CN105560445B - Willow leaf japanese spiraea root resisting rheumatoid disease active component and its preparation method and application - Google Patents
Willow leaf japanese spiraea root resisting rheumatoid disease active component and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of willow leaf japanese spiraea root resisting rheumatoid arthritis active components and its preparation method and application.The active component extracts through 60% alcohol reflux using willow leaf japanese spiraea root as raw material, ethyl alcohol is recovered under reduced pressure obtains extract, extract obtains total lignan active component through purification with macroreticular resin.(+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7 in the active componentR,8S) -5- melonia dehydrodiconiferyl alcohol and (7S,8RThe sum of weight of)-dihydro dehydrodiconiferyl alcohol 20% ~ 25%.The present invention determines active component in the method that modern separation means and Pharmacological Activity Screening combine, gained active component is at distinguishing one from the other, active component content is high, and through inside and outside the experimental results showed that, willow leaf japanese spiraea root resisting rheumatoid arthritis active component provided by the invention inhibits pro-inflammatory cytokine tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 generation to reach treatment rheumatoid arthritis effect.
Description
Technical field
The invention belongs to field of medicaments, are related to willow leaf japanese spiraea root resisting rheumatoid arthritis active component and its preparation side
Method further relates to willow leaf japanese spiraea root resisting rheumatoid arthritis active component in preparation treatment rheumatoid arthritis product
Purposes.
Background technique
Rheumatoid arthritis be it is a kind of it is common with joint tissue chronic inflammation lesion be itself exempting from of mainly showing
Epidemic disease disease.Its main pathological change is articular synovial cells infiltration, and synovial membrane screen is formed, the erosion of cartilage and bone tissue.Class
The purpose of rheumatic arthritis treatment is to prevent disease, i.e. prevention arthritis and destruction process, prophylactic function is lost.Though
There are many right remedy measures, but drug therapy is still basis.Therapeutic agent includes non-steroidal anti-inflammatory, steroidal anti-inflammatory medicine and disease at present
Adjusting antirheumatic.It is continuous to the understanding of aetology mechanism with to inflammatory process, molecular genetics and biotech development
Deeply, researcher strives to find the therapeutic agent with specificity.Mainly it is as target spot, with inflammatory mediator using inflammatory cytokine
Target spot, using immunocompetent cell surface antigen as target spot, using signal transduction pathway as target spot.Cell factor is one group and is exempted from by body
The small-molecule substance that epidemic disease cell and the synthesis of nonimmune cell, secretion generate, has multiple biological function.With rheumatoid joint
Scorching closely related cell factor is most of to be secreted by monocyte, macrophage, fibroblast and T cell, wherein tumour
Necrosis factor-alpha, Interleukin -1β, interleukin-6 are virulence factor, and interleukin-10, interleukin-4 are protective factors.
Hao little Jiang study group is to rosaceae Spiraea pink blossom meadow sweet chemical component and bioactivity
The research of system.Pink blossom meadow sweetSpiraea japonicaL. f. variability is strong, has 7 mutation in China, constitutes one again
It is gregarious, including oval leaf mutationS. japonica var. ovalifolia Franch., anxious sharp mutationS. japonica
Var.acutaYu, tapering mutationS. japonica var. acuminateFranch., light leaf mutationS. japonica
var. fortunei(Planchon) Rehd., hairless mutationS. japonica var. Glabra (Rege1)
Koidz., star flower mutationS. japonicaVar.stellaris Rehd. with decomposite leaf mutationS. japonicavar.incisaYu).As traditional herbal medicine, pink blossom meadow sweet is mainly used for wind dispelling of catching a cold, hemostasis promoting blood circulation, cough-relieving of easing pain, improving eyesight benefit
Urine, irregular menstruation and are removed necrosis and promoted granulation at subdhing swelling and detoxicating.The chemical constitution study of the plant starts from 1964, the former Russian scholar
Frolova etc. reports the diterpene alkaloid component in the plant, between more than 30 years henceforth, the former Soviet Union, Japan and China
Scholar reports dozens of diterpene alkaloid and Diterpene in succession.Hao little Jiang study group is from 1987 to the domestic compound group
The chemical component of each mutation of plant has carried out the chemical research of system, and successively separation identifies 53 new diterpene and diterpene biology
Alkali, and find that this kind of alkaloid has anti-inflammatory, platelet aggregation-against and neuroprotective activity.Rosaceae Spiraea willow leaf
Meadow sweet is resourceful, and through retrieving to existing technical literature, but the research of its rare chemical component and pharmacological action is reported.
Summary of the invention
Inventor sends out in the research of Changbai Mountain Spiraea resource resisting rheumatoid arthritis screening active ingredients
On the basis of the existing potential resisting rheumatoid arthritis effect of willow leaf japanese spiraea root extract, using the guiding under activity index guidance
Clastotype determines active component and invalid target, has studied the main chemical compositions and content of active component, thus completes
The present invention.
Object of the present invention is to overcome the deficiencies of the prior art and provide a kind of clear active principle, activity and mechanism clearly
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component, another object of the present invention are to provide willow leaf japanese spiraea root resisting rheumatoid disease
Property arthritis active component preparation method and preparation treatment rheumatoid arthritis product in application.
In order to achieve the goal above, the technical scheme adopted by the invention is as follows:
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component of the invention is to extract to divide from willow leaf japanese spiraea root
From total lignan active component, wherein (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7R,8S)-
5- melonia dehydrodiconiferyl alcohol and (7S,8RThe sum of weight of)-dihydro dehydrodiconiferyl alcohol 20% ~ 25%, by following
Method is prepared: willow leaf japanese spiraea root add 60% alcohol reflux extract three times, each dosage be by kilogram in terms of willow leaf japanese spiraea root
The volume in litres of 8 times of weight, 2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water
Make to dissolve, dosage be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, through large pore resin absorption column D101
Chromatography, filling macroporous absorbent resin D101 volume be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, use
Water elution is closely colourless to eluent, discards eluent, closely colourless to eluent with 30% ethanol elution, eluent is discarded, with 70%
Ethanol elution is closely colourless to eluent, collects eluent, is concentrated under reduced pressure, dry willow leaf japanese spiraea root resisting rheumatoid arthritis
Active component.
The present invention determines active component and invalid target using the guiding clastotype under activity index guidance, using macropore
Absorption resin D101 technology by 60% ethanol extract of willow leaf japanese spiraea root be separated into water elution position, 30% alcohol elution,
70% alcohol elution and 95% alcohol elution, wherein the rat that 70% alcohol elution induces Freund's complete adjuvant
Arthritis model primary inflammatory and secondary inflammation have apparent inhibiting effect.
Main component includes (+)-different fallen leaves in willow leaf japanese spiraea root resisting rheumatoid arthritis active component of the invention
Rosin element, 5- methoxyl group-(+)-isolariciresinol, (7R,8S) -5- melonia dehydrodiconiferyl alcohol, (7S,8R)-two
Hydrogen dehydrodiconiferyl alcohol, (7R,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan and (7R,
8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan.
The present invention is using modern separation identification technology to willow leaf japanese spiraea root resisting rheumatoid arthritis active component chemistry
Ingredient further separates identification, including 8- hydroxyl rosin spirit [Yeo H, et al.Arch Pharm Res, 2004,27:287-
290.], 8- hydroxyl -7'- table rosin spirit [Tsukamoto H, et al.Chem Pharm Bull, 1985,33:1232-
1241.]、(7R,8S) -5- melonia dehydrodiconiferyl alcohol [Meng J X, et al.Org Biomol Chem, 2010,
8:107-113.], (7S,8R)-dihydro dehydrodiconiferyl alcohol [Lee D Y, et al.Arch Pharm Res, 2007,30:
402-407.]、(7R,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan [Matsuda N, et
al. Chem Pharm Bull, 1996,44:1676-1679], (7R,8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -
8-O-4'- neolignan [Sinkkonen J, et al.Mag Reson Chem, 2006,44:633-636.], (+)-is different falls
Leaf rosin element [Julio D, et al. Blood, 2003,655:459-466.], 5- methoxyl group-(+)-isolariciresinol
[Zhang Z Z, et al.Phytochemistry, 1999,51:469-472.] and ring olive element [Zuo Guoying waits the Yunnan
Plant research, 2005,27:101 ~ 106.].
Chemical structure is as follows:
The present invention uses high effective liquid chromatography for measuring (+)-isolariciresinol, the different larch turpentine of 5- methoxyl group-(+)-
Element, (7R,8S) -5- melonia dehydrodiconiferyl alcohol and (7S,8RThe content of)-dihydro dehydrodiconiferyl alcohol.
The present invention provide willow leaf japanese spiraea root resisting rheumatoid arthritis active component preparation method, this method include with
Lower step: willow leaf japanese spiraea root adds 60% alcohol reflux to extract three times, each dosage be by kilogram in terms of willow leaf japanese spiraea root weight
8 times of volumes in litres, 2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, it is molten that extract adds water to make
Solution, dosage be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, through large pore resin absorption column D101 color
Spectrum, filling macroporous absorbent resin D101 volume be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, use water
It is closely colourless to be eluted to eluent, discards eluent, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, with 70% second
It is closely colourless that alcohol is eluted to eluent, collects eluent, is concentrated under reduced pressure, dry that willow leaf japanese spiraea root resisting rheumatoid arthritis has
Imitate position.
The preparation method of willow leaf japanese spiraea root resisting rheumatoid arthritis active component provided by the invention, preparation it is effective
(+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7 in positionR,8S) the double pines of -5- melonia dehydrogenation
Cypress pure and mild (7S,8RThe sum of weight of)-dihydro dehydrodiconiferyl alcohol 20% ~ 25%.
The preparation method of willow leaf japanese spiraea root resisting rheumatoid arthritis active component provided by the invention is embroidered from willow leaf
The total lignan active component of separation is extracted in line chrysanthemum root, main component includes (+)-isolariciresinol, 5- methoxyl group-(+)-
Isolariciresinol, (7R,8S) -5- melonia dehydrodiconiferyl alcohol, (7S,8R)-dihydro dehydrodiconiferyl alcohol, (7R,
8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan and (7R,8S) tetrahydroxy-3-4,7,9,9'-,
3'- dimethoxy -8-O-4'- neolignan.
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component of the invention treats rheumatoid arthritis in preparation
It is applied in product, is especially preparing pro-inflammatory cytokine tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 generation suppression
It is applied in formulation products.
Model of adjuvant arthritis in rats is the classical mould evaluating resisting rheumatoid disease arthritis drug and acting on immune inflammation
Type, after injecting Freund's complete adjuvant sensitization in rat toes, lesion is divided into primary and two kinds secondary: novo lesions
It is mainly shown as that early stage causes the inflammatory reaction of scorching part, 18 days right foot swelling reach to peak values after cause is scorching gradually mitigate after continuing 3 days；
Secondary arthritis comes across after cause inflammation 15 days or so, shows as the swelling of opposite side (left hind) and forelimb foot, and ear and tail portion occur
" arthritis " brief summary, allergic pannus and weight loss etc..Cell factor is one group by immune cell and nonimmune
The small-molecule substance that cell synthesis, secretion generate, has multiple biological function.With closely related thin of rheumatoid arthritis
Intracellular cytokine is most of to be secreted by monocyte, macrophage, fibroblast and T cell, wherein tumor necrosis factor-alpha, Bai Jie
- 1 β of element and interleukin-6 are virulence factor, and interleukin-4 and interleukin-10 are protective factors.Tumor necrosis factor-alpha is a kind of
Cell factor with multiple biological activities, is mostly derived from monocyte and macrophage.Mature tumor necrosis factor-alpha point
Son amount is about 17kd, is divided into cross-module type and secreting type, with trimeric form in conjunction with cell surface receptor.Tumor necrosis factor-alpha
Synovial cell and cartilage cell is promoted to synthesize and discharge prostaglandin E2And clostridiopetidase A, cause the absorption of bone and cartilage to destroy, promotees
Into the hyperplasia of fibroblast；Promote the absorption of proteoglycan in cartilage and inhibit its synthesis, makes cartilage degradation；In activity
The activity and aggregation of blood platelet can be improved in rheumatoid arthritis, and fibroblast is promoted to discharge adhesion molecule such as cell adhesion
Molecule.Interleukin-1 is mainly by monocyte and macrophages secrete.In the serum and joint fluid of patient with rheumatoid arthritis
High-caliber interleukin-1, horizontal activity and tectology feature such as synovial hyperplasia, leucocyte with disease can be detected
Infiltration etc. is closely related.Interleukin-1 adjusts the expression of cytokine profiles, cell adhesion molecule, immune modulatory molecules, in class
It plays a significant role in the bone erosion and cartilage destruction of rheumatic arthritis.Interleukin-1 promotes synovial cell and lymphocyte
Proliferation and differentiation, promote synovial cell and cartilage cell to synthesize and discharge prostaglandin E2 and clostridiopetidase A, prostaglandin E2 and glue
Protoenzyme can cause the disintegration of the inflammatory reaction of synovial membrane, cartilage matrix, cause joint injury, and the immune complex of part and free
The decomposition products stimulation interleukin-1 such as collagen synthesis；Interleukin-1 stimulates synovial cell to generate high-caliber matrix metal egg
White enzyme inhibits joint collagen and proteoglycan synthesis, leads to the destruction and reconstruction of extracellular matrix, accelerate rheumatoid joint
Scorching destruction process；Interleukin-1 stimulates fibroblast, endotheli ocytosis, promotes synovial membrane to thicken and is formed with pannus.It is white
Interleukin -6 is generated by T lymphocyte, monocyte and fibroblast, is had in inflammatory cytokine network and Immune Regulative Network
There is critical effect.Interleukin-6 induces acute phase reactive protein such as c reactive protein；Induction B cell is divided into can IgG secretion
The thick liquid cell of type antibody；Mediate the generation of the prostanoid factor.Willow leaf embroider line is studied using model of adjuvant arthritis in rats
The effect of chrysanthemum root resisting rheumatoid arthritis active component, the results showed that, willow leaf japanese spiraea root resisting rheumatoid arthritis is effective
1000 mgkg of position-1Dosage group has obvious inhibiting effect to adjuvant arthritic rats primary inflammatory and secondary inflammation；It adopts
It is effective with lipopolysaccharide-induced 264.7 model of mouse macrophage strain RAW research willow leaf japanese spiraea root resisting rheumatoid arthritis
Position generates inhibiting effect to pro-inflammatory cytokine tumor necrosis factor-alpha, Interleukin -1β and interleukin-6, the results showed that,
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component can obviously inhibit lipopolysaccharide-induced mouse macrophage strain RAW
264.7 pro-inflammatory cytokine tumor necrosis factor-alphas, Interleukin -1β and interleukin-6 generate.The above results show that willow leaf is embroidered
Line chrysanthemum root resisting rheumatoid arthritis active component can be applied in preparation treatment rheumatoid arthritis product, especially as
Pro-inflammatory cytokine tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 formation inhibitor treat rheumatoid in preparation
It is applied in arthritis product.It can also exist containing the pharmaceutical composition of willow leaf japanese spiraea root resisting rheumatoid arthritis active component
It is applied in preparation treatment rheumatoid arthritis product, especially as pro-inflammatory cytokine tumor necrosis factor-alpha, Bai Jie
- 1 β of element and interleukin-6 formation inhibitor are applied in preparation treatment rheumatoid arthritis product.
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component advantage of the invention is with modern separation means and medicine
The method that reason screening active ingredients combine determines active component, and gained active component is at distinguishing one from the other, and active component content is high, and through body
It is inside and outside the experimental results showed that, willow leaf japanese spiraea root resisting rheumatoid arthritis active component provided by the invention obviously inhibits proinflammatory
Property the cytokines Tumor Necrosis factor-α, Interleukin -1β and interleukin-6 generation reach treatment rheumatoid arthritis effect, this
The willow leaf japanese spiraea root resisting rheumatoid arthritis active component that invention provides can be convenient and pharmaceutically acceptable carrier system
The standby drug at various dosage forms, facilitates clinic to take.
Willow leaf japanese spiraea root resisting rheumatoid arthritis preparation method advantage of the invention is simple process, easy to operate,
Technology is easily grasped, and energy consumption is small, and solvent is recyclable to be recycled, and production cost is low.
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component of the invention can be used for preparing treatment resisting rheumatoid
Arthritis product advantage is clear for mechanism of action, based on inhibit pro-inflammatory cytokine tumor necrosis factor-alpha, Interleukin -1β and
Interleukin-6 generates.
The present invention also provides with willow leaf japanese spiraea root resisting rheumatoid arthritis active component of the invention and pharmaceutically
Acceptable carrier or the pharmaceutical preparation of excipient preparation.These pharmaceutical preparations are selected from following dosage form: tablet, sugar coated tablet, thin
Film coated tablet, enteric coated tablet, effervescent tablet, sublingual tablets, capsule, hard capsule, soft capsule, microcapsules, microspheres agent,
Granule, pill, pill, powder, paste, oral solution, suspension, solution, aerosol, injection, emulsion for injection, freeze-drying
Powder needle can also be prepared into sustained release or controlled release preparation as needed.
Of the invention contains willow leaf japanese spiraea root resisting rheumatoid arthritis effective fraction medicine preparation, in the drug system of preparation
Pharmaceutically acceptable carrier can be added when agent, the pharmaceutically acceptable carrier comes from: antioxidant, chelating agent, surfactant,
Filler, disintegrating agent, wetting agent, solvent, slow-release material, enteric material, pH adjusting agent, corrigent, pigment etc., common carrier
Such as: mannitol, dextran, lactose, glucose, sorbierite, xylitol, water for injection, injection ethyl alcohol, sodium chloride, silicon
Derivative, cellulose, cellulose derivative, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, poly- second two
Alcohol, cyclodextrin, phospholipid material, talcum powder, magnesium stearate, calcium stearate etc..
Oral dose changes, dose according to the age of patient, weight, coincident with severity degree of condition and other similar factor
40 ~ 60 mg based on the daily willow leaf japanese spiraea root resisting rheumatoid arthritis active component weight of every 1 kg body weight, point 3 clothes
With.
Below by way of specific embodiment, the present invention is further illustrated, not limitation of the invention, according to ability
The prior art well known to domain, embodiments of the present invention are not limited to specific embodiment.
Specific embodiment
Embodiment 1
The screening of willow leaf japanese spiraea root resisting rheumatoid arthritis active component
The preparation of willow leaf japanese spiraea root extract and separated part: 10 Kg of willow leaf japanese spiraea root adds 60% alcohol reflux to mention
It takes three times, each dosage is 80L, and 2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water
Make to dissolve, dosage 5L, through large pore resin absorption column D101 chromatography, filling macroporous absorbent resin D101 volume is 5L, is washed with water
It takes off closely colourless to eluent, collects eluent, be concentrated under reduced pressure, dry 264 g of willow leaf japanese spiraea root water elution position；With 30%
Ethanol elution is closely colourless to eluent, collects eluent, is concentrated under reduced pressure, dry 30% alcohol elution of willow leaf japanese spiraea root
86 g；It is closely colourless to eluent with 70% ethanol elution, eluent is collected, is concentrated under reduced pressure, dry 70% second of willow leaf japanese spiraea root
Alcohol elutes 117 g of position；It is closely colourless to eluent with 95% ethanol elution, eluent is collected, is concentrated under reduced pressure, it is dry that willow leaf is embroidered
95% alcohol elution of line chrysanthemum root, 19 g.
Each separated part of willow leaf japanese spiraea root extract is to adjuvant arthritic rats primary inflammatory and secondary inflammation
It influences: in addition to Normal group, the intradermal injection H containing inactivation of every group of right hind foot of rat toe37RV Mycobacterium hominis's atoleine emulsion
0.1 ml(8 mgH37RV Mycobacterium hominis/ml atoleine), Normal group injects 0.1ml atoleine.Modeling rat 60
Only, it is divided into 6 groups, respectively model control group, tripterygium glycosides (25 mg/kg) group, willow leaf japanese spiraea root water elution portion at random
Position (1000 mg/kg) group, 30% alcohol elution of willow leaf japanese spiraea root (1000 mg/kg) group, 70% second of willow leaf japanese spiraea root
Alcohol elutes position (1000 mg/kg) group, 95% alcohol elution of willow leaf japanese spiraea root (1000 mg/kg) group.Each group rat causes
Continuous gavage (ig) is administered 21 days after quick, and Normal group and model control group give same volume (10 ml/kg) distilled water.Respectively
Toes volume, difference before and after calculating swelling observe drug to primary inflammatory to group rat behind the right side of measurement in 3 days and 5 days after sensitization
Influence.It the results are shown in Table table 1.The result shows that 70% alcohol elution of willow leaf japanese spiraea root is to adjuvant arthritic rats primary
Inflammation has obvious inhibiting effect, tripterygium glycosides, willow leaf japanese spiraea root water elution position, 30% ethanol elution of willow leaf japanese spiraea root
Position and the effect of 95% alcohol elution of willow leaf japanese spiraea root are unobvious.The measurement in 17 days and 21 days after sensitization of each group rat is left
Toes volume afterwards calculates difference before and after swelling, observes influence of the drug to secondary inflammation.It the results are shown in Table 2.The result shows that willow
70% alcohol elution of leaf japanese spiraea root has obvious inhibiting effect to adjuvant arthritic rats secondary inflammation, more with tripterygium wilfordii
25 mg/kg of glycosides effect is close, and willow leaf japanese spiraea root water elution position, 30% alcohol elution of willow leaf japanese spiraea root and willow leaf are embroidered
The effect of 95% alcohol elution of line chrysanthemum root is unobvious.
Influence of the 1 each separated part of willow leaf japanese spiraea root extract of table to adjuvant arthritic rats primary inflammatory
Compare with model control group:**P < 0.01
Influence of the 2 each separated part of willow leaf japanese spiraea root extract of table to adjuvant arthritic rats secondary inflammation
Compare with model control group:**P < 0.01
Embodiment 2
HPLC method measures (+)-isolariciresinol, 5- first in willow leaf japanese spiraea root resisting rheumatoid arthritis active component
Oxygroup-(+)-isolariciresinol, (7R,8S) -5- melonia dehydrodiconiferyl alcohol and (7S,8RThe double pines of)-dihydro dehydrogenation
The method of cypress alcohol content
Chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1 phosphoric acid solution as mobile phase
B, linear gradient elution: 0 min(A 20%, B 80%, volume ratio) → 10 min(A40%, B 60%, volume ratio) → 25 min
(A 40%, B 60%, volume ratio) → 50 min(A 80%, B 20%)；Flow velocity: 1.0 mLmin-1；Detection wavelength is 280
nm.Theoretical cam curve is calculated by (+)-isolariciresinol peak, is not less than 6000.
The preparation of solution
The preparation of reference substance solution
Precision weighs (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7R,8S) -5- methoxyl group two
Hydrogen dehydrodiconiferyl alcohol and (7S,8R)-dihydro dehydrodiconiferyl alcohol reference substance is appropriate, adds methanol that every 1 mL is made and falls containing (+)-is different
Leaf rosin element 80μG, 5- methoxyl group-(+)-isolariciresinol 30μg、(7R,8S) -5- melonia dehydrodiconiferyl alcohol
50 μG and (7S,8R)-dihydro dehydrodiconiferyl alcohol 50μThe solution of g.
The preparation of test solution
100 mg of willow leaf japanese spiraea root resisting rheumatoid arthritis active component is taken, it is accurately weighed, add methanol to dissolve, shifts
Into 100 ml measuring bottles, adds methanol to scale, shake up, filter, take subsequent filtrate as test solution.
Sample measuring method
Precision draws test solution and control solution each 20μL is measured, one point external standard method meter by chromatographic condition sample introduction
Calculate (+)-isolariciresinol, the different fallen leaves of 5- methoxyl group-(+)-in willow leaf japanese spiraea root resisting rheumatoid arthritis active component
Rosin element, (7R,8S) -5- melonia dehydrodiconiferyl alcohol and (7S,8RThe content of)-dihydro dehydrodiconiferyl alcohol.
Embodiment 3
10 prosperous of the place of production Kg(Jilin of willow leaf japanese spiraea root), add 60% alcohol reflux to extract three times, each dosage is 80L,
2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water to make to dissolve, dosage 5L, through macropore
Adsorption resin column D101 chromatography, filling macroporous absorbent resin D101 volume are 5L, are eluted with water closely colourless to eluent, discard and wash
De- liquid, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, it is closely colourless to eluent with 70% ethanol elution, it collects
Eluent is concentrated under reduced pressure, dry 117 g of willow leaf japanese spiraea root resisting rheumatoid arthritis active component.Using embodiment 2
Method measurement, the different larch turpentine of 5- methoxyl group-(+)-of (+)-isolariciresinol, 4.15% weight containing 8.17% weight
(the 7 of element, 6.03% weightR,8S) -5- melonia dehydrodiconiferyl alcohol and 6.17% weight (7S,8RThe dehydrogenation of)-dihydro is double
Coniferyl alcohol.
Embodiment 4
10 place of production Kg(Changbai of willow leaf japanese spiraea root), add 60% alcohol reflux to extract three times, each dosage is 80L,
2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water to make to dissolve, dosage 5L, through macropore
Adsorption resin column D101 chromatography, filling macroporous absorbent resin D101 volume are 5L, are eluted with water closely colourless to eluent, discard and wash
De- liquid, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, it is closely colourless to eluent with 70% ethanol elution, it collects
Eluent is concentrated under reduced pressure, dry 145 g of willow leaf japanese spiraea root resisting rheumatoid arthritis active component.Using embodiment 2
Method measurement, the different larch turpentine of 5- methoxyl group-(+)-of (+)-isolariciresinol, 3.25% weight containing 7.76% weight
(the 7 of element, 4.70% weightR,8S) -5- melonia dehydrodiconiferyl alcohol and 4.86% weight (7S,8RThe dehydrogenation of)-dihydro is double
Coniferyl alcohol.
Embodiment 5
10 place of production Kg(Jilin Erdaobaihe River of willow leaf japanese spiraea root), add 60% alcohol reflux to extract three times, each dosage is
80L, 2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, and extract adds water to make to dissolve, dosage 5L, warp
Large pore resin absorption column D101 chromatography, filling macroporous absorbent resin D101 volume are 5L, and, abandoning closely colourless to eluent is eluted with water
Eluent is removed, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, it is closely colourless to eluent with 70% ethanol elution,
Eluent is collected, is concentrated under reduced pressure, dry 153 g of willow leaf japanese spiraea root resisting rheumatoid arthritis active component.Using implementation
The method of example 2 measures, the different fallen leaves of 5- methoxyl group-(+)-of (+)-isolariciresinol, 3.14% weight containing 7.96% weight
(the 7 of rosin element, 6.85% weightR,8S) -5- melonia dehydrodiconiferyl alcohol and 3.20% weight (7S,8R)-dihydro is de-
The double coniferyl alcohols of hydrogen.
Embodiment 6
10 place of production Kg(Jilin Fusong of willow leaf japanese spiraea root), add 60% alcohol reflux to extract three times, each dosage is 80L,
2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water to make to dissolve, dosage 5L, through macropore
Adsorption resin column D101 chromatography, filling macroporous absorbent resin D101 volume are 5L, are eluted with water closely colourless to eluent, discard and wash
De- liquid, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, it is closely colourless to eluent with 70% ethanol elution, it collects
Eluent is concentrated under reduced pressure, dry 131 g of willow leaf japanese spiraea root resisting rheumatoid arthritis active component.Using embodiment 2
Method measurement, the different larch turpentine of 5- methoxyl group-(+)-of (+)-isolariciresinol, 4.72% weight containing 8.44% weight
(the 7 of element, 3.65% weightR,8S) -5- melonia dehydrodiconiferyl alcohol and 4.77% weight (7S,8RThe dehydrogenation of)-dihydro is double
Coniferyl alcohol.
Embodiment 7
The separation and identification of willow leaf japanese spiraea root resisting rheumatoid arthritis active component chemical component
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component is isolated through ODS column chromatography and preparative liquid chromatography
9 compounds, using ESI-MS,1H-NMR、13The structure of C-NMR data authentication compound is 8- hydroxyl rosin spirit (1), 8- hydroxyl
Base -7'- table rosin spirit (2), (7R,8S) -5- melonia dehydrodiconiferyl alcohol (3), (7S,8RThe double pine and cypress of)-dihydro dehydrogenation
Alcohol (4), (7R,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan (5), (7R,8S)-4,7,9,
9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan (6), (+)-isolariciresinol (7), 5- methoxyl group-(+) -
Isolariciresinol (8) and ring olive are plain (9).
Compound 1:1H-NMR(500 MHz, CD3OD)δ7.05(1H, s, H-2'), 7.04(1H, d,J =1.4 Hz, H_
2), 6.86(1H, dd,J =8.5,1.8 Hz, H_6'), 6.85(1H, dd,J=8.6,1.8 Hz, H-6), 6.79(1H, d,J
=8.1, H_5), 6.79(1H, d,J =8.1, H_5'), 4.83(1H, d,J =5.4 Hz, H-7'), 4.67(1H, s, H-7),
4.45(1H, dd,J =8.6,8.6 Hz, Ha _9'), 4.03(1H, d,J =9.3 Hz, Ha _9), 3.86(1H, d,J = 9.3
Hz, Hb- 9), 3.86(3H, s, 3-OCH3), 3.85(3H, s, 3'-OCH3), 3.75(1H, dd,J=9.2,6.2 Hz, Hb-
9'), 3.04(1H, ddd,J =7.8,5.9,5.9 Hz, H-8')；13C-NMR(125 MHz, CD3OD)δ149.08(C-3'),
148.69(C-3), 147.48(C-4), 147.40(C-4'), 133.65(C-1') and, 129.08(C-1), 121.54(C-6),
120.49(C-6'), 116.07(C-5'), 115.69(C-5), 112.79(C-2) and, 111.35(C-2'), 92.77(C-8),
89.22(C-7), 87.77(C-7'), 76.13(C-9), 71.96(C-9') and, 62.36(C-8'), 56.43(3,3'-OCH3),
ESI-MS m/z 397 [M+Na]+。
Compound 2:1H-NMR(500 MHz, CD3OD)δ7.04(1H, d,J =1.7 Hz, H-2), 6.94(1H, br s,
H_2'), 6.85(1H, dd,J =8.1,1.7 Hz, H_6), 6.78(3H, overlap, H-5,5', 6'), 5.17(1H, d,J =
6.4 Hz, H-7'), 4.39(1H, s, H-7), 4.19(1H, d,J =9.1 Hz, Ha _9), 3.90(1H, dd,J =9.2,9.2
Hz, Ha _9'), 3.86(3H, s, 3-OCH3), 3.86(3H, s, 3'-OCH3), 3.61(1H, d,J=9.2 Hz, Hb- 9), 3.22
(1H, dd,J=9.1,9.1 Hz, Hb- 9'), 3.09(1H, ddd,J =8.8,8.8,6.5 Hz, H-8')；13C-NMR(125
MHz, CD3OD)δ148.91(C-3'), 148.69(C-3), 147.54(C-4), 146.73(C-4') and, 131.08(C-1'),
129.03(C-1), 121.64(C-6), 119.30(C-6'), 116.09(C-5') and, 115.66(C-5), 112.88(C-2),
110.51(C-2'), 91.59(C-8), 90.73(C-7), 82.76(C-7') and, 76.82(C-9), 69.05(C-9') and, 58.73
(C-8'), 56.46(3-OCH3), 56.42(3'-OCH3), ESI-MS m/z 397 [M+Na]+。
Compound 3:1H-NMR(500 MHz, CD3OD)δ6.73(2H, s, H-2,6), 6.68(2H, s, H_2', 6'), 5.50
(1H, d,J =6.2 Hz, H-7), 3.87(3H, s, 3'-OCH3), 3.84(1H, dd,J =11.0,5.8 Hz, Ha _9), 3.81
(6H, s, 3,5-OCH3), 3.76(1H, dd,J=11.1,7.3 Hz, Hb- 9), 3.57(2H, t,J=6.4 Hz, H _9'),
3.47(1H, dt,J=6.2,6.0 Hz, H-8), 2.63(2H, t,J=7.4 Hz, H _7'), 1.82(2H, m, H _8')；13C-NMR(125 MHz, CD3OD)δ149.38(C-3,5), 147.55(C-3') and, 145.23(C-4'), 137.01(C-1'),
136.52(C-4), 134.05(C-1), 129.87(C-5'), 117.96(C-6') and, 114.23(C-2'), 104.23(C-2,6),
89.12(C-7), 65.03(C-9), 62.24(C-9'), 56.84(C-8) and, 56.80(3,5-OCH3), 55.59(3'-OCH3),
35.80(C-8'), 32.90(C-7'), ESI-MS m/z 413 [M+Na]+。
Compound 4:1H-NMR(500 MHz, CD3OD)δ6.94(1H, d,J =1.8 Hz, H-2), 6.81(1H, dd,J =
8.2,1.9 Hz, H-6), 6.75(1H, d,J =8.2, H_5), 6.73(1H, br s, H_6'), 6.72(1H, br s, H_2'),
5.48(1H, d,J =6.3 Hz, H-7), 3.85(3H, s, 3'-OCH3), 3.84(1H, dd,J =11.2,5.6 Hz, Ha _9),
3.80(3H s, 3-OCH3), 3.76(1H, dd,J=11.2,7.2 Hz, Hb- 9), 3.57(2H, t,J=6.5 Hz, H _
9'), 3.47(1H, dt,J=6.1,5.8 Hz, H-8), 2.63(2H, t,J=8.0 Hz, H _7'), 1.82(2H, m, H _
8')；13C-NMR(125 MHz, CD3OD)δ149.37(C-3), 148.41(C-4'), 147.57(C-4), 145.20(C-3'),
136.88(C-1'), 134.20(C-1), 130.02(C-5'), 119.84(C-6) and, 117.96(C-5), 116.40(C-6'),
114.20(C-2'), 110.64(C-2), 89.10(C-7), 65.01(C-9) and, 62.26(C-9'), 56.81(3'-OCH3),
56.38(3-OCH3), 55.38(C-8), 35.79(C-8'), 32.90(C-7') and, ESI-MS m/z 383 [M+Na]+。
Compound 5:1H-NMR(500 MHz, CD3OD)δ7.02(1H, d,J =1.4 Hz, H-2), 6.97(1H, d,J =
8.2, H_5'), 6.86(1H, dd,J=8.8,1.8 Hz, H-6), 6.85(1H, d,J=1.4 Hz, H_2'), 6.76(1H, d,J =8.1 Hz, H-5), 6.71(1H, dd,J =8.0,1.4 Hz, H_6'), 4.88(1H, d,J =6.0 Hz, H-7),
4.20(1H, dd,J =9.8,5.2 Hz, H-8), 3.85(3H, s, 3'-OCH3), 3.82(3H, s, 3-OCH3), 3.71(1H,
Dd,J =12.0,4.0 Hz, Ha _9), 3.56(2H, t,J=6.4 Hz, H _9'), 3.46(1H, dd,J=11.9,5.2
Hz, Hb- 9), 2.62(2H, t,J=7.5 Hz, H _7'), 1.81(2H, m, H _8')；13C-NMR(125 MHz, CD3OD)δ
151.69(C-3'), 148.87(C-3), 147.59(C-4'), 147.28(C-4) and, 138.24(C-1'), 133.74(C-1),
122.04(C-6'), 120.81(C-6), 119.64(C-5'), 115.90(C-5) and, 114.00(C-2'), 111.80(C-2),
87.72(C-8), 74.18(C-7), 62.20(C-9'), 61.91(C-9) and, 56.55(3'-OCH3), 56.38(3-OCH3),
35.51(C-8'), 32.69(C-7'), ESI-MS m/z 379 [M+H]+。
Compound 6:1H-NMR(500 MHz, CD3OD)δ7.00(1H, d,J =1.8 Hz, H-2), 6.82(1H, dd,J =
8.2,1.8 Hz, H_6), 6.82(1H, d,J=8.2 Hz, H-5'), 6.79(1H, d,J=1.8 Hz, H_2'), 6.73(1H,
D,J =8.1 Hz, H-5), 6.66(1H, dd,J =8.1,2.0 Hz, H_6'), 4.83(1H, overlap, H-7), 4.28
(1H, m, H-8), 3.84(1H, dd,J =11.9,5.7 Hz, Ha _9), 3.80(3H, s, 3-OCH3), 3.78(3H, s, 3'-
OCH3), 3.75(1H, dd,J=11.9,3.7 Hz, Hb- 9), 3.55(2H, t,J=6.6 Hz, H _9'), 2.60(2H, t,J
=7.5 Hz, H _7'), 1.80(2H, m, H _8')；13C-NMR(125 MHz, CD3OD)δ151.87(C-3'), 148.72(C-
3), 147.22(C-4), 147.00(C-4'), 138.11(C-1') and, 134.17(C-1), 121.88(C-6') and, 121.00(C-
6), 119.65(C-5'), 115.68(C-5), 114.07(C-2') and, 111.86(C-2), 86.64(C-8) and, 74.15(C-7),
62.20(C-9,9'), 56.51(3-OCH3), 56.37(3'-OCH3), 35.52(C-8'), 32.68(C-7'), ESI-MS m/z
379 [M+H]+。
Compound 7:1H-NMR(500 MHz, CD3OD)δ6.74(1H, d,J=8.0 Hz, H-5), 6.68(1H, d,J =
1.8 Hz, H-2), 6.65(1H, s, H-2'), 6.61(1H, dd,J =8.0,1.8 Hz, H-6), 6.19(1H, s, H-5'),
3.79(3H s, 3-OCH3), 3.78(1H, d,J=10.4 Hz, H-7), 3.76(3H, s, 3'-OCH3), 3.68(1H, m, H-
9', Ha _9), 3.40(1H, dd,J=11.2,4.2 Hz, Hb _9), 2.77(2H, d,J =7.6 Hz, H _7'), 2.00(1H,
M, H_8'), 1.77(1H, m, H_8)；13C-NMR(125 MHz, CD3OD)δ149.01(C-3), 147.19(C-3'), 145.95
(C-4), 145.28(C-4'), 138.60(C-1), 134.18(C-6') and, 129.02(C-1'), 123.20(C-6) and, 117.36
(C-5'), 116.01(C-5), 113.87(C-2), 112.44(C-2') and, 66.02(C-9'), 62.36(C-9), 56.39(3,
3'-OCH3), 48.49(C-7,8), 40.07(C-8'), 33.55(C-7').ESI-MS m/z 361 [M+H]+。
Compound 8:1H-NMR(500 MHz, CD3OD)δ6.66(1H, s, H-2'), 6.43(2H, s, H-2,6), 6.21
(1H, s, H-5'), 3.81(3H, s, 3'-OCH3), 3.78(1H, d,J =10.4 Hz, H-7), 3.78(6H, s, 3,5-OCH3),
3.69(3H m, H_9', Ha- 9), 3.41(1H, dd,J =11.2,4.0 Hz, Hb _9), 2.78(2H, d,J =7.7 Hz, H _
7'), 2.01(1H, m, H_8'), 1.79(1H, m, H_8)；13C-NMR(125 MHz, CD3OD)δ149.30(C-3,5), 147.30
(C-3'), 145.36(C-4'), 137.78(C-1), 135.19(C-4) and, 134.04(C-6'), 129.02(C-1') and, 117.31
(C-5'), 112.48(C-2'), 107.82(C-2,6), 65.98(C-9') and, 62.32(C-9), 56.79(3,5-OCH3),
56.44(3'-OCH3), 48.49(C-7), 47.93(C-8), 40.01(C-8') and, 33.57(C-7').ESI-MS m/z 391
[M+H]+。
Compound 9:1H-NMR(500 MHz, CD3OD)δ6.76(1H, d,J=8.0 Hz, H-5), 6.70(1H, d,J =
1.8 Hz, H-2), 6.66(1H, dd,J =8.0,1.9 Hz, H-6), 6.63(1H, s, H-2'), 6.19(1H, s, H-5'),
4.02(1H, d,J =11.6 Hz, H-7), 3.82(1H, m, Ha- 9), 3.80(3H, s, 3'-OCH3), 3.79(1H, m, Hb- 9),
3.78(1H m, Ha- 9'), 3.78(3H, s, 3-OCH3), 3.58(1H, m, Hb- 9'), 3.22(1H, d,J =16.6 Hz, Ha-
7'), 2.61(1H, d,J =16.7 Hz, Hb- 7'), 2.03(1H, m, H-8)；13C-NMR(125 MHz, CD3OD)δ149.15
(C-3), 147.51(C-3'), 146.15(C-4), 145.33(C-4') and, 138.48(C-1), 133.58(C-6') and, 126.44
(C-1'), 123.58(C-6), 117.36(C-5'), 116.04(C-5) and, 113.99(C-2), 113.01(C-2') and, 74.96(C-
8'), 69.43(C-9'), 60.86(C-9), 56.42(3-OCH3), 56.39(3'-OCH3), 47.62(C-8), 44.91(C-7),
39.94(C-7')；ESI-MS m/z 377 [M+H]+。
Embodiment 8
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component is to lipopolysaccharide-induced mouse macrophage strain RAW
264.7 pro-inflammatory cytokine tumor necrosis factor-alphas, Interleukin -1β and interleukin-6 generate inhibitory activity measurement
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
It is added the culture medium for the willow leaf japanese spiraea root resisting rheumatoid arthritis active component that 20 μ l contain various concentration in each hole, 30
μ l contains the culture medium of 100 ng/mL LPS, is placed in containing 5%CO237 DEG C of constant incubators in cultivate 12 hours, supernatant
With ELISA kit detection tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content.It the results are shown in Table 3.
3 willow leaf japanese spiraea root resisting rheumatoid arthritis active component of table is to lipopolysaccharide-induced mouse macrophage strain
RAW 264.7
The influence that proinflammatory cytokine is generated by tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Embodiment 9
(+)-isolariciresinol is to lipopolysaccharide-induced 264.7 proinflammatory cytokine of mouse macrophage strain RAW because of tumour
Necrosis factor-alpha, Interleukin -1β and interleukin-6 generate inhibitory activity measurement
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
The culture medium of (+)-isolariciresinol that 20 μ l contain various concentration is added in each hole, 30 μ l contain 100 ng/mL LPS
Culture medium, be placed in containing 5%CO237 DEG C of constant incubators in cultivate 12 hours, supernatant is detected swollen with ELISA kit
Tumor necrosis factor-α, Interleukin -1β and interleukin-6 content.It the results are shown in Table 4.
Table 4 (+)-isolariciresinol is to lipopolysaccharide-induced mouse macrophage strain RAW 264.7
The influence that proinflammatory cytokine is generated by tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Embodiment 10
5- methoxyl group-(+)-isolariciresinol is to lipopolysaccharide-induced 264.7 proinflammatory of mouse macrophage strain RAW
Cell generates inhibitory activity measurement because of tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
5- methoxyl group-(+)-isolariciresinol culture medium that 20 μ l contain various concentration is added in each hole, 30 μ l contain 100
The culture medium of ng/mL LPS, is placed in containing 5%CO237 DEG C of constant incubators in cultivate 12 hours, supernatant is tried with ELISA
Agent box detects tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content.It the results are shown in Table 5.
5 5- methoxyl group of table-(+)-isolariciresinol is to lipopolysaccharide-induced mouse macrophage strain RAW 264.7
The influence that proinflammatory cytokine is generated by tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Embodiment 11
(7R,8S) -5- melonia dehydrodiconiferyl alcohol is to lipopolysaccharide-induced mouse macrophage strain RAW 264.7
Proinflammatory cytokine generates inhibitory activity measurement because of tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
20 μ l are added in each hole and contain (the 7 of various concentrationR,8S) -5- melonia dehydrodiconiferyl alcohol culture medium, 30 μ l contain
There is the culture medium of 100 ng/mL LPS, is placed in containing 5%CO237 DEG C of constant incubators in cultivate 12 hours, supernatant use
ELISA kit detects tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content.It the results are shown in Table 6.
Table 6 (7R,8S) -5- melonia dehydrodiconiferyl alcohol is to lipopolysaccharide-induced mouse macrophage strain RAW
264.7
The influence that proinflammatory cytokine is generated by tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Embodiment 12
(7S,8R)-dihydro dehydrodiconiferyl alcohol is thin to lipopolysaccharide-induced mouse macrophage strain 264.7 proinflammatory of RAW
Born of the same parents generate inhibitory activity measurement because of tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
20 μ l are added in each hole and contain (the 7 of various concentrationS,8RThe culture medium of)-dihydro dehydrodiconiferyl alcohol, 30 μ l contain 100
The culture medium of ng/mL LPS, is placed in containing 5%CO237 DEG C of constant incubators in cultivate 12 hours, supernatant is tried with ELISA
Agent box detects tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content.It the results are shown in Table 7.
Table 7 (7S,8R)-dihydro dehydrodiconiferyl alcohol is to lipopolysaccharide-induced mouse macrophage strain RAW 264.7
The influence that proinflammatory cytokine is generated by tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
Embodiment 13
(7R,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan is to lipopolysaccharide-induced small
264.7 proinflammatory cytokine of mouse macrophage strain RAW inhibits to live because tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 generate
Property measurement
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
20 μ l are added in each hole and contain (the 7 of various concentrationR,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- is new
The culture medium of lignanoid, 30 μ l contain the culture medium of 100 ng/mL LPS, are placed in containing 5%CO237 DEG C of constant incubators in
Culture 12 hours, supernatant detect tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content with ELISA kit.Knot
Fruit is shown in Table 8.
Table 8 (7R,8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan is to lipopolysaccharide-induced
Mouse macrophage strain RAW 264.7 proinflammatory cytokine generated because of tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
It influences
Embodiment 14
(7R,8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan is to lipopolysaccharide-induced small
264.7 proinflammatory cytokine of mouse macrophage strain RAW inhibits to live because tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 generate
Property measurement
Mouse macrophage strain RAW 264.7 is inoculated in (green containing 100 units/mL containing 10% fetal calf serum DMEM culture solution
Mycin, gentamicin 100uG/mL it in), sets containing 5%CO2Regular growth culture is carried out in 37 DEG C of constant incubators.When experiment
The good cell EDTA digestive juice digestion of growth conditions is taken, is counted.It is 8 × 10 by density5The mouse macrophage strain of a/ml
264.7 250 μ l of RAW is seeded on 48 well culture plates, is placed in containing 5 %CO237 DEG C of constant incubators in cultivate 2 days,
20 μ l are added in each hole and contain (the 7 of various concentrationR,8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- is new
The culture medium of lignanoid, 30 μ l contain the culture medium of 100 ng/mL LPS, are placed in containing 5%CO237 DEG C of constant incubators in
Culture 12 hours, supernatant detect tumor necrosis factor-alpha, Interleukin -1β and interleukin-6 content with ELISA kit.Knot
Fruit is shown in Table 9.
Table 9 (7R,8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan is to lipopolysaccharide-induced
Mouse macrophage strain RAW 264.7 proinflammatory cytokine generated because of tumor necrosis factor-alpha, Interleukin -1β and interleukin-6
It influences
Embodiment 15
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component pharmaceutically acceptable carrier or excipient preparation
Pharmaceutical preparation.These pharmaceutical preparations are selected from following dosage form: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, effervesce
Tablet, sublingual tablets, capsule, hard capsule, soft capsule, microcapsules, microspheres agent, granule, pill, pill, powder,
Paste, oral solution, suspension, solution, aerosol, injection, emulsion for injection, freeze-dried powder can also be prepared as needed
At sustained release or controlled release preparation, pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier can be added when preparing pharmaceutical preparation
It comes from: antioxidant, chelating agent, surfactant, filler, disintegrating agent, wetting agent, solvent, slow-release material, enteric material, pH
Regulator, corrigent, pigment etc., common carrier such as: mannitol, dextran, lactose, glucose, sorbierite, xylitol,
Water for injection, injection ethyl alcohol, sodium chloride, silicon derivative, cellulose, cellulose derivative, gelatin, polyvinylpyrrolidone,
Glycerol, Tween 80, agar, calcium carbonate, polyethylene glycol, cyclodextrin, phospholipid material, talcum powder, magnesium stearate, calcium stearate.
Above-mentioned preparation process is this field routine operation, is not added repeats herein.
Embodiment 16
Willow leaf japanese spiraea root resisting rheumatoid arthritis active component is that pharmaceutical preparation made from raw material not only includes independent
It further include the addition anti-class of willow leaf japanese spiraea root using the pharmaceutical preparation of willow leaf japanese spiraea root resisting rheumatoid arthritis active component
Pharmaceutical preparation of the rheumatic arthritis active component as active constituent.
Claims (6)
1. a kind of willow leaf japanese spiraea root resisting rheumatoid arthritis active component, it is characterised in that extracted from willow leaf japanese spiraea root
Isolated total lignan active component, wherein (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7R,
8S) the sum of the weight of -5- melonia dehydrodiconiferyl alcohol and (7S, 8R)-dihydro dehydrodiconiferyl alcohol accounts for willow leaf meadow sweet
The 20%~25% of root resisting rheumatoid arthritis active component total weight, is prepared by the following method to obtain: willow leaf meadow sweet
Root adds 60% alcohol reflux to extract three times, each dosage be by kilogram in terms of 8 times of willow leaf japanese spiraea root weight volumes in litres,
2 hours every time, filtration, filtrate merge, ethyl alcohol is recovered under reduced pressure and obtains extract, extract adds water to make to dissolve, dosage be by kilogram in terms of
The volume in litres of 0.5 times of willow leaf japanese spiraea root weight fills macroporous absorbent resin through large pore resin absorption column D101 chromatography
D101 volume be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, be eluted with water it is closely colourless to eluent,
Eluent is discarded, it is closely colourless to eluent with 30% ethanol elution, eluent is discarded, with 70% ethanol elution to the nearly nothing of eluent
Color collects eluent, is concentrated under reduced pressure, dry willow leaf japanese spiraea root resisting rheumatoid arthritis active component.
2. willow leaf japanese spiraea root resisting rheumatoid arthritis active component according to claim 1, it is characterised in that main
Ingredient includes (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, the dehydrogenation pair of (7R, 8S) -5- melonia
Coniferyl alcohol, (7S, 8R)-dihydro dehydrodiconiferyl alcohol, (7R, 8R) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'-
Neolignan and (7R, 8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-O-4'- neolignan.
3. a kind of preparation method of willow leaf japanese spiraea root resisting rheumatoid arthritis active component, it is characterised in that including following step
Rapid: willow leaf japanese spiraea root adds 60% alcohol reflux to extract three times, each dosage be by kilogram in terms of 8 times of willow leaf japanese spiraea root weight
Volume in litres, 2 hours every time, filtration, filtrate merged, and ethyl alcohol is recovered under reduced pressure and obtains extract, and extract adds water to make to dissolve, and used
Amount for by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, through large pore resin absorption column D101 chromatography, filling
Macroporous absorbent resin D101 volume be by kilogram in terms of 0.5 times of willow leaf japanese spiraea root weight volume in litres, be eluted with water to
Eluent is closely colourless, discards eluent, closely colourless to eluent with 30% ethanol elution, discards eluent, is washed with 70% ethyl alcohol
It takes off closely colourless to eluent, collects eluent, be concentrated under reduced pressure, dry the effective portion of willow leaf japanese spiraea root resisting rheumatoid arthritis
Position.
4. the preparation method of willow leaf japanese spiraea root resisting rheumatoid arthritis active component according to claim 3, special
Sign is (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol, (7R, 8S) -5- first in the active component prepared
The sum of the weight of oxygroup dihydro dehydrodiconiferyl alcohol and (7S, 8R)-dihydro dehydrodiconiferyl alcohol accounts for the anti-class wind of willow leaf japanese spiraea root
The 20%~25% of wet arthritis active component total weight.
5. the preparation method of willow leaf japanese spiraea root resisting rheumatoid arthritis active component according to claim 3, special
Sign be prepare active component main component include (+)-isolariciresinol, 5- methoxyl group-(+)-isolariciresinol,
(7R, 8S) -5- melonia dehydrodiconiferyl alcohol, (7S, 8R)-dihydro dehydrodiconiferyl alcohol, (7R, 8R) -4,7,9,9'- four
Hydroxyl -3,3'- dimethoxy -8-O-4'- neolignan and (7R, 8S) -4,7,9,9'- tetrahydroxy -3,3'- dimethoxy -8-
O-4'- neolignan.
6. willow leaf japanese spiraea root resisting rheumatoid arthritis active component described in claim 1 is closed in preparation treatment rheumatoid
Save the application in scorching product.
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