CN1405177A - Rabdosia lophanthide extractive, natural medicine for treating hepatitis - Google Patents

Abstract

The invention relates to a natural medicine Xihuangcao extract for treating hepatitis and a preparation method thereof. The drug uses Xihuangcao as raw material, adopts hot water extraction method, supercritical carbon dioxide extraction method or organic solvent extraction and other processes to extract, and is made into Xihuangcao Extract by decompression concentration, vacuum drying, spray drying and other methods. It contains characteristic active ingredients such as antiviral active ingredients 2-hydroxyursolic acid, 2,3-dihydroxyursolic acid, and anti-oxidative active ingredient rosmarinic acid; it can inhibit the replication of hepatitis B virus and protect immune liver damage , Improving cellular immunity, increasing the weight of spleen and thymus and increasing bile excretion in immunocompromised experimental animals. The Xihuangcao extract is used as the active ingredient, combined with suitable excipients, to make various pharmaceutical preparations, including oral dosage forms such as granules, tablets, capsules, oral liquids, and injection dosage forms, for various viruses The treatment of chronic hepatitis, especially hepatitis B, has broad application prospects.

Description

A kind of natural drug Herba Rabdosiae Lophanthoidis extract for the treatment of hepatitis

Technical field

The present invention relates to a kind of natural medicinal herbs extract, particularly can treat the natural drug Herba Rabdosiae Lophanthoidis extract of hepatitis.The said Linearstripe Rabdosia Herb of the present invention, refering in particular to formal name used at school is that isodon lophanthoides is the plant of Isodonlophanthoides (Buch.-Ham.ex D.Don) Hara var.gerardianus (Benth.) Hara.

Background technology

Viral hepatitis is the major disease that threatens human health.Up to the present, found first, second, third, fourth, penta, oneself, heptan etc. 7 types of viral hepatitis.And with the hazardness maximum of hepatitis B (HepatitisB).Hepatitis B is worldwide disease, and the whole world has 3.5 hundred million patients approximately.China is hepatitis B big country, and the positive person of hepatitis B surface antigen (HBsAg) accounts for 1/10 of total population.Imbalance is the key link of hepatitis B morbidity in view of hepatitis B virus (HBV) persistent infection and body's immunological function, thereby antiviral therapy is praised highly the basic method for hepatitis treatment.

Interferon, rabbit and lamivudine are the two best class medicines of anti-HBV effect of generally acknowledging at present, but some people is insensitive to Interferon, rabbit, and use back recurrence rate height, and side effect is big, costs an arm and a leg.Lamivudine is stronger than Interferon, rabbit to the inhibition effect of hbv replication, but also has easily knock-on after the drug withdrawal, makes virus variation, and shortcoming such as develop immunity to drugs.So, seek new anti-hepatic-B virus medicine and remain the severe task that medical scholar faces.

Summary of the invention

The objective of the invention is limitation at existing viral hepatitis treatment medicine (if any making virus variation; potential collision hazard develops immunity to drugs; or recurrence rate height, side effect big, cost an arm and a leg etc.), have clear and definite antiviral, protection liver cell for society provides a kind of, improve the natural drug Herba Rabdosiae Lophanthoidis extract of effects such as immunizing power.

The object of the present invention is achieved like this:

A kind of natural drug Herba Rabdosiae Lophanthoidis extract for the treatment of hepatitis, it is characterized in that one of preparation method is for getting Linearstripe Rabdosia Herb dry product meal with methyl alcohol or alcohol reflux, extracting solution reduction vaporization or concentrated removing are desolvated, fully extract with sherwood oil after adding suitable quantity of water, the undissolved part of sherwood oil eliminates solvent through evaporation or concentrating under reduced pressure, lyophilize, promptly obtains Herba Rabdosiae Lophanthoidis extract; The preparation method two for getting Linearstripe Rabdosia Herb dry product meal, (include 2 with supercritical carbon dioxide fluid.5%-10% ethanol), 50 ℃ with down to room temperature, extract in supercritical carbon dioxide extraction still internal recycle, the extract solvent removed by evaporation at reduced pressure, add the abundant degreasing decoloring of sherwood oil, the undissolved part of sherwood oil eliminates solvent through the underpressure distillation lyophilize, promptly gets Herba Rabdosiae Lophanthoidis extract; The preparation method three for getting the boiling of Linearstripe Rabdosia Herb dry product meal, extracting solution is through evaporation or concentrating under reduced pressure, vacuum lyophilization or spraying drying eliminate solvent, promptly get Herba Rabdosiae Lophanthoidis extract.

---described Herba Rabdosiae Lophanthoidis extract contains anti-hepatitis B virus activity composition 2-hydroxyl ursolic acid and 2, and 3-dihydroxyl ursolic acid also contains protection liver cell, oxidation-resistant active ingredient rosmarinic acid.

---described Herba Rabdosiae Lophanthoidis extract is used to have the manufacturing of the various viral hepatitis of the treatment medicine that especially hepatitis B is relevant of following effect:

(a) suppress the dhbv dna dna replication dna, suppress the 2.2.15 cell expressing and produce b hepatitis mark (Australia antigen(AA));

(b) the mouse immune liver damage there is provide protection;

(c) effect that improves cellular immunity is arranged;

(d) can increase spleen and the thymic weight of immunocompromised laboratory animal;

(e) significantly increase the rat bile excretion.

---described Herba Rabdosiae Lophanthoidis extract technology is routinely made oral Pharmaceutical dosage forms such as tablet, capsule, particle, oral liquid or the injection that meets the preparation requirement.

Outstanding progress of the present invention and unusual effect are that Linearstripe Rabdosia Herb is the common herbal medicine that is easy to get in South China, and manufacture craft is simple and easy, and is with low cost, have clear and definite antiviral, protection liver cell for society provides a kind of, improve the natural drug of effects such as immunological competence.

Embodiment

Linearstripe Rabdosia Herb activity extract of the present invention, and the extract pharmacologically active is by represented method manufacturing of following embodiment or discovery.Involved method is the technique means that those skilled in the art can grasp and use.But following examples must not be interpreted as the restriction to claim of the present invention of going up in all senses.

Embodiment 1: the preparation of Herba Rabdosiae Lophanthoidis extract (method one)

Linearstripe Rabdosia Herb (isodon lophanthoides) dry product meal 4kg extracts 3 times each 2 hours with methanol eddy, filter, merging filtrate removes methyl alcohol under reduced pressure, and residue (methyl alcohol general extractive) adds suitable quantity of water, fully extracts with sherwood oil (bp60-90 ℃), reclaim solvent and get petroleum ether soluble matter and petroleum ether insoluble, insolubles eliminates solvent through concentrating under reduced pressure, lyophilize, obtains the Linearstripe Rabdosia Herb dry extract, pulverizes, cross 80 mesh sieves, promptly.

Embodiment 2: the preparation of Linearstripe Rabdosia Herb activity extract (method two)

Linearstripe Rabdosia Herb (isodon lophanthoides) dry product 20kg adds water, decocts 2 times, and the 1st time 2 hours, the 2nd time 1 hour, united extraction liquid, (15000rpm) centrifugal insolubles of removing at a high speed.Supernatant liquor gets Herba Rabdosiae Lophanthoidis extract through concentrating under reduced pressure, spraying drying, crosses 80 mesh sieves, promptly.

Embodiment 3: the preparation of Linearstripe Rabdosia Herb activity extract (method three)

Linearstripe Rabdosia Herb (isodon lophanthoides) dry product meal 2kg, with supercritical carbon dioxide fluid (including 5% ethanol) as entrainment agent, under 40 ℃, the condition of pressure 10Mpa, extraction circulates in the supercritical carbon dioxide extraction still, extract after 2 hours, at room temperature, adjust extraction-container temperature, pressure 2Mpa and discharge carbonic acid gas.Gained medicinal extract is the supercritical carbon dioxide extraction extract of sweet wormwood.Get this extract and add the abundant degreasing decoloring of sherwood oil, the undissolved part of sherwood oil eliminates solvent through underpressure distillation, lyophilize, obtains Herba Rabdosiae Lophanthoidis extract, pulverizes, and crosses 80 mesh sieves, promptly.

Embodiment 4: the isolation identification that has the effective constituent of antiviral or hepatocyte protection effect in the Linearstripe Rabdosia Herb activity extract

The separation of efficient part chemical ingredients

Get embodiment 1 gained petroleum ether insoluble, add suitable quantity of water, add ethyl acetate successively and propyl carbinol fully extracts, respectively combined ethyl acetate extracting solution and n-butanol extracting liquid.Evaporated under reduced pressure gets ethyl acetate extract and n-butanol extract respectively.

Get ethyl acetate extract (40g) and use dissolve with methanol, be scattered in proper silica gel, dry method dress silica gel (200-300 order) post.Use chloroform-methanol (98: 2-8: 2) wash-out, every part of 150ml successively.With the TLC monitoring, merge the cut of same composition.Get 2 single component X-1 (210mg) and X-2 (90mg) from 98: 2 eluates, be colourless particulate state crystal; Pass through several silica gel medium pressure (200-300 order) column chromatography again from 90: 10 eluates and obtain 2 single component X-3 (230mg) and X-4 (110mg), be colourless particulate state crystal; Get 1 single component X-5 (70mg) from 8: 2 eluates, be colourless particulate state crystal.

Get n-butanol portion (17.5g) and use dissolve with methanol, be scattered in proper silica gel, dry method dress silica gel (200-300 order) post.Use chloroform-methanol (9: 1-8: 2) wash-out, every part of 150ml successively.With the TLC monitoring, merge the cut of same composition.Get 1 single component X-6 (230mg) from 85: 15 eluates; Get 1 single component X-7 (70mg) from 8: 2 eluates.

Structure is identified

1 X-1, C ₃₅H ₆₀O ₆, white unformed powder, its IR, MS, ¹H NMR data are consistent with the daucosterol standard reference material; TLC shows purple dot, and the Rf value is identical with reference substance.Be accredited as daucosterol (daucosterol).

2 X-2, C ₃₀H ₄₈O ₅, colourless granular crystal, mp274-276 ℃; TLC displaing amaranth spot, the Rf value is identical with standard reference material; IR ν ^KBr _MaxCm ^-1: 3425 (ν _OH); 2927 and 2872 (ν _CH); 1693 (ν _C=O); 1458,1386,1277,1030,997.

EIMS(70eV)m/z(rel.abundance)：438(2)[M ^+.-H ₂O]，300(4)，248(60)[frag.C ₁₆H ₂₄O ₂ ^+.from?RDA?cleavage]，219(8)，203(40)[248-COOH]，189(15)，133(65)，105(20)，81(22％)，69(43)，55(100)，43(100)：

More than consistent with the spectroscopic data of ursolic acid standard reference material;

¹H?NMR(400MHz，DMSO-d ₆，δin?ppm)：5.11(1H，br.s，12-H)，4.30(1H，d，J＝6Hz，3α-H)，2.10(1H，br.d，J＝12Hz，18β-H)，1.03(3H，s，CH ₃)，0.88-0.85(6H，s，2×CH ₃)，0.80?and?0.89(3H?each，s，two?CH ₃)，0.73?and0.66(3H?each，s，two?CH ₃)；

X-2's ¹³C NMR data (wide-band decoupling spectrum, 100MHz, DMSO-d ₆, δ in ppm) and see Table 1, because 18 β-H signal δ _HHigher, 13 δ _CHigher of signal, 12 δ _CSignal is on the low side, also meets black bearberry acid type pentacyclic triterpene constitutional features, and all data is consistent with standard reference material, is ursolic acid (ursolic acid) so identify X-2.

3 X-3, C ₃₀H ₄₈O ₄, colourless granular crystal, mp 255-258 ℃, TLC shows the bluish voilet spot.IR ν ^KBr _MaxCm ^-1: 3430 (broad) (ν _OH); 2973,2938 and 2875 (ν _CH); 1693 (ν _C=O); 1658 (weak absorption, ν _C=C); 1454,1384,1314,1278,1236,1187,1110,1046,997,962,927.

EIMS(70eV)m/z(rel.abundance)：454(1)[M ^+.]，248(60)[frag.C ₁₆H ₂₄O ₂ ^+.from?RDA?cleavage]，219(8)，203(42)[248-COOH]，133(35)，105(20)，81(22％)，69(43)，55(100)，43(100)；

FABMS?m/z(rel.abundance)：632(6)，496(12)，495(43)，437(6)，409(9)，329(6)，301(7)，248(18)，203(21)，119(70)，91(97)，55(100)；

¹H?NMR(500MHz，C ₅D ₅N，δin?ppm)：5.455(1H，m，12-H)，4.077(1H，ddd，J＝9.5，7.0，4.0Hz，2β-H)，3.387(1H，d，J＝9.5Hz，3α-H)，2.614(1H，d，J＝11.5Hz，18β-H)，1.267、1.209、1.066?and?1.038(each?3H，s，4×CH ₃)，0.980(6H，s，2×CH ₃)，0.960(3H，d，J＝6.5Hz，19-CH ₃)

¹³C NMR data (wide-band decoupling spectrum and DEPT spectrum, 125MHz, C ₅D ₅N, δ in ppm) (table 1) shows that X-3 also is black bearberry acid type triterpene, Duos a hydroxyl [δ than X-2 _C68.54 (CH), with the hydrocarbon (δ of 2 β-H) _H4.07] replace, show that with the coupling relation of 3 α-H this hydroxyl is 2 α-OH; Comprehensive various spectroscopic analysis result thinks that X-3 should be consistent with 2 (that is corosolic acid) [1];

The acetylate AcX-3 of X-3, colourless needle, mp237-238 ℃, TLC shows the bluish voilet spot; Its spectroscopic data and structural analysis are as follows:

IR ν ^KBr _MaxCm ^-1: 2973,2945 and 2875 (ν _CH); 1743 and 1693 (ν _C=O); 1658 (ν _C=C); 1454,1370,1250,1152,1110,1039,962,920.

FABMS?m/z：580(8)，579(23)，511(6)，437(16)，391(6)，248(18)，201(24)，189(28)，119(70)，91(94)，55(100)；

¹H NMR (500MHz, CDCl ₃, δ in ppm): 5.227 (1H, m, 12-H), 5.100 (1H, ddd, J=10.5,8.0,4Hz, 3 α-H), 4.753 (1H, d, J=10.5,2 β-H), 2.184 (1H, d, J=11.5Hz, 18 β-H), 2.057 and 1.985 (each 3H, s, 2 * CH ₃CO), 1.070 (6H, s, 2 * CH ₃), 0.955,0.900,0.892 and 0.731 (each 3H, s, 4 * CH ₃), 0.847 (3H, d, J=7.0Hz, 19-CH ₃); Show that with the hydrogen spectrum contrast of X-3 2 hydroxyls that are acetylation are 2 α-OH and 3 β-OH; To sum up analyze, X-3 can be defined as 2 (2 α-hydroxy ursolic acid).

4X-4, C ₃₀H ₄₈O ₅, colourless granular crystal, mp240-243 ℃, TLC shows the bluish voilet spot, and polarity is more bigger than X-5.Its spectroscopic data and structural analysis are as follows:

IR ν ^KBr _MaxCm ^-1: 3571 and, 3423 (ν _OH), 2973,2938 and 2875 (ν _CH); 1686 (ν _C=O); 1651 (weak absorption, ν _C=C); 1461,1384,1271,1236,1154,1110,1046,997,962,934.

EIMS(70eV)m/z(rel.abundance)：442(2)，246(2)，219(2)，205(5)，146(8)，119(5)，105(6)，72(24)，55(38)，43(100)；

FABMS?m/z(rel.abundance)：512(4)，511(12)，397(6)，329(15)，307(15)，279(12)，235(5)，205(13)，176(58)，154(100)，107(50)，77(56)，55(65)；

¹H?NMR(500MHz，C ₅D ₅N，δin?ppm)：5.581(1H，m，12-H)，4.953(1H，s，)，4.090(1H，ddd，J＝9.5，7.0，4.0Hz，2β-H)，3.370(1H，d，J＝9.5Hz，3α-H)，3.118(1H，ddd，J＝9.5，7.0，4.0Hz，)3.039(1H，s，18β-H)，1.705、1.432、1.265、1.104、1.078?and?1.017(each?3H，s，6×CH ₃)，1.120(3H，d，J＝6.5Hz，CH ₃)

The acetylate AcX-4 of X-4, colourless needle, mp228-230 ℃, TLC shows the bluish voilet spot; Its spectroscopic data and structural analysis are as follows:

IR ν ^KBr _MaxCm ^-1: 2973,2938 and 2875 (ν _CH); 1743 (ν _C=O); 1651 (weakabsorption, ν _C=C); 1454,1370,1250,1152,1039,962.

FABMS?m/z：692(6)，596(10)，595(30)，526(6)，453(9)，407(11)，187(24)，145(38)，91(90)，55(100)；

¹H NMR (500MHz, CDCl ₃, δ in ppm): 5.329 (1H, m, 12-H), 5.100 (1H, ddd, J=10.5,8.0,4Hz, 3 α-H), 4.757 (1H, d, J=10.5,2 β-H), 2.531 (1H, s, 18 β-H), 2.060 and 1.984 (each 3H, s, 2 * CH ₃CO), 1.254,1.200,1.061 and 0.715 (each 3H, s, 4 * CH ₃), 0.948 (3H, d, J=7.0Hz, 19-CH ₃), 0.899 (6H, s, 2 * CH ₃); Show that with the hydrogen spectrum contrast of X-4 2 hydroxyls that are acetylation are 2 α-OH and 3 β-OH;

With X-4 and X-3 ¹³C NMR data (table 1) contrasts finds that X-4 Duos a δ than X-3 _C72.71 quaternary carbon, simultaneously its 18 β-H signal becomes unimodal and to low field displacement to 3.090, illustrates that 19 adjacent α-H is substituted, and becomes 19 α-OH[δ _C72.71 (C)], comprehensive various spectroscopic analysis results, think X-4 should with 2 α, 19 alpha-dihydroxy-ursolic acid (that is corosolic acid) ^[1]Consistent.

5X-5, C ₁₈H ₁₆O ₈, colourless grain is brilliant; Polarity is bigger than X-4, and TLC shows black to 10% ethanol solution of sulfuric acid, and 5% iron trichloride is shown black-and-blue.The molion signal does not appear in its EI MS, benzyl diphenol fragmention [(C occurred in the big mass fragments district ₇H ₇O ₂) ⁺, m/z 123] and base peak and dihydroxycinnamic acid fragment [(C ₉H ₈O ₄) ⁺, m/z 180, relative abundance 20%] exist. ¹H NMR (500MHz, C ₅D ₅N, δ in ppm) data analysis is as follows:

9.638,9.163,8.788 and 8.730 (1H each, s, 4 * OH); 7.454 (1H, d, J=15.6Hz) and 6.233 (1H, d J=15.6Hz) show the alkene hydrogen that contains trans cinnyl; 7.045 and 6.665 small split branchs bimodal (be 1H, J=1.5Hz) and 7.000 (1H, dd, J=8.4,1.5Hz), 6.757 (1H, d, J=8.0Hz); 6.626 (1H, dd, J=8.0Hz), 6.516 (1H, dd, J=8.4,1.5Hz) two groups of proton signals show two 1,2, the existence of 4-substituted benzene ring; In addition 5.020 (1H, dd, J=8,4Hz) and 2.920 (1H, ddd, J=12.5,8,2Hz) disclose and contain ArCH ₂CH (OH) fragment, correspondingly X-5 ¹³18 carbon signals of C NMR spectrum data presentation wherein have 2 saturated carbon (methylene radical of the secondary alcohol of δ 72.8 and δ 32.1).Consistent (table 1) of each chemical displacement value and rosmarinic acid; So infer X-7 and be rosmarinic acid (Rosmarinicacid) [Sun Wenji, the golden house-owner of rope compile. the simple and clear handbook of active skull cap components, 488 pages, Chinese Medicine science and technology press, Beijing, 1998].For from the Rabdosia plant, separating the natural organic-compound that obtains first.

Through the 2.2.15 test cell line, 2 and 2 α, 19 alpha-dihydroxy-ursolic acid respectively under 125 and 250 μ g/ml concentration the secretion to hepatitis B surface antigen HBsAg obvious restraining effect is arranged, inhibiting rate is respectively 74.34%-85.84% and 88.71%-97.58%, and therapeutic index is respectively 0.57 and 1.08; But to the not obviously influence of secretion of hepatitis B virus e antigen HBeAg, this is similar to the effect characteristics of positive reference substance virazole on the 2.2.15 cell model.

Rosmarinic acid then has very strong antioxygenation.It is to by vitamins C-Triphosphopyridine nucleotide, reduced or by Fe ²⁺The rat brain that-halfcystine brings out, liver, the MC lipid peroxidation of kidney all have strong restraining effect, and this effect is 10 of a vitamin-E ²-10 ³Doubly; And short fibrinolytic activity is arranged; To hsv also have certain restraining effect [Sun Wenji, the golden house-owner of rope compile. the simple and clear handbook of active skull cap components, 488 pages, Chinese Medicine science and technology press, Beijing, 1998].Three terpene components in table 1 Linearstripe Rabdosia Herb (isodon lophanthoides) ¹³C NMR (DEPT) data

C???????X-2??????????X-3????????X-4??????????X-5

1???????27.0?????????47.89CH ₂?47.84CH ₂????165.9C

2???????27.5?????????68.54CH????68.60CH??????121.6CH

3???????76.8?????????83.73CH????83.88CH??????148.6CH

4???????38.5?????????39.76C?????39.52C???????1’-170.8C

5???????54.8?????????55.85CH????55.98CH??????2’-72.8CH

6???????18.0?????????18.77CH ₂?19.00CH ₂????3’-36.1CH ₂

7???????32.7?????????33.45CH ₂?33.55CH ₂????1”-127.3C

8???????38.4?????????39.97C?????39.84C???????2”-120.1CH

9???????46.8?????????48.03C?????48.28C???????3”-144.9C

10??????36.5?????????39.76C?????40.44C???????4”-145.9C

11??????23.3?????????23.67CH ₂??24.13CH ₂???5”-116.7CH

12??????124.6????????125.49CH???127.97CH?????6”-114.9CH

13??????138.2????????139.27C????139.97C??????1-125.4C

14??????41.6?????????42.48C?????42.16C???????2-121.6CH

15??????28.2?????????28.57CH ₂??28.47CH ₂???3-144.0C

16??????23.8?????????24.83CH ₂??24.13CH ₂???4-145.6C

17??????47.0?????????47.97C?????48.28C???????5-115.8CH

18??????52.4?????????53.45CH????54.60CH??????6-113.3CH

19??????38.6?????????39.42CH????72.71C

20??????38.2?????????39.34CH????42.37CH

21??????30.2?????????31.02CH ₂??33.55CH ₂

22??????36.3?????????37.42CH ₂??38.47CH ₂

23??????28.2?????????29.30CH ₃??29.35CH ₃

24??????17.0?????????16.97CH ₃??16.77CH ₃

25??????15.2?????????17.45CH ₃??17.89CH ₃

26??????16.1?????????17.41CH ₃??17.26CH ₃

27??????22.8?????????23.86CH ₃??24.70CH ₃

28??????178.3????????179.80C????180.61C

29??????16.9?????????17.62CH ₃??27.12CH ₃

30 21.1 21.35CH ₃21.35CH ₃

X-2 ursolic acid (ursolic acid) R1=R2=HX-3 2 (2 α-hydroxy ursolic acid) R1=OH, R2=H

X-4 2 α, 19-dihydroxyl ursolic acid (2 α, 19 α-dihydroxy ursolic acid) R1=R2=OH

X-5 rosmarinic acid (rosmarinic acid) R=H

Embodiment 5: the antivirus action of Herba Rabdosiae Lophanthoidis extract

(1) external hepatitis B virus resisting experiment

1 material and method

1.1 medicine: isodon lophanthoides water extract, isodon lophanthoides alcohol are got and are carried thing, Linearstripe Rabdosia Herb alcohol extract ethyl acetate solvend, are self-control; Ribavirin (Ribavirin, virazole) is provided by Hubei Province's institute of Pharmaceutical Industry.

1.2 2215 cells: draw 302 hospital Viral Laboratories from Beijing, with DMEM nutrient solution (GBICO company product), nutrient solution adds 10% calf serum, 100u/ml penicillin, the 100u/ml Streptomycin sulphate, G418 100ug/ml (GBICO company product), 0.03% glutaminase is transferred pH to 6.8 with 0.238%Hepes.

1.3 medicinal application: 2215 cells are dispersed into the individual cells suspension with 0.6% trypsinase, plant in 96 orifice plates by 3 * 104 cells/well branch, use the pastille nutrient solution behind the 3-4d instead, each concentration adds four holes, medicine and cytosis add 10%DMEM (containing G418) 0.1ml/ hole after four days again, suct in the 8th day to be ELISA clearly and to measure HBsAg, HBeAg, remaining cell is measured drug cell toxicity with mtt assay, and nutrient solution blank group four holes are established in experiment in addition.

1.4 mtt assay is measured the half toxic concentration of medicine cell growth: medicine and cytosis are after four days, add 10%DMEM (containing G418) 0.1ml/ hole again, sucted in the 8th day and be ELISA mensuration HBsAg clearly, HBeAg, remaining cell adds 400ug/ml MTT0.1ml/ hole (Sigma company product), 37 ℃ of 5%CO2 were hatched 4 hours, as seen yellow black first a ceremonial jade-ladle, used in libation particle, discard MTT liquid, add 100%DMSO 0.1ml/ hole, after treating that first a ceremonial jade-ladle, used in libation particle dissolves (about 10 minutes) fully, measure the absorbance A value with ultraviolet spectrophotometer 570nm wavelength, blank adds 100%DMSO 0.1ml/ hole, four holes.The drug level of cell survival rate (%)=(experimental port A value/control wells A value) * when 100%, 50% toxic concentration (TC50) is control wells 50% for the experimental port survivaling cell.Therapeutic index (TI) is for estimating the parameter of clinical drug application prospect, TI=TC50/IC50.

1.5 the detection of HBsAg, HBeAg: adopt magnificent public ELISA to measure box and detect.Inhibiting rate (%)=[(control wells P/N value one experimental port P/N value)/(control wells P/N value-2.1)] * 100%, 50% inhibition concentration (IC50) is 50% o'clock drug level for HBsAg or HBeAg inhibiting rate.

2.. result

2.1 select HBsAg, HBeAg preliminary screening index as effect of drugs, measure the cytotoxicity concentration of medicine by mtt assay, and the corresponding therapeutic index that calculates estimates the clinical drug application prospect, wherein TI＞2 are effective low toxicity, 1＜TI＜2 are that poor efficiency is poisonous, and TI＜1 is a toxic action.Result of study shows that it is less influenced by the survival of reagent thing pair cell, and therapeutic index all belongs to effective low toxicity greater than 2 (tables 1), and isodon lophanthoides extract is restraining effect to the secretion of HBsAg and HBeAg.

Table 1: three kinds of extracts of isodon lophanthoides are to inhibition effect and the cytotoxicity thereof of HBsAg and HBeAg

Medicine	????TC50	??????????HBsAg		???????HBeAg
							??IC50	??TI	??IC50	??TI
	The Linearstripe Rabdosia Herb aqueous extract (1)	????＞100	??5.29	??＞18.90	??8.51						??＞11.75
The Linearstripe Rabdosia Herb alcohol extract (1)						????＞100	??0.34	??＞294.11	??＞100	??-
	Linearstripe Rabdosia Herb alcohol extract ethyl acetate solvend	????＞100	??0.16	??＞628.9	??63.1						??＞1.58

Annotate: concentration unit is mg/ml, and (1) represents that all the other are in sample size in the crude drug amount.

(2) anti-dhbv dna experiment in the body

1 material and method

1.1 medicine: isodon lophanthoides water extract, isodon lophanthoides alcohol are got and are carried thing, Linearstripe Rabdosia Herb alcohol extract ethyl acetate solvend, are self-control; Acycloguanosine (ACV) is provided by Hubei Province's institute of Pharmaceutical Industry.

1.2 animal: a male age in days Guangzhou sheldrake, available from Xin Cun hatcher, Guangzhou, lot number 96-4-17 and 97-12-4, body weight 40-50 gram/only, every group of 5-11 is only.

1.3 virus: duck hepatitis B virus DHBV-DNA strong positive serum, pick up from the Shanghai sheldrake ,-70 ℃ of preservations.

1.4 experiment material: duck hepatitis B virus (DHBV) DNA plasmid draws from Chinese medicine institute Institute of Medicinal Biological Technique; The NC film is available from Amersham company; The nick translation medicine box is a Promega company product; The nick translation medicine box is available from promega company; α-32p-dCTP is available from the inferior brightness in Beijing company; Sephadex G-50 is available from Pharmacia company; 96 holes hybridization sample applicator is a U.S. Bio-rad company product; Geiger counter is a U.S. S.E.International company product.

1.5 DHBV virus infection and pharmacological agent test: an age in days Guangzhou sheldrake, through leg intravenous injection Shanghai sheldrake DHBV-DNA positive serum, every 0.2ml got blood in back 13 days in infection, separation of serum ,-70 ℃ of preservations are to be checked.DHBV infect duckling after 13 days random packet carry out the pharmacological agent test, every group of 5-11 only do not wait, administration component isodon lophanthoides aqueous extract and medicinal powder capsule, each dosage (10g/kg/ days) group, and isodon lophanthoides alcohol extract ethyl acetate solvend group, dosage is 10mg/kg/ days; All oral.1 day 2 times, 14 days is a course of treatment.Other establishes virus control group (DHBV), with the physiologic saline for substitute medicine, positive drug is oral with acycloguanosine, administration 200mg/kg/ days, 1 day 2 times, 14 days one courses of treatment, the 13rd day is (T0) before the medication after infection, after medication (T7) medication in the 7th day the 14th day (T14) and the drug withdrawal the 3rd day (P3), get blood from duck leg shin vein, separation of serum-70 ℃ preservation is to be checked.

1.6 DHBV-DNA detection method: get above-mentioned duck serum, every batch with the time point film, measure the variation of DHBV-DNA level in the duck serum, press nick translation test kit specification sheets method, with 32P mark DHBV-DNA probe, and make duck serum dot hybridization, radioautograph diaphragm spot, on enzyme mark detector, measure the 490nm OD of place value, calculate serum DHBV-DNA density.

2.. result

Table 2 isodon lophanthoides restraining effect experiment group duck to DHBV-DNA in the duck body is counted OD ₄₉₀Value (batch (only) T of X ± SD) ₀T ₇T ₁₄P ₃

The narrow basic leaf strain line fragrant 11 1.36 ± 0.29 1.24 ± 0.33 0.97 ± 0.35 in virus control group 5 1.53 ± 0.11 1.37 ± 0.43 1.36 ± 0.21 1.10 ± 0.331 ^{* Δ}1.13 ± 0.41

The dish aqueous extract

(10g/kg)

ACV200mg/kg???5?????1.75±0.22???1.66±0.47 ^*??????1.19±0.24 ^**?????1.75±0.20

The narrow baseline line scented tea 8 0.90 ± 0.22 0.89 ± 0.23 0.72 ± 0.35 0.53 ± 0.36 in virus control group 8 0.84 ± 0.37 0.91 ± 0.24 0.85 ± 0.26 0.78 ± 0.292 ^*

The dish capsule

(10g/kg)

ACV200mg/kg???7?????0.95±0.24???0.26±0.13 ^**ΔΔ?????0.08±0.07 ^**ΔΔ????0.63±0.19 ^*

Annotate: ^*With (T0) contrast P＜0.05 before the self administration, ^*With (T0) contrast P＜0.01 before the self administration, Δ and concurrent control group be P＜0.05 relatively; Δ Δ and concurrent control group be P＜0.01 relatively.The result shows: have during 10g/kg/ days dosage groups of isodon lophanthoides aqueous extract administration 14 days (T14) and reduce DHBV infected duck serum DHBV-DNA horizontal force in the remarkable body, administration 7 days (T7) also can reduce DHBV infected duck serum DHBV-DNA level, and 3 days (P3) backs of drug withdrawal virus levels has knock-on slightly; And can reduce DHBV infected duck serum DHBV-DNA level when 10g/kg/ days dosage groups of isodon lophanthoides capsule administration 7 days (T7) and administration 14 days (T14), drug withdrawal had remarkable reduction DHBV-DNA horizontal force in 3 days after (P3), do not see rebound phenomenon.

Table 3 isodon lophanthoides extract in the duck body to the restraining effect of DHBV-DNA

The group duck is counted OD ₄₉₀Value (X ± SD)

(only) T ₀T ₅T ₁₀P ₃

Virus control group 8 1.48 ± 0.28 0.98 ± 0.39 1.39 ± 0.35 1.46 ± 0.34

Linearstripe Rabdosia Herb alcohol extract 8 1.57 ± 0.26 1.07 ± 0.45 1.13 ± 0.47 ^*1.07 ± 0.48 ^*

Ethyl acetate is solvable

Thing (10mg/kg)

ACV200mg/kg????6???????1.56±0.48????0.13±0.07 ^**ΔΔ??????0.05±0.02 ^**ΔΔ????1.84±0.31

Annotate: ^*With (T0) contrast P＜0.05 before the self administration, ^*With (T0) contrast P＜0.01 before the self administration, Δ and concurrent control group be P＜0.05 relatively; Δ Δ and concurrent control group be P＜0.01 relatively.

The result shows: have when 5 days (T5) of 10g/kg/ days dosage groups of Linearstripe Rabdosia Herb alcohol extract ethyl acetate solvend administration, 10 days (T5) and drug withdrawal 3 days (P3) and reduce DHBV infected duck serum DHBV-DNA horizontal force in the remarkable body, do not see that the virus levels rebound phenomenon was arranged in 3 days after the drug withdrawal.

Embodiment 6: Herba Rabdosiae Lophanthoidis extract is to the provide protection of immune liver injury

1. materials and methods

1.1 medicine: narrow baseline line scented tea grass aqueous extract is self-control, and bacille Calmette-Guerin vaccine is a purchase.

1.2 with the experiment mice random packet, every each injection of BCG vaccine of tail vein of modeling group and medication group (15g/Kg body weight) (BCG, about 5 * 106/ of bacteria containing amount) 0.2ml, control group injection isometric(al) physiological saline 0.2ml.Every tail vein injection lipopolysaccharides (LPS, the about 10ug/ tail of concentration) 0.2ml after 12 days, medication group administration every day 1 time was infected behind the BCG 12 days, and behind the last administration 2h, the ivLPS10ug/ mouse is surveyed the serum transaminase level behind the 16h.

2. result

Experimental result sees the following form

The protective effect of the mouse immune hepatic injury that table isodon lophanthoides water extract is induced BCG+LPS (group number of animals (only) dosage (g/Kg) ALT (U/L) BCG+LPS control group 11-117.36 ± 81.90 solvent control groups 12-51.27 ± 16.48* DDB group 10 0.75 73.50 ± 65.00* administration group 8 15 59.75 ± 74.10* of x ± s)

Annotate: ^*Compare p＜0.01 with the BCG+LPS control group.

Experimental result shows; obviously raise with BCG+LPS inductive immunological liver injury model mouse serum ALT activities; and narrow basic aqueous extract can significantly reduce Serum ALT levels, and near the solvent control group, illustrates that the isodon lophanthoides aqueous extract has good provide protection to mouse.

Cholagogic, the immunization of example 7 Linearstripe Rabdosia Herb aqueous extracts

1, material and method

1.1 medicine: narrow baseline line scented tea grass aqueous extract is self-control; Phytohemagglutinin (PHA) is provided by biology department of Ji'nan University, lot number 950425; Sodium deoxycholate is provided by Chinese biological chemical reagent shop, Shanghai, lot number 910710; Prednisone acetate is provided by south China pharmaceutical factory of Guangdong Province's pharmaceutical industry company, lot number 940703.

1.2 animal: the NIH mouse, available from Department of Health of Guangdong Province laboratory animal field.

1.3 instrument: the 200-10 of Hitachi spectrophotometer.

1.4 influence experiment to immunizing power

1.4.1 the influence of pair cell immunity

Get 50 of body weight 18-22g NIH mouse, ♀ ♂ half and half, be divided into five groups at random, press 20ml/kg body weight gastric infusion, once a day, continuous 15 days, after administration 7 days, give each mouse intramuscular injection PHA 10ml/kg body weight, once a day, for three days on end, wherein four groups gavage Prednisone acetate 0.05ml/kg simultaneously, every day 1 time, modeling in continuous 6 days, and continue to be administered to 15 days, after the last administration 12 hours, stimulating lymphocyte transformation test by PHA---revulsion (3) is got blood in the mouse body, and microscopy also calculates the lymphocyte transformation situation.

1.4.2 influence to humoral immunization

Grouping is identical with 2.1.1 with medication, after administration 7 days, the physiological saline chicken red blood cell suspension 0.2ml of ip 5% carries out immunity, wherein four groups gavage the same 2.1.1 of Prednisone acetate simultaneously, and continue to be administered to 15 days, made immunogenic hemolysin assay method (3) by chicken red blood cell in 12 hours after the last administration and get blood, with each mouse hemolysin content of the 200-10 of Hitachi spectrophotometric determination.

1.4.3 influence to non-specific immunity

1.4.3.1 influence to the mononuclear phagocyte function

Grouping and the same 2.1.1 of medication, behind medicine 7 days, wherein four groups gavage the same 1.4.1 of Prednisone acetate 0.05ml/kg modeling, and continue to be administered to 15 days, after the last administration 12 hours, get blood by mouse carbon clearance method (3), measure down with the 200-10 of Hitachi spectrophotometer and calculate each mouse phagocytic index (k) and activate the phagocytic capacity (a).

1.4.3.2 select the 16-20g mouse for use, grouping, administration, the same 2.2.1 of modeling method, after the last administration 12 hours, by immune organ weight method (3) clip and weighing immune organ weight.

1.5 choleretic effect experiment

Select 220-250g ♂ SD rat for use, press the courage method secretory volume that anesthetized rat courage method secretory volume assay method (3) is measured 30min before the rat medicine,, measure behind the medicine 30,60 respectively, the courage method secretory volume of 90min then by duodenal administration.

2. experimental result

2.1 influence to immunizing power

2.1.1 the influence of pair cell immunity

Experimental result shows, isodon lophanthoides can significantly improve Prednisone acetate and cause mouse immune lymphocytic transformation efficiency when low, with model group relatively, P＜0.01 illustrate that isodon lophanthoides has the effect of raising cellular immunity, the results are shown in Table one.

Show the influence that a pair of Prednisone acetate causes mouse immune power lymphocyte transformation rate when low (n=10, group dosage lymphocyte % cell % of the excessive phase lymphocyte transformation rate % of X ± SD)

(g/kg body weight) normal group 20ml 45.23 ± 6.23 38.93 ± 5.24 84.16 ± 45.52 models+water group 20ml 30.40 ± 25.20 ± 55.60 ± 4.38 ^###

7.14 ^###7.98 ^###Model+isodon lophanthoides group 4.00 42.00 ± 31.00 ± 9.07 ^*72.90 ± 8.23 ^{* *}

9.94 ^***

Annotate: # refers to P value and normal group relatively ^*# P＞0.05

^*Refer to that P value and model group are relatively ^*## P＜0.05

^{* * *}### P＜0.01 (down together)

2.1.2 influence to humoral immunization

Experimental result prompting, isodon lophanthoides Dichlorodiphenyl Acetate prednisone cause mouse immune when low the content of hemolysin do not make significant difference P＞0.05; Explanation is little to the humoral immunization influence, the results are shown in Table two.

Table two Dichlorodiphenyl Acetate prednisone causes influence (n=10, the group dosage hemolysin O D value of X ± SD) of mouse immune power hemolysin when low

(g/kg body weight) normal group 20ml 0.42 ± 0.13 model+water group 20ml 0.21 ± 0.15 ^###Model+isodon lophanthoides group 4.0 0.18 ± 0.09*

2.1.3 influence experiment to non-specific immunity

Experimental result shows that isodon lophanthoides can increase thymic weight, compares with model group, and P＜0.05 the results are shown in Table three, four.

Table three Dichlorodiphenyl Acetate prednisone causes influence (n=10, the group dosage phagocytic index K activate the phagocytic capacity a of X ± SD) of mouse immune power phagocytic cell phagocytic power when low

(g/kg body weight) normal group 20ml 0.0201 ± 0.0032 5.2915 ± 0.2765 model+water group 20ml 0.0058 ± 0.0012 ^###3.4364 ± 0.6819 ^###Model+isodon lophanthoides group 4.00 0.0101 ± 0.0028 ^{* *}3.7640 ± 0.7873 ^*

Table four Dichlorodiphenyl Acetate prednisone causes influence (n=10, the group dosage spleen thymus gland of X ± SD) of mouse immune power immune organ when low

4.00 group 42.73 ± 8.13 on (g/kg body weight) (mg/kg body weight) (mg/kg body weight) normal group 20ml model+water group 20ml 39.11 ± 8.39 13.96 ± 2.66 models+isodon lophanthoides ^*17.35 ± 4.04 ^*2.1.4 choleretic effect experiment

Experimental result shows that isodon lophanthoides can significantly increase the rat bile excretion, compares with control group, and P＜0.05 illustrates that isodon lophanthoides has certain choleretic effect.The results are shown in Table five.

The influence of table five pair rat bile excretion (n=10, the group dosage bile lifting rate of X ± SD)

(difference behind the g/kg medicine prodrug

Body weight) 30min 60min 90min distilled water group 10.00 0.192 ± 0.029 ↓ 0.010 ± 0.057 ↓ 0.026 ± 0.076

↓ 0.002 ± 0.067 ^#Isodon lophanthoides 6.00 0.190 ± 0.057

↑ 0.080 ± 0.124 ^#↑ 0.092 ± 0.076 ^##↑ 0.092 ± 0.079 ^##Group sodium deoxycholate group 0.06 0.162 ± 0.067 ↑ 0.132 ±

↑0.144±0.034 ^###??↑0.104±0.069 ^##

0.074 ^###

Claims (4)

1. A natural medicine Xihuangcao extract for treating hepatitis, which is characterized in that one of the preparation methods is to take the dried Xihuangcao coarse powder and extract it with methanol or ethanol under reflux, the extract is evaporated under reduced pressure or concentrated to remove the solvent, and after adding an appropriate amount of water Fully extract with petroleum ether, the insoluble part of petroleum ether is evaporated or concentrated under reduced pressure, freeze-dried to remove the solvent, and then the extract of Xihuangcao is obtained; the second preparation method is to take the dry powder of Xihuangcao, and use supercritical carbon dioxide fluid (Contains 2.5%-10% ethanol), at below 50°C to room temperature, circulate and extract in a supercritical carbon dioxide fluid extraction kettle, evaporate the extract under reduced pressure to remove the solvent, add petroleum ether to fully degrease and decolorize, and the insoluble part of petroleum ether The solvent is removed by vacuum distillation and freeze-drying to obtain the Xihuangcao extract; the third preparation method is to take the dried Xihuangcao coarse powder and add water to decoct, the extract is evaporated or concentrated under reduced pressure, and vacuum freeze-dried or spray-dried to remove all the solvents. Solvent, that is, the extract of Xihuangcao. 2. The extract according to claim 1, characterized in that the Xihuangcao extract contains anti-hepatitis B virus active ingredients 2-hydroxyursolic acid and 2,3-dihydroxyursolic acid, and also contains liver cell protection , Antioxidant active ingredient rosmarinic acid. 3. The extract according to claim 1, characterized in that the Xihuangcao extract is used for the manufacture of drugs related to the treatment of various viral hepatitis, especially hepatitis B, with the following effects: (a) inhibit duck hepatitis B virus DNA replication, inhibit 2.2.15 cell expression and produce hepatitis B marker (hepatitis B antigen); (b) It has a protective effect on immune liver injury in mice; (c) It has the effect of improving cellular immunity; (d) It can increase the weight of spleen and thymus in immunocompromised experimental animals; (e) Significantly increased bile excretion in rats. 4. The extract according to claim 1, characterized in that the Xihuangcao extract can be made into oral pharmaceutical dosage forms such as tablets, capsules, granules, oral liquids or injections that meet the requirements of formulations according to conventional processes.