CN103919835A - Method for preparing tripterygium wilfordii extract by using triterpenes as main components and pharmaceutical application of tripterygium wilfordii extract - Google Patents
Method for preparing tripterygium wilfordii extract by using triterpenes as main components and pharmaceutical application of tripterygium wilfordii extract Download PDFInfo
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Abstract
The invention discloses a method for preparing a tripterygium wilfordii extract by using triterpenes as main components and a pharmaceutical application of the triterpenes as main components. The method for preparing the tripterygium wilfordii extract by using triterpenes as main components comprises the following steps: (1) crushing a dried tripterygium wilfordii root medicinal material, screening through a screen of 10-40 meshes, and weighing 150-200g of powder; (2) performing ultrasonic extraction with an extracting solvent, wherein the usage ratio of the extracting solvent to the medicinal materials is (8-16)ml:1g; filtering the extract, reclaiming the filtrate at reduced pressure to extract the solvent to obtain concentrate, and drying to obtain a coarse product of tripterygium wilfordii extract; and (3) adding 5.0-15.0g of the coarse product of tripterygium wilfordii extract into a solvent for re-dissolving, wherein the usage ratio of the solvent to the coarse product of tripterygium wilfordii extract is (10-15)ml:1g, filtering, and reclaiming the solvent at reduced pressure to obtain the tripterygium wilfordii extract. The invention also discloses a medicament prepared from the tripterygium wilfordii extract for treating rheumatoid arthritis. The content of total triterpenes of the prepared tripterygium wilfordii extract is 50-75 percent, so that the utilization of a tripterygium wilfordii medicinal material is improved, and the toxicity of the tripterygium wilfordii medicinal material is reduced.
Description
Technical field
The invention belongs to Chinese medicine ingredients extractive technique field, be specifically related to a kind of extraction optimization method and application in treatment medicine for treating rheumatoid arthritis thereof of take the Radix Tripterygii Wilfordii extract powder that total triterpene is main component.
Background technology
Radix Tripterygii Wilfordii (Tripterygium wilfordii Hook.f) is Celastraceae tripterygium plant, mainly originates in the ground such as Zhejiang, Anhui, Jiangxi, Hunan, Fujian and Yunnan.Bitter in the mouth, pungent, cool in nature, malicious greatly.Return liver, kidney channel.Effect expelling wind and removing dampness, removing obstruction in the collateral to relieve pain, reducing swelling and alleviating pain, removing toxic substances parasite killing.For damp and hot tuberosity, the long-pending poison of carcinoma, with it, treat lepra reaction, rheumatoid arthritis etc. clinically.Be everlasting in the Chinese patent medicines such as Radix Tripterygii Wilfordii tablet, Tripterygium wilfordii Polyglycosidium Tablets, Glucosidorum Tripterygll Totorum, tripterygium total terpenoids tablets, thunder network ester sheet, Triptolide ointment and apply.
The main chemical compositions of Radix Tripterygii Wilfordii comprises Diterpenes, triterpenes, sesquiterpenoids, alkaloids, tannin, organic acid and polysaccharide etc.Radix Tripterygii Wilfordii can act on lymphocyte comprehensively, reduces the generation of the inflammation factor, Immunosuppression cell proliferation, and cell death inducing, thereby Immunosuppression, to treatment, RA has certain curative effect.Radix Tripterygii Wilfordii complicated component, the prepared from active ingredients of tripterygium wilfordii of at present generally acknowledged treatment RA is mainly Diterpenes, triterpenes and alkaloids.Wherein, Diterpenes content in tripterygium wilfordii is low, and toxicity is large; Triterpenes content is higher, easily extracts.There is document (Miao Kangli, Xu Xuehua, Wei Chaohui, Rui Chaosong. the research of active ingredients of thunder god vine Demethylzeylasteral. contemporary Chinese clinical medicine, 2005,4(10): 7-10.) the median lethal dose(LD 50) LD50 (mice) of report triterpenes DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is 1295mg/kg, does not present cytotoxicity, immunosuppressive action and its dosage are proportionate and in certain dosage range.
Current existing Radix Tripterygii Wilfordii extract patent is mainly with being object for extracting a certain single compound of Radix Tripterygii Wilfordii, take the purification of Radix Tripterygii Wilfordii lactone alcohol and structure of modification as main, as: Chinese Patent Application No. 201010603887.5 has been introduced a kind of employing and has been prepared the method that high performance liquid chromatogram extracts DEMETHYLZEYLASTERAL DZY T-96 TZ-93 in Radix Tripterygii Wilfordii, and the purity that makes DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is 98.5%.200810209679.X has introduced a kind of method of extracting first element from Radix Tripterygii Wilfordii, obtains containing the pure triptolide Radix Tripterygii Wilfordii extract powder that triptolide purity is 1.21%.201110139090.9 have introduced a kind of preparation method of Radix Tripterygii Wilfordii effective site powder, but its total triterpene contents is only 15%.
The patent Introduction of the patent No. 201110139090.9 a kind of Radix Tripterygii Wilfordii effective site powder that is used for the treatment of chronic nephritis prepare separation method, the terpenoid content of the effective site powder of this invention gained is greater than 50%, wherein total triterpene contents is in 15% left and right, the larger sesquiterpene alkaloids class component of toxicity is in 40% left and right, and indication is chronic nephropathy.
Summary of the invention
The present inventor studies through long-term experiment, finds that triterpenoids class material has significant curative effect aspect treatment rheumatic Bi syndrome.Therefore, the Radix Tripterygii Wilfordii extract powder that total triterpene of take if can make is main component, the utilization that can improve tripterygium wilfordii resource, reduces the toxicity of Radix Tripterygii Wilfordii and then improves the efficacy and safety that it treats rheumatoid arthritis.Based on this, the invention provides a kind of preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component, with and purposes in drugs for rheumatoid arthritis.
The Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion that contains provided by the present invention is compared with undressed Radix Tripterygii Wilfordii extract powder, in the drug action that suppresses the aspects such as arthritis synovial cell propagation, significantly improves, and toxicity reduction, can realize suitability for industrialized production.
For achieving the above object, the present invention takes following technical scheme:
A preparation method for the Radix Tripterygii Wilfordii extract that the triterpenes of take is main component, its concrete steps are as follows:
(1) the full root herb of dry Radix Tripterygii Wilfordii is pulverized, crossed 10 order～40 mesh sieves, take powder 150g～200g;
(2) with alcoholic solution, ethyl acetate or acetone equal solvent, carry out supersound extraction, extracting solvent and medical material amount ratio is 8ml～16mL:1g, extracting liquid filtering, and filtrate decompression reclaims solvent, obtains concentrated solution, dry, obtains Radix Tripterygii Wilfordii extract powder crude product;
(3) gained extract powder crude product 5.0g～15.0g in step (2), add ethyl acetate, ethanol, dehydrated alcohol or redissolve with the acid ethanol solution equal solvent of salt acid for adjusting pH, the amount ratio of solvent and extract powder crude product is 10～15mL:1g, filter, decompression and solvent recovery, obtains Radix Tripterygii Wilfordii extract powder.
Preferably, in step (2), described alcoholic solution concentration is 80%～95%, and ultrasonic number of times is 1～3 time, each 0.5～2.0h.
Preferably, in step (2), described being dried as 55～75 ℃ of vacuum dryings.
Preferably, in step (2), described supersound extraction power is 250W～500W, mixing speed 750～1250rpm, and ultrasonic temperature is 25～50 ℃.
The invention also discloses the medicine for the preparation of rheumatoid arthritis by said extracted method gained Radix Tripterygii Wilfordii extract powder.
The present invention is usingd the less total Triterpenoid of terpenoid component toxic as object, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is as effective monomer component, in the extract making, total triterpene contents is 50%～75%, and effective monomer component DEMETHYLZEYLASTERAL DZY T-96 TZ-93 content is 10% left and right, and indication is rheumatoid arthritis.
Inventor finds to take that total triterpene that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is representative difference in solubility in the different solvent of polarity is very large after deliberation, and its dissolubility is subject to the impact of pH, utilize this dissolution characteristics can realize containing the preparation of the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion.Adopt technical scheme of the present invention, the total triterpene contents in the Radix Tripterygii Wilfordii extract powder after processing with determination of color is 50%～75%.
The present inventor, through long-term Radix Tripterygii Wilfordii Related Experimental Study, find that in Radix Tripterygii Wilfordii, triterpenes and Diterpenes are effective ingredient aspect treatment rheumatic Bi syndrome, but wherein the triptolide of Diterpenes is but main toxic component.When the content of triptolide surpasses certain limit, very easily cause intoxicating phenomenon, the triterpene substance that the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of take is representative is not found obvious toxicity.
Research based on to the toxicity of Radix Tripterygii Wilfordii and active ingredient, the invention provides a kind of main component is the preparation method of the Radix Tripterygii Wilfordii extract powder of total triterpene, in the Radix Tripterygii Wilfordii extract powder making by the method, contain higher total triterpenes material, and can be exclusively used in treatment rheumatic Bi syndrome, the waste of tripterygium wilfordii resource and the safety of Tripterygium Preparations, the narrow difficult problem of clinical application range have effectively been solved, improved the bioavailability of medicine, for the application of Radix Tripterygii Wilfordii Chinese crude drug provides new technological development direction.
Through preparation method of the present invention, make containing the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion, find that after measured total triterpene contents is 50～75%; The present invention proposes take total triterpene as effective ingredient treatment rheumatoid arthritis; The total triterpenes Radix Tripterygii Wilfordii extract powder that utilizes the inventive method to make, the total triterpene in Radix Tripterygii Wilfordii of take is object, improved the availability of tripterygium wilfordii, and toxicity obviously reduces.
The specific embodiment
Below in conjunction with embodiment, further illustrate technical scheme of the present invention, but these embodiment do not limit the scope of the invention.
Embodiment 1: preparation contains the method for the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion:
(1) get the full root herb of Radix Tripterygii Wilfordii, pulverize, cross 40 mesh sieves, take 150g;
(2) with 2400mL ethanol ultrasonic (ultrasonic power 250W, rotating speed 750rpm, 20 ℃ of temperature), extract 1 time, extract 2h, extracting liquid filtering, filtrate decompression reclaims solvent, obtains concentrated solution, and 75 ℃ of vacuum dryings, obtain Radix Tripterygii Wilfordii extract powder crude product 10.30g;
(3) above-mentioned extract powder crude product is added ethyl acetate 103mL redissolve, filter, filtrate decompression reclaims solvent, obtains Radix Tripterygii Wilfordii extract powder 9.28g, and by UV method, measuring wherein total triterpene contents is 54.7%, and with HPLC, measuring wherein DEMETHYLZEYLASTERAL DZY T-96 TZ-93 content is 6.2%.
Embodiment 2: preparation contains the method for the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion:
(1) get the full root herb of Radix Tripterygii Wilfordii, pulverize, cross 10 mesh sieves, take 200g;
(2) use respectively 1600mL ethyl acetate ultrasonic (ultrasonic power 500W, rotating speed 1250rpm, temperature 50 C) to extract 3 times, each 0.5h, merge extractive liquid,, filters, and filtrate decompression reclaims solvent, obtain concentrated solution, 55 ℃ of vacuum dryings, obtain Radix Tripterygii Wilfordii extract powder crude product 6.46g;
(3) above-mentioned extract powder crude product is added dehydrated alcohol 85mL redissolve, filter, filtrate decompression reclaims solvent, obtains Radix Tripterygii Wilfordii extract powder 5.89g, and by UV method, measuring wherein total triterpene contents is 72.8%, and with HPLC, measuring wherein DEMETHYLZEYLASTERAL DZY T-96 TZ-93 content is 10.5%.
Embodiment 3: preparation contains the method for the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion:
(1) get the full root herb of Radix Tripterygii Wilfordii, pulverize, cross 24 mesh sieves, take 200g;
(2) use respectively 2000mL acetone ultrasonic (ultrasonic power 400W, rotating speed 1000rpm, 40 ℃ of temperature) to extract 2 times, each 1.0h, merge extractive liquid,, filters, and filtrate decompression reclaims solvent, obtain concentrated solution, 70 ℃ of vacuum dryings, obtain Radix Tripterygii Wilfordii extract powder crude product 5.98g;
(3) above-mentioned extract powder crude product is added acidic ethanol (hydrochloric 0.1%) 55mL with salt acid for adjusting pH redissolve, filter, filtrate decompression reclaims solvent, obtain Radix Tripterygii Wilfordii extract powder 5.32g, by UV method, measuring wherein total triterpene contents is 68.4%, and with HPLC, measuring wherein DEMETHYLZEYLASTERAL DZY T-96 TZ-93 content is 8.6%.
Embodiment 4: preparation contains the method for the Radix Tripterygii Wilfordii extract powder of total triterpene at high proportion:
(1) get the full root herb of Radix Tripterygii Wilfordii, pulverize, cross 24 mesh sieves, take 200g;
(2) use respectively 2400mL80% ethanol ultrasonic (ultrasonic power 450W, rotating speed 1000rpm, 40 ℃ of temperature) to extract 2 times, each 0.5h, merge extractive liquid,, filters, and filtrate decompression reclaims solvent, obtain concentrated solution, 70 ℃ of vacuum dryings, obtain Radix Tripterygii Wilfordii extract powder crude product 11.39g;
(3) above-mentioned extract powder crude product is added ethyl acetate 250mL redissolve, filter, decompression and solvent recovery, obtains Radix Tripterygii Wilfordii extract powder 9.35g, and by UV method, measuring wherein total triterpene contents is 52.5%, and with HPLC, measuring wherein DEMETHYLZEYLASTERAL DZY T-96 TZ-93 content is 7.5%.
Embodiment 5: the Mus ear swelling experiment of Radix Tripterygii Wilfordii extract powder:
Select embodiment 1, the Radix Tripterygii Wilfordii extract powder of gained carried out to preliminary animal antiinflammatory evaluating drug effect:
1. experiment material
1.1 materials and instrument: CMC ?Na(CP, lot number F20110609, Chemical Reagent Co., Ltd., Sinopharm Group), dimethylbenzene (CP, lot number 20030501, chemical reagent work of Juhua Group Co.), (100 slices/bottle of indometacin enteric-coated tablet, lot number 110101, upper Hisense's friendship the Yellow River pharmaceutical Co. Ltd), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 reference substance (1g, lot number FY18540201, Fei Yu bio tech ltd, Nantong), 6mm diameter card punch.
1.2 animals: Male Kunming strain mice (animal occupancy permit number: SCXK (Zhejiang) ?20080033)
2. method and result
2.1 samples: take Radix Tripterygii Wilfordii extract powder, DEMETHYLZEYLASTERAL DZY T-96 TZ-93, indometacin enteric-coated tablet appropriate, add 15mL0.5%CMC ?Na solution, after stirring, the ultrasonic suspendible that makes is even, as following table 1,2.
The preparation of table 1 DEMETHYLZEYLASTERAL DZY T-96 TZ-93 test of pesticide effectiveness group medicinal liquid
The preparation of table 2 Radix Tripterygii Wilfordii extract powder test of pesticide effectiveness group medicinal liquid
| Group | Extract powder concentration/c(mg/mL) | Dosage (mg/kg/d) |
| High dose | 6.34 | 127.08 |
| Middle dosage | 1.43 | 28.46 |
| Low dosage | 0.47 | 9.45 |
2.2 DEMETHYLZEYLASTERAL DZY T-96 TZ-93 antiinflammatory effect experiments:
Water is can't help in mice administration fasting evening before that day.The grouping of mice: mice is divided into 3 groups according to body weight, the mice of getting at random again each body weight group is divided into 8 groups, every group 10, five groups are respectively blank group (0.5%CMC ?Na solution), positive controls (indomethacin), high dose group, middle dosage group, low dose group, as shown in table 1,2.Every day gavage 0.4mL, for three days on end.Auris dextra mice after last administration 1h is smeared 0.05mL dimethylbenzene with syringe, and left ear does not deal with.After 4h, the de-neck of mice is put to death, along auricle baseline, cut two ears, with 6mm diameter card punch, at the same position of left and right ear, lay round auricle respectively, weigh.Calculate swelling.
Heavy (the mg)-auris dextra sheet of swelling (mg)=left auricle heavy (mg)
Inhibitory rate of intumesce (%)=(the average swelling of the average swelling-administration of matched group group) average swelling of/matched group
The different Radix Tripterygii Wilfordii medicines of table 3 and the indomethacin swelling suppression ratio result to Mus ear swelling
| Group | Suppression ratio % |
| Blank | 0 |
| Indomethacin | 32.92±2.665 |
| High dose (DEMETHYLZEYLASTERAL DZY T-96 TZ-93) | 31.43±1.949 |
| Middle dosage (DEMETHYLZEYLASTERAL DZY T-96 TZ-93) | 20.05±1.749 |
| Low dosage (DEMETHYLZEYLASTERAL DZY T-96 TZ-93) | 5.94±1.324 |
| High dose (Radix Tripterygii Wilfordii extract powder) | 68.41±1.247** |
| Middle dosage (Radix Tripterygii Wilfordii extract powder) | 37.76±0.851 |
| Low dosage (Radix Tripterygii Wilfordii extract powder) | 14.11±2.345 |
Note: with indomethacin comparison, * P < 0.05, * * P < 0.01.
From the above results: DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and Radix Tripterygii Wilfordii extract powder all have obvious anti-inflammatory activity; With contrast the comparison of medicine indomethacin, when high concentration, the antiinflammatory action of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and indomethacin are close, the Radix Tripterygii Wilfordii extractum that the present invention makes antiinflammatory action and indomethacin when in high concentration and middle concentration are more than or equal to the antiphlogistic effects of indomethacin.
Embodiment 6: the acute toxicity test of Radix Tripterygii Wilfordii extract powder to mice
Select embodiment 3, the extract powder of gained carried out to preliminary toxicity assessment:
1. experiment material
1.1 materials and instrument: CMC ?Na(CP, lot number F20110609, Chemical Reagent Co., Ltd., Sinopharm Group), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 (making by patent 201010603887.5 methods),
1.2 animals: Male Kunming strain mice (animal occupancy permit number: SCXK (Zhejiang) ?20080033)
2. method and result
2.1 sample preparations: take Radix Tripterygii Wilfordii extract powder, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 add 0.5%CMC ?Na solution, after stirring, the ultrasonic suspendible that makes evenly obtains acute toxicity reagent, as table 4.
Table 4 DEMETHYLZEYLASTERAL DZY T-96 TZ-93, Radix Tripterygii Wilfordii extract powder acute toxicity test medicinal liquid
| Numbering | Extract powder concentration (mg/mL) | Dissolving situation |
| Blank | 0 | ‐‐‐ |
| DEMETHYLZEYLASTERAL DZY T-96 TZ-93 1 | 63.56 | Suspendible |
| DEMETHYLZEYLASTERAL DZY T-96 TZ-93 2 | 135.75 | Suspendible |
| DEMETHYLZEYLASTERAL DZY T-96 TZ-93 3 | 260.35 | Drug solution is thicker |
| Radix Tripterygii Wilfordii extract powder 1 | 387.44 | Drug solution is thicker |
| Radix Tripterygii Wilfordii extract powder 2 | 502.63 | Drug solution is thicker |
| Radix Tripterygii Wilfordii extract powder 3 | 795.84 | Seldom, extract powder is wet face state to solvent |
2.2 acute toxicity test methods and result:
Water is can't help in mice administration fasting evening before that day.The grouping of mice: according to body weight, mice is divided into 3 groups, then the mice of getting at random each body weight group is divided into 7 groups, 10 every group, blank group (0.5%CMC ?Na solution), as shown in table 4.Gavage 0.2mL.Statistical computation LD
50, in Table 5.
Table 5 Radix Tripterygii Wilfordii the acute toxicity tests
| Group | LD 50（mg/kg） |
| DEMETHYLZEYLASTERAL DZY T-96 TZ-93 | 1357.50 |
| Radix Tripterygii Wilfordii extract powder | 5235.81 |
From the above results: the LD of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 in this experiment
50approach with the 1295mg/kg of bibliographical information; Compare the prepared Radix Tripterygii Wilfordii extractum of the present invention LD with DEMETHYLZEYLASTERAL DZY T-96 TZ-93
50be greater than the LD of DEMETHYLZEYLASTERAL DZY T-96 TZ-93
50,, its toxicity is lower than pure DEMETHYLZEYLASTERAL DZY T-96 TZ-93.
Embodiment 7: the inhibited proliferation of Radix Tripterygii Wilfordii extract powder to arthritis synovial cell
Select and select embodiment 3, the Radix Tripterygii Wilfordii extract powder of gained is carried out to preliminary evaluating drug effect:
1. experiment material
1.1 samples: take Radix Tripterygii Wilfordii extract powder, triptolide, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 appropriate, dissolve with cell culture fluid, obtain pastille culture fluid as table 6.
The pastille culture fluid (mg/mL) of each group of table 6
1.2 reagent: hyclone (Gibco company, lot number: 623311); MTT(sigama subpackage, lot number: M2128), DMEM low sugar culture fluid (Ji Nuo biological medicine technology company limited, lot number: 12102204).
1.3 instruments: inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), the long microplate reader (M4 type, MD company) of all-wave.
1.4 cells: primary HFLS ?RA cell, be purchased from Cell Applications company, have the cultivation of going down to posterity of this laboratory.
2. method and result
Get the 5th generation HFLS ?RA cell, in the 96 every holes of porocyte culture plate, add 100 μ l cell suspension, cell number is 1 * 10
4/ hole.Cultivate 24h and make after cell attachment, add respectively Radix Tripterygii Wilfordii extract powder, triptolide and the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 (in Table 6) of 200 μ l A, B, C, tetra-groups of concentration of D.Each concentration is established 3 multiple holes.Using do not add medicine containing cell DMEM culture fluid as standard control, using not celliferous DMEM culture fluid as blank.Administration is cultivated after 24h, and every hole adds MTT working solution 200 μ l; Cultivate after 4h, remove MTT working solution, more every hole adds 200 μ l DMSO, shaking table 180rpm, lucifuge, incubated at room 10min; Under wavelength 570nm, by microplate reader, measure absorbance.
Calculate cell inhibitory rate, suppression ratio computing formula is as follows: suppression ratio %=[1 ?(sample well absorbance/standard control hole absorbance)] * 100%, its result of calculation is as table 7.
The inhibition of table 7 Radix Tripterygii Wilfordii different component to HFLS-RA cell proliferation
n=3
| Group | Triptolide | DEMETHYLZEYLASTERAL DZY T-96 TZ-93 | Radix Tripterygii Wilfordii extract powder |
| A group | 98.19±1.43 | 70.77±4.48** | 72.28±3.43 |
| B group | 93.11±3.55 | 50.32±2.87** | 55.50±6.52 |
| C group | 82.25±4.09 | 46.87±4.90** | 50.22±5.31** |
| D group | 63.37±7.21 | 18.85±6.34** | 22.13±4.08** |
Note:
Under concentration level group, with the comparison of triptolide group, * P < 0.05, * * P < 0.01; With the comparison of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 group,
△p < 0.05,
△ △p < 0.01.
Above-mentioned experimental result shows: triptolide and DEMETHYLZEYLASTERAL DZY T-96 TZ-93 have proliferation inhibiting effect to arthritis synovial cell, and reduces and weaken with dosage.Adopt the product (being Radix Tripterygii Wilfordii extract powder) making in the present invention, there is the effect of obvious treatment rheumatic Bi syndrome, and suitable with pure DEMETHYLZEYLASTERAL DZY T-96 TZ-93 effect, but the purer DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of preparation technology simply, easily go.
Embodiment 8: the inhibited proliferation of Radix Tripterygii Wilfordii extract powder to RAW264.7 cell
Select and select embodiment 3, the Radix Tripterygii Wilfordii extract powder of gained is carried out to preliminary antiinflammatory action evaluation:
1. experiment material
1.1 to take Radix Tripterygii Wilfordii extract powder, triptolide, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 appropriate, with cell culture fluid, dissolves, and obtains pastille culture fluid as table 8.
The pastille culture fluid (mg/mL) of each group of table 8
1.2 reagent: hyclone (Tian Hang bio tech ltd, Zhejiang, lot number: 121112); MTT(sigama subpackage, lot number: M2128), DMEM high sugared culture fluid (Ji Nuo biological medicine technology company limited, lot number: 13040204).
1.3 instruments: inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), the long microplate reader (M4 type, MD company) of all-wave.
1.4 cells: RAW264.7 cell, be purchased from Chinese Academy of Sciences's Shanghai cell bank, there is the cultivation of going down to posterity of this laboratory.
2. method and result
Get RAW264.7 cell, in the 96 every holes of porocyte culture plate, add 100 μ l cell suspension, cell number is 1 * 104/ hole.Cultivate 24h and make after cell attachment, add respectively Radix Tripterygii Wilfordii extract powder, triptolide and the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 (in Table 8) of 200 μ l A, B, C, tetra-groups of concentration of D.Each concentration is established 3 multiple holes.Using do not add medicine containing cell DMEM culture fluid as standard control, using not celliferous DMEM culture fluid as blank.Administration is cultivated after 24h, and every hole adds MTT working solution 200 μ l; Cultivate after 4h, remove MTT working solution, more every hole adds 200 μ l DMSO, shaking table 180rpm, lucifuge, incubated at room 10min; Under wavelength 570nm, by microplate reader, measure absorbance.
Calculate cell inhibitory rate, suppression ratio computing formula is as follows: suppression ratio %=[1 ?(sample well absorbance/standard control hole absorbance)] * 100%, its result of calculation is as table 9.
The inhibition of table 9 Radix Tripterygii Wilfordii different component to RAW264.7 cell proliferation
n=3
| Group | Triptolide | DEMETHYLZEYLASTERAL DZY T-96 TZ-93 | Radix Tripterygii Wilfordii extract powder |
| A group | 100.19±1.67 | 101.77±1.48 | 98.28±9.13 |
| B group | 94.11±3.26 | 86.32±3.67 | 90.50±6.34 |
| C group | 85.25±6.09 | 50.87±4.70** | 63.22±5.89* △ |
| D group | 83.52±5.21 | 23.85±7.34** | 40.13±4.72** △△ |
Note:
Under concentration level group, with the comparison of triptolide group, * P < 0.05, * * P < 0.01; With DEMETHYLZEYLASTERAL DZY T-96 TZ-93 comparison,
△p < 0.05,
△ △p < 0.01.
Above-mentioned experimental result shows: when high concentration, RAW264.7 cell is had to proliferation inhibiting effect to Radix Tripterygii Wilfordii extract powder and pure DEMETHYLZEYLASTERAL DZY T-96 TZ-93 does not have significant difference, and when low concentration, Radix Tripterygii Wilfordii extract powder has proliferation inhibiting effect to RAW264.7 cell and is better than pure DEMETHYLZEYLASTERAL DZY T-96 TZ-93, and inhibition has significant difference.The purer DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of Radix Tripterygii Wilfordii extract powder that the present invention makes is easy to make, and is conducive to suitability for industrialized production and clinical practice.
Embodiment 9: the inhibited proliferation of Radix Tripterygii Wilfordii component to HaCaT cell
Select and select embodiment 2, the Radix Tripterygii Wilfordii extract powder of gained is carried out to preliminary toxic action evaluation:
1. experiment material
1.1 samples: take with embodiment triptolide, Radix Tripterygii Wilfordii extract powder and tripterygium total diterpene (containing total diterpene 70%), dissolve with cell culture fluid, obtain pastille culture fluid as table 10.
The pastille culture fluid (mg/mL) of each group of table 10
| Group | Triptolide | Tripterygium total diterpene | Radix Tripterygii Wilfordii extract powder |
| A group | 0.0128 | 0.0135 | 0.0129 |
| B group | 0.0064 | 0.0067 | 0.0064 |
| C group | 0.0032 | 0.0034 | 0.0032 |
| D group | 0.0016 | 0.0017 | 0.0016 |
| E group | 0.0008 | 0.0008 | 0.0008 |
1.2 reagent: hyclone (Tian Hang bio tech ltd, Zhejiang, lot number: 121112); MTT(sigama subpackage, lot number: M2128), DMEM high sugared culture fluid (Ji Nuo biological medicine technology company limited, lot number: 13040204).
1.3 instruments: inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), the long microplate reader (M4 type, MD company) of all-wave.
1.4 cells: HaCaT cell, be purchased from Wuhan cell bank company, there is the cultivation of going down to posterity of this laboratory.
2. method and result
Get HaCaT cell, in the 96 every holes of porocyte culture plate, add 100 μ l cell suspension, cell number is 1 * 10
4/ hole.Cultivate 24h and make after cell attachment, add respectively 200 μ l containing the pastille culture fluid (in Table 10) of the different proportionings of strychnine strychnine.Each concentration is established 3 multiple holes.Using do not add medicine containing cell DMEM culture fluid as standard control, using not celliferous DMEM culture fluid as blank.Administration is cultivated after 24h, and every hole adds MTT working solution 200 μ l; Cultivate after 4h, remove MTT working solution, more every hole adds 200 μ l DMSO, shaking table 180rpm, lucifuge, incubated at room 10min; Under wavelength 570nm, by microplate reader, measure absorbance.
Calculate cell inhibitory rate, suppression ratio computing formula is as follows: suppression ratio %=[1 ?(sample well absorbance/standard control hole absorbance)] * 100%, its result of calculation is as table 11.
The inhibition of the different Radix Tripterygii Wilfordii components of table 11 to HaCaT cell proliferation
| Group | Triptolide | Tripterygium total diterpene | Radix Tripterygii Wilfordii extract powder |
| A group | 73.94±3.74 | 75.05±4.34 | 68.79±4.68 |
| B group | 70.95±5.13 | 69.17±5.29 | 69.62±1.58 |
| C group | 76.30±3.34 | 73.29±3.23 | 60.34±3.57* △ |
| D group | 71.76±5.89 | 70.26±6.52 | 46.12±9.55* △ |
| E group | 76.79±1.95 | 64.32±5.08 | 21.57±9.97* △ |
Note:
With triptolide comparison, * * P < 0.01; With the comparison of tripterygium total diterpene,
△ △p < 0.01.
Above-mentioned experimental result shows: at triptolide, HaCaT cell is had to proliferation inhibiting effect strong, and reduce and there is no significant difference with concentration, illustrate that the cytotoxicity of triptolide is larger; Tripterygium total diterpene, owing to containing triptolide, equates substantially with triptolide group toxicity.Radix Tripterygii Wilfordii extract powder is with the reduction of concentration, the toxicity of HaCaT cell is reduced, when low concentration and triptolide and tripterygium total diterpene group have significant difference.From table 9, also proved the product of method for extraction and purification gained the present invention (i.e. " Radix Tripterygii Wilfordii extract powder " group), the effect aspect for the treatment of rheumatic Bi syndrome can reach efficiently, the effect of low toxicity, can be used for the treatment of for a long time rheumatoid rheumatic arthritis.
Claims (7)
1. a preparation method for the Radix Tripterygii Wilfordii extract that the triterpenes of take is main component, its concrete steps are as follows:
(1) the full root herb of dry Radix Tripterygii Wilfordii is pulverized, crossed 10 order～40 mesh sieves, take powder 150g～200g;
(2) to extract solvent, carry out supersound extraction, extracting solvent and medical material amount ratio is 8ml～16mL:1g, extracting liquid filtering, and filtrate decompression reclaims extracts solvent, obtains concentrated solution, dry, obtains Radix Tripterygii Wilfordii extract powder crude product;
(3) gained Radix Tripterygii Wilfordii extract powder crude product 5.0g～15.0g in step (2), adds solvent to redissolve, and the amount ratio of this solvent and Radix Tripterygii Wilfordii extract powder crude product is 10～15mL:1g, filters, and this solvent of reclaim under reduced pressure, obtains Radix Tripterygii Wilfordii extract powder.
2. preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component as claimed in claim 1, is characterized in that: step (2), and described alcoholic solution concentration is 80%～95%, ultrasonic number of times is 1～3 time, each 0.5～2.0h.
3. preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component as claimed in claim 1 or 2, is characterized in that: step (2), described is dried as 55～75 ℃ of vacuum dryings.
4. preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component as claimed in claim 1 or 2, is characterized in that: step (2), and described supersound extraction power is 250W～500W, mixing speed 750～1250rpm, ultrasonic temperature is 25～50 ℃.
5. preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component as claimed in claim 1 or 2, is characterized in that: step (2), described extraction solvent is selected from alcoholic solution, ethyl acetate or acetone.
6. preparation method of take the Radix Tripterygii Wilfordii extract that triterpenes is main component as claimed in claim 1 or 2, is characterized in that: step (3), described solvent is selected from ethyl acetate, ethanol, dehydrated alcohol or with the acid ethanol solution of salt acid for adjusting pH.
7. the medicine for the preparation of rheumatoid arthritis by claim 1-6 preparation method gained Radix Tripterygii Wilfordii extract powder.
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| CN104225386A (en) * | 2014-08-29 | 2014-12-24 | 浙江中医药大学附属第三医院 | Compound tripterygium wilfordii gel paste with high effect and low toxicity and preparation method thereof |
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| US20040018260A1 (en) * | 2002-06-19 | 2004-01-29 | Novemed Group Limited | Novel botanical extract of Tripterygium Wilfordii Hook F. |
| CN1506365A (en) * | 2002-12-10 | 2004-06-23 | 浙江得恩德制药有限公司 | Prepn process of tripterygium extractive |
| CN103908484A (en) * | 2014-03-20 | 2014-07-09 | 浙江工业大学 | Tripterygium wilfordii plastic for treating rheumatoid arthritis and preparation method thereof |
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| CN104225386A (en) * | 2014-08-29 | 2014-12-24 | 浙江中医药大学附属第三医院 | Compound tripterygium wilfordii gel paste with high effect and low toxicity and preparation method thereof |
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