CN1730448A - Cimicifuga rhizome organic acid extraction method and novel uses - Google Patents
Cimicifuga rhizome organic acid extraction method and novel uses Download PDFInfo
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Abstract
The invention discloses a process for extracting cimicifuga rhizome organic acid and its novel use, The process for preparing the cimicifuga rhizome liposoluble organic acid consists of charging ethanol and immersing, heating for backflow, filtering, dissolving the extract, charging hydrochloric acid, extracting with acetic ether, combining the upper layer phase solution, extracting with sodium hydroxide, adjusting pH of the lower layer alkali liquor, extracting with acetic ether, combining the upper layer phase solution, scouring and removing acetic ether. The process for preparing the cimicifuga rhizome water-soluble organic acid consists of charging distilled water, heating, filtering, concentrating the filter liquor and charging ethanol, stewing, centrifuging, charging distilled water into the filter liquor, passing through resin columns, eluting and gathering the effluent liquid, decompressing and evaporating to dryness, charging distilled water and evaporating to dryness repeatedly. The invention also discloses the use of the cimicifuga rhizome organic acid in preparing medicaments for treating hepatitis B.
Description
Invention field
The present invention relates to a kind of Chinese medicine organic acid extracting method and new purposes.Particularly relate to rattletop organic acid extracting method and new purposes.
Background technology
2000 editions P55 of pharmacopeia record: rattletop be the big three leaf rattletop Cimicifugaheracleifolia Kom. of ranunculaceae plant the rattletop Cimicifuga dahurica of Xingan (Turcz.) Maxim. or the dry rhizome of rattletop Cimicifu foetida L..Excavate autumn.Nature and flavor with return through: hot, little sweet, be slightly cold.Return lung, stomach, large intestine channel.Function with cure mainly: deliver promoting eruption, clearing heat and detoxicating, the elevate a turnable ladder yang-energy.Be used for headache due to pathogenic wind-heat, dentalgia, aphtha, swelling and pain in the throat, measles without adequate eruption, the sun poison is sent out spot; Prolapse of the anus, uterine prolapse.Usage and consumption: 3-9 gram.
Hepatitis B is called serum hepatitis, hepatitis B (abbreviation hepatitis B) again, is caused, is served as main and can cause a kind of transmissible disease of multiple organ injury with the liver inflammatory lesion by hepatitis B virus (HBV).By blood and body fluid communication, has chronic carrier state.China is the most popular country of hepatitis B, and crowd infection rate's height reaches more than 35% at the certain areas infection rate, is the most serious transmissible disease of current harm people ' s health. China HBsAg carries population more than 100,000,000, accounts for whole world HBsAg and carries 50% of population.According to China's hepatitis investigation result, estimate that China hepatitis B patient has 2,700 ten thousand people approximately, annual New Development patient has 9,000,000 approximately.
Hepatitis B clinical manifestation variation, easily develop into chronic hepatitis and liver cirrhosis, a few patients can be converted into primary hepatocarcinoma. and hepatic fibrosis is the total pathological change that many chronic hepatopathys develop into the liver cirrhosis process, and chronic, persistence liver injury is the prerequisite that hepatic fibrosis forms.Cause the factor of liver injury a lot, generally because of medicine, a large amount of alcohol, allergy etc. cause acute liver damage, hepatitis B, liver cirrhosis, long clothes some drugs etc. can cause acute and chronic liver injury.
The medicine that is used for hepatitis B in the market, Interferon, rabbit are drugs of first choice, are the comparatively effectively antiviral of generally acknowledging both at home and abroad.Because there is big individual difference in body to the reaction of Interferon, rabbit, many results of study show that American-European countries's patient's curative effect is preferable, and east (as China, Japan) patient's curative effect is relatively poor, and Most patients all has side effect in various degree during interferon therapy.The nucleoside medicine tool improves the effect of liver histological pathological change and liver function in addition, and its security and tolerance are better, but occur the variation of virogene in the prolonged application process, thereby the sensitivity that reduces medicine produces the resistance phenomenon, and sickness rate is quite high after the drug withdrawal.
The curative effect of China's treatment by Chinese herbs hepatitis B is by a large amount of clinical confirmations; Chinese medicine all can be brought into play curative effect preferably at aspects such as antiviral, transaminase lowering, adjustment immunity of organisms, protection liver cells; but marketed drug mostly is compound Chinese medicinal preparation at present; the medicine that the present invention treats hepatitis B is the single medicinal material extract formulation; side effect is little, has many-sided pharmacological action simultaneously.
Summary of the invention
One object of the present invention is open rattletop organic acid extracting method; Another object of the present invention is open rattletop organic acid hepatitis B virus resisting, the new purposes of prevention and treatment hepatitis B.
The present invention seeks to be achieved through the following technical solutions:
A: fat-soluble organic acid: get rattletop medicinal material 1000 weight parts, add 70-80% ethanol and make liquid level cover medicinal material, soak post-heating backflow in 0-16 hour 2-4 time, each 6000-8000 parts by volume is filtered; Filtrate is concentrated, dry, gets extract 100-140 weight part; Get this extract 25-35 weight part, with its dissolving and transfer in the liquid distributing device, adding 5-10 parts by volume 28-37% hydrochloric acid also stirs with 50-70 ℃ of 600-1000 parts by volume distillation moisture 2-6 time; With ethyl acetate extraction 2-6 time, each 200-500 parts by volume merges upper strata phase solution; Upper strata phase solution takes off a layer alkali lye with 2-6% sodium hydroxide solution 200-600 parts by volume extraction 2-5 time; After lower floor's alkali lye was regulated pH to 2-3 with the 28-37% hydrochloric acid soln, the ethyl acetate extraction of each 200-500 parts by volume extracted 2-6 time; Merge upper strata phase solution, with distilled water wash, each 50-200 parts by volume is pH4-6 until last distilled water elutant; This organic layer phase solution obtains Rhizoma Cimicifugae liposolubility organic acid 3-6 weight part after removing vinyl acetic monomer;
B: water-soluble organic acid: get rattletop medicinal material 1000 weight parts, adding distil water 5000-8000 parts by volume, heated and boiled 1-4 time keeps the 5000-8000 parts by volume at every turn, seethes with excitement 1-3 hour; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate, be concentrated to the 800-1200 parts by volume, put cold after, add the 90-100% ethanol of 2000-4000 parts by volume, the limit edged stirs to make and mixes, and leaves standstill 8-48 hour, with the centrifugal 10-50 of 1500-4000rpm minute, inclining filtrate; Filtrate recycling ethanol is not to there being the alcohol flavor, and the adding distil water adjusted volume is to the 800-1200 parts by volume; Get this extracting solution,,, collect effluent liquid till effluent liquid is pH6-7 with the distilled water wash-out by pretreated cation exchange resin column; This effluent liquid is all by pretreated anion-exchange resin column, with the distilled water wash-out, treat that effluent liquid is pH8-7 after, use 0.3-0.7N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till; This effluent liquid adds 10-100 parts by volume distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-6, promptly get water-soluble organic acid 5-15 weight part.
The alleged rattletop organic acid of the present invention be meant by the fat-soluble organic acid of aforesaid method gained and (or) water-soluble organic acid.Rattletop organic acid of the present invention adds that the auxiliary material that pharmaceutics is accepted makes clinical acceptable various formulations, as formulations such as tablet, capsule, granule, injection, lyophilized injectable powder, subcutaneous administration preparation, suppositorys.
Weight part/parts by volume is corresponding with grams per milliliter among the present invention.
The rattletop organic acid proves to have good anti-HBV effect through pharmacological research, can alleviate further developing of hepatitis B.The medicine that the rattletop organic acid is made has more significantly to hepatitis B virus resisting, liver function protecting effect.The pharmacodynamic study of following experimental example is used to further specify but is not limited to new purposes of the present invention.
Pharmacodynamic study
Experimental example 1: external restraining effect test to hepatitis B virus
Be subjected to the reagent thing: rattletop organic acid (fat-soluble organic acid, water-soluble organic acid) is provided by China Academy Of Traditional Chinese Medicine Traditional Chinese Medicine Research Institute.Be mixed with the mother liquor of 10mg/ml before the experiment in vitro with high purity water, filtration sterilization, 4 ℃ of preservations are standby.2.2.15 cell: the 2.2.15 system of the human liver cancer cell (Hep-G2) of hepatitis B virus DNA clone transfection is provided by the Peking University first affiliated hospital Viral Laboratory.Reagent: hepatitis B surface antigen(HBsAg) detection kit, hepatitis B e antigen detection kit are Huamei Bio-Engrg Co.,'s product; Gene diagnosis center, hepatitis B virus DNA fluorescent quantificationally PCR detecting kit Anda product, G418; The Engls nutrient solution, japanese product.Microplate reader 750, Bayer company product; The ABI5700 quantitative real time PCR Instrument.Positive control drug: lamivudine, be made into the original liquid of 20mg/ml before the experiment of Ge Lan SmithKline pharmaceutical Co. Ltd product with high purity water, filter-sterilized is standby.
Influence to 2.2.15 cell culture system secretion HBsAg, HBeAg
Get the 2.2.15 Tissue Culture Plate (24 hole) that grows up to monolayer cell, outwell nutrient solution, add the following corresponding dilution soup of non-toxic concn, concentration is respectively: 0.625,0.312,0.156 and 0.078mg/ml, the 1ml/ hole, each extent of dilution is done 6 multiple holes, establishes lamivudine contrast medicine and cell contrast simultaneously.Put 37 ℃ of 5%CO
2Cultivate in the incubator, collected supernatant on the 8th day, two batches of collected supernatants of experiment are measured HBsAg, HBeAg simultaneously.Adopt the enzyme linked immunological adsorption method to measure, calculate inhibiting rate.
The result shows: to 2.2.15 emiocytosis HBsAg, 4 dosage that Rhizoma Cimicifugae extract tried in two batches of experiments all have the obvious suppression effect, with control group relatively have significant difference (P＜0.01 〉, and present good dose-effect dependency.The positive control drug lamivudine is to 2.2.15 emiocytosis HBsAg unrestraint effect.The results are shown in Table 1.
To 2.2.15 emiocytosis HBeAg, 4 dosage that Rhizoma Cimicifugae extract tried in two batches of experiments all have the obvious suppression effect, first experimental group and control group relatively have significant difference (P＜0.05 〉, second batch of experiment is because indivedual contrast ghost growth conditions difference inhibiting rate is lower, and statistics is meaningless.The positive control drug lamivudine is to 2.2.15 emiocytosis HBeAg unrestraint effect.The results are shown in Table 2.
Table 1 Rhizoma Cimicifugae extract is to the influence of 2.2.15 culturing cell secretion HBsAg
| Batch | The cytopathy (CPE) of medicine index different concns medicine |
| 625(ug/ml) 312(ug/ml) 156(ug/ml) 78(ug/ml) 16(ug/ml) 7.8(ug/ml) 3.9(ug/ml) | |
| First | Fat-soluble OD value 0.25 ± 0.06** 0.84 ± 0.09** 1.00 ± 0.09* 0.90 ± 0.04** inhibiting rate % 72.00 32.47 19.53 27.86 lamivudine OD values 0.20 ± 0.05 0.14 ± 0.01 0.13 ± 0.02 0.12 ± 0.06 inhibiting rate %-39.97 0.36 12.58 13.81 cell contrast 1.25 ± 0.17 0.14 ± 0.06 |
| Second batch | Fat-soluble OD value 1.33 ± 0.23** 1.59 ± 047** 2.05 ± 0.29** 2.22 ± 0.18** inhibiting rate % 50.06 40.24 23.32 17.01 lamivudine OD values 0.15 ± 0.03 0.17 ± 0.04 0.10 ± 0.02 0.08 ± 0.03 inhibiting rate %-3.89-16.89 28.83 41.38 cells contrast 2.67 ± 0.28 0.14 ± 0.06 |
| Water-soluble 7.8 (ug/ml) 3.9 (ug/ml), 1.95 (ug/ml) 0.98 (ug/ml) OD values, 0.08 ± 0.00**, 0.10 ± 0.04**, 0.09 ± 0.03**, 0.09 ± 0.01** inhibiting rate %, 64 54.8 62.27 61.79 cells contrast 0.24 ± 0.07 |
Compare with the cell control group: * * P＜0.01 * P＜0.05
The result shows in the table: 4 dosage that Rhizoma Cimicifugae extract tried in two batches of experiments all have the obvious suppression effect to 2.2.15 emiocytosis HBsAg, with control group relatively have significant difference (P＜0.01 〉, and present good dose-effect dependency.The positive control drug lamivudine is to 2.2.15 emiocytosis HBsAg unrestraint effect.
Table 2 Rhizoma Cimicifugae extract is to the influence of 2.2.15 culturing cell secretion HBeAg
| Batch | The cytopathy of different concns medicine (CPE) |
| Medicine index 625 (ug/ml) 312 (ug/ml) 156 (ug/ml) 78 (ug/ml) 16 (ug/ml) 7.8 (ug/ml) 3.9 (ug/ml) | |
| First | Fat-soluble OD value 0.26 ± 0.13* 0.45 ± 0.10 0.43 ± 0.23 0.25 ± 0.09** inhibiting rate % 57.14 27.89 30.96 59.33 lamivudine OD values 0.50 ± 0.11 0.37 ± 0.11 0.36 ± 0.12 0.27 ± 0.08 inhibiting rate % 19.83 39.70 41.18 55.34 cells contrast 0.62 ± 0.16 0.62 ± 0.16 |
| Second batch | Fat-soluble OD value 0.27 ± 0.13 0.34 ± 0.04 0.35 ± 0.09 0.37 ± 0.12 inhibiting rate %, 39.00 25.43 23.19 18.10 lamivudine OD values, 0.47 ± 0.12 0.48 ± 0.15 0.36 ± 0.06 0.28 ± 0.15 inhibiting rate %-3.28-7.00,19.14 37.85 cells contrast 0.45 ± 0.18 0.45 ± 0.18 |
| Water-soluble 7.8 (ug/ml) 3.9 (ug/ml), 1.95 (ug/ml) 0.98 (ug/ml) OD values, 0.59 ± 0.16**, 0.21 ± 0.58**, 0.81 ± 0.31**, 0.69 ± 0.23** inhibiting rate %, 61.38 21.18 47.13 54.60 cells contrast 1.53 ± 0.48 |
Compare with the cell control group: * * P＜0.01 * P＜0.05
The result shows in the table: 4 dosage that Rhizoma Cimicifugae extract tried in two batches of experiments all have the obvious suppression effect to 2.2.15 emiocytosis HBeAg, first experimental group and control group relatively have significant difference (P＜0.05 〉, the second batch of experiment since indivedual contrast ghost growth conditions difference inhibiting rates than the, statistics is meaningless.The positive control drug lamivudine is to 2.2.15 emiocytosis HBeAg unrestraint effect.
Experimental example 2: the pharmacodynamics test of the anti-duck hepatitis B virus of Rhizoma Cimicifugae extract
Be subjected to the reagent thing: rattletop organic acid (fat-soluble organic acid, water-soluble organic acid), provide by China Academy Of Traditional Chinese Medicine Traditional Chinese Medicine Research Institute, brown ceramic powder is with No. 2 capsulation 3mg～12mg/ capsules.The positive control medicine: lamivudine, Glaxo Wellcome Britain action company limited produces, and the crushing back is from the capsule of making the 5mg/ grain.Animal: 1 age in days duckling of the egg incubation that the Chongqing sheldrake of employing healthy adult produces, through abdominal cavity inoculation 0.1mlDHBV DNA positive-virus serum.After inoculating for 1 week, respectively external jugular vein blood drawing detects to filter out through dot hybridization with the DHBV dna probe of digoxigenin labeled and infects positive duck, raise to 2 all ages as laboratory animal.
Test method:
To infect 55 of positive ducks and be divided into 5 groups at random: virus control group (11): use the starch capsule, the oral cavity was irritated and was fed 1 time every day, and dosage is 500mg/.Positive drug control group (12): use the lamivudine capsule, the oral cavity was irritated and was fed 1 time every day, and dosage is 50mg.Kg
-1.D
-1Extract small dose group (10): the oral cavity was irritated and was fed 1 time every day, and dosage is 60mg.Kg
-1.D
-1The heavy dose of group of extract (12): the oral cavity was irritated and was fed 1 time every day, and dosage is 120mg.Kg
-1.D
-1
The experimental drug time is 28 days, and drug withdrawal was observed 7 days.Observation index is as follows:
Serum DHBV DNA changes situation:
Before medication, medication 7 days, 14 days, 21 days, 28 days, drug withdrawal external jugular vein blood drawing respectively in 7 days, separation of serum is to be checked in-20 ℃ of preservations.Adopt spot hybridization, prepare with the digoxigenin labeled test kit of Roche Holding Ag that the DHBV dna probe is unified to be detected, carry out diaphragm with VuegoScan (Brisa-620ST) scanner and scan; With the Discovery SeriesQuantity One software spot is carried out quantitative analysis, the spot value is volume (volume=intensity * mm2).Statistics adopts spss software, paired t-test.
Serum DHBsAg changes situation: before medication, medication 7 days, 14 days, 21 days, 28 days, drug withdrawal external jugular vein blood drawing respectively in 7 days, separation of serum is to be checked in-20 ℃ of preservations.Serum before and after the medication is unified comparison and detection, adopt ELISA rapid method, enzyme mark reading apparatus (Bio-TEK company) 490nm to read the O.D value.Statistics adopts spss software, paired t-test.
Each organizes serum DHBsAg O.D value change situation before, during and after the medication:
As time goes on, each is organized DHBsAg O.D value and presents downtrending, learns the relatively change not statistically significant of virus control group serum DHBsAg O.D value by statistics; And the obviously reduction (P＜0.01) of serum DHBsAg O.D value all appears in the different administration times of each dosage group of lamivudine group and extract, relatively still has lasting reduce (P＜0.01) to the medication of 7 days serum DHBsAg of drug withdrawal O.D value.The results are shown in Table 3:
Table 3 is respectively organized (the X ± S) of serum DHBsAgO.D value change situation before and after the medication
| Medication medication in 7 days medication in 14 days medication in 21 days drug withdrawal in 28 days is 7 days before the group medication |
| Water-soluble heavy dose of 0.996 ± 0.265 0.833 ± 0.604 0.567 ± 0.464**, 0.445 ± 0.679**, 0.441 ± 0.311**, the 0.302 ± 0.129** of fat-soluble heavy dose of 0.896 ± 0.623 0.842 ± 0.508 0.891 ± 0.750 0.769 ± 0.381 0.597 ± 0.198**, 0.491 ± 0.353** low dose, 1.583 ± 0.748 1.008 ± 0.3200**, 0.8710 ± 0.560**, 0.806 ± 0.54**, 0.7258 ± 0.577**, the 0.356 ± 0.364** of virus control group 0.503 ± 0.423 0.595 ± 0.540 0.556 ± 0.472 0.438 ± 0.338 0.331 ± 0.281 0.232 ± 0.137 lamivudine group 0.479 ± 0.278 0.392 ± 0.196 0.425 ± 0.194 0.272 ± 0.130**, 0.213 ± 0.097**, 0.161 ± 0.072** |
Annotate: upward table institute classifies as and respectively organizes DHBsAgO.D value mean number and standard deviation, and statistics adopts paired t-test.(" * ": P＜0.05, " * ": P＜0.01) is the comparison of DHBsAg0.D value before different time DHBsAgO.D value and the medication on the same group
Serum DHBV DNA titre changes situation before, during and after the medication:
Serum DHBV DNA titre does not have obvious reduction before and after the medication of virus control group, and presents slight ascendant trend.The lamivudine group medication begins to occur serum DHBV DNA titre and reduces (P＜0.01) after 7 days, DHBV DNA rise phenomenon is arranged after the drug withdrawal.Each dosage group of extract began to occur serum DHBV DNA titre in 14 days respectively at medication and reduces (P＜0.05, P＜0.01), and DHBV DNA rise phenomenon is not obvious after the drug withdrawal; The results are shown in Table 4:
Serum DHBV DNA titre variation before and after the medication of table 4 Rhizoma Cimicifugae extract (X ± S)
| DHBV DNA(volume) |
| Medication medication in 7 days medication in 14 days medication in 21 days drug withdrawal in 28 days is 7 days before the group medication |
| 1167.01±208.39 1184.44±282.22 1233.14±262.75 1272.21±260.11 1241.69±249.30 1347.79±236.66 1092.79±256.75 908.20±221.82** 947.99±317.20* 920.43±161.61** 931.59±97.63* 1207.23±247.09 1229.65±146.82 1163.37±264.28 1098.57±247.97* 1011.17±222.00* 1042.86±134.81** 1037.36±121.14* 1161.08±158.85 867.44±82.73** 844.16±121.07** 798.18±78.86** 722.94±112.37** 770.48±133.12** 1545.27±183.92 1270.02±222.10 1200.65±124.54** 1034.64±254.77** 1009.86±213.21** 980.06±75.14 1451.08±144.85 1356.44±81.72 1025.16±103.07** 912.18±98.86** 900.94±122.37** 900.48±183.22** |
Annotate: go up table mean number and the standard deviation of respectively organizing DNA spot volume value of classifying as, statistics employing paired t-test (" * ": P＜0.05: " * * ": P＜0.01), be different time DNA spot value and the comparison of DNA spot value before the medication on the same group.
Embodiment 1:The preparation of Rhizoma Cimicifugae liposolubility organic acid
Get rattletop medicinal material 1000 grams, add 75% ethanol and make liquid level cover medicinal material, soak 5 hours post-heating and reflux 2 times, each 6500 milliliters, filter, get filtrate.Concentrate, drying gets extract 120 grams.Get this extract 30.0 grams, it is dissolved as far as possible and transfer in 2000 milliliters of separating funnels, add 8 milliliter of 36% hydrochloric acid and stir with 850 milliliters 60 ℃ distillation moisture 4 times.With ethyl acetate extraction 5 times, each 300 milliliters, merge upper strata phase solution.Upper strata phase solution is with 500 milliliters of 3% sodium hydroxide solutions, 400 milliliters and 300 milliliters of coextractions 3 times.Merge lower floor's alkali lye, regulate pH to 2-3 with 36% hydrochloric acid after, with ethyl acetate extraction 4 times, each 350 milliliters.Merge clarifying upper strata phase solution,, each 140 milliliters, be pH4-5 until last distilled water elutant with the distilled water repetitive scrubbing.This organic layer phase solution obtains fat-soluble organic acid 4.6 grams, yield 1.84% after removing vinyl acetic monomer.
Embodiment 2:The preparation of water soluble organic acid of Rhizoma Cimicifugae
Get rattletop medicinal material 1000 grams, add 6000 ml distilled waters, heated and boiled 2 hours.It is many with the consumption that keeps water often to augment distilled water between boiling period.Filter, get filtrate.The dregs of a decoction add 5000 ml distilled waters and carry out the heated and boiled 2 hours second time, filter, and get filtrate.Merging filtrate is concentrated to 1000 milliliters, slowly adds 95% ethanol of 2.8 times of volumes (2800 milliliters), and the limit edged stirs to make and mixes, and leaves standstill 16 hours, with 3000rpm centrifugal 20 minutes, gets filtrate.This filtrate through decompression recycling ethanol to there not being the alcohol flavor, adding distil water adjusted volume to 1000 milliliter.Get 600 milliliters of this extracting solutions,,, collect effluent liquid till effluent liquid is pH neutrality, collect about 5000 milliliters of effluent liquid altogether with the distilled water wash-out by pretreated 732 type strong acid cation exchange resin columns (H type).This effluent liquid is all by 717 pretreated type strongly basic anion exchange resin posts (OH type), with the distilled water wash-out, treat that effluent liquid is pH neutrality after, use 0.5N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till.This effluent liquid adds a small amount of distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-5, promptly get water-soluble organic acid 5.7 grams, yield 0.95%.
Embodiment 3:The preparation of Rhizoma Cimicifugae liposolubility organic acid
Get rattletop medicinal material 1000kg, add 75% ethanol and make liquid level cover medicinal material, soak 5 hours post-heating and reflux 3 times, each 7000 liters, filter; Filtrate concentrates, and drying gets extract 110kg; Get this extract 30kg, with its dissolving and transfer in the liquid distributing device, add 9.6 liter of 30% hydrochloric acid and stir with 800 liters 60 ℃ distillation moisture 3 times; With ethyl acetate extraction 4 times, each 300 liters, merge upper strata phase solution; Upper strata phase solution takes off a layer alkali lye with 300 liters of extractions of 3% sodium hydroxide solution 5 times; After lower floor's alkali lye is regulated pH to 2-3 with 30% hydrochloric acid, each 300 liters ethyl acetate extraction, coextraction 3 times; Merge upper strata phase solution,, each 100 liters, be pH5 until last distilled water elutant with the distilled water repetitive scrubbing; After removing vinyl acetic monomer, this organic layer phase solution obtains Rhizoma Cimicifugae liposolubility organic acid 4kg.
Embodiment 4: the preparation of Rhizoma Cimicifugae liposolubility organic acid
Get rattletop medicinal material 1000kg, add 80% ethanol and make liquid level cover medicinal material, soak 10 hours post-heating and reflux 4 times, each 7500 liters, filter; Filtrate concentrates, and drying gets extract 120kg; Get this extract 35kg, with its dissolving and transfer in the liquid distributing device, add 8 liter of 35% hydrochloric acid and stir with 900 liters 65 ℃ distillation moisture 5 times; With ethyl acetate extraction 5 times, each 400 liters, merge upper strata phase solution; Upper strata phase solution takes off a layer alkali lye with 500 liters of extractions of 4% sodium hydroxide solution 4 times; After lower floor's alkali lye is regulated pH to 3 with 35% hydrochloric acid, each 400 liters ethyl acetate extraction, coextraction 4 times; Merge upper strata phase solution,, each 150 liters, be pH6 until last distilled water elutant with the distilled water repetitive scrubbing; After removing vinyl acetic monomer, this organic layer phase solution obtains Rhizoma Cimicifugae liposolubility organic acid 5kg.
Embodiment 5: the preparation of water soluble organic acid of Rhizoma Cimicifugae
Get rattletop medicinal material 1000kg, adding distil water, heated and boiled 2 times keeps 6000 liters at every turn, seethes with excitement 2 hours; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate, be concentrated to 1000 liters, put cold after, slowly add 3000 liters 95% ethanol, the limit edged stirs to make and mixes, and leaves standstill 15 hours, with 2500rpm centrifugal 20 minutes, inclining filtrate; This filtrate recycling ethanol is not to there being the alcohol flavor, adding distil water adjusted volume to 1000 liter; Get this extracting solution,,, collect effluent liquid till effluent liquid is pH6 with the distilled water wash-out by pretreated cation exchange resin column; This effluent liquid is all by pretreated anion-exchange resin column, with the distilled water wash-out, treat that effluent liquid is pH8-7 after, use 0.4N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1 till; This effluent liquid adds 30 liters of distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH6, promptly get water-soluble organic acid 8kg.
Embodiment 6: the preparation of water soluble organic acid of Rhizoma Cimicifugae
Get rattletop medicinal material 1000kg, adding distil water, heated and boiled 3 times keeps 7000 liters at every turn, seethes with excitement 2.5 hours; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate, be concentrated to 1100 liters, put cold after, slowly add 3500 liters 95% ethanol, the limit edged stirs to make and mixes, and leaves standstill 30 hours, with 3500rpm centrifugal 40 minutes, inclining filtrate; This filtrate recycling ethanol is not to there being the alcohol flavor, adding distil water adjusted volume to 1100 liter; Get this extracting solution,,, collect effluent liquid till effluent liquid is pH7 with the distilled water wash-out by pretreated cation exchange resin column; This effluent liquid is all by pretreated anion-exchange resin column, with the distilled water wash-out, treat that effluent liquid is pH7 after, use 0.6N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH2 till; This effluent liquid adds 60 liters of distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH5, promptly get water-soluble organic acid 12kg.
Embodiment 7:Prepare fat-soluble organic acid
Get the rattletop medicinal material, get fat-soluble organic acid 100-300 gram by above-mentioned fat-soluble organic acid extracting method, make 1000 of capsules according to a conventional method, hepatitis is taken, and calculates each 1-2 grain, every day 2 times by adult's 60 kg body weight.
Embodiment 8:The preparation water-soluble organic acid
Get the rattletop medicinal material, get water-soluble organic acid 100-300 gram by above-mentioned water-soluble organic acid extracting method, make 1000 in tablet according to a conventional method, hepatitis is taken, and calculates each 1-2 sheet, every day 2 times by adult's 60 kg body weight.
Embodiment 9:Prepare fat-soluble organic acid
Get the rattletop medicinal material, get fat-soluble organic acid 100-300 gram by above-mentioned fat-soluble organic acid extracting method, make 1000 bags of granules according to a conventional method, hepatitis is taken, and calculates each 1-2 bag, every day 2 times by adult's 60 kg body weight.
Embodiment 10:The preparation water-soluble organic acid
Get the rattletop medicinal material, get water-soluble organic acid 100-300 gram, make 1000 of injection liquids according to a conventional method by above-mentioned water-soluble organic acid extracting method, the 10-20 milliliter/, the hepatitis intravenous drip is calculated by adult's 60 kg body weight, and each 1-2 props up every day 1 time.
Embodiment 11:Prepare fat-soluble organic acid
Get the rattletop medicinal material, get fat-soluble organic acid 100-300 gram by above-mentioned fat-soluble organic acid extracting method, make 1000 of freeze-dried powders according to a conventional method, the hepatitis intravenous drip is calculated by adult's 60 kg body weight, and each 1-2 props up every day 1 time.
Embodiment 12:The preparation water-soluble organic acid
Get the rattletop medicinal material, get water-soluble organic acid 100-300 gram by above-mentioned water-soluble organic acid extracting method, make 1000 of suppositorys according to a conventional method, the hepatitis anum administration calculates by adult's 60 kg body weight, and each 1, every day 1-2 time.
Claims (10)
1, a kind of rattletop organic acid, it is characterized in that this organic acid is to be extracted by following method to obtain: get rattletop medicinal material 1000 weight parts, add 70-80% ethanol and make liquid level cover medicinal material, soak post-heating backflow in 0-16 hour 2-4 time, each 6000-8000 parts by volume is filtered; Filtrate concentrates, and drying gets extract 100-140 weight part; Get this extract 25-35 weight part, with its dissolving and transfer in the liquid distributing device, adding 5-10 parts by volume 28-37% hydrochloric acid also stirs with 50-70 ℃ of 600-1000 parts by volume distillation moisture 2-6 time; With ethyl acetate extraction 2-6 time, each 200-500 parts by volume merges upper strata phase solution; Upper strata phase solution takes off a layer alkali lye with 2-6% sodium hydroxide solution 200-600 parts by volume extraction 2-5 time; After lower floor's alkali lye is regulated pH to 2-3 with 28-37% hydrochloric acid, the ethyl acetate extraction of each 200-500 parts by volume, coextraction 2-6 time; Merge upper strata phase solution, with the distilled water repetitive scrubbing, each 50-200 parts by volume is pH4-6 until last distilled water elutant; This organic layer phase solution obtains Rhizoma Cimicifugae liposolubility organic acid 3-6 weight part after removing vinyl acetic monomer.
2, rattletop organic acid as claimed in claim 1, it is characterized in that this organic acid is to be extracted by following method to obtain: get rattletop medicinal material 1000 weight parts, adding 75% ethanol makes liquid level cover medicinal material, soaking 5 hours post-heating refluxes 2 times, each 6500 parts by volume are filtered, and get filtrate concentrating, drying gets extract 120 weight parts; Get this extract 30.0 weight parts, it is dissolved as far as possible and transfer in the 2000 parts by volume liquid distributing devices, add 8 parts by volume concentrated hydrochloric acids and stir with 60 ℃ of 850 parts by volume distillation moisture 4 times; With ethyl acetate extraction 5 times, each 300 parts by volume, merge upper strata phase solution; Upper strata phase solution is with 3% sodium hydroxide solution, 500 parts by volume, 400 parts by volume and 300 parts by volume coextractions 3 times; Merge lower floor's alkali lye, regulate pH to 2-3 with concentrated hydrochloric acid after, with ethyl acetate extraction 4 times, each 350 parts by volume; Merge clarifying upper strata phase solution, with the distilled water repetitive scrubbing, each 140 parts by volume are pH4-5 until last distilled water elutant; This organic layer phase solution obtains fat-soluble organic acid 4.6 weight parts after removing vinyl acetic monomer.
3, a kind of rattletop organic acid is characterized in that this organic acid is to be extracted by following method to obtain:
Get rattletop medicinal material 1000 weight parts, adding distil water 5000-8000 parts by volume, heated and boiled 1-4 time keeps the 5000-8000 parts by volume at every turn, seethes with excitement 1-3 hour; Filter, get filtrate, be concentrated to the 800-1200 parts by volume, put cold after, add the 90-100% ethanol of 2000-4000 parts by volume, the limit edged stirs to make and mixes, and leaves standstill 8-48 hour, with the centrifugal 10-50 of 1500-4000rpm minute, inclining filtrate; This filtrate recycling ethanol is not to there being the alcohol flavor, and the adding distil water adjusted volume is to the 800-1200 parts by volume; Get this extracting solution,,, collect effluent liquid till effluent liquid is pH6-7 with the distilled water wash-out by pretreated cation exchange resin column; This effluent liquid is all by pretreated anion-exchange resin column, with the distilled water wash-out, treat that effluent liquid is pH8-7 after, use 0.3-0.7N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till; This effluent liquid adds 10-100 parts by volume distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-6, promptly get water-soluble organic acid 5-15 weight part.
4, rattletop organic acid as claimed in claim 3 is characterized in that this organic acid is to be extracted by following method to obtain: get rattletop medicinal material 1000 weight parts, add 6000 parts by volume distilled water, heated and boiled 2 hours; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate; The dregs of a decoction add 5000 parts by volume distilled water and carry out the heated and boiled 2 hours second time, filter, and get filtrate; Merging filtrate is concentrated to 1000 parts by volume, adds 95% ethanol of 2800 parts by volume, and the limit edged stirs to make and mixes, and leaves standstill 16 hours, with 3000rpm centrifugal 20 minutes, gets filtrate; To there not being the alcohol flavor, the adding distil water adjusted volume is to 1000 parts by volume through decompression recycling ethanol for this filtrate; Get this extracting solution 600 parts by volume,,, collect effluent liquid till effluent liquid is pH neutrality, collect about 5000 parts by volume of effluent liquid altogether with the distilled water wash-out by 732 pretreated type strong acid cation exchange resin column H types; This effluent liquid is all by 717 pretreated type strongly basic anion exchange resin post OH types, with the distilled water wash-out, treat that effluent liquid is pH neutrality after, use 0.5N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till; This effluent liquid adds a small amount of distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-5, promptly get water-soluble organic acid 5.7 weight parts.
5, a kind of rattletop organic acid extracting method is characterized in that this method comprises in the following method any:
A, get rattletop medicinal material 1000 weight parts, add 70-80% ethanol and make liquid level cover medicinal material, soak post-heating backflow in 0-16 hour 2-4 time, each 6000-8000 parts by volume is filtered; Filtrate concentrates, and drying gets extract 100-140 weight part; Get this extract 25-35 weight part, with its dissolving and transfer in the liquid distributing device, adding 5-10 parts by volume 28-37% hydrochloric acid also stirs with 50-70 ℃ of 600-1000 parts by volume distillation moisture 2-6 time; With ethyl acetate extraction 2-6 time, each 200-500 parts by volume merges upper strata phase solution; Upper strata phase solution takes off a layer alkali lye with 2-6% sodium hydroxide solution 200-600 parts by volume extraction 2-5 time; After lower floor's alkali lye is regulated pH to 2-3 with 28-37% hydrochloric acid, the ethyl acetate extraction of each 200-500 parts by volume, coextraction 2-6 time; Merge upper strata phase solution, with the distilled water repetitive scrubbing, each 50-200 parts by volume is pH4-6 until last distilled water elutant; This organic layer phase solution obtains Rhizoma Cimicifugae liposolubility organic acid 3-6 weight part after removing vinyl acetic monomer;
B, get rattletop medicinal material 1000 weight parts, adding distil water, heated and boiled 1-4 time keeps the 5000-8000 parts by volume at every turn, seethes with excitement 1-3 hour; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate, be concentrated to the 800-1200 parts by volume, put cold after, slowly add the 90-100% ethanol of 2000-4000 parts by volume, the limit edged stirs to make and mixes, and leaves standstill 8-48 hour, with the centrifugal 10-50 of 1500-4000rpm minute, inclining filtrate; This filtrate recycling ethanol is not to there being the alcohol flavor, and the adding distil water adjusted volume is to the 800-1200 parts by volume; Get this extracting solution,,, collect effluent liquid till effluent liquid is pH6-7 with the distilled water wash-out by pretreated cation exchange resin column; This effluent liquid is all by pretreated anion-exchange resin column, with the distilled water wash-out, treat that effluent liquid is pH8-7 after, use 0.3-0.7N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till; This effluent liquid adds 10-100 parts by volume distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-6, promptly get water-soluble organic acid 5-15 weight part.
6, rattletop organic acid extracting method as claimed in claim 5 is characterized in that this method comprises any in the following method:
A, get rattletop medicinal material 1000 weight parts, add 75% ethanol and make liquid level cover medicinal material, soak 5 hours post-heating and reflux 2 times, each 6500 parts by volume are filtered, and get filtrate concentrating, drying, extract 120 weight parts; Get this extract 30.0 weight parts, it is dissolved as far as possible and transfer in the 2000 parts by volume liquid distributing devices, add 8 parts by volume concentrated hydrochloric acids and stir with 60 ℃ of 850 parts by volume distillation moisture 4 times; With ethyl acetate extraction 5 times, each 300 parts by volume, merge upper strata phase solution; Upper strata phase solution is with 3% sodium hydroxide solution, 500 parts by volume, 400 parts by volume and 300 parts by volume coextractions 3 times; Merge lower floor's alkali lye, regulate pH to 2-3 with concentrated hydrochloric acid after, with ethyl acetate extraction 4 times, each 350 parts by volume; Merge clarifying upper strata phase solution, with the distilled water repetitive scrubbing, each 140 parts by volume are pH4-5 until last distilled water elutant; This organic layer phase solution obtains fat-soluble organic acid 4.6 weight parts after removing vinyl acetic monomer;
B, get rattletop medicinal material 1000 weight parts, add 6000 parts by volume distilled water, heated and boiled 2 hours; It is many with the consumption that keeps water often to augment distilled water between boiling period; Filter, get filtrate; The dregs of a decoction add 5000 parts by volume distilled water and carry out the heated and boiled 2 hours second time, filter, and get filtrate; Merging filtrate is concentrated to 1000 parts by volume, adds 95% ethanol of 2800 parts by volume, and the limit edged stirs to make and mixes, and leaves standstill 16 hours, with 3000rpm centrifugal 20 minutes, gets filtrate; To there not being the alcohol flavor, the adding distil water adjusted volume is to 1000 parts by volume through decompression recycling ethanol for this filtrate; Get this extracting solution 600 parts by volume,,, collect effluent liquid till effluent liquid is pH neutrality, collect about 5000 parts by volume of effluent liquid altogether with the distilled water wash-out by 732 pretreated type strong acid cation exchange resin column H types; This effluent liquid is all by 717 pretreated type strongly basic anion exchange resin post OH types, with the distilled water wash-out, treat that effluent liquid is pH neutrality after, use 0.5N hydrochloric acid soln wash-out instead, and begin to collect effluent liquid when effluent liquid is pH1-2 till; This effluent liquid adds a small amount of distilled water and evaporated under reduced pressure after evaporated under reduced pressure, adding distil water and evaporate to dryness repeatedly repeatedly till final product is pH4-5, promptly get water-soluble organic acid 5.7 weight parts.
7,, it is characterized in that the auxiliary material that this organic acid adds that pharmaceutics is accepted makes clinically or pharmaceutically acceptable formulation: tablet, capsule, granule, injection, lyophilized injectable powder, subcutaneous administration preparation or suppository as the described rattletop organic acid of claim 1-4.
8, as the application of the described rattletop organic acid of claim 1-4 in the medicine of preparation treatment or prevention B type pneumonia.
9, as the application of the described rattletop organic acid of claim 1-4 in preparation inhibition hepatitis B virus medicaments.
10, has application in the medicine that suppresses HBsAg in serum, HBeAg and the effect of HBV DNA titre as the described rattletop organic acid of claim 1-4 in preparation.
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| EP2339030A1 (en) * | 2008-05-30 | 2011-06-29 | Golden Biotechnology Corporation | Screening method for liver x receptor agonists and application thereof |
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| CN1111427C (en) * | 1999-02-08 | 2003-06-18 | 山东绿叶制药股份有限公司 | Extract of cimicifuga rhizome and its preparation process and application |
| CN1475269A (en) * | 2003-07-31 | 2004-02-18 | 凯 曹 | Rhizoma cimicifugae extract and its preparation method |
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| EP2339030A1 (en) * | 2008-05-30 | 2011-06-29 | Golden Biotechnology Corporation | Screening method for liver x receptor agonists and application thereof |
| EP2339030A4 (en) * | 2008-05-30 | 2011-11-16 | Golden Biotechnology Corp | Screening method for liver x receptor agonists and application thereof |
| CN102060693A (en) * | 2010-12-20 | 2011-05-18 | 大兴安岭嘉迪欧营养原料有限公司 | Method for enriching and purifying ferulic acid in cimicifugae foetidae |
| CN102060693B (en) * | 2010-12-20 | 2013-11-13 | 大兴安岭嘉迪欧营养原料有限公司 | Method for enriching and purifying ferulic acid in cimicifugae foetidae |
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