CN1164314C - Achillea total flavone capsule - Google Patents

Abstract

The present invention relates to an achillea total flavone capsule. The present invention uses achillea as a raw material; the achillea is thoroughly pulverized, is extracted by ethanol, is degreased by petroleum ether, is extracted by ethyl acetate, and is concentrated into extract, wherein the content of total flavone and ketonic acid of effective regions is from 50 to 80%; then the extract is dried in vacuum to prepare dry paste, and starch is added to prepare particles; the particles are dried at a low temperature, are finished, and then are filled into capsules to prepare finished products. The capsule is an oral medicine used for treating viral hepatitis and various kinds of liver damage, and has the advantages of small doses and high therapeutic effect. Animal experiments and clinical observation show that the present invention does not have toxic or side effect, and the total effective rate of treatment is 91.0%.

Description

Achillea total flavone capsule

Technical field

The present invention relates to a kind ofly prepare Achillea total flavone capsule, be used to cure mainly the medicine of viral hepatitis and all kinds of hepatic injury diseases by Chinese herbal medicine.

Background technology

Viral hepatitis, hepatic 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 hepatic 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 hepatic 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 Chinese and western medicine curative effect of this class.For example, the Western medicine interferon 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 heat-clearing and toxic substances removing, and what have focuses on activating blood circulation to dissipate blood stasis, the nourishing the liver and kidney that has 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 injury, and are unfavorable to the hepatopathy recovery from illness on the contrary.

The present invention is directed to the deficiency that in the Drug therapy hepatopathy, exists at present,, developed a kind of oral medicine Achillea total flavone capsule that is used for the treatment of viral hepatitis and all kinds of hepatic injury in conjunction with Chinese medicine and both length of national medicine theory.

Summary of the invention

The object of the invention is, the oral medicine Achillea total flavone capsule of development is to be raw material with the Herba Achilleae, with the Herba Achilleae herb pulverize, with ethanol extraction, defat with petroleum ether, ethyl acetate extraction, concentratedly make extractum, wherein effective site total flavones and keto acid class content are 50%-80%; Then extractum is made dried cream through vacuum drying, add starch and make granule,, incapsulate after the arrangement and get product through cold drying.This capsule is used for the treatment of the oral medicine of viral hepatitis and all kinds of hepatic injury, and it is little to have dosage, and the curative effect height has no side effect through animal experiment and clinical observation, and the treatment total effective rate is 91.0%.

Achillea total flavone capsule of the present invention is to be raw material with the Herba Achilleae, the Herba Achilleae herb is pulverized, made the extractum that relative density is 1.10-1.30 with 95% ethanol extraction, defat with petroleum ether, ethyl acetate extraction, concentration step, and wherein effective site total flavones and keto acid class content are 50 ℃-80%; Then extractum is made dried cream through vacuum drying, add starch and make granule,, incapsulate after the arrangement and get product through cold drying.

The preparation method of Achillea total flavone capsule, press routine step and carry out:

A, at first the Herba Achilleae herb is ground into powder, the alcohol reflux with 95% three times, 70 ℃-80 ℃ of reflux temperatures carry out defat with the alcohol extract after extracting with petroleum ether, filter, and the elimination petroleum ether layer keeps its residue;

B, the residue that keeps is extracted three times with ethyl acetate, extraction temperature is a room temperature, filter, elimination ethyl acetate insoluble matter, remaining ethyl acetate layer is decoloured with the 0.5%-1% active carbon, it is the extractum of 1.10-1.30 that concentrating under reduced pressure becomes relative density, and the effective site of this extractum is total flavones and keto acid class, and its content is 50%-80%;

C, the extractum after will concentrating gets dry extract through 40 ℃ of-60 ℃ of vacuum dryings then, dried cream is added starch be modulated into granule, and through 40 ℃ of-60 ℃ of cold drying, arrangement incapsulates and gets product.

Achillea total flavone capsule of the present invention, wherein the pharmacological properties of Herba Achilleae is:

The dry herb of Herba Achilleae Artemisia rupestris L..Seven, tap August, dry.10-50 centimetre of this product total length.Root and root are cylindrical, and khaki is to taupe, and length is with minority short hairs root, and section is light yellow.Stem single or several, in the bending of rhizome place, diameter 1.5-3 centimetre, the close tomentellate of sprout and spray top, the bottom of old branch or branch is smooth, inapparent again stria, the surface is an aubergine often, section white, hollow.Function with cure mainly: heat-clearing and toxic substances removing, invigorating the stomach and promoting digestion, expelling wind and activating blood circulation.Be used for flu, allergy, food stagnation, snakebite, furuncle.

The pharmaceutical research of Achillea total flavone capsule of the present invention:

Herba Achilleae is the Uygur medicine medicinal herbs most in use, and bibliographical information Herba Achilleae herb contains chemical compounds such as flavonoid, keto acid class, aminoacid, glycoside, polysaccharide, volatile oil, polypeptide, alkaloid.We have analyzed the Herba Achilleae effective ingredient, and experimentatioies such as p53 and Bcl expression of gene when having carried out hepatoprotective effect, treatment autoallergic, antioxidation, the anti-allergic effects research of Achillea total flavone capsule on this basis and having induced apoptosis of human hepatoma cell, and adopt electron paramagnetic resonance method detection Achillea total flavone capsule to O ₂ ^-, OH inhibitory action, be intended to illustrate the scientific meaning of this medicine disease preventing and treating.

One, experiment material

1. medicine: Achillea total flavone capsule is by the invention provides.

2. reagent: histamine phosphate, Shanghai Inst. of Biochemistry, Chinese Academy of Sciences's product; Freeze Dried Bacillus Calmette-Guerin Vaccine, Lanzhou Institute of Biological Products's product; Radix Trichosanthis, pharmacology teaching and research room of preclinical medicine institute of Xinjiang Medicine University provides; Carbon tetrachloride, the new Photochemical agents in Beijing factory product; D-Gal ammonia hydrochloric acid salt, Fine Chemistry Division of Beijing Science and Technology Coorporation Centre's product; Lipopolysaccharide, SIGMA company product; 5-N-nitride (DMPO) is available from U.S. Sigma company.Dimethyl sulfoxide (DMSO), chemically pure reagent, Shanghai Sulphuric Acid Plant product.Sodium hydroxide, analytical reagent, Shanghai reagent three factory's products.NaH ₂PO ₄, K ₂HPO ₄, H ₂O ₂, FeSO ₄.7H ₂O, EDTA is homemade analytical reagent.QGY-7701 (people's hepatocarcinoma) cell is provided by Shanghai cell biological institute of Chinese Academy of Sciences cell bank; Burnt ethylene two fat (DEPC), random primer (Oligo[dT] ₁₅Primer), deoxyribonuclease inhibitor (Rnasin), deoxynucleoside triphosphate (dNTP) are the Promega product; Reverse transcription (M-MuLV ReverseTranscriptase), MBI Fermentas product; Taq archaeal dna polymerase (5u), pcr amplification buffer, worker (Sangon) product is given birth in Shanghai; Marker (φ χ 174/Hinc IIdigest) is Japan (TOYOBO) company product.

3. animal: Wistar rat, body weight 200 ± 20g, secondary; The NIH mice, body weight 20 ± 1.4g, secondary, male and female are partly individual, all available from Department of Public Health of autonomous region medical experiment animal center.

4. instrument: 721-type spectrophotometer, Shanghai the 3rd analytical tool factory; Vital-200 type full-automatic biochemical analyzer, Dutch Rittal GmbH product; ER200D-SRC type electron paramagnetic resonance instrument and ER4111-VT changing device, German Bruker company product; PCR thermal cycler (Gene Amp PCR System 9700 types), Perkin-Elmer company; Ultraviolet photometer (Gene Quant ^TMII RNA/DNA Calculator type), Amersham pharmaciabiotech company.

Two, method and result

(1) Achillea total flavone capsule antiallergic research

1. the homology models of passive skin irritability of rats is reacted the influence of (PCA)

Get 5 of healthy male rats, the every sufficient sole of the foot injection 0.1ml Radix Trichosanthis of each Mus-gel aluminum hydroxide suspension (5mg/ml), ip 0.1ml bacillus calmette-guerin vaccine is strengthened heart extracting blood after 15 days, the centrifugal antiserum that obtains simultaneously.With preceding antiserum is diluted 10 times with normal saline.Other gets rat peace table 1 random packet, at each Mus back depilation place subcutaneous injection antiserum 0.1ml sensitization, totally 3 points.0.5% azovan blue solution of iv 5me (5mg) Radix Trichosanthis is attacked behind the 48h.In sensitization beginning in preceding 1 day administration, successive administration 3 days.Antigen is attacked back 0.5h, and the sacrificed by decapitation rat is centrifugal with the digestion of skin locus coeruleus, gets supernatant spectrophotometric determination trap (A).The results are shown in Table 1.

The influence that table 1 Achillea total flavone capsule reacts P of Rats CA (X ± SD)

Group number of animals dosage (g/kg) trap A suppression ratio (%)

Blank group 5-0.96 ± 0.14-

Dexamethasone group 5 0.01 0.04 ± 0.02 ^{* *}95.8

Administration 6 0.50 0.38 ± 0.07 ^{* *}60.1

Administration 6 1.00 0.11 ± 0.02 ^{* *}88.5

Compare with the blank group ^{* *}P＜0.01.AR represents Herba Achilleae

The result shows that Achillea total flavone capsule has the effect of obvious suppression homology P of Rats CA reaction, and has the direct ratio dependency relationships between concentration and the inhibitory action.

2. the influence that capillary permeability due to the histamine is increased

Get 26 of rats, the ig administration, once a day, totally 5 days, 15h rat back cropping place intradermal injection histamine phosphate normal saline solution 0.1ml (750 μ g/ml) after the last administration, every Mus 2 points, iv 1ml 0.5% azovan blue solution simultaneously, 0.5h back sacrificed by decapitation rat is pressed the trap (A) that the PCA method is measured each reflecting point of skin.The results are shown in Table 2.

The influence that table 2 Achillea total flavone capsule increases capillary permeability due to the histamine phosphate

( X±SD)

Group number of animals dosage (g/kg) trap A suppression ratio (%)

Blank group 7-0.92 ± 0.29-

Dexamethasone group 6 0.01 0.38 ± 0.11 ^{* *}58.7

Administration group 7 0.50 0.73 ± 0.16 ^{* *}20.6

Administration group 6 1.00 0.70 ± 0.22 ^{* *}23.6

Compare with the blank group ^{* *}P＜0.01.

The result shows, Achillea total flavone capsule has the effect that capillary permeability increases due to the obvious suppression histamine, and has the direct ratio dependency relationships between concentration and the inhibitory action.

3. to the influence of the antibody-mediated mast cell degranulation of rat allogenic cell

Get 5 of rats, prepare rat anti Radix Trichosanthis serum by the PCA method.Other gets 5 of rats, preparation rat mesentery specimen, 10 of every Mus, every 1cm ²Put into Tai Shi nutritional solution 2ml, 1 of mesentery specimen and rat anti Radix Trichosanthis serum 0.2ml wash specimen 3 times with 37 ℃ of Tai Shi nutritional solutions behind 37 ℃ of water-bath incubation 0.5h, add Tai Shi nutritional solution 2ml again, 37 ℃ of water-bath incubation 10min press the dosing in pipe of table 3 dosage, continue incubation 10min, adding Radix Trichosanthis antigen 0.1ml (1.1mg/ml) attacks, with the specimen formaldehyde fixed, normal dyeing and mounting are with microscopic examination and count the mast cell degranulation situation behind 37 ℃ of incubation 20min.The results are shown in Table 3.

Table 3 Achillea total flavone capsule rings the antibody-mediated mastocyte of rat allogenic cell

( X±SD)

Group number of animals (only) dosage (μ g/ml) takes off granule rate (%) suppression ratio (%)

Blank group 10-71.7 ± 8.2-

Dexamethasone group 10 50 34.2 ± 9.6 ^{* *}52.9

Administration group 10 50 30.1 ± 8.7 ^{* *}30.1

Administration group 10 100 35.8 ± 7.6 ^{* *}50.1

Compare with the blank group ^{* *}P＜0.01.

The result shows that Achillea total flavone capsule has inhibitory action to the antibody-mediated mast cell degranulation of rat allogenic cell, and with the increase of drug level, inhibitory action is also along with increase.

(2) Achillea total flavone capsule protects the liver antihepatitic activity research

1. Achillea total flavone capsule causes the protective effect of chmice acute hepatic injury to carbon tetrachloride

Get 60 of Kunming mouses, be divided into 6 groups at random, ig administration, every day 1 time, totally 7 days by table 1.1h after the last administration, other respectively organizes ip in mice 0.1% carbon tetrachloride peanut oil solution 10ml/kg except that the blank group.Eyeball is got blood behind the 24h, and conventional separation of serum is measured ALT content, the results are shown in Table 4.

Table 4 Achillea total flavone capsule causes the influence of hepatic injury mice serum ALT to carbon tetrachloride

( X±SD)

Group number of animals (only) dosage (g/kg) ALT (i μ/L)

Blank group 9-34.06 ± 11.60

Liver injury model group 9-128.80 ± 57.36 ^Δ

Radix Glycyrrhizae glucin sheet group 9 0.3 18.86 ± 8.81 ^{* *}

Low dose of administration group 9 1.0 42.74 ± 25.69 ^{* *}

Middle dosed administration group 9 2.0 21.81 ± 7.43 ^{* *}

Heavy dose of administration group 9 4.0 16.98 ± 7.98 ^{* *}

Annotate: compare with the blank group ^ΔCompare with the liver injury model group p＜0.01 ^{* *}P＜0.01.

The result shows that Achillea total flavone capsule obviously reduces CCl ₄Cause the Serum ALT content (p＜0.01) of hepatic injury mice, effect is better than Radix Glycyrrhizae glucin sheet.

2. Achillea total flavone capsule causes the protective effect of rat acute toxic liver injury to carbon tetrachloride

Get 42 of Wistar rats, be divided into 6 groups at random, ig administration, every day 1 time, totally 7 days by table 2.Other respectively organized rat sc 25% carbon tetrachloride 0.5ml/100g except that the blank group in the 3rd day, the 7th day after the administration.Put to death rat heart behind the last administration 12h and get blood, conventional separation of serum is measured ALT content, the results are shown in Table 5.

Table 5 Achillea total flavone capsule causes acute poisoning liver damage rat blood serum ALT to carbon tetrachloride

Influence (X ± SD)

Group number of animals (only) dosage (g/kg) ALT (i μ/L)

Blank group 7-20.50 ± 12.82

Liver injury model group 7-180.07 ± 10.40 ^Δ

Radix Glycyrrhizae glucin sheet group 7 0.2 155.67 ± 50.98 ^*

Low dose of administration group 7 0.75 147.97 ± 25.78 ^*

Middle dosed administration group 7 1.5 115.57 ± 35.03 ^{* *}

Heavy dose of administration group 7 3.0 129.63 ± 21.43 ^{* *}

Annotate: compare with the blank group ^ΔCompare with the liver injury model group p＜0.01 ^*P＞0.05, ^*P＜0.05, ^{* *}P＜0.01.

The result shows that the Achillea total flavone capsule of various dose obviously reduces CCl ₄Cause the Serum ALT content (p＜0.01) of acute poisoning liver damage rat, effect is better than Radix Glycyrrhizae glucin sheet

3. Achillea total flavone capsule is to the protective effect of the salt-induced chmice acute hepatic injury of D-Gal ammonia hydrochloric acid

Get 60 of Kunming mouses, be divided into 6 groups at random, ig administration, every day 1 time, totally 7 days by table 3.1h 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 ALT content, the results are shown in Table 6.

The result shows that the Achillea total flavone capsule of various dose obviously reduces the Serum ALT content (p＜0.01) of the salt-induced Rats with Acute Liver Injury of D-Gal ammonia hydrochloric acid

Table 6 Achillea total flavone capsule is to the salt-induced acute liver damage mice of D-Gal ammonia hydrochloric acid

The influence of Serum ALT (X ± SD)

Group number of animals (only) dosage (g/kg) ALT (i μ/L)

Blank group 9-53.89 ± 19.23

Liver injury model group 9-81.13 ± 40.18 ^Δ

Radix Glycyrrhizae glucin sheet group 8 0.15 54.28 ± 10.12 ^*

Low dose of administration group 8 1.0 57.38 ± 15.53 ^*

Middle dosed administration group 9 2.0 56.83 ± 20.60 ^*

Heavy dose of administration group 8 4.0 50.50 ± 17.78 ^*

Annotate: compare with the blank group ^ΔCompare with the liver injury model group p＜0.01 ^*P＜0.05.

4. Achillea total flavone capsule is to the preventive effect of mouse immune hepatitis

Get 51 of Kunming mouses, ♀ ♂ half and half is divided into 5 groups at random by table 4, 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/Mus, iv LPS 7.5 μ g/ Mus again behind the 12d are caused the autoallergic model, and eyeball is got blood behind the 10h, and conventional separation of serum is measured ALT content, the results are shown in Table 7.As a result, Achillea total flavone capsule does not have preventive effect in ivLPS administration in preceding 5 days to the mouse immune hepatitis.

Table 7 Achillea total flavone capsule is to the preventive effect of mouse immune hepatitis (X ± SD)

Group number of animals (only) dosage (g/kg) ALT (i μ/L)

Blank group 11-55.20 ± 8.90

Hepatitis model group 11-79.10 ± 22.80 ^Δ

Radix Glycyrrhizae glucin sheet group 10 0.3 76.90 ± 32.10 ^*

Low dose of administration group 10 1.0 72.60 ± 21.90 ^*

Middle dosed administration group 9 2.0 77.10 ± 21.70 ^*

Annotate: compare with the blank group ^ΔCompare with the hepatitis model group p＜0.01 ^*P＞0.05.

5. Achillea total flavone capsule is to the therapeutical effect of mouse immune hepatitis

Get 51 of Kunming mouses, ♀ ♂ half and half is divided into 5 groups at random by table 5.Other respectively organizes mouse tail vein iv BCG 5 * 10 except that the blank group ⁶Individual bacterium/Mus, ivLPS 7.5 μ g/ Mus again after 10d days are caused the autoallergic model.Each is organized mice and began ig administration (normal saline of capacity such as normal control group and hepatitis model group mice ig) on the same day of iv BCG, every day 1 time, 11d continuously.Eyeball is got blood behind iv LPS 10h, and conventional separation of serum is measured ALT content, the results are shown in Table 8.

As a result, Achillea total flavone capsule continues administration behind ivLPS, to the Serum ALT content (p＜0.01) of autoallergic mice.

Table 8 Achillea total flavone capsule is to the therapeutical effect of mouse immune hepatitis (X ± SD)

Group number of animals (only) dosage (g/kg) ALT (i μ/L)

Blank group 11-26.45 ± 11.81

Hepatitis model group 11-31.45 ± 21.83 ^Δ

Low dose of administration group 10 1.0 35.70 ± 8.29

Middle dosed administration group 10 2.0 39.00 ± 8.10

Heavy dose of administration group 10 5.0 13.36 ± 2.93 ^{* *}

Annotate: compare with the blank group ^ΔCompare with the hepatitis model group p＜0.05 ^{* *}P＜0.01.

Above experimental result shows that Achillea total flavone capsule is to CCl ₄Or the salt-induced acute liver damage of D-Gal ammonia hydrochloric acid has protective effect: and can treat autoallergic.

(3) Achillea total flavone capsule Study on antioxidation

1. to O ₂ ^-Inhibitory action

The DMSO and the H that at room temperature, will contain saturated air ₂O, the quantitative mixing of NaOH solution.NaOH is timing once adding, reaction 30min.Before the measurement, quantitatively draw reactant liquor in the hyaloid QC of diameter 3mm, carry out ESR and measure when temperature 130K, can detect O this moment ₂ ^-Characteristic signal, this is as the blank pipe.As test sample and O ₂ ^-Do the time spent, add quantity of sample medicinal liquid (5%-100%) in producing O ₂ ^-Model system in, other condition is the same.

EPR operating parameter: probe temperature (TE) 130K, microwave frequency (SF) 9.67GHz, microwave power (SP) 20mW, modulating frequency (MF), modulation amplitude (MA) 5.0G, time constant (TC) 500ms, field sweep time (TI) 100s, central magnetic field (CF) 3360G, field sweep width (SW) 300G.

2. to the inhibitory action of OH

Utilize the Fenton reaction to produce OH free radical, DMPO final concentration 100 μ M in the reaction system, PH7.4NaH ₂PO ₄/ K ₂HPO ₄Buffer final concentration 40mM, H ₂O ₂Final concentration 100mM, the sample medicinal liquid (is annotated: then add 100 μ lH during as standard ₂O) 5%-100%, FeSO ₄.7H ₂O+EDTA mixes liquid final concentration 300 μ M, adds DMPO, buffer, H successively at plastic tube during operation ₂O ₂, medicinal liquid (or water) adds Fe at last ²⁺Initiation reaction is carried out EPR and is measured behind the 5min.

EPR operating parameter: SW=100G, SF=9.82GHz, MF=100kHz, MA=0.8G, TE=297K.

The result: according to measured EPR spectroscopic signal, the interaction factor E between calculation sample and the free radical.According to the situation of change of E, each concentration of judgement sample is to the effect of pressing down that increases of free radical.The results are shown in Table 9, Fig. 1.

Table 9 Achillea total flavone capsule is to O ₂ ^-, the OH free radical increase the effect of pressing down (relation of concentration and interaction factor E)

Interaction factor E

Sample concentration (g/ml)

The ultra-oxygen anion free radical hydroxy radical

0.10 -0.9999 -0.9889

0.08 -0.9999 -0.9885

0.06 -0.4858 -0.9577

0.04 -0.3053 -0.9020

0.02 0.9655 -0.7327

0.01 0.8963 -0.6659

0.005 0.4825 -0.4855

By table 9, Fig. 1 as seen, Achillea total flavone capsule is to O ₂ ^-Effect show as the effect that high concentration suppresses, low concentration increases.But the OH free radical is shown as concentration dependent ground inhibitory action, and the regression relation between concentration (M) and the interaction factor (E) is:

E＝-1.4199-0.1709 lnM(r＝0.9887)，IC ₅₀＝0.00460g/ml。

(4) expression of Achillea total flavone capsule mediator hepatoma cell apoptosis gene p53, Fas and Bc1-2

1. primer sequence has designed two primers by p53 gene order in document and the Gen Bank storehouse, p1:5 '-GGTCGACTGACACGCTTCCCTGGATTGG-3 ', p2:5 '-GGATCCTGCTTCTGACGCACACCTATTG-3 ' (295bp fragment).Having designed Bcl-2 and Fas gene order: Bcl-2 by document [1] is 5 '-GCGTCAACCGGGAGATGTCGCCC, 3 '-TTTCTTAAACAGCCTGCAGCTTTG (348bp fragment); Fas is 5 '-GTACAGAAAACATGCAGAAAGCAC, 3 '-CTCTGCAAGAGTACAAAGATTGGC (342 bp fragmen t).

2.mRNA extract and reverse transcription: the cell of collection is washed 2 times with PBS solution, 200ul RNA extracts that buffer is centrifugal to discard the precipitation that not cell lysis and nucleus are formed, and supernatant is with the E.C. 3.4.21.64 of 50 μ g/ml, and 37 ℃ digest 30min.Then, add the water-saturated phenol of 1 volume and the chloroform of 0.2 volume: isoamyl alcohol (24: 1).1h is put at least with isopropanol precipitating RNA and under-20 ℃ in centrifugal back, and centrifugation RNA washes one time with 70% ethanol, and vacuum drying is also used the heavy molten RNA of DEPC water.In the reverse transcription buffer of 20 μ l, add 2 μ, 1 strands [dT] ₁₅Primer, 2.5 μ l dNTP (each 10mmol/L), 0.5 μ lRNasin (1000u/ml), then, 70 ℃ of heating 5min, cooled on ice.Brief 1.2 μ l Moloney MuLV virus reverse transcription (GIBCO company) and the 37 ℃ insulation 1h down that adds after centrifugal, 90 ℃ of degeneration 5min, the cDNA sample preservation that obtains is in-70 ℃.

3.PCR amplification: the gene amplification system of 0.2ml contains 2.5 μ l cDNA, 50mmol/L KCL, 10mmol/L Tris-HCl, 2.5mmol/L MgCl ₂, 0.2mmol/L dNTPs, 0.5mmol/L 5 ' → 3 ' oligonucleotide primers and 2.5u Taq enzyme (Sangon).30 circulations are carried out in PCR reaction, and at 94 ℃ of degeneration 30s, annealing and extend 2min in 72 ℃ on the corresponding annealing temperature of each primer.The PCR product detects with 1.5% agarose gel electrophoresis, and establishes negative control group with deionized water.Just using/anti-665 egative films according to the system that resembles, DNA is with video picture under uviol lamp behind the ethidium bromide staining.

5. result: this research is that model is used extremely sensitive RT-PCR (reverse transcription polymerase chain reaction) method research apoptogene expression with the hepatoma carcinoma cell, the result shows 5 μ g/ml, the Achillea total flavone capsule of 10 μ g/ml is handled hepatoma carcinoma cell 24h, a large amount of p53 (Fig. 1: 4 is arranged when 36h and 72h, No. 7 swimming lanes, Fig. 2: 2,3,4, No. 5 swimming lanes) gene expression, and bcl-2 and Fas gene do not see Table and reach (Fig. 1: 2,3,5, No. 6 swimming lanes, Fig. 2: 6,7,8,9,10,11,12, No. 13 swimming lanes), above-mentioned three kinds of genes there is no expression (Fig. 2: 14 before the same drug treating, 15, No. 16 swimming lanes).

6. discover that after Achillea total flavone capsule acted on hepatoma carcinoma cell, Fas gene and Bcl-2 gene were not expressed, and wild type p53 gene expression strengthens.Increase with the closely-related wild type p53 expression of gene of natural death of cerebral cells, the Bcl-2 gene relevant with propagation with cell differentiation do not expressed, and illustrates that AR effective site may activate the hepatoma cell apoptosis gene and make cell generation apoptosis.

(5) studies show that, Achillea total flavone capsule has hepatoprotective effect, treatment autoallergic, antiallergic, but and inducing tumor cell differentiation, the increment of tumor cell and DNA synthetic there is the obvious suppression effect, simultaneously ultra-oxygen anion free radical, hydroxy radical are had in various degree inhibitory action, have the direct ratio dependency relationships between drug level and the inhibitory action.

The acute toxicity test of Achillea total flavone capsule

Get 8 Kunming mouses, divide 4 groups at random, every group of 2 mices by 30%, 32%, 36%, 40% concentration, with 1 administration of 0.2ml/10g dosage, were observed 3 days continuously.None death as a result.

Increase dosage according to its result, count per 10 gram 0.4ml, oral at twice in 1.5 hours, observed continuously 3 days.The most complete works of unlikely dead amount (D of result _Minute) be 13g/kg, minimum full lethal dose (D _Max) be 17g/kg.

Get 60 of Kunming mouses, body weight 20 ± 1.8g, male and female dual-purpose, random packet, 10 every group.The component dose ratio is 1: 1.7, and Pharmaceutical press 40ml/10g by geometric ratio (1: 1.07) dilution, gastric infusion at twice in 1.5 hours, observation every day, continuous 7 days.Record animal toxicity response situation and dead animal distribute.The result is as follows:

Dosage (mg/kg)	17000	16000	15000	14000	13000	12000
							Death toll (r/n)	10/10	6/10	2/10	0/10	0/10	0/10

With CASIO fx-180p computer, program is pressed the Bliss method and is calculated LD ₅₀Deng related parameter is arranged.

D	P	Ye	Y	n	W	A	B	Yr	Yr-Y
										17000	1.00	6.96	6.73	10	0.208	10.633	-3.459	6.83	0.10
16000	0.60	5.25	5.53	10	0.581	2.840	3.536	5.48	-0.05
										15000	0.20	4.16	4.26	10	0.532	3.202	3.525	4.04	-0.22
14000	0.00	3.04	2.89	10	0.110	22.736	2.494	2.50	-0.39

| Yr-Y|＞0.2 replaces Y to carry out second with Yr and takes turns calculating:

D	P	Y	n	W	A	B	Yr	Yr-Y
									17000	1.00	6.83	10	0.180	12.666	-5.411	6.89	0.06
16000	0.60	5.48	10	0.581	2.840	3.536	5.48	0.00
									15000	0.20	4.04	10	0.439	4.133	3.344	3.98	-0.06
14000	0.00	2.50	10	0.050	57.051	2.146	2.73	-0.13

| Yr-Y|＜0.2, regression result is satisfied, and result of calculation is:

LD ₅₀＝15673.75mg/kg

S ₅₀＝0.00528

95% credibility interval PL=15304.75-16051.65mg/kg

Conclusion: this measuring result, the LD of Achillea total flavone capsule ₅₀Be 15674 ± 0.0103mg/kg.

Achillea total flavone capsule process of the present invention is to 123 routine patient's medication clinical observations, wherein male 80 examples, women 43 examples; Oldest 67 years old, minimum 14 years old; Medical history is the longest 10 years, and is the shortest 1 year; Matched group 60 examples, wherein male 34 examples, women 26 examples; The oldest 63 years old, minimum 15 years old; Medical history is the longest 11 years, the shortest 9 months.All case all meets the diagnostic criteria of hepatitis B.Through the medication treatment, wherein experimental group uses Achillea total flavone capsule, and every day 3 times, each 3,3 months is a course of treatment.Matched group is taken capsule for recovering liver and spleen, and every day 3 times, each 5,3 months is a course of treatment.Therapeutic outcome: two courses of treatment of medication, produce effects 86 examples in 123 examples, 26 examples that take a turn for the better, invalid 4 examples are organized in treatment; Produce effects 31 examples in matched group 60 examples, 20 examples that take a turn for the better, invalid 9 examples.Treatment group total effective rate 91.0%; The matched group total effective rate is 85.0%.

Description of drawings

Referring to accompanying drawing

Fig. 1 is the graph of a relation between sample concentration and the interaction factor E in the antioxidation experiment of the present invention.

Fig. 2 transfers the expression figure (24 hours) of die gene p53, Fas and Bcl-2 for mediator's hepatoma carcinoma cell of the present invention, and p53 (Fig. 2: gene expression 4, No. 7 swimming lanes), and bcl-2 and Fas gene do not see Table and reach (Fig. 2: 2,3,5, No. 6 swimming lanes).

Fig. 3 transfers the expression figure (36 hours and 72 hours) of die gene p53, Fas and Bcl-2 for mediator's hepatoma carcinoma cell of the present invention, p53 (Fig. 3: gene expression 2,3,4, No. 5 swimming lanes), (Fig. 3: 6,7,8,9,10,11,12, No. 13 swimming lanes), above-mentioned three kinds of genes there is no expression (Fig. 3: 14,15, No. 16 swimming lanes) before the same drug treating and bcl-2 and Fas gene do not see Table and reach.

The specific embodiment

Embodiment 1

A, at first Herba Achilleae herb 10kg is ground into powder, with 5 times 95% alcohol reflux three times, 70 ℃ of reflux temperatures carry out defat with the alcohol extract after extracting with the 400ml petroleum ether, filtration, and the elimination petroleum ether layer keeps its residue;

B, the residue that keeps is extracted three times with the 400ml ethyl acetate, extraction temperature is a room temperature, filter, elimination ethyl acetate insoluble matter, remaining ethyl acetate layer is decoloured with 0.5% active carbon, it is 1.10 extractum that concentrating under reduced pressure becomes relative density, and the effective site of this extractum is total flavones and keto acid class, and its content is 50%;

C, the extractum after will concentrating gets dry extract through 45 ℃ of vacuum dryings then, dried cream is added starch be modulated into granule, and through 45 ℃ of cold drying, arrangement incapsulates and gets product.

Use Achillea total flavone capsule, every day 3 times, each 3,3 months is a course of treatment, and total effective rate is 91.0%, and wherein the total effective rate to hepatitis B is 88.0%, and the total effective rate of alcoholic liver injury is 94.0%.

Embodiment 2

A, at first Herba Achilleae herb 10kg is ground into powder, with 6 times 75% alcohol reflux three times, 75 ℃ of reflux temperatures carry out defat with the alcohol extract after extracting with the 500ml petroleum ether, filtration, and the elimination petroleum ether layer keeps its residue;

B, the residue that keeps is extracted three times with the 500ml ethyl acetate, extraction temperature is a room temperature, filter, elimination ethyl acetate insoluble matter, remaining ethyl acetate layer is decoloured with 0.8% active carbon, it is 1.25 extractum that concentrating under reduced pressure becomes relative density, and the effective site of this extractum is total flavones and keto acid class, and its content is 70%;

C, the extractum after will concentrating gets dry extract through 60 ℃ of vacuum dryings then, dried cream is added starch be modulated into granule, and through 50 ℃ of cold drying, arrangement incapsulates and gets product.

Use Achillea total flavone capsule, every day 3 times, each 3, half an hour ante cibum is oral, and 3 months is a course of treatment, and total effective rate is 89.0%, and wherein the total effective rate to hepatitis B is 87.0%, and the total effective rate of alcoholic liver injury is 91.0%.

Embodiment 3

A, at first Herba Achilleae herb 10kg is ground into powder, with 6 times 85% alcohol reflux three times, 80 ℃ of reflux temperatures carry out defat with the alcohol extract after extracting with the 600ml petroleum ether, filtration, and the elimination petroleum ether layer keeps its residue;

B, the residue that keeps is extracted three times with the 600ml ethyl acetate, extraction temperature is a room temperature, filter, elimination ethyl acetate insoluble matter, remaining ethyl acetate layer is decoloured with 1% active carbon, it is 1.30 extractum that concentrating under reduced pressure becomes relative density, and the effective site of this extractum is total flavones and keto acid class, and its content is 80%;

C, the extractum after will concentrating gets dry extract through 50 ℃ of vacuum dryings then, dried cream is added starch be modulated into granule, and through 60 ℃ of cold drying, arrangement incapsulates and gets product.

Use Achillea total flavone capsule, every day 3 times, each 3, half an hour ante cibum is oral, and 3 months is a course of treatment, and total effective rate is 87.0%, and wherein the total effective rate to hepatitis B is 85.0%, and the total effective rate of alcoholic liver injury is 89.0%.

Claims (2)

1, a kind of Achillea total flavone capsule, it is characterized in that 95% ethanol extraction is pulverized, used to the Herba Achilleae herb, with the alcohol extract defat with petroleum ether after extracting, with the residue that keeps with ethyl acetate extraction after, filter, decolouring, concentrating under reduced pressure is made the extractum that relative density is 1.10-1.30, and wherein effective site total flavones and keto acid class content are 50%-80%; Then extractum is made dried cream through vacuum drying, add starch and make granule,, incapsulate after the arrangement and get product through cold drying.

2, a kind of preparation method of Achillea total flavone capsule is characterized in that pressing routine step and carries out:

A, at first the Herba Achilleae herb is ground into powder, the alcohol reflux with 95% three times, 70 ℃-80 ℃ of reflux temperatures carry out defat with the alcohol extract after extracting with petroleum ether, filter, and the elimination petroleum ether layer keeps its residue;

B, the residue that keeps is extracted three times with ethyl acetate, extraction temperature is a room temperature, filter, elimination ethyl acetate insoluble matter, remaining ethyl acetate layer is decoloured with the 0.5%-1% active carbon, concentrating under reduced pressure becomes the extractum of relative density 1.10-1.30, and the effective site of this extractum is total flavones and keto acid class, and its content is 50%-80%;

C, the extractum after will concentrating gets dry extract through 40 ℃ of-60 ℃ of vacuum dryings then, dried cream is added starch be modulated into granule, and through 40 ℃ of-60 ℃ of cold drying, arrangement incapsulates and gets product.