times amount 70% 4 times,
Each 3 hours, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. the extractum after concentrating in step S1 is dissolved in 8L water, uses D101 macroporous adsorptive resins that it is carried out eluting, washes
De-agent is ethanol and the volume ratio of water is 10:90,3 column volumes of eluting, collects eluent, and named MM-1 is standby；
S3. with anti-phase ODS column chromatography, the flow point MM-1 collected in step S2 being carried out eluting, eluant is methanol-water system,
Its volume ratio is 30:70,18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collects 6 in order
Individual flow point, is respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 10ml/min, flowing is methanol-water-acetic acid system mutually, 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. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, methanol: water: the volume ratio of acetic acid is 15:85:0.01,
Collect eluent, after recrystallization, obtain described phenylpropanoids.
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. take Flemingia macrophylla 50kg, with root as raw material, be dried, be cut into small pieces.The alcohol reflux of 8 times amount 60% 2 times,
Each 1.5 hours, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. the extractum after concentrating in step S1 is dissolved in 6L water, uses D101 macroporous adsorptive resins that it is carried out eluting, washes
De-agent is ethanol and the volume ratio of water is 5:95,3 column volumes of eluting, collects eluent, and named MM-1 is standby；
S3. with anti-phase ODS column chromatography, the flow point MM-1 collected in step S2 being carried out eluting, eluant is methanol-water system,
Its volume ratio is 25:75,18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collects 6 in order
Individual flow point, is respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 10ml/min, flowing is methanol-water-acetic acid system mutually, 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. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, methanol: water: the volume ratio of acetic acid is 15:85:0.01,
Collect eluent, after recrystallization, obtain described phenylpropanoids.
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. take Flemingia macrophylla 50kg, with root as raw material, be dried, be cut into small pieces.The alcohol reflux of 8 times amount 80% 2 times,
Each 1.5 hours, extracting solution is merged, is concentrated into without alcohol taste, obtains extractum standby；
S2. the extractum after concentrating in step S1 is dissolved in 6L water, uses D101 macroporous adsorptive resins that it is carried out eluting, washes
De-agent is ethanol and the volume ratio of water is 10:90,3 column volumes of eluting, collects eluent, and named MM-1 is standby；
S3. with anti-phase ODS column chromatography, the flow point MM-1 collected in step S2 being carried out eluting, eluant is methanol-water system,
Its volume ratio is 28:72,18 column volumes of eluting, collects the eluent of a flow point by every 3 column volumes, collects 6 in order
Individual flow point, is respectively designated as: MM-11, MM-12, MM-13, MM-14, MM-15, MM-16 are standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 10ml/min, flowing is methanol-water-acetic acid system mutually, 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. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, preparative liquid chromatography post is: YMC, 20mm*
250mm, flow velocity: 5ml/min, flowing is methanol-water-acetic acid system mutually, methanol: water: the volume ratio of acetic acid is 15:85:0.01,
Collect eluent, after recrystallization, obtain described phenylpropanoids.
Compound embodiment 1 to embodiment 5 prepared carries out mass spectrum, proton nmr spectra, carbon-13 nmr spectra
Detection, result prove gained compound be: 4-glucosyl group-11-methyl-8,9-benzene hexene.Its structural formula such as formula I institute
Show:
(I).
Its mass spectrum, proton nmr spectra, carbon-13 nmr spectra spectral data as follows:
HR-ESIMS shows [M+Na]+for m/z 375.1984, and in conjunction with nuclear-magnetism feature, can obtain molecular formula is C19H28O6, unsaturated
Degree is 6.
1H-NMR (600 MHz, CD3OD): 6.89 (d,J=1.8 Hz, 2H), 5.82 (d,J = 1.8Hz, 2H),
4.94 (d,J=7.6Hz, 1H), 4.86 (d, 2H), 2.71 (t,J=7.0 Hz, 2H), 2.44 (t,J=7.0 Hz,
2H),2.40(s, 3H), 1.95 (m,1H),1.17(s,3H),3.30-4.00 (glc-H)。
13C-NMR (150MHz, CD3OD): 141.4 (C-4),130.2(C-1), 119.6(C-2), 115.4 (C-
3), 114.4(C-5),111.3(C-6),101.3(C-1'),109.3(C-9),102.5(C-8),61.5-77.8(C2''-
C6''),45.0(C-7),28.8(C-10),18.7(C-11),17.3(C-12),14.5(-CH3)。
Experimental example 6 application test
RAW 264.7 oxidative macrophage that LPS is induced by compound of the present invention stress be with the impact of inflammation.(for reality
During testing, record is convenient, is numbered by the phenylpropanoids shown in formula I of the present invention below: medicine MM-122, i.e. originally
Medicine MM-122 described in invention i.e. refers to phenylpropanoids shown in formula I of the present invention.)
1 materials and methods
1.1 medicines and instrument
Lipopolysaccharide (lipopolysaccharide, LPS), MTT is purchased from Sigma company；Mouse macrophage Raw 264.7 purchases
The refined cell bank from 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 detects examination
Agent box, lot number: 2014/06 (96T)；Mouse nitrous oxide (NO) ELISA detection kit, lot number: 2014/10 (96T)；Little
Mus 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 DMSO content in final concentration be less than 1 ‰.
1.3 cells are cultivated
Mouse macrophage Raw 264.7 is incubated at containing 10% heat the inactivation hyclone (FBS) of (56 DEG C, 30min), 10U/mL
Penicillin sodium, 100 μ g/mL streptomycins DMEM culture medium in, 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 hatch in 96 orifice plates (1 × 104/hole)
24h, resynchronization 24h, then act on cell 2h by the medicine of variable concentrations, and then adding LPS (30 μ g/mL) stimulates
24h, inhales and abandons former culture medium, and every hole adds the MTT (0.5mg/mL) of 100 μ L and continues to hatch 4h, inhales and abandons culture medium, and every hole adds
The DMSO of 150 μ L, shaking table shaking 10min, measure absorbance at 490nm.
1.5 NO assays
Raw 264.7 cell is inoculated in 96 orifice plate 24h, resynchronization 24h, then the medicine of variable concentrations is acted on cell
2h, then adds LPS(30 μ g/mL) stimulate 24h, finally collect supernatant, and be centrifuged 5min, subpackage supernatant in 10000rpm
It is placed in-80 DEG C to save 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, the amount of IL-1 β, IL-6
By mice TNF-α, IL-1 β, IL-6 test kit measures.
1.7 OH assays
Sample takes 1.5 samples prepared for OH factor determination.By OH kit measurement content.
1.8 statistical analysis
Using SPSS17.0 software, experimental data represents with x ± s；The data obtained is by by one factor analysis of variance, homogeneity of variance
Checking with LSD, heterogeneity of variance Dunnett T3 checks.
2 experimental results
2.1 cell viability
The impact of cell viability is evaluated by medicine by mtt assay.As it is shown on figure 3, medicine MM-122 is at 1.50-13.50 μ g/mL
In concentration range, Raw 264.7 cell viability is had no significant effect；Therefore the drug level under this range of concentrations is for rear
Continuous experiment is suitable.
The generation of 2.2 Drug inhibition NO
As shown in Figure 4, stimulating Raw 264.7 cell by LPS, it produces NO (65.81 ± 2.93 IU/mL) content with normal
Group NO (33.61 ± 2.19IU/mL) compares significantly raised (p ＜ 0.01).The inflammation that LPS is caused under this concentration by medicine MM-122
In disease model, NO content is raised and there is no obvious inhibitory action.
2.3 Drug inhibition TNF-α, the generation of IL-1 β, IL-6
As shown in Figures 5 to 7, Raw 264.7 cell, Raw 264.7 cellular inflammation factor TNF-α are stimulated by LPS
(132.16 ± 5.28pg/mL), IL-1 β (358.80 ± 24.64 pg/mL), IL-6 (198.39 ± 5.97 pg/mL) contain
Amount and normal group TNF-α (65.41 ± 6.29 pg/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 Raw 264.7 cell to produce a large amount of inflammation
The factor.
LPS is stimulated Raw 264.7 cell caused scorching in the range of concentration (7.50-13.50 μ g/mL) by medicine MM-122
Inflammation factor TNF-α content has obvious inhibitory action (p ＜ 0.05), and shows obvious dose-dependence；In concentration
Under 13.50 μ g/mL, inflammatory factor IL-1 β content is had obvious inhibitory action (p ＜ 0.05)；At concentration (10.50-13.50 μ
G/mL) in the range of, inflammatory factor IL-6 content is had obvious inhibitory action (p ＜ 0.05), shows obvious dose-dependant and close
System.
The generation of 2.4 Drug inhibition OH
As shown in Figure 8, stimulating Raw 264.7 cell by LPS, it produces OH (113.58 ± 6.03 ng/mL) content with normal
Group OH (63.40 ± 1.19ng/mL) compares significantly raised (p ＜ 0.01).
The OH content that LPS is caused by medicine MM-122 at various concentrations raises does not has obvious inhibitory action.
This experiment, through In vitro culture, have studied medicine MM-122 to mouse macrophage NO, TNF-α, IL-1 β, IL-
6, the OH impacts generated.
Medicine MM-122 without obvious inhibiting effect, but significantly inhibits TNF-α to the generation of factor NO under middle and high concentration,
IL-1 β, IL-6 content, illustrate that it mainly affects is TNF-α, the approach of IL-1 β, IL-6, thus plays antiphlogistic effects,
It is obvious to OH unrestraint effect, illustrates that it is substantially without antioxidant activity.
Embodiment 7
The preparation of tablet: first prepare the phenylpropanoids shown in formula I as embodiment 1 method, by itself and excipient weight
Excipient, pelletizing press sheet is added than the ratio for 1:10.
Embodiment 8
The preparation of powder: first prepare the phenylpropanoids shown in formula I, routinely powder preparation method system as embodiment 1 method
Become powder.
Embodiment 9
Capsule or the preparation of granule: first prepare the phenylpropanoids shown in formula I as embodiment 1 method, by its with
Excipient weight adds excipient than the ratio for 1:10, makes capsule or granule.
Embodiment 10
The preparation of injection: first prepare the phenylpropanoids shown in formula I, routinely water for injection as embodiment 1 method,
Fine straining, injection is made in embedding sterilizing.
Embodiment 12
A kind of pharmaceutical composition, prepares the phenylpropanoids shown in formula I, and Radix Rosae Laevigatae, list containing embodiment 1 method
The powder that face pin, 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, and Radix Rosae Laevigatae, list containing embodiment 1 method
Face pin, Caulis Spatholobi, Caulis Mahoniae, Herba Andrographis, Radix Angelicae Sinensis, the extract of Radix Codonopsis, and adjuvant.Extract is by patent announcement number
CN1078079C、CN1170549C、CN1158087C、CN1330335C、CN1296071C、CN1321631C、CN1296072C、
In any one of CN1296073C or several patent documents, extracting method prepares.
The ultimate principle of the present invention and principal character and the advantage of the present invention have more than been shown and described.The technology of this area
Personnel, it should be appreciated that the present invention is not restricted to the described embodiments, simply illustrating this described in above-described embodiment and description
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and this is to this
Being apparent from for skilled person, these changes and improvements both fall within scope of the claimed invention.This
Bright 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 such as formula
(I) shown in,
(I)；
Described preparation method comprises the steps:
S1. the root taking Flemingia macrophylla is raw material, is dried, stripping and slicing, extracts through ethanol solution, is merged by extracting solution, is concentrated into
Taste without alcohol, obtains extractum standby；
S2. being dissolved in water by gained extractum in step S1, use macroporous adsorptive resins that it is carried out eluting, eluant is second
Alcohol-water system, collects the eluent of front 3 column volumes, and named MM-1 is standby；
S3. the flow point MM-1 reversed material ODS column chromatography collected in step S2 being carried out eluting, eluant is methanol-water
System, 18 column volumes of eluting, collect the eluent of a flow point by every 3 column volumes, collect 6 flow points in order, respectively
Named MM-11, MM-12, MM-13, MM-14, MM-15, MM-16, standby；
S4. by the flow point MM-12 collected in step S3 with preparing liquid phase separation, flowing is methanol-water-acetic acid system mutually, presses
Peak sequence collects eluent, collects 7 flow points altogether, is respectively designated as MM-121, MM-122, MM-123, MM-124, MM-
125, MM-126, MM-127, standby；
S5. being purified with preparing liquid phase by the flow point MM-122 collected in step S4, flowing is methanol-water-acetic acid system mutually, receives
Collection eluent, obtains described phenylpropanoids after recrystallization.
Preparation method the most according to claim 1, it is characterised in that: in step S1, the concentration of ethanol solution is 50 ~ 80 bodies
The concentration of long-pending %, preferably ethanol solution is 60 volume %.
Preparation method the most according to claim 1, it is characterised in that: in step S1, the extraction time of ethanol solution is 2 ~ 4
Secondary, extract 1 ~ 3 hour every time, the preferably extraction time of ethanol solution is 3 times, each 2 hours.
Preparation method the most according to claim 1, it is characterised in that: in step S2, macroporous adsorbent resin uses D101 big
Macroporous adsorbent resin.
Preparation method the most according to claim 1, it is characterised in that: in step S2, eluant ethanol and the volume ratio of water
For 0:100 ~ 15:85.
Preparation method the most according to claim 1, it is characterised in that: in step S3, eluant methanol and the volume ratio of water
For 20:80 ~ 30:70, preferably 25:75.
Preparation method the most 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, preferably 15:85:0.01.
Preparation method the most according to claim 1, it is characterised in that: in step S4, the chromatographic column of preparation liquid phase is YMC,
20mm*250mm, flow rate of mobile phase is 5 ~ 10mL/min, preferably flow velocity 5mL/min.
Preparation method the most 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。
Preparation method the most according to claim 1, it is characterised in that: in step S5, the chromatographic column of preparation liquid phase is YMC,
20mm*250mm, flow rate of mobile phase is 5mL/min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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