CN1380277A - Method for separating, extracting and purifying nonyl alcohol from plant and its application - Google Patents
Method for separating, extracting and purifying nonyl alcohol from plant and its application Download PDFInfo
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Abstract
The present invention relates to a method for separating, extracting and purifying compound add nonanol from medicinal plant siegesbeckia and the application of said compound in preparation of medicine. The medicine preparation made up by using add nonanol as leading compound possesses the effect of resisting rheumatoid arthritis, stopping pain, resisting bacteria, antimalarial, reducing blood pressure and relaxing blood vessel, etc. and possesses the advantages of low toxicity and obvious medicine effect.
Description
Technical field
The present invention relates to a kind of from plant the method for separation and Extraction and purifying compounds kirenol (kirenol), particularly from the method for Yao with separation and Extraction and purifying nonyl alcohol the Zhi Wu pig Xian leather, the invention still further relates to the application of kirenol, particularly in medicines such as resisting rheumatoid arthritis, analgesia, use.
Background technology
Herba Siegesbeckiae (Herbasiegesbeckiae) is that (compositae, asteraceae) pig Xian belongs to (siegesbeckiae) plant to composite family.Be China's traditional Chinese medicine, China mainly contains 3 kinds, i.e. Dong Fang pig Xian (Siegesbeckia orientails L.), Herba Siegesbeckiae (Siegesbeckia pubescensMakino) and siegesbeckia glabrescens Makino (Siegesbeckia glabrescens Makino).
According to " Pharmacopoeia of People's Republic of China " (committee of pharmacopeia of Ministry of Health of the People's Republic of China compiles, Chemical Industry Press, P325 of nineteen ninety-five version) record ， Herba Siegesbeckiae is the dry aerial parts of feverfew Dong Fang pig Xian, Herba Siegesbeckiae or siegesbeckia glabrescens Makino.Nature and flavor suffering, hardship, tremble with fear, return liver, kidney channel.Major function is wind-damp dispelling, sharp joint, detoxifcation with curing mainly.Be used for that rheumatic arthralgia, muscles and bones are unable, soreness of the waist and knees, four paralysis, hemiplegia, the wet sore of rubellas.
Rheumatoid arthritis (UA) is a kind of chronic general immunoreactivity diseases associated with inflammation of unknown cause, and the immune response pilosity is born in synovium of joint, is modal connective tissue disease.
The drug treatment of rheumatoid arthritis mainly is at present:
1. anti-inflammatory analgesic class
1. acetylsalicylic acid has anti-inflammatory analgesic action, the joint is steamed positive effect is housed.
2. the INDOMETHACIN effect is strong than acetylsalicylic acid, but digestive tract ulcer and hepatic and kidney function obstacle person avoid usefulness.
But 3. Phenylbutazone 1 produce effects in week, but can cause granulocyte and thrombopenia.
4. this medicine anti-inflammatory action of acidum clofenamicum is strong, and side effect is little, and headache, dizziness are arranged once in a while.
2. glucocorticosteroid is big because of hormone side effect, and the easily recurrence of back of stopping using is so generally just use when other wants invalid.
3. golden preparation disodium aurothiomalate alloy sulfur sugar, deep intramuscular injection, this medicine can bring out dermatitis, if any kidney function damage Ying Shenyong.
4., can add and use immunosuppressor if immunosuppressor is undesirable with said medicine.As endoxan.
5. but chloroquine medication 1-3 month produce effects should be noted retinopathy during application.
The main at present Western medicine that uses all has toxic side effect or other side effect in various degree, the present invention has overcome the inconvenient part of directly using the treatment by Chinese herbs disease in the prior art, the method that obtains the effective component kirenol from traditional Chinese medicine is provided, and with kirenol as lead compound, for treatment of diseases such as resisting rheumatoid arthritis, analgesia, antibiotic, antimalarial, step-down, vasodilators, provide the good medicine that toxicity is low, drug effect is obvious.
Summary of the invention
De involved in the present invention method and step of separation and Extraction and purifying nonyl alcohol Cong Herba Siegesbeckiae is:
1. get raw material medicinal material Herba Siegesbeckiae east side pig Xian; one or both in Herba Siegesbeckiae or the siegesbeckia glabrescens Makino or three kinds, after crude drug dry product or fresh pulverizing medicinal materials, take by weighing dry product or fresh crude drug, to wherein adding sherwood oil or hexanaphthene refluxing extraction 2-3 time, each return time is 1-4 hour, the adding volume of each sherwood oil or hexanaphthene rises the ratio=6-12 of number and crude drug dry product weight kilogram number: 1, the adding volume of sherwood oil or hexanaphthene rises the ratio=4-8 of number and fresh medicinal material weight kilogram number: 1, all carry out filtration treatment after each the backflow, remove phegma, filter residue is refluxed next time, last filter residue is stand-by again.
2. with the filter residue that obtains in solvent refluxing extraction or the immersion diacolation extraction step 1.Solvent can be 30-95% aqueous ethanolic solution, chloroform, methylene dichloride, hexyl hexanoate, acetone, methyl alcohol or the water liquid that contains acetone 40-80%; The add-on of solvent is calculated according to the raw material add-on that adds in the step 1, and the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1; The method of refluxing extraction is: add solvent in filter residue in proportion, refluxing extraction is 2-5 time repeatedly, and each return time is 1-4 hour; The method of soaking diacolation is: directly with above-mentioned solvent, at normal temperatures filter residue is carried out 1-4 diacolation according to the above ratio and extracts, diacolation 4-6 days at every turn, with the extracting liquid filtering that obtains, collection filtrate; Being lower than under 80 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4.
3. the extract A to obtaining in the step 2, add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 1-4 time, solvent I is sherwood oil or comprises hexane, hexanaphthene, normal hexane is at interior C6-C7 alkane, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, water layer is again with solvent II extraction 1-4 time, solvent II is ethyl acetate or comprises methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin is at interior halon or comprise the ether of ether or comprise butylacetate, pentyl acetate is at interior organic acid ester or the Fatty Alcohol(C12-C14 and C12-C18) of C3-C5, and each water is 1 with solvent volume ratio mutually: 0.2-3; Merge the solvent II liquid after each time extracts, get solvent liquid B, the wherein main kirenol that contains.
4. the solvent liquid B to collecting in the step 3 carries out concentrating under reduced pressure being lower than under 80 ℃ of conditions, obtains extracting solid concentrates B1.
5. the extraction solid concentrates B1 to obtaining in the step 4 carries out one or many purification by chromatography of the same race or not of the same race with chromatography, or the product of chromatography is carried out recrystallization purifying again, just obtains the pure product of kirenol.The concrete steps of chromatography chromatography purification are: get chromatography column, adorn post with filler, use the elutriant wash-out, collect the position that contains kirenol, obtain the said purifying active compound of the present invention kirenol; For the different fillers that use in the chromatography, take different conditions, be specially: 1) silica gel column chromatography: get the normal pressure glass chromatography column, with 60-400 order silica gel is filler, extract solid concentrates B1 and silica gel ratio of weight and number 1: 10-1: between 300, with volume ratio 1: 0.5-1: the sherwood oil between 5 and the mixed solvent of ethyl acetate are elutriant, or with volume ratio 1: 0.5-1: the sherwood oil between 5 and the mixed solvent of acetone are elutriant, with the elutriant of collecting that contains kirenol, concentrating under reduced pressure becomes solid under 50 ℃ of temperature being lower than again; 2) D101 macroporous resin chromatography column: get the normal pressure glass chromatography column, with the D101 macroporous resin is filler, the ratio of weight and number that extracts solid concentrates B1 and filler is 1: 20-1: between 200, it is colourless that water is eluted to the wash-out effluent liquid, and then be 10% with volume percent successively, 20%, 30%, 40%, 50%, 60%, 70%, 80% aqueous ethanolic solution carries out gradient elution, be eluted to the colourless gradient that increases again of wash-out effluent liquid at every turn, collect the wash-out effluent liquid of 60%-80%, the wherein main kirenol that contains becomes solid with wash-out effluent liquid concentrating under reduced pressure being lower than under 60 ℃ of temperature again; 3) neutral alumina chromatography column: get normal pressure or vacuum glass chromatography column, with 60-300 order neutral alumina is filler, the ratio of weight and number that extracts solid concentrates B1 and neutral alumina is 1: 20-1: between 200, with volume ratio 1: 0.5-1: the sherwood oil between 5 and the mixed solvent of ethyl acetate are gradient eluent, or with volume ratio 1: the sherwood oil between the 0.5-5 and the mixed solvent of acetone are gradient eluent, promptly successively with 2: 1,1: 1,1: 2,1: 3,1: 4,1: 5 polarity gradient increases, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of kirenol, controlled temperature is lower than 60 ℃, and wash-out effluent liquid concentrating under reduced pressure is become solid; 4) polymeric amide chromatography post: get the normal pressure glass chromatography column, with 60-300 order polymeric amide is filler, the ratio of weight and number that extracts enriched material B1 and polymeric amide is 1: 20-1: between 200, with volume ratio 1: 1-1: the sherwood oil between 10 and the mixed solvent of ethyl acetate are that gradient eluent is promptly successively with 1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9, be gradient eluent at 1: 10, or with volume ratio 1: the sherwood oil between the 0.5-10 and the mixed solvent of acetone are that gradient eluent is promptly successively with 2: 1,1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9,1: 10 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of kirenol, controlled temperature is lower than 60 ℃, and wash-out effluent liquid concentrating under reduced pressure is become solid; 5) reversed-phase silica gel chromatography post: get the normal pressure post or comprise the preparation type pressured column of Lobar post, with 100-300 order reverse phase silica gel C-18 or C-8 is filler, the ratio of weight and number that extracts solid concentrates B1 and reverse phase silica gel C-18 or C-8 is 1: 20-1: between 200, with volume ratio 1: 9-4: the mixed solvent of the first alcohol and water between 1 is an elutriant, collection contains the elutriant of kirenol, or with volume ratio 0: 1-1: the methanol-water mixture between 0 is a gradient eluent, promptly successively with 0: 1,1: 9,2: 8,3: 7,4: 6,5: 5,6: 4,7: 1,8: 2,9: 1, be gradient eluent at 1: 0, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collect the wash-out effluent liquid that contains kirenol; Controlled temperature is lower than 60 ℃, the wash-out effluent liquid concentrating under reduced pressure of collecting is become solid then;
Described recrystallization, exactly with the solid that obtains behind the chromatography chromatography purification, the organic solvent acetic acid ethyl ester or the acetone of analytical pure level comprise the C1-C4 alcohol of ethanol or water in carry out recrystallization 1-4 time, obtain the pure product of kirenol crystallization.
According to the kirenol that the aforesaid method separation and Extraction arrives, its purity is not less than 95%.
The molecular structural formula of kirenol is:
According to the compound kirenol that aforesaid method and step obtain, its physicochemical characteristics and character in methyl alcohol is the water white transparency prismatic crystal, is dissolvable in water hexyl hexanoate, acetone, chloroform, methylene dichloride, methyl alcohol, propyl carbinol, is partially dissolved in water.Molecular composition is C
20H
34O
4, mp.201 ℃～202 ℃, results of elemental analyses is C 71.012%; H 10.061%; Infrared spectrum IR (KBr) max cm-1:3314,2914,1650,1450,1397,1059,1033,857; Carbon-13 nmr spectra 13CNMR (C
5D
5N) ppm:49.2 (C-1), 63.8 (C-2), 45.6 (C-3), 40.9 (C-4), 55.4 (C-5), 22.6 (C-6), 36.7 (C-7), 138.0 (C-8), 51.3 (C-9), 39.7 (C-10), 19.1 (C-11), 32.7 (C-12), 37.9 (C-13), 129.7 (C-14), 76.6 (C-15), 63.8 (C-16), 23.3 (C-17), 28.2 (C-18), 64.8 (C-19), 16.9 (C-20); Proton nmr spectra 1HNMR (C
5D
5N) ppm:5.50 (1H, br, s, 14-H), 4.14 (1H, d, J=11.8Hz, 19-Ha), 3.64 (1H, d, J=11.8Hz, 19-Hb), δ 4.01 (1H, t, J=11.7Hz, 16-CH), 3.42 (d, J=11.7Hz, 2H, H-17), 4.19 (1H, m, 1H), 2.85 (1H, br, d), 1.26 (3H, s, 18-CH3), 1.15 (3H, s, 15-CH4), 0.81 (3H, s, C20-CH3); Mass spectrum EIMSm/z:277[M-CH (OH)-CH2OH]+, 259,241,151,121,109,81,43.FABMSm/z:339 (M+1)+.
The kirenol that aforesaid method and step are obtained carries out purity test, tR=4.08min, and 215nm detects, purity 98.28%.
Retention time peak width peak height peak area per-cent 1 1.350 0.0530 187.32345 1.68372 4.057 0.1545 1364.72534 98.21383. 1.210 0.0324 152.34754 0.10243 in peak area per-cent peak annotate: 2 is the dissolvent residual peak, and 2 is the kirenol peak
Silica gel tlc detection assay result is:
Point sample amount 100 μ l do not see obvious impurity spot.
Exhibition distance: 9cm
Developer: 10% sulfuric acid liquid, heat 110 ℃, 5min.Rf value: 0.46
The present invention also provides the application of kirenol in the resisting rheumatoid arthritis medicine.The formulation of medicine is decided according to the clinical application mode, can be tablet, pill, capsule, electuary, sprays, oral liquid, suspensoid, externally applied liniment, transdermal absorption formulation or injection.
The present invention from plant, particularly from Yao with separation and Extraction the Zhi Wu Herba Siegesbeckiae be purified into kirenol, kirenol has antibiotic, anti-inflammatory action, the effect of step-down and diastole blood, immunosuppressive action, antimalarial effect, antifertility action etc.With the kirenol is the medicine that effective constituent is made, and rheumatoid arthritis is had obvious effect.
Embodiment
Below by embodiment the present invention is further specified.
Embodiment 1:
By following method and step separation and Extraction and purifying nonyl alcohol:
1. get 1 kilogram of the seasoning over-ground part of raw material medicinal material Herba Siegesbeckiae Herba Siegesbeckiae, through pulverizing, add in the reflux bottle, add 8 liters of sherwood oils again, be warming up to backflow, return time is 2 hours for the first time; Cooling is fallen and is removed phegma in the B filtration under diminished pressure.Add 6 liters of sherwood oils again in filter residue, repeat above-mentioned backflow 1 time, filtration under diminished pressure is removed phegma.
2. the filter residue that obtains in the step 1 is added 6 liters of 95% ethanol in Backflow bottle, be warming up to and refluxed 2 hours, cooling, filtration under diminished pressure removes phegma, and filter residue repeats above-mentioned reflux course 2 times, merging filtrate again.Filtrate 45 ℃ of controlled temperature on Rotary Evaporators are removed alcohol through concentrating under reduced pressure, proportion is 1.3 medicinal extract shape extract A 120 grams.
3. the extract A 120 gram suspendibles that obtain in the step 2 are dissolved in 360 ml waters, in separating funnel, extract 1 time for 100 milliliters with sherwood oil, receive water layer, repeat above-mentioned extraction process 1 time again, receive water layer, water layer is used the ethyl acetate re-extract 3 times again, uses 100 milliliters of ethyl acetate at every turn, merge the acetic acid ethyl acetate extract B after each time extracts, the wherein main kirenol that contains.
4. the solvent acetic acid ethyl acetate extract B to collecting in the step 3 carries out concentrating under reduced pressure under 40 ℃ of temperature, must extract about 40 grams of solid concentrates B1.
5. the extraction enriched material B140 that obtains in the step 4 is restrained, with 10 grams, 100 order silica gel mixed samples, airing is stand-by; Dry method dress 200-300 order silica gel 400 grams in 5 * 60cm glass column, to mix sample silica gel again is loaded in the post, add 2 gram silica gel above again, with volume ratio is that 1: 1 sherwood oil and ethyl acetate mixed solvent is elutriant, collection contains the kirenol position, controlled temperature is 40 ℃ on Rotary Evaporators, obtains about 3.5 grams of kirenol coarse crystallization.The kirenol coarse crystallization that obtains is dissolved in the ethyl acetate, and recrystallization 2 times obtains about 2 grams of the pure product of kirenol crystallization.
The kirenol purity that aforesaid method and step obtain, detecting with HPLC is 98%.
Embodiment 2:
By following method and step separation and Extraction and purifying nonyl alcohol:
1. get 1 kilogram of the seasoning over-ground part of raw material medicinal material Herba Siegesbeckiae Herba Siegesbeckiae, through pulverizing, add in the reflux bottle, add 8 liters of hexanaphthenes again, be warming up to backflow, return time is 2 hours for the first time; Cooling is fallen and is removed phegma in the B filtration under diminished pressure.Add 6 liters of hexanaphthenes again in filter residue, repeat above-mentioned backflow 1 time, filtration under diminished pressure is removed phegma.
2. the filter residue that obtains in the step 1 is added 6 liters in solvent acetone in Backflow bottle, be warming up to and refluxed 2 hours, cooling, filtration under diminished pressure removes phegma, and filter residue repeats above-mentioned reflux course 2 times, merging filtrate again.Filtrate 40 ℃ of controlled temperature on Rotary Evaporators are removed acetone through concentrating under reduced pressure, proportion is 1.6 about 100 grams of medicinal extract shape extract A.
3. the extract A 100 gram suspendibles that obtain in the step 2 are dissolved in 300 ml waters, in separating funnel, extract 1 time for 100 milliliters with hexanaphthene, receive water layer, repeat above-mentioned extraction process 1 time again, receive water layer, water layer is used the ethyl acetate re-extract 3 times again, uses 100 milliliters of ethyl acetate at every turn, the acetic acid ethyl fluid that merges after each time extracts extracts B, the wherein main kirenol that contains.
4. the solvent acetic acid ethyl acetate extract B to collecting in the step 3 carries out concentrating under reduced pressure under 40 ℃ of temperature, must extract about 35 grams of solid concentrates B1.
5. the extraction enriched material B1 35 that obtains in the step 4 is restrained, with 10 grams, 100 order silica gel mixed samples, airing is stand-by; Dry method dress 200-300 order silica gel 350 grams in 5 * 60cm glass column, to mix sample silica gel again is loaded in the post, add 2 gram silica gel above again, with volume ratio is that 2: 1 sherwood oil and acetone mixed solvent is elutriant, collection contains the kirenol position, controlled temperature is 40 ℃ on Rotary Evaporators, obtains about 3.5 grams of kirenol coarse crystallization.The kirenol coarse crystallization that obtains is dissolved in the anhydrous propanone, and recrystallization 2 times obtains about 2 grams of the pure product of kirenol crystallization.
The kirenol purity that aforesaid method and step obtain, detecting with HPLC is 98%.
Embodiment 3:
By following method and step separation and Extraction and purifying nonyl alcohol:
1. get 1 kilogram of the seasoning over-ground part of raw material medicinal material Herba Siegesbeckiae Herba Siegesbeckiae, through pulverizing, add in the reflux bottle, add 8 liters of hexanaphthenes again, be warming up to backflow, return time is 2 hours for the first time; Cooling is fallen and is removed phegma in the B filtration under diminished pressure.Add 6 liters of hexanaphthenes again in filter residue, repeat above-mentioned backflow 1 time, filtration under diminished pressure is removed phegma.
2. the filter residue that obtains in the step 1 is added 6 liters of methyl alcohol in Backflow bottle, be warming up to and refluxed 2 hours, cooling, filtration under diminished pressure removes phegma, and filter residue repeats above-mentioned reflux course 2 times again, merges phegma.Filtrate 45 ℃ of controlled temperature on Rotary Evaporators are removed methyl alcohol through concentrating under reduced pressure, proportion is 1.8 about 110 grams of medicinal extract shape extract A.
3. the about 110 gram suspendibles of the extract A that obtains in the step 2 are dissolved in 350 ml waters, in separating funnel, extract 1 time for 100 milliliters with hexanaphthene, receive water layer, repeat above-mentioned extraction process 1 time again, receive water layer, water layer is used the methylene dichloride re-extract 3 times again, uses 100 milliliters of methylene dichloride at every turn, merge the dichloromethane extract B after each time extracts, the wherein main kirenol that contains.
4. the dichloromethane extract B to collecting in the step 3 carries out concentrating under reduced pressure under 40 ℃ of temperature, must extract about 40 grams of solid concentrates B1.
5. the solids extract enriched material B1 40 that obtains in the step 4 is restrained, be dissolved in 20% (volume ratio) alcohol-water of 60 milliliters, the elimination insolubles, in order to 60-300 order polymeric amide is filler, on 30 * 200 centimetres glass chromatography column, adds through pretreated D101 polymeric amide 1000 grams, interface with water saturation, 60 milliliters 20% alcohol-water that adds B1, leave standstill 3 hours after, water is eluted to colourless; Use 10% respectively again, 20%, 30%, 40%, 50%, the alcohol-water gradient polarity wash-out of 60%, 70%, 80% (volume ratio), the elution amount of every degree of polarity is looked elutriant increases polarity gradient again after colourless, collection contains the elutriant of kirenol, and controlled temperature carries out concentrating under reduced pressure to the elutriant of collecting and becomes solid B2 at 45 ℃ then; To thickened solid thing B2 5 grams that obtain, with 10 grams, 100 order silica gel mixed samples, airing is stand-by; Dry method dress 200-300 order silica gel 250 grams in 5 * 60cm glass column, to mix sample silica gel again is loaded in the post, on add again 2 the gram silica gel, with volume ratio is that 2: 1 solvent sherwood oil and acetone mixed solvent is elutriant, collection contains the kirenol position, controlled temperature is 40 ℃ on Rotary Evaporators, obtains about 3.0 grams of kirenol coarse crystallization.The kirenol coarse crystallization is dissolved in 10 milliliters of dehydrated alcohols, and stand at low temperature in refrigerator is carried out recrystallization 1-4 time, obtains about 2 grams of the pure product of kirenol crystallization.
The kirenol purity that aforesaid method and step obtain, detecting with HPLC is 98%.
Embodiment 4:
Present embodiment is an Application Example.
With the pure product of the kirenol that obtains among the embodiment 1, carry out resisting rheumatoid arthritis and analgesic drug efficacy study, test method and result are as follows:
Material and method:
1. medicine and reagent: the Fu Shi Freund's complete adjuvant (Freund ' s Adjuvant complete), the RPMI-1640 powder is a U.S. Gibco company product, tetramethyl-azo azoles salt (MTT), and canavaline (Con A), lipopolysaccharides (LPS) is Sigma company product.
2. animal grouping: SD rat male and female half and half, 2-3 monthly age, body weight (250 ± 30) gram, C
57The BL/6 mouse, the male and female dual-purpose, 6 ages in week, body weight (20 ± 3) gram, portion provides by the Department Of Medicine, Peking University laboratory animal.Rat is divided into 3 groups at random, every group 10, every of normal group every day gavage 4ml water, gave the left back sufficient sole of the foot intradermal injection 0.1ml physiological saline of rat on the 1st in feedwater, every of model group every day gavage 4ml (0.24g/ml) kirenol, model group and administration group be in feedwater, and administration the 1st day is respectively at left back sufficient sole of the foot intradermal injection 0.1ml Fu Shi Freund's complete adjuvant, animal was all put to death in the 18th, get spleen and peritoneal macrophage is standby.
Measurement result
1. increment has promoter action to kirenol to adjuvant type rats with arthritis lymphocyte, rat assist agent arthritis rat lymphocyte value-added functionality is low, relatively descend 28% with normal group, increment has promoter action to increase by 20% to kirenol to AA rat model lymphocyte.Concrete data are as shown in table 1.Table 1, strange king's alcohol are to the lymphopoietic influence of adjuvant arthritis rats (X ± S)
OD value (570nmm
The strange king's alcohol 7 1.400 ± 0.15 of Group Animal 5 * 106 splenGontrol 7 1.623 ± 0.42AA 7 1.164 ± 0.09## P＜0.05AA+
*
P＜0.05＜##: compare with normal group; * and AA group are relatively.
2. kirenol can promote the generation of AA rat splenic lymphocyte IL-2.The adjuvant arthritis rats lymphocyte produces the IL-2 hypofunction, relatively descends 28% with normal group, and the IL-2 that kirenol produces AA rat model lymphocyte after the administration was at 1: 2; 1: 4; Promoter action is all arranged at 1: 8, compare, increase by 50% respectively with model group; 52%; 65% (P＜0.05), and can return to the normal rat level.Concrete data are as shown in table 2.Table 2, strange king's alcohol are to the influence of AA rat splenic lymphocyte IL-2-(X ± S)
IL-2 OD value 570 (nm) Group Animal 1: 21: 41: 8Control 7 0.241 ± 0.023 0.175 ± 0.023 0.143 ± 0.012AA 7 0.174 ± 0.017 0.122 ± 0.011 0.077 ± 0.016AA+XXC 7 0.261 ± 0.009 0.186 ± 0.024 0.127 ± 0.021##: compare＜0.05 with normal group; * and AA group be P＜0.05 relatively.
3. kirenol can suppress the generation of LPS inductive AA rat abdominal cavity scavenger cell IL-1.
The adjuvant arthritis rats abdominal cavity cell produces the IL-1 hyperfunction, relatively rises 34% with normal group, and the IL-1 that kirenol produces AA rat model peritoneal macrophage after the administration was at 1: 10; 1: 20; The dilution poison all had restraining effect in 1: 40, compared with model group, descended 23% respectively; 33%; 57% (P＜0.05), and can return to the normal rat level.Concrete data are as shown in table 3.Table 3, strange king's alcohol are to the influence of AA rat abdominal cavity scavenger cell IL-1 (X ± S)
The strange king's alcohol 7 0.168 ± 0.020 of IL-1 OD value 570 (nm) Group Animal 1: 10 1: 20 1: 40Control 7 0.164 ± 0.013 0.097 ± 0.015 0.055 ± 0.014AA, 7 0.219 ± 0.016##, 0.141 ± 0.040##, 0.106 ± 0.063##AA+
*0.108 ± 0.023
*0.046 ± 0.009
*##: compare P＜0.05 with normal group;
*Compare P＜0.05 with the AA group.
4. kirenol is woven with restitution to the big genus ankle joint of AA partial groups.The local synovial bursa hyperplasia of cutting into slices of AA rat ankle joint, fiber oozes out, and inflammatory cell is invaded profit.After gavaging kirenol, hyperemia alleviates, and inflammatory cell oozes out minimizing.
Conclusion
Using the rat assist agent arthritis of Fu Shi Freund's complete adjuvant preparation, is the immune inflammation model, one of rheumatoid arthritis test model commonly used.Rheumatoid arthritis is a kind of autoimmune disorder, causes the pathology of joint and whole body, exempts to cause all relevant with body fluid and cell, and the T cell is participated in this pathology directly.Adopt above-mentioned immune inflammation model for this this test, inquire into the antiheumatic mechanism of action of strange king's alcohol.Cause scorching back rat ConA inductive spleen lymphocyte proliferation hypofunction, the interleukin II that splenic lymphocyte produces is active to descend, the interleukin 1 activity that LPS inductive rat abdominal cavity scavenger cell produces is hyperfunction, show the adjuvant arthritis rats cellular immune function decreased, this is consistent with bibliographical information.After giving strange king's alcohol, strange king's alcohol can promote the spleen lymphocyte proliferation function, and can make the interleukin II of its generation return to normal level, sees Table 2.The body's immunity of pointing out strange king's alcohol can pass through to strengthen the function of T cell and then adjust rat, thus modulation on immune status improved.The generation of bibliographical information rat assist agent arthritis is relevant with the abnormal activation of scavenger cell; the abnormal activation of scavenger cell can produce excessive interleukin 1, and interleukin 1 plays an important role in the carrying out property destruction of adjuvant-induced arthritis and arthritic joint cartilage of human rheumatoid and sclerotin.Make newborn normal cartilage protein-polysaccharide loss, stimulate synovial cell's propagation, thereby make cartilage and destruction of bone.
This test confirms that also the generation of adjuvant arthritis rats interleukin 1 is hyperfunction, show that simultaneously strange king's alcohol can suppress the interleukin 1 that adjuvant arthritis rats produces, see Table 3, thereby can stop the destruction process of interleukin 1, alleviate whole body and partial pathology process rat joint cartilage and sclerotin.
The adjuvant arthritis rats ankle swelling, limitation of activity.Histopathologic examination shows: the hyperplasia of ankle joint synovial bursa, inflammatory cell are invaded profit, Mierocrystalline cellulose oozes out.The AA rat ankle joint section that gavages strange king's alcohol shows that Mierocrystalline cellulose oozes out minimizing, and hyperemia alleviates.Above-mentioned test shows that tentatively alkene Xian grass can adjust the immunologic function of adjuvant arthritis rats, suppresses the local organization inflammatory cell and oozes out, and alleviates local pathology reflection, and adjuvant-induced arthritis is recovered.
What the strange king's alcohol of strange king's alcohol analgesic activity drug efficacy study can obviously reduce the pain model mouse turns round the body number of times, sees Table 4.Can alleviate the pain stimulation of acetic acid behind the strange king's alcohol of mouse gavaging, with model group significant difference be arranged relatively, the analgesia rate is 65%, shows that strange king's alcohol has analgesic activity preferably.The strange king's alcohol of table 4 is to the analgesic activity of pain mouse model
Number of animals dosage writhing response analgesia rate group (n) is (number of times/only) (%) physiological saline model 20 10ml 29 0 Srm-Rhotaards 20 0.01 0 100 strange kings pure 20 1.5 10 65 (g/kg)
Annotate: compare * P＜0.001 with the physiological saline group
The analgesic activity group number of animals concentration of table 5. east alkene Xian and gland stalk alkene Xian body occurs turning round for the first time and turns round body number of times analgesia rate (only) (g/ crude drug kg) time (min) ((%) control group 10 0 4.3 20.4 ± 8.4 east alkene Xian 10 28.39 3.2 12.1 ± 6.9 of a ± SD)
*40.7
10 9.46 4.4 15.4 ± 12.724.5 gland stalk alkene Xian 10 41.73 4.6 13.2 ± 6.2
*35.3
10 13.91 4.6 15.0 ± 7.426.5 annotates: * P＜0.05
As can be seen from Table 5, east alkene Xian and gland stalk alkene Xian all have analgesic activity to the stomachache that Glacial acetic acid brings out, and the effect of east alkene Xian is better than gland stalk alkene Xian.Toxicity result
Use according to tcm clinical practice, the present invention has carried out the determination test of maximum tolerated dose to kirenol, and laboratory animal is a kunming mice.
The maximum tolerated dose experimental result shows that kunming mice is respectively 880.7mg/kg to the maximum tolerated dose of kirenol, results suggest: the toxicity of kirenol is minimum, uses safer in recommending the pharmaceutical quantities scope.
Embodiment 5:
Present embodiment is an Application Example.
With the kirenol that embodiment 1 obtains, make medicament, advise that clinical recommendation consumption is 15mg/ day.Method for preparing tablet thereof is: get kirenol 150 grams, be ground into powder, cross 400 mesh sieves, add 1% Magnesium Stearate, mix, compacting is 10000 on tabletting machine, makes to contain the 15mg kirenol in every tablet, and the patient takes 1 every day.Main effect is a wind-damp dispelling, sharp joint, analgesia, detoxifcation.Be used for rheumatic arthralgia, muscles and bones is unable, soreness of the waist and knees, tetraplegia, hemiplegia, the rubella sore that wets.
Claims (8)
- One kind from medicine with the method for planting separation and Extraction and purifying compounds kirenol the thing Herba Siegesbeckiae, it is characterized in that this method and step are:(1) gets raw material medicinal material Herba Siegesbeckiae east side pig Xian; one or both in Herba Siegesbeckiae or the siegesbeckia glabrescens Makino or three kinds, after crude drug dry product or fresh pulverizing medicinal materials, take by weighing dry product or fresh crude drug, to wherein adding sherwood oil or hexanaphthene, refluxing extraction 2-3 time, each return time is 1-4 hour, the adding volume of each sherwood oil or hexanaphthene rises the ratio=6-12 of number and crude drug dry product weight kilogram number: 1, or the adding volume of sherwood oil or hexanaphthene rises the ratio=4-8 of number and fresh medicinal material weight kilogram number: 1, all carry out filtration treatment after each the backflow, remove phegma, filter residue is refluxed next time again, last filter residue is stand-by;(2) with the filter residue that obtains in solvent refluxing extraction or the immersion diacolation extraction step (1), solvent can be 30-95% (volume ratio) aqueous ethanolic solution, chloroform, methylene dichloride, hexyl hexanoate, acetone, methyl alcohol or the water liquid that contains acetone 40-80% (volume ratio); The add-on of solvent is calculated according to the raw material add-on that adds in the step (1), and the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1; The method of refluxing extraction is: add solvent in filter residue in proportion, refluxing extraction is 2-5 time repeatedly, and each return time is 1-4 hour; The method of soaking diacolation is: directly with above-mentioned solvent, at normal temperatures filter residue is carried out 1-4 diacolation extraction, diacolation 4-6 days at every turn according to the above ratio; With the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 80 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4;(3) extract A to obtaining in the step (2), add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 1-4 time, solvent I is sherwood oil or comprises hexane, hexanaphthene, normal hexane is at interior C6-C7 alkane, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, water layer is again with solvent II extraction 1-4 time, solvent II is ethyl acetate or comprises methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin is at interior halon or comprise the ether of ether or comprise butylacetate, pentyl acetate is at interior organic acid ester or the Fatty Alcohol(C12-C14 and C12-C18) of C3-C5, and each water is 1 with solvent volume ratio mutually: 0.2-3; Merge the solvent II liquid after each time extracts, get solvent liquid B, the wherein main kirenol that contains;(4) the solvent liquid B to collecting in the step (3) carries out concentrating under reduced pressure being lower than under 80 ℃ of conditions, obtains extracting solid concentrates B1;(5) the extraction solid concentrates B1 to obtaining in the step (4) carries out the of the same race of one or many or purification by chromatography not of the same race with chromatography, or the product of chromatography is carried out recrystallization purifying again, obtains the pure product of kirenol; The concrete steps of chromatography chromatography purification are: get chromatography column, adorn post with filler, use the elutriant wash-out, collect the position that contains kirenol, obtain sublimed kirenol; For the different fillers that use in the chromatography, take different conditions, be specially: 1) silica gel column chromatography: get the normal pressure glass chromatography column, with 60-400 order silica gel is filler, extract solid concentrates B1 and silica gel ratio of weight and number 1: 10-1: between 300, with volume ratio 1: 0.5-1: the mixed solvent of the sherwood oil between 5 and the mixed solvent of ethyl acetate or sherwood oil and acetone is an elutriant, and with the elutriant of collecting that contains kirenol, concentrating under reduced pressure becomes solid under 50 ℃ of temperature being lower than; 2) D101 macroporous resin chromatography column: get the normal pressure glass chromatography column, with the D101 macroporous resin is filler, the ratio of weight and number that extracts solid concentrates B1 and filler is 1: 20-1: between 200, it is colourless that water is eluted to the wash-out effluent liquid, and then be 10% with volume percent successively, 20%, 30%, 40%, 50%, 60%, 70%, 80% aqueous ethanolic solution carries out gradient elution, be eluted to the colourless gradient that increases again of wash-out effluent liquid at every turn, collect the wash-out effluent liquid of 60%-80%, the wherein main kirenol that contains becomes solid with wash-out effluent liquid concentrating under reduced pressure being lower than under 60 ℃ of temperature again; 3) neutral alumina chromatography column: get normal pressure or vacuum glass chromatography column, with 60-300 order neutral alumina is filler, the ratio of weight and number that extracts solid concentrates B1 and neutral alumina is 1: 20-1: between 200, with volume ratio 1: 0.5-1: the mixed solvent of the sherwood oil between 5 and the mixed solvent of ethyl acetate or sherwood oil and acetone is a gradient eluent, promptly successively with 2: 1,1: 1,1: 2,1: 3,1: 4,1: 5 polarity gradient increases, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of kirenol, controlled temperature is lower than 60 ℃, and wash-out effluent liquid concentrating under reduced pressure is become solid; 4) polymeric amide chromatography post: get the normal pressure glass chromatography column, with 60-300 order polymeric amide is filler, the ratio of weight and number that extracts enriched material B1 and polymeric amide is 1: 20-1: between 200, with volume ratio 1: 1-1: the sherwood oil between 10 and the mixed solvent of ethyl acetate are that gradient eluent is promptly successively with 1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9, be gradient eluent at 1: 10, or with volume ratio 1: the sherwood oil between the 0.5-10 and the mixed solvent of acetone are that gradient eluent is promptly successively with 2: 1,1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9,1: 10 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of kirenol, controlled temperature is lower than 60 ℃, and wash-out effluent liquid concentrating under reduced pressure is become solid; 5) reversed-phase silica gel chromatography post: get the normal pressure post or comprise the preparation type pressured column of Lobar post, with 100-300 order reverse phase silica gel C-18 or C-8 is filler, the ratio of weight and number that extracts solid concentrates B1 and reverse phase silica gel C-18 or C-8 is 1: 20-1: between 200, with volume ratio 1: 9-4: the mixed solvent of the first alcohol and water between 1 is an elutriant, collection contains the elutriant of kirenol, or with volume ratio 0: 1-1: the methanol-water mixture between 0 is that gradient eluent is promptly successively with 0: 1,1: 9,2: 8,3: 7,4: 6,5: 5,6: 4,7: 1,8: 2,9: 1, be gradient eluent at 1: 0, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collect the wash-out effluent liquid that contains kirenol; Controlled temperature is lower than 60 ℃, the wash-out effluent liquid concentrating under reduced pressure of collecting is become solid then;Described recrystallization, exactly with the solid that obtains behind the chromatography chromatography purification, the organic solvent acetic acid ethyl ester or the acetone of analytical pure level comprise the C1-C4 alcohol of ethanol or water in carry out recrystallization 1-4 time, obtain the pure product of kirenol crystallization.
- 2. the application of kirenol in the medicine of preparation treatment resisting rheumatoid arthritis.
- 3. the application of kirenol in the preparation anodyne.
- 4. the application of kirenol in the preparation antimicrobial drug.
- 5. the application of kirenol in the preparation antimalarial drug.
- 6. the application of kirenol in preparation depressor.
- 7. the application of kirenol in preparation vasodilator medicine.
- 8. the application of kirenol in preparation immunosuppressor medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101880217A (en) * | 2009-05-07 | 2010-11-10 | 北京大学 | Medicinal composition for treating immunological diseases, preparation and application thereof |
| CN102372606A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Method for extracting kirenol from glandularstalk st.paulswort herb |
| CN102406682A (en) * | 2011-11-23 | 2012-04-11 | 合肥七星医药科技有限公司 | Glandularstalk St.Paulswort herb total flavonoid medicine for preventing and treating cardiovascular and cerebrovascular diseases, nervous system diseases and various joint diseases and preparations and preparation method thereof |
| CN101439069B (en) * | 2007-11-22 | 2012-05-09 | 上海医药工业研究院 | Leaf extract of Herba siegesbeckiae, preparation method and uses thereof |
| CN106383194A (en) * | 2016-08-29 | 2017-02-08 | 贵州信邦制药股份有限公司 | Identification method of herba siegesbeckiae in anti-rheumatism medicinal liquor |
| KR20170134101A (en) * | 2016-05-27 | 2017-12-06 | 주식회사 엘지생활건강 | Composition for promoting the hair growth comprising kirenol |
| CN109761752A (en) * | 2017-11-10 | 2019-05-17 | 义守大学 | The method of purifying nonyl alcohol |
| CN110467520A (en) * | 2018-05-10 | 2019-11-19 | 上海现代药物制剂工程研究中心有限公司 | The method that nonyl alcohol isolates and purifies in Yi Zhong Common St.Paulswort Herb leaf |
| CN111233668A (en) * | 2020-02-10 | 2020-06-05 | 济南大学 | Preparation of methyl valerate derivatives and antibacterial application thereof |
| CN112587590A (en) * | 2020-12-25 | 2021-04-02 | 河北医科大学第二医院 | Traditional Chinese medicine composition for treating rheumatic arthritis and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101439069B (en) * | 2007-11-22 | 2012-05-09 | 上海医药工业研究院 | Leaf extract of Herba siegesbeckiae, preparation method and uses thereof |
| CN101880217A (en) * | 2009-05-07 | 2010-11-10 | 北京大学 | Medicinal composition for treating immunological diseases, preparation and application thereof |
| CN101880217B (en) * | 2009-05-07 | 2014-05-28 | 北京大学 | Medicinal composition for treating immunological diseases, preparation and application thereof |
| CN102372606A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Method for extracting kirenol from glandularstalk st.paulswort herb |
| CN102406682A (en) * | 2011-11-23 | 2012-04-11 | 合肥七星医药科技有限公司 | Glandularstalk St.Paulswort herb total flavonoid medicine for preventing and treating cardiovascular and cerebrovascular diseases, nervous system diseases and various joint diseases and preparations and preparation method thereof |
| KR20170134101A (en) * | 2016-05-27 | 2017-12-06 | 주식회사 엘지생활건강 | Composition for promoting the hair growth comprising kirenol |
| CN106383194A (en) * | 2016-08-29 | 2017-02-08 | 贵州信邦制药股份有限公司 | Identification method of herba siegesbeckiae in anti-rheumatism medicinal liquor |
| CN106383194B (en) * | 2016-08-29 | 2018-08-31 | 贵州信邦制药股份有限公司 | The discrimination method of anti-rheumatism Yao Jiu Zhong Common St.Paulswort Herbs |
| CN109761752A (en) * | 2017-11-10 | 2019-05-17 | 义守大学 | The method of purifying nonyl alcohol |
| CN110467520A (en) * | 2018-05-10 | 2019-11-19 | 上海现代药物制剂工程研究中心有限公司 | The method that nonyl alcohol isolates and purifies in Yi Zhong Common St.Paulswort Herb leaf |
| CN111233668A (en) * | 2020-02-10 | 2020-06-05 | 济南大学 | Preparation of methyl valerate derivatives and antibacterial application thereof |
| CN112587590A (en) * | 2020-12-25 | 2021-04-02 | 河北医科大学第二医院 | Traditional Chinese medicine composition for treating rheumatic arthritis and preparation method thereof |
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