CN101040891A - Method of preparing tripterygium hypoglaucum (Levl) hutch alkaloids - Google Patents
Method of preparing tripterygium hypoglaucum (Levl) hutch alkaloids Download PDFInfo
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Images
Abstract
The invention relates to a method for preparing Malus spectabilis alkaloid, and relative application, comprising that using Malus spectabilis extractive to immerge paste powder, adding acetic acid ethyl ester, chloroform or butanol to reflux or immerge for several times, until the extractive has not alkaloid reaction, extracting via acetic acid ethyl ester, chloroform or butanol for several times, until the water phase has not alkaloid reacton, taking the organic extractive liquid, concentrating and recycling solvent, to obtain the Malus spectabilis alkaloid compound, or using Malus spectabilis extractive to immerge paste powder, adding silica gel to be mixed uniformly to obtain material gel, penetrating in penetrating barrel via chloroform, until the penetrated liquid has no alkaloid reaction, washing the filter liquid via acid water, extracting via acetic acid ethyl ester, depressurizing, concentrating solvent, to obtain the final product. The inventive compound can be used to treat rheumatic arthritis, atrophic arthritis, Cazenave's lupus or the immunologic rejection after organ transplantation.
Description
Technical field
The invention belongs to plant extract technology and drug research field, specifically be from the Tripterygium hypoglaucum plant, extract alkaloidal method and the application of the alkaloid that extracts in pharmacy.
Background technology
Rheumatism is one group of commonly encountered diseases, and general reference influences more than 100 kind of disease of osteoarthrosis and surrounding soft tissue thereof.Systemic lupus erythematosus (sle) etiology unknown wherein, the mortality rate height; Rheumatic arthritis is common and obstinate, can cause the joint swelling and pain and the dysfunction of whole body, is one of main disabling disease.China has 5,000,000 patients to suffer the torment of these two kinds of diseases approximately.Though this sick direct crisis life, as untimely, rational therapy joint function disturbance, deformity will occur down to disabled, be called as " not dead cancer ".Preresearch estimates, direct losses such as medical fee that China is therefore sick and charge for loss of working time are no less than 50,000,000,000 yuans every year.
Rheumatic arthritis belongs to autoimmune disease, and the cause of disease it be unclear that at present, so also there is not specific drug to come out, doctor trained in Western medicine cooperates an amount of immunosuppressant based on the verification treatment of anti-inflammatory analgesic in the treatment, and is evident in efficacy at the acute stage of morbidity; But from the long-term effect of Chinese medicine to rheumatic and rheumatoid arthritis, curative effect is sure, is better than Western medicine; Come at arthritis clinical observation research summary according to China's recent two decades, therapy of combining Chinese and Western medicine, effect is remarkable, is that China's arthritis disease reaches the Therapeutic Method and the means of main flow from now at present.This course of disease can be touching lasting, can reach several years, many decades.Patient lives the misery life of " stubbornly refused, it is not prosperous to live " for a long time.Though should disease lack effective treatment means, early and adhere to that rational long-term treatment can make state of an illness long-term remission, control, and the course of disease is controlled at before the joint grievous injury, its meaning will be very far-reaching.For patient, adhere to treatment, and keep good, optimistic phychology, most patients can both survive to old and keep quality of life preferably.
Tripterygium hypoglaucum Tripterygium Hypoglaucum (Levl.) Hutch (being called for short THH) is the Celastraceae tripterygium plant, has another name called colguhoumia root, Radix Cissi hexangularis, Cortex tripterygii hypoglauci radicis, falls mao grass etc., mainly is distributed in China southwest.Its medicinal part is a root, has expelling wind and removing dampness, and effects such as relaxing muscles and tendons and activating QI and blood in the collateral, anti-inflammatory analgetic are mainly used in diseases such as treatment rheumatoid arthritis, rheumatic arthritis, lupus erythematosus clinically.Used clinically at present preparation has Tripterygium Hypoglaucum Hutch Tablet, for the THH root through 50% ethanol extraction, the extract powder of extract drying compacting gets (Tripterygium Hypoglaucum Hutch Tablet see " Chinese pharmacopoeia version in 2005) in flakes.
Tripterygium Hypoglaucum Hutch Tablet is the treatment rheumatoid arthritis that filters out in the clinical practice, the active drug of lupus erythematosus, but find also that in secular observation on Clinical Application this medicine has serious toxic and side effects, normal because of toxic and side effects influences the compliance of patient's medication, thus therapeutic effect influenced.Therefore, further such medicine is studied, screened, remove and produce the toxic and side effects material, active constituent-enriched, heighten the effect of a treatment, prospect is long-range.
Studies show that main component is terpenoid, lactone, pigment, saponin, phenolic acid in the Tripterygium hypoglaucum.Wherein, lactone (particularly triepoxides) and terpenoid are the main toxic components in the Tripterygium hypoglaucum clinical preparation, digestive system, blood circulation and genitourinary system there are more serious toxicity, can cause that blood cell reduces disease, thrombocytopenia, aplastic anemia and acute renal failure etc.; Can also act on the follicular cell of ovary, cause follicle secretion decrease in estrogen, negative feedback ground causes that hypothalamus secretory vacuole stimulin increases, and causes hypo-ovaria or depletion, causes the women ovarian amenorrhea; Can also suppress the proteic biosynthesis of sperm nucleus, spermatid nucleoprotein group type conversion is obstructed, make the sperm can not be ripe and cause male sterility.
Total alkaloid content is about 0.3%～4% in the Tripterygium hypoglaucum, and is evident in efficacy to immunologic rejection aspect after treatment of arthritis, tumor, the clinical organ transplantation, and toxicity is low more than its terpenoid and lactone.Therefore, by modern chemistry, biotechnology, from Tripterygium hypoglaucum, extract refined highly effective low toxicity alkaloid component, make appropriate formulation and be applied to clinical, be the technical problem that needs to be resolved hurrily in the secondary development of Tripterygium hypoglaucum new drug, have great social benefit and economic well-being of workers and staff prospect.
Summary of the invention
The objective of the invention is to overcome problems of the prior art, provide a kind of and from Tripterygium hypoglaucum, extract the alkaloidal method of refined highly effective low toxicity, and alkaloidal application.
The present invention utilizes Solid-Phase Extraction to combine with solvent extraction technology, alkaloid target component in the Tripterygium hypoglaucum is carried out selective enrichment make with extra care, and removes toxic component more than 96% and useless composition in the former Tripterygium hypoglaucum preparation.The gained alkaloid shows through pharmacological testing, immunologic rejection after arthritis, tumor, the organ transplantation is had significant therapeutical effect, and toxicity is extremely low.
Purpose of the present invention is achieved by the following technical programs.
Tripterygium hypoglaucum alkaloid preparation method may further comprise the steps:
(1) gets Tripterygium hypoglaucum extract extract powder (2005 editions " 490 pages of Chinese pharmacopoeia: be compacting Tripterygium hypoglaucum tablet raw material), adding weight is that 1～10 times of ethyl acetate (or chloroform or butanols) refluxes (or immersion) extraction for several times, does not show the alkaloid reaction until extracting solution;
(2) extracting liquid filtering with sour water (PH=1～5) washing for several times, no longer shows alkaloid to organic facies and reacts;
(3) get acid liquid in (2) step, add alkali liquor and transfer to PH=8～13, add ethyl acetate or chloroform or butanols extracted several times, no longer show alkaloid to water and react;
(4) get organic extractant phase liquid in (2) step, concentrate and reclaim solvent, get light brown, promptly get the Tripterygium hypoglaucum alkaloid composition to white powder;
Perhaps:
(1) get the Tripterygium hypoglaucum extract extract powder, add 2～4 times in 80～100 order silica gel, mixing must expect that glue is standby;
(2) elder generation is incorporated in 80～100 order silica gel of 2～3 times of extract powders in the percolation bucket, adds the material glue of mix homogeneously again, uses the chloroform percolation, till percolate does not show the alkaloid reaction;
(3) percolate is evaporated to about 30L, filters;
(4) filtrate is used acid rinsing 3～4 times, and shake well merges acid liquid;
(5) add alkali liquor in the acid liquid, constantly stir, transfer solution PH=13, add ethyl acetate extraction 3 times, each 10L, combined ethyl acetate liquid;
(6) concentrating under reduced pressure solvent gets the Tripterygium hypoglaucum alkaloid composition.
Above-mentioned gained Tripterygium hypoglaucum alkaloid composition can be used for the treatment of immunological rejection after rheumatic arthritis, rheumatoid arthritis, lupus erythematosus and kinds of tumors disease and the organ transplantation.
Above-mentioned gained Tripterygium hypoglaucum alkaloid adds an amount of pharmaceutical necessities, can make the preparation (tablet, capsule, injection, pellet, drop pill, aerosol, oral liquid) that is suitable for clinically.
The present invention is directed to former Tripterygium hypoglaucum clinical preparation poison, side effect is big, defectives such as medication dose is big, utilize solvent extraction that wherein alkaloid is separated, enrichment, refining, obtain the Tripterygium hypoglaucum alkaloid, pharmacological research shows, this compositions is to rheumatic arthritis, rheumatoid arthritis, lupus erythematosus, immunological rejection is evident in efficacy after kinds of tumors disease and the organ transplantation, toxic and side effects is minimum, improved the compliance that clinical patients is taken medicine for a long time greatly, and its technology is easy, the controllability height, the gained alkaloid is applied to pharmaceuticals industry, has good market prospect and social benefit.
Speed emaciation due to emotional upset (being the Tripterygium hypoglaucum alkaloid) antiinflammatory action experiment situation:
Relatively doses speed emaciation due to emotional upset (Herba Begoniae Yunnanensis total alkali SFX-04), Tripterygium hypoglaucum water portion (SFX-02) and fat portion (SFX-03) are contrast to the influence of inflammation swelling with the Tripterygium Hypoglaucum Hutch Tablet to cause the rat paw edema method with carrageenin.The result shows, total alkali has antiinflammatory action preferably, 4.8mg/kg body weight is given the rat oral gavage administration, all can significantly suppress rat paw edema degree due to the carrageenin in 1,2,4,6 hour causing scorching back, with the blank group than P＜0.05 and P＜0.01, this effect is better than the respective amount Tripterygium hypoglaucum: fat portion and water portion do not have significant antiinflammatory action.
1, test objective
Relatively doses speed emaciation due to emotional upset (SFX-04), Tripterygium hypoglaucum water portion (SFX-02) and fat portion (SFX-03) are to the influence of inflammation swelling, for new drug development provides experimental basis.
2, test material
2.1 supply test agent: test with fast emaciation due to emotional upset (Tripterygium hypoglaucum total alkali, SFX-04), Tripterygium hypoglaucum water portion (SFX-02) and fat portion (SFX-03) are provided by Kunming Fengshanjian Medicine Research Co., Ltd., the speed emaciation due to emotional upset is the khaki powder, water portion is brown powder, fat portion is the dark brown block, and total alkali during use, water portion be with a small amount of 0.5% carboxymethyl cellulose rope suspending, and fat portion is with a spot of Polyethylene Glycol-400 hydrotropy, drinking water is diluted to finite concentration, and rat is pressed 1ml/100g body weight ig.
2.2 laboratory animal: the SD rat is provided by unming Medical College's Experimental Animal Center, SPF level, laboratory animal production licence number: SCXK (Yunnan) 2005-0008 number.Barrier environment laboratory animal observation ward is observed.
2.3 main medicine and reagent and equipment: carrageenin (CARRAGEENAN) is a Sigma company product, and Tripterygium Hypoglaucum Hutch Tablet is produced by Yunnan Plant Pharmaceutical Industry Co., Ltd., lot number; 20060901, drainage volumetric measurement instrument.
3. experimental technique and result
3.1 the test dosage is selected
Tripterygium Hypoglaucum Hutch Tablet is total extract powder preparation of Herba Begoniae Yunnanensis, the 250mg/ sheet, with each two, three calculating every day, clinical consumption is 1500mg/d/ people, in average weight 60kg, the human body consumption is equivalent to 25mg/kg/d, because animal is human low to the sensitivity of medicine, enlarging 30 times, to experimentize be 750mg/kg/d.
Contain approximately in the 250mg Herba Begoniae Yunnanensis extract powder after measured:
Total alkali 1.6mg, the 750mg extract powder contains total alkali 4.8mg.
Water portion 90%, moisture 675mg of 750mg extract powder.
Fat portion 10%, the 750mg extract powder contains the 75mg of fat portion.
This experiment compares each effective site physiologically active with this dosage.
3.2 date processing
(x ± s) expression adopts SPSS 11.5 statistical softwares to carry out variance analysis to determination data, and P＜0.05 is for having significant difference between group with mean ± standard deviation.
3.3 influence to rat paw edema due to the carrageenin
50 of SD rats, ♂, body weight 160～210g is divided into 5 groups at random by body weight, be fast emaciation due to emotional upset (SFX-04) group, Tripterygium hypoglaucum water portion (SIX-02) group, Tripterygium hypoglaucum fat portion (SFX-03) group, Tripterygium Hypoglaucum Hutch Tablet matched group and blank group (to medicine group equal volume drinking water).Every day, ig was administered once, continuous five days, after the last administration 1 hour in the right back sufficient plantar subcutaneous injection 1% carrageenin 0.1ml of rat, measure before the injection carrageenin respectively and injection back I, 2,4,6 hours right back sufficient volumes with volumetric method. the difference of injecting forward and backward volume is the swelling degree, between organizing relatively.
The result shows, 1 hour right foot begins swelling behind the injection proinflammatory agent, reached the peak in 2～4 hours, speed emaciation due to emotional upset (SFX-04) group causes 1,2,4, the 6 hour right paw swelling in scorching back and significantly is lower than blank group (P＜0.05, P＜0.01), Tripterygium Hypoglaucum Hutch Tablet also has certain inflammation-inhibiting swelling effect, cause scorching back 6 hours with the blank group than difference remarkable (P＜0.05), but its inflammation-inhibiting swelling effect the results are shown in Table 1 than the low 20-30% of total alkali.Water portion and fat portion do not have remarkable inflammation-inhibiting swelling effect.
The influence of rat paw edema due to fast emaciation due to emotional upset of table 1 and Tripterygium hypoglaucum water portion and the fat portion on Carrageenan
| Group | Give medicament (mgkg -1 ·d -1) | Swelling degree (ml) | |||
| 1h | 2h | 4h | 6h | ||
| Blank group speed wind tripterygium hypoglaucum hutcs fat section of (SFX-04) tripterygium hypoglaucum hutcs water section (SFX-02) (SFX-03) tripterygium hypoglaucum hutch tablet that disappears | - 4.8 675 75 750 | 0.207±0.069 0.125±0.084 b 0.149±0.093 0.118±0.082 b 0.162±0.073 | 0.359±0.110 0.164±0.077 a 0.275±0.141 b 0.356±0.106 0.245±0.129 | 0.528±0.110 0.348±0.086 b 0.474±0.176 0.442±0.132 0.450±0.161 | 0.472±0.095 0.291±0.089 a 0.455±0.107 0.432±0.112 0.364±0.159 b |
N=10, x ± s, and with time point blank group ratio,
aP＜0.01,
bP＜0.05
Conclusion: experimental result shows, in three samples, speed emaciation due to emotional upset (SFX-04) has rat paw edema effect due to the remarkable inhibition carrageenin, its effect obviously is better than Tripterygium Hypoglaucum Hutch Tablet, and Tripterygium hypoglaucum water portion (SIX-02) and Tripterygium hypoglaucum fat portion (SFX-03) only had certain inflammation-inhibiting swelling effect in back 2 hours, 1 hour causing inflammation respectively.
Description of drawings
Fig. 1 is process chart of the present invention (the literal declaratives is not the concrete qualification to technology among the figure, only are the exemplary illustration effect).
The specific embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail, but be not limitation of the invention.
Embodiment 1:
Get Tripterygium hypoglaucum extract extract powder 5kg, add 5 times of amounts (W/V) ethyl acetate, reflux, extract, 3 times, each 1 hour, merge extractive liquid, filtered; Acetic acid ethyl fluid adds sour water 10L with (phosphoric acid) sour water (PH=2～3) washing 6 times at every turn, and shake well merges acid liquid; Add NaOH liquid in the acid liquid, constantly stir, transfer solution pH value to 10, add ethyl acetate extraction 3 times, each 5L, combined ethyl acetate solution; The concentrating under reduced pressure solvent gets Tripterygium hypoglaucum alkaloid composition 40.5g; Get said composition 8g, add starch 142g, mixing is granulated, drying, and granulate is loaded capsule, makes 1000.
Embodiment 2:
Get Tripterygium hypoglaucum extract extract powder 5kg, add 2 times of amounts (W/V) butanols merceration 5 times, each 8 hours, merge extractive liquid, filtered; Butanols liquid adds sour water 10L with (hydrochloric acid) sour water (PH=2～3) washing 5 times at every turn, and shake well merges acid liquid; Add KOH (potassium hydroxide) in the acid liquid, solution pH value to 10 is transferred in constantly stirring, adds butanols extraction 4 times, and each 10L merges butanol solution, and the concentrating under reduced pressure solvent gets Tripterygium hypoglaucum alkaloid composition 42.8g.Get said composition 8g, be dissolved in the vegetable oil, be filled into soft capsule, make 1000.
Embodiment 3:
Get Tripterygium hypoglaucum extract extract powder 5kg, add 80～100 order silica gel 15kg, mixing must be expected glue; Earlier add 80～100 order silica gel 10kg in the percolation bucket then, add the material glue of mix homogeneously again, use the chloroform percolation, till percolate did not show the alkaloid reaction, percolate was evaporated to about 30L, filtered; Filtrate is washed 3 times with (sulphuric acid) sour water (PH=2), each 10L, and shake well merges acid liquid; Add ammonia in the acid liquid, constantly stir, transfer solution PH=13, add ethyl acetate extraction 3 times, each 10L, combined ethyl acetate liquid; The concentrating under reduced pressure solvent gets Tripterygium hypoglaucum alkaloid composition 38.7g.Get said composition 10g, be dissolved in the 500ml water for injection, fully stir, constantly drip 10% hydrochloric acid solution, to dissolving fully, add the injection water to 1000ml, transfer solution PH=3～6 with 5%NaOH liquid, filter, 1000 (bottle) injection are made in fill.
Embodiment 4:
Repeat embodiment 1 or 2 or 3, following distinctive points is arranged: described extraction or extractant are chloroform, perhaps the mixed solvent of ethyl acetate, chloroform, butanols two or three arbitrary proportion.Also available acid or glacial acetic acid of used sour water or citric acid.
Claims (6)
1, the alkaloidal preparation method of a kind of Tripterygium hypoglaucum is characterized in that carrying out according to the following steps:
(1) get the Tripterygium hypoglaucum extract extract powder, adding weight is 1～10 times of ethyl acetate or chloroform or butanols backflow or immersion extraction several, does not show alkaloid until extracting solution and reacts;
(2) extracting liquid filtering with acid rinsing for several times, no longer shows alkaloid to organic facies and reacts;
(3) get acid liquid in (2) step, add alkali liquor and transfer to PH=8～13, add ethyl acetate or chloroform or butanols extracted several times, no longer show alkaloid to water and react;
(4) get organic extractant phase liquid in (2) step, concentrate and reclaim solvent, get light brown, promptly get the Tripterygium hypoglaucum alkaloid composition to white powder;
Perhaps:
(1) get the Tripterygium hypoglaucum extract extract powder, add 2～4 times in 80～100 order silica gel, mixing must expect that glue is standby;
(2) elder generation is incorporated in 80～100 order silica gel of 2～3 times of extract powders in the percolation bucket, adds the material glue of mix homogeneously again, uses the chloroform percolation, till percolate does not show the alkaloid reaction;
(3) percolate is evaporated to about 30L, filters;
(4) filtrate is used acid rinsing 3～4 times, and shake well merges acid liquid;
(5) add alkali liquor in the acid liquid, constantly stir, transfer solution PH=13, add ethyl acetate extraction 3 times, each 10L, combined ethyl acetate liquid;
(6) concentrating under reduced pressure solvent gets the Tripterygium hypoglaucum alkaloid composition.
2, the alkaloidal application of the described Tripterygium hypoglaucum of claim 1, it is characterized in that getting the Tripterygium hypoglaucum alkaloid and add an amount of pharmaceutical necessities, the preparation that is suitable for clinically be can make, tablet, capsule, injection, pellet, drop pill, aerosol, oral liquid comprised.
3, according to the alkaloidal application of the described Tripterygium hypoglaucum of root claim 2, it is characterized in that getting the Tripterygium hypoglaucum alkaloid composition, add starch, mixing is granulated, drying, and granulate is loaded capsule, corning.
4, the alkaloidal application of Tripterygium hypoglaucum according to claim 2 is characterized in that the Tripterygium hypoglaucum alkaloid composition, is dissolved in the vegetable oil, be filled into soft capsule.
5, the alkaloidal application of the described Tripterygium hypoglaucum of claim 1 is characterized in that can be used for immunological rejection after rheumatic arthritis, rheumatoid arthritis, lupus erythematosus, kinds of tumors disease and the organ transplantation.
6, the alkaloidal application of Tripterygium hypoglaucum according to claim 2, it is characterized in that getting the Tripterygium hypoglaucum alkaloid composition, be dissolved in the water for injection, fully stir, constantly drip 10% hydrochloric acid solution, to dissolving fully, add the injection water to required amount, transfer solution PH=3～6, filter with 5%NaOH liquid, injection is made in fill.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406670A (en) * | 2010-09-21 | 2012-04-11 | 中国人民解放军第三军医大学 | Extraction method of total alkaloids from tripterygium hypoglaucum and application of extract |
| CN102727558A (en) * | 2012-07-04 | 2012-10-17 | 重庆市中药研究院 | Tripterygium Hypoglaucum Hutch root extract, and bi-layer extended release tablet and application thereof |
| CN109806291A (en) * | 2019-03-22 | 2019-05-28 | 楚雄医药高等专科学校 | The method for extraction and purification of effective component in a kind of Yi nationality's medicine tripterygium hypoglaucum hutcs |
| CN111494465A (en) * | 2019-09-19 | 2020-08-07 | 重庆市中药研究院 | Pharmaceutical composition containing artesunate and herba Erodii seu Geranii, preparation and preparation method thereof |
-
2007
- 2007-04-16 CN CN200710065808A patent/CN101040891B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406670A (en) * | 2010-09-21 | 2012-04-11 | 中国人民解放军第三军医大学 | Extraction method of total alkaloids from tripterygium hypoglaucum and application of extract |
| CN102727558A (en) * | 2012-07-04 | 2012-10-17 | 重庆市中药研究院 | Tripterygium Hypoglaucum Hutch root extract, and bi-layer extended release tablet and application thereof |
| CN109806291A (en) * | 2019-03-22 | 2019-05-28 | 楚雄医药高等专科学校 | The method for extraction and purification of effective component in a kind of Yi nationality's medicine tripterygium hypoglaucum hutcs |
| CN111494465A (en) * | 2019-09-19 | 2020-08-07 | 重庆市中药研究院 | Pharmaceutical composition containing artesunate and herba Erodii seu Geranii, preparation and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN101040891B (en) | 2010-05-19 |
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Assignee: KUNMING SINOWAY NATURAL PHARMACEUTICALS Co.,Ltd. Assignor: KUNMING FENGSHANJIAN MEDICINE RESEARCH Co.,Ltd. Contract record no.: 2011530000072 Denomination of invention: Method of preparing tripterygium hypoglaucum (Levl) hutch alkaloids Granted publication date: 20100519 License type: Exclusive License Open date: 20070926 Record date: 20110914 |
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