CN100339353C - Preparation method and use of alpine yarrow herb ketoacid - Google Patents
Preparation method and use of alpine yarrow herb ketoacid Download PDFInfo
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- CN100339353C CN100339353C CNB2004100848182A CN200410084818A CN100339353C CN 100339353 C CN100339353 C CN 100339353C CN B2004100848182 A CNB2004100848182 A CN B2004100848182A CN 200410084818 A CN200410084818 A CN 200410084818A CN 100339353 C CN100339353 C CN 100339353C
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Abstract
The present invention relates to a preparation method and use for rupestonic acid of a monomeric compound extracted by alpine yarrow herb. Rupestonic acid is prepared with the methods of conventional chromatography and liquid phase combination; firstly, raw materials are processed; the whole alpine yarrow herb is pulverized and then is refluxed and extracted with ethanol; extract liquids are merged, decompressed and concentrated into a paste shape and are extracted with ethyl acetate; extracted liquids are concentrated, mixed with silica gel and arranged on a silica gel column with 100 to 200 meshes and are eluted with petroleum ether-ethyl acetate; eluted products are concentrated for use; chromatographic crude products are dissolved in a methanol solution with ultrasonic waves and are purified with the semi-preparing high performance liquid chromatography; reserved components are collected and are dried in a constant temperature drying box after decompressed and concentrated; colorless rod-shapes crystal is obtained; the rupestonic acid is used for preparing medicine for inhibiting hepatitis B viruses and protecting a liver.
Description
Technical field
The present invention relates to the preparation method and the pharmaceutical applications of rupestonic acid, especially at the inhibition of preparation hepatitis B virus and the pharmaceutical use of hepatoprotective effect.
Background technology
Viral hepatitis, liver injury are the diseases of infringement human health, and wherein viral hepatitis is the global infectious disease of serious harm human health, patient's poor prognosis, and most patient transfers to chronic, may develop into liver cirrhosis etc.The liver injury that viral hepatitis causes is mainly mediated by the immune response reaction, behind the activated viral body immune system, produces primed lymphocyte and specific antibody, particularly virus specificity cell toxicity T cell.They not only can react with the body inner virus and be killed, and cause liver injury because of the surface of hepatocytes that is infected by the virus has virus antigen, cause hepatocellular degeneration swelling and necrosis.At present, very not satisfactory for the sick traditional Chinese and western medicine curative effect of this class.For example, the Western medicine Interferon, rabbit is eliminated hepatitis virus certain curative effect, but undesirable through clinical effectiveness, can not kill virus, and also administration time is long, and expenses for medicine is higher.Each is different in the treatment viral hepatitis with aspect protecting the liver for the Chinese patent medicine class, and the Chinese medicine that has biases toward clearing heat and detoxicating, and focusing on of having activates blood circulation and disperses blood clots, and what have is nourishing liver and kidney etc., and these medicines all respectively have its deficiency.Clinical clothes bitter cold medicine of a specified duration, but impairment of the spleen kidney reduce appetite, digestion and absorption.In addition, many medicines must carry out oxidation, decomposition, drainage in liver, and what have also can directly cause liver damage, and are unfavorable to the hepatopathy recovery from illness on the contrary.
Summary of the invention
The preparation method and the pharmaceutical applications that the purpose of this invention is to provide rupestonic acid specifically, are to adopt conventional chromatography and liquid phase bonded method to prepare rupestonic acid, and in the inhibition of preparation hepatitis B virus and the purposes of hepatoprotective effect medicine.
The Preparation method and use of rupestonic acid of the present invention, adopt conventional chromatography and liquid phase bonded method to prepare rupestonic acid, at first carrying out raw material handles: the Herba Achilleae herb is pulverized the back with 95% alcohol reflux 4 times, time is respectively 1-5h, 30min-4h, 15min-2h, 30min-5h, extracting solution is merged, concentrating under reduced pressure becomes paste again, uses ethyl acetate extraction 3 times, extraction liquid concentrates mixes 100-200 order silicagel column on the silica gel, with sherwood oil 30-60 ℃-ethyl acetate 3: 1, V/V wash-out, the product that elutes concentrates standby; The standard substance preparation: the crude product of chromatography is dissolved in methanol solution, and ultrasonic 30min is with half preparative high-performance liquid chromatographic method purifying, collecting retention time is the component of 18.5min, with 60 ℃ of rearmounted thermostatic drying chamber inner drying 24h of its concentrating under reduced pressure, 40 ℃, obtain colourless bar-shaped crystallization at last.
Rupestonic acid is at the inhibition of preparation hepatitis B virus and the pharmaceutical use of hepatoprotective effect.
Artemisia rupestris L. (Artemisia rupestris L.) is a feverfew, originates in domestic Tianshan Mountains, Xinjiang Uygur Autonomous Regions and mountain range, Altai Mountains, is kazakh's folk tradition medication, has detoxifcation, antianaphylaxis, antibiotic, antiviral and effect such as protect the liver.The clinical treatment that is used for hepatitis, flu, pharyngitis, tonsillitis, ophthalmia.Contain compounds such as flavonoid, ketone acid class, amino acids, glucoside, polyose, volatile oil, polypeptide, alkaloid in the Herba Achilleae.Ketone acid is one of main effective constituent of Herba Achilleae, and the institute of materia medica, Xinjiang has carried out separation and structure to rupestonic acid and identified, employing be the normal pressure silica gel column method.But have not yet to see the report that rupestonic acid is separated preparation with half preparative chromatography.Therefore for Herba Achilleae being carried out drug quality control, this paper adopts conventional chromatography and high performance liquid phase bonded method to prepare rupestonic acid.Method is simple, and product purity can reach more than 98%.
The structure of rupestonic acid
Rupestonic acid is at the inhibition of preparation hepatitis B virus and the pharmaceutical use of hepatoprotective effect.
Rupestonic acid is provided by Xinjiang Uygur medicine institute, for studying its inhibition and hepatoprotective effect to hepatitis B virus, this experiment detects the restraining effect of rupestonic acid to hepatitis B virus HBsAg, HBeAg and HBV-DNA expression in external 2.2.15 cell culture system.Adopt the 2.2.15 system of the human liver cancer cell (Hep-G2) of hepatitis B virus DNA clone transfection in this experiment, observation post's reagent thing rupestonic acid under different concns to the influence of 2.2.15 emiocytosis HBsAg, HBeAg and HBV-DNA.The result shows: 4 dosage that rupestonic acid tried in three batches of experiments all have the obvious suppression effect to 2.2.15 emiocytosis HBsAg.With control group significant difference (P＜0.01) is arranged relatively, and in 250ug/ml, 125ug/ml, 62ug/ml, 31ug/ml dosage range, present good dose-effect dependency.The positive control drug lamivudine is to 2.2.15 emiocytosis HBsAg unrestraint effect.Observe the protecting the liver of rupestonic acid, immunomodulatory and anti-radical action, rupestonic acid experimentation on animals gastric infusion 80,160,320mg/kg, qd, 5-7d obviously reduces CCL
4Cause serum GPT of liver injury mouse and the content of GOT; Significantly reduce serum GPT, GOT and the ALP content of the salt-induced acute liver damage mouse of D-Gal ammonia hydrochloric acid; And can reduce the content of the serum GPT of mouse immune liver damage mouse.Prompting, rupestonic acid has tangible hepatoprotective effect.
Description of drawings
Fig. 1 is sample half preparative high-performance liquid chromatographic figure of the present invention
Fig. 2 is the analysis mode high-efficient liquid phase chromatogram of ketone acid standard substance of the present invention
Fig. 3 is each administration group HBV-DNA fluorescence quantitative PCR detection graphic representation of the present invention
Fig. 4 is a quantitative fluorescent PCR canonical plotting of the present invention
Fig. 5 is a quantitative fluorescent PCR canonical plotting of the present invention
Embodiment
Embodiment
(1) Preparation method and use of rupestonic acid, adopt conventional chromatography and liquid phase bonded method to prepare rupestonic acid, at first carrying out raw material handles: the Herba Achilleae herb is pulverized the back with 95% alcohol reflux 4 times, time is respectively 1-5h, 30min-4h, 15min-2h, 30min-5h, extracting solution is merged, concentrating under reduced pressure becomes paste again, uses ethyl acetate extraction 3 times, extraction liquid concentrates mixes 100-200 order silicagel column on the silica gel, with sherwood oil 30-60 ℃-ethyl acetate 3: 1, V/V wash-out, the product that elutes concentrates standby; The standard substance preparation: the crude product of chromatography is dissolved in methanol solution, and ultrasonic 30min is with half preparative high-performance liquid chromatographic method purifying, collecting retention time is the component of 18.5min, with 60 ℃ of rearmounted thermostatic drying chamber inner drying 24h of its concentrating under reduced pressure, 40 ℃, obtain colourless bar-shaped crystallization at last.
1 experimental technique
1.1 instrument and reagent: Dionex half preparative high-performance liquid chromatographic instrument, DionexUVD170U/340U UV/VIS detector, Foxy Jr. run tank, 10ml quantitatively encircles.Waters2690 analysis mode high performance liquid chromatograph, the 996PDA detector.RE-52C type rotatory evaporator (Gongyi City's English gives magnificent instrument plant), Yanaco fusing point instrument, silica gel and silica-gel plate (Haiyang Chemical Plant, Qingdao), chromatographically pure methyl alcohol, acetonitrile (Fisher company), the secondary redistilled water, other reagent are analytical pure.
1.2 raw material is handled: Herba Achilleae herb 10kg pulverizes the back with 95% alcohol reflux 4 times, is respectively 1-5h, 30min-4h, and 15min-2h, 30min-5h.Extracting solution is merged, and concentrating under reduced pressure becomes paste, and with ethyl acetate extraction 3 times, extraction liquid concentrates mixes 100-200 order silicagel column on the silica gel, and sherwood oil (30-60 ℃)-ethyl acetate (3: 1, V/V) wash-out, the product that elutes concentrates standby.
1.3 chromatographic condition
1.3.1 half preparation HPLC: YMC-Pack ODS-A post (20mmI.D. * 250mm, 5 μ m); Column temperature: room temperature; Detect wavelength: 245nm; Moving phase: methyl alcohol-0.2% aqueous formic acid (65: 35, V/V); Flow velocity: 8.0ml/min.
1.3.2 analysis mode HPLC:YMC-Pack ODS-A post (4.6mmI.D. * 250mm, 5 μ m); Column temperature: room temperature; Detect wavelength: 210--400nm; Moving phase: methyl alcohol-0.2% aqueous formic acid (70: 30, V/V); Flow velocity: 1.0ml/min.
1.4 standard substance preparation: the crude product of chromatography is dissolved in methanol solution, ultrasonic 30min, with half preparative high-performance liquid chromatographic method purifying, collecting retention time is the component of 18.5min, sees Fig. 1.With the rearmounted thermostatic drying chamber inner drying 24h of its concentrating under reduced pressure (below 60 ℃) (40 ℃), obtain colourless bar-shaped crystallization at last.
Result and discussion
2.1 the purity detecting of preparation sample
Fusing point (mp) is measured: 132-133 ℃ of thin-layer chromatography detects: adopt two kinds of expansion systems to detect thin layer plate silica gel G F
254Plate.Developping agent A is petroleum ether-ethyl acetate-acetate (volume ratio is 20: 10: 1), R
FABe 0.45; Developping agent B is normal hexane-ether-acetate (volume ratio is 5: 10: 1), R
FBBe 0.61.Show color spot point under the ultraviolet 254nm, the inclusion-free point.
HPLC detects: product type high performance liquid chromatography by analysis detects, and sees Fig. 2, adopts normalization method quantitative, and its purity is greater than 98%.
Structure is identified
IRν
kBr max(cm
-1)：3200，2900，1704，1658，1616，950。
1H-NMR(400MHz，CDCl
3)：δ0.65(3H，d，J＝7.1Hz，H-15′，15″，15)，0.83(1H，q，J＝16.33Hz，H-10)，1.60(3H，s，H-14′，14″，14)，1.75(2H，m，J＝32.69Hz，H-9′，9″)，2.10(2H，m，J＝51.43Hz，H-8′，8″)，2.45(2H，q，J＝31.7Hz，H-2′，2″)，2.60(1H，dd，J＝18.80Hz，6.41Hz，H-7)，2.88(2H，q，J＝41.42Hz，H-6′，6″)，5.73(1H，d，J＝40.14Hz，H-13″)，6.36(1H，d，J＝23.77Hz，H-13′)。
13C-NMR(400MHz，CDCl
3)：δ208.536(C-12)，174.693(C-3)，171.454(C-5)，145.598(C-4)，137.798(C-11)，125.521(C-13)，45.950(C-14)，41.314(C-2)，38.353(C-6)，37.690(C-7)，36.564(C-8)，35.284(C-1)，31.589(C-9)，12.124(C-10)，8.022(C-15)。
2.3 the selection of moving phase
Selected multiple moving phase condition in the experiment.Select the acetonitrile-water system for use, the ketone acid retention time is too short, and hangover; Select the methanol-water system that does not add acid for use, except retention time was too short, main peak and impurity peaks overlapping phenomenon were serious.We select methyl alcohol-0.2% aqueous formic acid for use at last, and formic acid boiling point and water are near, and Yi Yushui steams together, can not cause that product purity reduces.And best proportioning methyl alcohol-0.2% aqueous formic acid when having determined preparation (65: 35, V/V), best proportioning methyl alcohol-0.2% aqueous formic acid during analysis (70: 30, V/V), make main peak and impurity peaks reach baseline separation like this.
2.4HPLC normalization method is surveyed the condition of content and is selected
Because ketone acid is the quantitative standard substance of using of first batch of markization, thus 245nm selected, 254nm, four of 238nm and 230nm detect wavelength.Selected for use acetonitrile-water (70: 30, V/V) and methyl alcohol-0.2% aqueous formic acid (67: 33, V/V) two kinds of solvent systemss are done the normalization method assay, with several measuring method results' mean value as the assay result.
3, conclusion
The method for preparing rupestonic acid that this experiment is set up, easy to operate, favorable reproducibility, the product purity height of preparation.
(2) rupestonic acid is at the inhibition of preparation hepatitis B virus and the pharmaceutical use of hepatoprotective effect.
Experiment material:
1. be subjected to the reagent thing: rupestonic acid, institute provides by the Xinjiang Uygur medicine, dark brown powder.Be mixed with the mother liquor of 20mg/ml before the experiment with high purity water, filtration sterilization, 4 ℃ of preservations are standby.Be mixed with desired concn with the 2.2.15 cell culture fluid during experiment.
2.2.2.15 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.By the preservation of going down to posterity of my chamber.
3. reagent: hepatitis B surface antigen(HBsAg) detection kit, hepatitis B e antigen detection kit, lot number; 040210, be Huamei Bio-Engrg Co.,'s product; Gene diagnosis center, hepatitis B virus DNA fluorescent quantificationally PCR detecting kit Anda product, lot number: 2004008; G418; The Engls nutrient solution, japanese product.Microplate reader 750, Bayer company product; The ABI5700 quantitative real time PCR Instrument.
4. positive control drug: lamivudine, Ge Lan SmithKline pharmaceutical Co. Ltd lot identification mark: 03100055.Be made into the original liquid of 20mg/ml before the experiment with high purity water, filter-sterilized is standby.
Method and result
1. determining 2.2.15 culturing cell non-toxic concn
Get eugonic 2.2.15 cell, be inoculated in 96 porocyte culture plates, every hole 100ul after waiting to grow up to individual layer, to falling nutrient solution, adds different dilution soups, and each extent of dilution is done 4 multiple holes simultaneously, puts 37 ℃ of 5% CO
2Cultivate in the incubator, changed one time soup on the 4th day, observation of cell form under the every day inverted microscope, continuous 8 days, determines that the minimum extension rate (peak concentration) of obvious regression does not appear in cell, postpones during experiment and accomplishes minimum effective concentration (maximum dilution multiple).Calculate 50% toxic concentration (TC by the Reed-Muench method
50) and maximal non-toxic concentration (TC
0).Cytopathy is judged by six grade standards.
-: the cell growth is normal, and no pathology occurs;
±: cytopathy is less than 10% of whole individual layer;
1: cytopathy accounts for below 25% of whole monolayer cell;
2: cytopathy accounts for below 50% of whole monolayer cell;
3: cytopathy accounts for below 75% of whole monolayer cell;
4: cytopathy accounts for more than 75% of whole monolayer cell.
Table 1 rupestonic acid is to the toxicity of 2.2.15 culturing cell
| Medicine | Index | The cytopathy of different concns medicine (CPE mg/ml) | ||||||
| 1 | 0.5 | 0.25 | 0.125 | 0.062 | 0.031 | 0.015 | ||
| Rupestonic acid | CPE pathology % | 4444 100％ | 4444 100％ | ---- 0％ | ---- 0％ | ---- 0％ | ---- 0％ | ---- 0％ |
| Lamivudine | CPE pathology % | 4444 100 ％ | 4444 100％ | 4444 100％ | 4444 100％ | 2222 50％ | ---- 0％ | ---- 0％ |
Table 2 rupestonic acid is to the TC0 TC50 (mg/ml) of 2.2.15 culturing cell
| Soup | Mother liquor | TC 0 | TC 50 |
| The rupestonic acid lamivudine | 20mg/ml 20mg/ml | 0.25 0.031 | 0.187 0.062 |
Table 1,2 results show: rupestonic acid is to the TC of 2.2.15 culturing cell
0Be 0.25mg/ml, TC
50Therefore when testing for 0.187mg/ml with it as the peak concentration and 4 concentration that postpone.
2. the 2.2.15 cell culture system is secreted the influence of HBsAg, HBeAg and HBV-DNA
Get 2.215 Tissue Culture Plates (24 hole) that grow up to monolayer cell, outwell nutrient solution, add the following corresponding dilution soup of non-toxic concn, concentration is respectively: 0.25,0.125,0.062 and 0.031mg/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, three batches of collected supernatants of experiment are measured HBsAg, HBeAg and HBV-DNA simultaneously.HBsAg, HBeAg adopt the enzyme linked immunological adsorption method to measure, and HBV-DNA adopts fluorescence quantitative PCR method, calculate inhibiting rate.
See Table 3-table 5
Table 3 AR capsule is to the influence of 2.2.15 culturing cell secretion HBsAg
| Batch | Medicine | Index | The cytopathy of different concns medicine (CPE) | ||||||
| 250 (ug/ml) | 125 (ug/ml) | 62(ug/ml) | 31(ug/ml) | 16(ug/ml) | 7.8 (ug/ml) | 3.9(ug/ml) | |||
| First | The contrast of AR capsule lamivudine cell | OD value inhibiting rate % OD value inhibiting rate % | 0.10±0.03** 51.39 0.21±0.05 | 0.10±0.01** 51.47 | 0.11±0.02** 46.89 | 0.11±0.02** 44.72 0.20±0.05 -39.97 0.14±0.06 | 0.14±0.01 0.36 | 0.13±0.02 12.58 | 0.12±0.06 13.81 |
| Second batch | The contrast of AR capsule lamivudine cell | OD value inhibiting rate % OD value inhibiting rate % | 0.09±0.04** 56.79 0.20±10.05 | 0.09±0.01** 55.65 | 0.11±0.02** 45.19 | 0.15±0.05** 28.52 0.15±0.03 -3.89 0.14±0.06 | 0.17±0.04 -16.89 | 0.10±0.02 28.83 | 0.08±0.03 41.38 |
| The 3rd batch | The contrast of AR capsule lamivudine cell | OD value inhibiting rate % OD value inhibiting rate % | 0.09±0.04** 55.89 0.20±0.05 | 0.09±0.02** 55.73 | 0.11±0.03** 44.88 | 0.09±0.01** 58.62 0.15±0.03 -4.00 0.14±0.06 | 0.11±0.02 21.88 | 0.10±0.03 32.42 | 0.09±0.02 37.23 |
Compare with the cell control group:
*P＜0.01
*P＜0.05 AR represents Herba Achilleae
Table 3 result shows: 4 dosage that the AR capsule is tried in three batches of experiments all have the obvious suppression effect to 2.2.15 emiocytosis HBsAg, with control group significant difference (P＜0.01) is arranged relatively, and in 250ug/ml, 125ug/ml, 62ug/ml, 31ug/ml dosage range, present good dose-effect dependency.The positive control drug lamivudine is to 2.2.15 emiocytosis HBsAg unrestraint effect.
Table 4 AR capsule is to the influence of 2.2.15 culturing cell secretion HBeAg
| Batch | Medicine | Index | The cytopathy of different concns medicine (CPE) | ||||||
| 250 (ug/ml) | 125 (ug/ml) | 62(ug/ml) | 31(ug/ml) | 16(ug/ml) | 7.8 (ug/ml) | 3.9(ug/ml) | |||
| First | The contrast of AR capsule lamivudine cell | OD value inhibiting rate % OD value inhibiting rate % | 0.26±0.13* 57.14 0.62±0.16 | 0.45±0.10 27.89 | 0.43±0.23 30.96 | 0.25±0.09** 59.33 0.50±0.11 19.83 0.62±0.16 | 0.37±0.11 39.70 | 0.36±0.12 41.18 | 0.27±0.08 55.34 |
| Second batch | The contrast of AR capsule lamivudine cell | OD value inhibiting rate % OD value inhibiting rate % | 0.27±0.13 39.00 0.45±0.18 | 0.34±0.04 25.43 | 0.35±0.09 23.19 | 0.37±0.12 18.10 0.47±0.12 -3.28 0.45±0.18 | 0.48±0.15 -7.00 | 0.36±0.06 19.14 | 0.28±0.15 37.85 |
| The 3rd batch | The contrast of AR capsule cell | OD value inhibiting rate % | 0.84±0.46* 50.36 1.71±0.21 | 0.86±0.48* 49.42 | 0.91±0.50* 46.46 | 0.93±0.49* 45.58 | |||
Compare with the cell control group:
*P＜0.01
*P＜0.05 AR represents Herba Achilleae
Table 4 result shows: 4 dosage that the AR capsule is tried in three batches of experiments all have the obvious suppression effect to 2.2.15 emiocytosis HBeAg, the first, the 3rd batch of 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.
Table 5 AR capsule is to HBV-DNA synthetic influence in the 2.2.15 culturing cell system
| Batch | Medicine | Index | The HBV-DNA content (copy/ml) of different concns medicine | |||||
| 250(ug/ml) | 125(ug/ml) | 62(ug/ml) | 31(ug/ml) | 16(ug/ml) | 7.8(ug/ml) | |||
| First | The contrast of AR capsule lamivudine cell | Copy value inhibiting rate % copy value inhibiting rate % | 1.88×10 5 58.80** 4.58×10 5 | 1.37×10 5 70.01** | 1.25×10 5 72.73** | 1.23×10 5 73.06** 2.32×10 4 94.92** | 1.93×10 4 95.79** | 1.41×10 4 92.50** |
| Second batch | The contrast of AR capsule lamivudine cell | Copy value inhibiting rate % copy value inhibiting rate % | 3.44×10 5* 34.72 5.27×10 5 | 1.75×10 5 66.79** | 8.43×10 4 84.75** | 2.03×10 5 61.38*8 2.32×10 4 95.58** | 2.56×10 4 95.13** | 2.10×10 4 93.88*8 |
Compare with the cell control group:
*P＜0.01
*P＜0.05 AR represents Herba Achilleae
Table 5 result shows: the AR capsule has the obvious suppression effect to HBV-DNA in the 2.2.15 clone synthetic in the two batches of experiments, with the cell control group significant difference P＜0.01 P＜0.05 is arranged relatively.The positive control rummy is told pyridine also has the obvious suppression effect to synthesizing of HBV-DNA in the 2.2.15 clone.
(3) anti-duck hepatitis B virus test in the rupestonic acid body
Adopt Chongqing sheldrake hepatitis B animal model, observe the effect of anti-duck hepatitis B virus (DHBV) in the rupestonic acid body.Rupestonic acid adopts three dosage: 15mg.Kg in the test
-1.D
-1, 30mg.Kg
-1.D
-1, 60mg.Kg
-1.D
-1, oral administration 28 days, drug withdrawal was observed 7 days, detect medication before, the change situation of DHBV DNA, DHBsAg in the serum after different time points and the drug withdrawal after the medication.The result shows: the big or middle dosage group of rupestonic acid respectively at medication 14 days, began to occur serum DHBV DNA titre in 21 days and reduce (P＜0.05), small dose group occurred serum DHBV DNA titre in 28 days in medication and reduces (P＜0.01), drug withdrawal after 7 days DHBV DNA all do not have the rebound significantly phenomenon; During the medication except that virus control group (starch capsule), the administration of the big or middle dosage group of rupestonic acid after 7 days serum DHBsAgO.D value obviously reduce (P＜0.01), small dose group administration after 28 days serum DHBsAgO.D value obviously reduce (P＜0.01), to drug withdrawal 7 days serum DHBsAgO.D value and medication, relatively still have lasting reduction (P＜0.01).Test-results shows that rupestonic acid has the effect that suppresses duck hepatitis B virus in the duck body, and good dose-effect dependency is arranged.
Test objective:
Observe the effect of anti-duck hepatitis B virus (DHBV) in the rupestonic acid body, for treatment hepatitis B patient provides the preclinical study data.
Experiment material
1. be subjected to the reagent thing: rupestonic acid, institute provides by the Xinjiang Uygur medicine, dark brown powder, lot number: YZH031222.With No. 2 capsulation 3mg～12mg/ capsules.
2. positive control medicine: lamivudine, lot number: B038238, Glaxo Wellcome Britain action company limited produces, and the crushing back is from the capsule of making the 5mg/ grain.
3. animal: adopt 1 age in days duckling of the egg incubation that the Chongqing sheldrake of healthy adult produces, through abdominal cavity inoculation 0.1ml DHBV 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.
Method and result:
1, method:
To infect 55 of positive ducks and be divided into 5 groups at random:
(1) virus control group (11): use the starch capsule, the oral cavity was irritated and was fed 1 time every day, and dosage is 500mg/.。
(2) 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
-1
(3) rupestonic acid small dose group (10): the oral cavity was irritated and was fed 1 time every day, and dosage is 15mg.Kg
-1.D
-1
(4) dosage group (10) in the rupestonic acid: the oral cavity was irritated and was fed 1 time every day, and dosage is 30mg.Kg
-1.D
-1
(5) the heavy dose of group of rupestonic acid (12): the oral cavity was irritated and was fed 1 time every day, and dosage is 60mg.Kg
-1.D
-1
The experimental drug time is 28 days, and drug withdrawal was observed 7 days.Observation index is as follows:
(1) 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 Vuego Scan (Brisa-620ST) scanner and scan; With the Discovery Series Quantity One software spot is carried out quantitative analysis, the spot value is volume (volume=intensity * mm
2).Statistics adopts spss software, paired t-test.
(2) 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.
The result:
(1) serum DHBV DNA titre changes situation before, during and after the medication: serum DHBV DNA titre obviously raise before than medication (P＜0.01) during virus control group (starch capsule) medication.The lamivudine group medication begins to have serum DHBVDNA titre and reduces (P＜0.01) after 7 days, have DHBV DNA that rise trend is arranged after the drug withdrawal, but with medication before relatively, still have obvious reduction (P＜0.01).The big or middle dosage group of rupestonic acid respectively at medication 14 days, began to have serum DHBV DNA titre in 21 days and reduce (P＜0.05), small dose group occurred serum DHBV DNA titre in 28 days in medication and reduces (P＜0.01), drug withdrawal after 7 days DHBV DNA all do not have the rebound significantly phenomenon.See Table 6.
Serum DHBV DNA titre change situation before and after table 6 medication (x ± S)
| Group | DHBV DNA(volume) | ||||||||
| Before the | Medication | 7 | Medication | 14 days | Medication 21 | Medication | 28 | Drug withdrawal | 7 days |
| The heavy dose of group of dosage group rupestonic acid in the starch Capsules group lamivudine group rupestonic acid small dose group rupestonic acid | 2118.87± 362.68 2168.25± 460.65 2610.60± 521.08 2525.92± 501.77 2906.33± 267.27 | 2357.63± 411.44** 1872.74± 377.00** 2538.80± 699.39 2266.32± 614.76 2656.14± 509.63 | 2543.40± 506.19** 1939.27± 432.92* 2755.97± 796.88 2267.01± 721.83 2641.14± 388.94* | 2722.19± 556.65** 1635.60± 264.03** 2375.61± 769.29 2084.23± 561.52* 2495.21± 485.19** | 2651.80± 518.38** 1549.34± 257.01** 2033.46± 622.31** 1991.35± 516.50* 2312.86± 542.91** | 2572.17± 460.56** 1735.17± 255.06** 1996.23± 522.44** 1847.35± 410.37** 2213.52± 410.84** | |||
Annotate: go up table mean number and the standard deviation of respectively organizing DNA spot volume value of classifying as, statistics adopts paired t-test (" * ": P＜0.05; " * * ": P＜0.01), be the comparison of DNA spot value before different time DNA spot value and the medication on the same group.
(2) respectively organize serum DHBsAgO.D value change situation before, during and after the medication:
As time goes on, each is organized the DHBsAgO.D value and presents downtrending, learns relatively the change not statistically significant of serum DHBsAgO.D value before and after virus control group (starch capsule) medication by statistics.The administration of the big or middle dosage group of rupestonic acid after 7 days serum DHBsAgO.D value obviously reduce (P＜0.01), small dose group administration after 28 days serum DHBsAgO.D value obviously reduce (P＜0.01), to drug withdrawal 7 days serum DHBsAgO.D value and medication, relatively still have lasting reduction (P＜0.01).See Table 7.
Table 7 is respectively organized (the x ± S) of serum DHBsAgO.D value change situation before and after the medication
| Group | Before the | Medication | 7 | Medication | 14 days | Medication 21 | Medication | 28 | Drug withdrawal | 7 days |
| The heavy dose of group of dosage group AR capsule in the starch Capsules group lamivudine group AR capsule small dose group AR capsule | 0.503±0.423 0.479±0.278 0.837±0.492 1.253±0.487 0.905±0.665 | 0.595±0.540 0.392±0.196 0.814±0.508 1.058±0.500 ** 0.733±0.604 * | 0.556±0.472 0.425±0.194 0.856±0.470 0.890±0.460 ** 0.587±0.483 ** | 0.438±0.338 0.272±0.130 ** 0.679±0.391 0.806±0.432 ** 0.473±0.379 ** | 0.331±0.281 0.213±0.097 ** 0.456±0.268 ** 0.708±0.417 ** 0.429±0.311 ** | 0.232±0.137 0.161±0.072 ** 0.397±0.263 ** 0.393±0.174 ** 0.203±0.119 ** |
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 DHBsAgO.D value before different time DHBsAgO.D value and the medication on the same group.AR represents Herba Achilleae
Brief summary:
Study human hepatitis B pathogeny, virus replication and screen effective medicine as the hepatitis B animal model with the DHBV infected duck, generally acknowledged by Chinese scholars.1-3 age in days duckling infects DHBV and can keep long-term viremia and not have the tangible phenomenon of turning out cloudy naturally, therefore, we use 1 age in days duckling and set up the examination that the duck hepatitis-B animal model carries out the antiviral curative effect of rupestonic acid through the method for abdominal cavity infection DHBV, and expectation filters out the medicine of anti-hepatitis B virus effect.And use and have obvious inhibiting nucleotide analog lamivudine to contrast as positive drug to hepatitis B virus.
This animal test results shows: rupestonic acid is big, middle dosage group was respectively at medication 14 days, began to have serum DHBV DNA titre in 21 days and reduce (P＜0.05), small dose group occurred serum DHBV DNA titre in 28 days in medication and reduces (P＜0.01), drug withdrawal after 7 days DHBV DNA all do not have the rebound significantly phenomenon, in addition, rupestonic acid is big, middle dosage group administration is the obviously reduction (P＜0.01) of serum DHBsAgO.D value after 7 days, the small dose group administration is the obviously reduction (P＜0.01) of serum DHBsAgO.D value after 28 days, to drug withdrawal 7 days serum DHBsAgO.D value and medication, relatively still have lasting reduce (P＜0.01).The above results explanation rupestonic acid has the effect that suppresses duck hepatitis B virus in the duck body, and good dose-effect relationship is arranged.
(4) hepatoprotective effect of rupestonic acid research
Test objective: observe protecting the liver of rupestonic acid, face research data before the window for the clinical study of medicine provides.
Material
1, be subjected to the reagent thing: rupestonic acid provides lot number by Xinjiang Uygur medicine institute: 030502.This product is brown to chocolate brown powder; Gas perfume (or spice), mildly bitter flavor has water absorbability.
2, positive control drug: kurarinone capsule, its content are white or off-white powder and particle, and five is smelly, bitter; Jiangsu Zhengda Tianqing Drug Industry Co., Ltd produces, lot number: 030605.
3, experiment reagent: tetracol phenixin, the new Photochemical agents in Beijing factory product; Fine Chemistry Division of Beijing Science and Technology Coorporation Centre's product;
4, laboratory apparatus: automatic clinical chemistry analyzer, Italian AUTOLAB; DT200 type multifunction electronic balance, Shanghai balance factory; The TGL-16G refrigerated centrifuge, Shanghai scientific instrument factory product.
5, experimental animal: Kunming mouse, body weight 20 ± 2g, the SPF level, male and female half and half are all purchased Shanghai Slac Experimental Animal Co., Ltd./Chinese Academy of Sciences's Shanghai Experimental Animal Center.
6, laboratory animal room: laboratory animal room of Uigur Medical Inst., Uigur Autonomous Region, Xinjiang scope of application is barrier environment (rodents), uses animal to be the SPF level.Laboratory animal occupancy permit number: SYXK (newly) 2003-0002.
1. rupestonic acid causes the provide protection of chmice acute liver injury to tetracol phenixin
Get 80 of Kunming mouses, according to the form below is divided into 8 groups at random, ig administration, every day 1 time, totally 7 days.2h after the last administration, other respectively organizes ip in mice 0.1% tetracol phenixin peanut oil solution 10ml/kg except that the blank group.Eyeball is got blood behind the 16h, and conventional separation of serum is measured GPT, GOT content.
Table 8 rupestonic acid causes the influence (x ± SD) of liver injury mice serum GPT, GOT to tetracol phenixin
| Group | Number of animals | Dosage (mg/kg) | GPT(iμ/L) | GOT(iμ/L) |
| Blank group liver injury model group Oxymatrine Capsule group rupestonic acid rupestonic acid rupestonic acid rupestonic | 10 10 10 10 10 10 10 10 | - - 50 80 160 320 640 1280 | 45.70±9.88 218.90±104.99 △△△ 62.20±21.36 *** 82.40±34.62 *** 55.60±15.97 *** 60.00±17.96 *** 49.30±12.83 *** 56.00±16.12 *** | 145.00±21.96 521.10±213.05 △△△ 163.70±32.88 *** 172.60±39.48 *** 162.10±37.98 *** 158.90±33.09 *** 135.20±25.42 *** 128.30±20.08 *** |
Annotate: compare with normal group
△ △ △Compare with model group p＜0.01
*P＞0.05,
* *P＜0.01.
The result shows: the rupestonic acid of various dose obviously reduces CCL
4Cause serum GPT of liver injury mouse and the content of GOT.
2. rupestonic acid is to the provide protection of the salt-induced chmice acute liver injury of D-Gal ammonia hydrochloric acid
Get 60 of Kunming mouses, according to the form below is divided into 6 groups at random, ig administration, every day 1 time, totally 7 days.1h after the last administration, other respectively organizes ip in mice D-Gal 800mg/kg except that the blank group, and eyeball is got blood behind the 24h, and conventional separation of serum is measured GPT, GOT, ALP content.
Table 9 rupestonic acid is to the influence of the salt-induced acute liver damage mice serum of D-Gal ammonia hydrochloric acid GPT, GOT, ALP (x ± SD)
| Group | Number of animals | Dosage (mg/kg) | GPT (iμ/L) | GOT (iμ/L) | ALP (iμ/L) |
| Blank group liver injury model group Oxymatrine Capsule group rupestonic acid rupestonic | 10 10 10 10 10 10 | - - 50 80 160 320 | 30.16±6.39 209.16±87.90 △△△ 82.25±35.38 *** 157.25±49.50 *** 133.16±22.90 *** 144.08±21.40 *** | 125.00±17.69 816.92±89.28 △△△ 173.33±45.27 *** 510.75±155.31 *** 413.92±76.72 *** 356.42±61.24 *** | 273.66±49.82 881.92±174.94 △△△ 413.75±109.20 *** 493.58±171.22 *** 363.25±95.36 *** 392.66±54.44 *** |
Annotate: compare with the blank group
△ △ △Compare with model group p＜0.01
* *P＜0.01.
The result shows: the rupestonic acid of three various dose obviously reduces serum GPT, GOT and the ALP content of the salt-induced acute liver damage mouse of D-Gal ammonia hydrochloric acid.
3. rupestonic acid is to the prophylactic effect of mouse immune hepatitis
Get 72 of Kunming mouses, ♀ ♂ half and half, according to the form below are divided into 6 groups at random, in iv LPS administration in preceding 5 days, every day 1 time, totally 5 days.Other respectively organizes mouse tail vein iv BCG 5 * 10 except that the blank group
6Individual bacterium/mouse, iv LPS 7.5 μ g/ mouse again behind the 12d are caused the autoallergic model, and eyeball is got blood behind the 12h, and conventional separation of serum is measured GPT, GOT content.
Table 10 rupestonic acid is to the prophylactic effect of mouse immune hepatitis (x ± SD)
| Group | Number of animals (only) | Dosage (mg/kg) | GPT (iu/L) | GOT (iμ/L) |
| Blank group model group kurarinone Capsules group rupestonic acid rupestonic | 12 12 12 12 12 12 | - - 50 80 160 320 | 17.08±3.45 59.75±18.30 △△△ 33.58±15.88 *** 42.83±25.33 * 45.58±22.37 * 27.33±11.38 *** | 140.92±27.84 192.92±92.45 156.67±54.92 198.50±46.33 181.92±35.92 154.75±44.99 |
Annotate: compare with the blank group
△ △ △Compare with model group p＜0.01
*P＞0.05,
* *P＜0.01.
The result shows: the rupestonic acid of three various dose can reduce the content of the serum GPT of mouse immune liver damage mouse.
Conclusion
Rupestonic acid experimentation on animals gastric infusion 80,160,320mg/kg, qd, 5-7d obviously reduces CCL
4Cause serum GPT of liver injury mouse and the content of GOT; Significantly reduce serum GPT, GOT and the ALP content of the salt-induced acute liver damage mouse of D-Gal ammonia hydrochloric acid; And can reduce the content of the serum GPT of mouse immune liver damage mouse.Prompting, rupestonic acid has tangible hepatoprotective effect.
Claims (2)
1, a kind of preparation method of the monomeric compound rupestonic acid that from Herba Achilleae, extracts, it is characterized in that adopting conventional chromatography and high performance liquid chromatography bonded method to prepare rupestonic acid, at first carrying out raw material handles: the Herba Achilleae herb is pulverized the back with 95% alcohol reflux 4 times, time is respectively 1-5h, 30min-4h, 15min-2h, 30min-5h, again extracting solution is merged, concentrating under reduced pressure becomes paste, uses ethyl acetate extraction 3 times, and extraction liquid concentrates mixes 100-200 order silicagel column on the silica gel, under 30-60 ℃, volume ratio is 3: 1 a petroleum ether-ethyl acetate wash-out, and the product that elutes concentrates standby; The standard substance preparation: the crude product of chromatography is dissolved in methanol solution, and ultrasonic 30min is with half preparative high-performance liquid chromatographic instrument purifying, collecting retention time is the component of 18.5min, it at 60 ℃ of following concentrating under reduced pressure, then at 40 ℃ of thermostatic drying chamber inner drying 24h, is obtained colourless bar-shaped crystallization at last.
2, preparation method as claimed in claim 1, the rupestonic acid that wherein makes is used to prepare the medicine that suppresses hepatitis B virus and hepatoprotective effect.
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| CN101337882B (en) * | 2008-08-07 | 2011-05-11 | 中国科学院新疆理化技术研究所 | Method for purifying rupestonic acid and chrysosplenetin B by high speed reversed flow chromatography |
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| CN1380094A (en) * | 2002-04-29 | 2002-11-20 | 斯拉甫·艾白 | Achillea total flavone capsule |
| CN1454664A (en) * | 2003-05-29 | 2003-11-12 | 斯拉甫·艾白 | Liver-recovering capsule |
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| CN1380094A (en) * | 2002-04-29 | 2002-11-20 | 斯拉甫·艾白 | Achillea total flavone capsule |
| CN1454664A (en) * | 2003-05-29 | 2003-11-12 | 斯拉甫·艾白 | Liver-recovering capsule |
Non-Patent Citations (2)
| Title |
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| 《新疆一枝蒿化学成分的研究》 王燕 等，《新疆医科大学学报》，第27卷第4期 2004 * |
| 《新疆一枝蒿新倍半萜成分－一枝蒿酮酸的结构》 徐广顺 等，《药学学报》，第23卷第2期 1988 * |
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