CN101186627A - Method for preparing picroside I - Google Patents

Method for preparing picroside I Download PDF

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CN101186627A
CN101186627A CNA2007101643215A CN200710164321A CN101186627A CN 101186627 A CN101186627 A CN 101186627A CN A2007101643215 A CNA2007101643215 A CN A2007101643215A CN 200710164321 A CN200710164321 A CN 200710164321A CN 101186627 A CN101186627 A CN 101186627A
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kutkin
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CN101186627B (en
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周亚伟
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Beida Shijia Technology Development Co ltd
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Abstract

The invention relates to the research and development technical field of new Chinese medicine, in particular to a preparation method of kutkoside I, which uses advanced extraction, separation and purification technique to extract the effective component kutkoside I from picrorhiza rhizome medicinal material for treating hepatitis B, while the content of the kutkoside I is higher than 90%. The inventive preparation method has low cost, simple operation and industrialization production suitability.

Description

A kind of preparation method of kutkin I
Technical field
The present invention is dividing an application of 200410098646.4 applications for a patent for invention, and the original bill applying date is on December 15th, 2004, and the original bill denomination of invention is application and preparation and the preparation method of kutkin I in preparation treatment hepatitis B medicament.The invention belongs to new Chinese medicine research and development field, be specifically related to a kind of preparation method of kutkin I.
Background technology
As everyone knows, China is a hepatitis B diseases big country.At present, the whole world estimates at the medicine that is used for the treatment of viral hepatitis more than 600 kinds, but does not still have gratifying medicine so far.World Health Organization West Pacific region eternal honor chairman doctor Han Xiangtai represent, current the most frequently used treatment hepatitis B medicament is cell Interferon, rabbit (Cytokine Interferon) and nucleoside analog (Nucleoside Analogues).Cell Interferon, rabbit commonly used comprises interferon-' alpha ', and Interferon, rabbit must be injected every day, and side effect is more, comprise flu symptom, myalgia, depression and diarrhoea etc. such as headache and fever occur, whether medication also depends on liver function, if liver function is badly damaged, then is not suitable for using Interferon, rabbit.Another kind of cell Interferon, rabbit Peg-intron is also bringing into use.Nucleoside analog as for commonly used then comprises lamivudine (Lamivudine) and Adefovir (Adefovir).Lamivudine is used as the treatment chronic hepatitis B, belongs to oral pharmaceutical, and few side effects suppresses viral usefulness height, resistance can occur but take for a long time, produces the virus of mutation.The Adefovir toxic side effect is few, and this medicine also is used to suppress the research of HIV (human immunodeficiency virus), but discovers that large dose oral administration can influence renal function, and renal function bad person should not use.Use cell Interferon, rabbit and one of shortcoming of examining the beanstalk analogue, be that all life-time service can cause severe side effect.Part regulate immune medicine for example Zadaxin (Thymosin) also be found therapeutic efficiency, but be not widely used as treating hepatitis B at present because detailed therapeutic efficiency is clear not as good as other medicines, price is also quite expensive.
Up to the present, there is not a kind of medicine absolute utility to be arranged for suppressing hepatitis B.Doctor Han Xiangtai thinks, treats in future on the method for hepatitis B, and herbal medicine can be taken on an important role.The World Health Organization also with the treatment by Chinese herbs hepatitis B as newly leading future, and wish with the therapy as future blending the east and the west.Therefore people focus on sight on the Chinese herbal treatment hepatitis B gradually.
The habitual always in the past Chinese herbal treatment hepatitis B of the traditional Chinese medical science is used treatment by Chinese herbs hepatitis and has been experienced secular practice, has also accumulated rich experience.Chinese herbal treatment hepatitis B price is cheap, curative effect is reliable and side effect is little, more and more becomes the main medicine of China's treatment hepatitis B.The treatment by Chinese herbs of hepatitis B comprises following several method: (1) clearing liver dampness removing promotes that jaundice disappears; (2) liver microcirculation that improves promoting blood circulation and removing blood stasis; (3) QI and blood regulating is transferred the general immunity function; (4) tonifying speen and tonifying kidney improves the clinical symptom of hepatitis; (5) various complication are treated in enriching yin, tonifying Qi.But herbal medicine all is being short of science data aspect validity for the treatment of hepatitis B and the security.For this reason, press for to numerous hepatitis B disease patients provides pharmacological action clear and definite drug effect height, the medicine of the treatment hepatitis B that toxic side effect is low.
The Chinese medicine Rhizoma Picrorhizae is former calls its " Great Cold " for Tang Materia Medica, " main hectic fever due to yin labor heat, the tonifying spleen courage makes eye bright "." Xinxiu Bencao " say " main hectic fever due to yin labor heat, the tonifying liver courage makes eye bright ... thick stomach "." Kaibao Bencao " calls its " bitter, flat, nontoxic ".In " the thunder fire is boiled in water for a while, then dress with soy, vinegar, etc. pharmacy and separated ", it is returned through " going into liver, courage, stomach three warps ".Miao Xiyong " Bencao Jingshu " say " Rhizoma Picrorhizae, kind remove damp and hot ... all are damp and hot, heat symptoms caused by an exopathgen, the cloudy latent heat institute that divides is sick all, there's no one who doesn't or isn't eliminates." " medicine justice " say: " Rhizoma Picrorhizae is solely gone into blood system and heat-clearing." " wet fire knot is poly-, non-this disease institute that can not go directly for " book on Chinese herbal medicine justice " speech." Rhizoma Picrorhizae kind remove damp and hot, disappear and gather, the tonifying liver courage can be removed the damp and hot disease and evil of chronic hepatitis B, protects the liver courage helping except that the epidemic disease poison, for thorough this disease of treatment provides foundation.
Rhizoma Picrorhizae is a goatweed Rhizoma Picrorhizae Picrorhiza scrophulariifloraPennell dry rhizome, be per nnial herb, grow on the rock of extremely frigid zones and in the mound, perhaps the area without shade of shallow soil layer, be distributed in western Sichuan, northwestern Yunnan Province, southern Tibet.It is documented, contain iridoid glycosides, cucurbitacine, phenolic glycoside class in the Rhizoma Picrorhizae, also contain a spot of aromatic acid and D-N.F,USP MANNITOL in addition, wherein kutkin I (Picroside I) belongs to the iridoid glycoside compounds.The preparation method of relevant kutkin I and the application in the medicine of preparation treatment hepatitis B thereof yet there are no report.
The inventor adopts Traditional Chinese medical theory and modern science achievement to combine, adopt advanced extraction separation purification technique, from Chinese medicine Rhizoma Picrorhizae medicinal material, extract the effective constituent kutkin I of its treatment hepatitis B, make its content surpass 90%, and kutkin I and corresponding pharmaceutical preparation thereof have been carried out the pharmacodynamics of treatment hepatitis B, pharmacological toxicology is learned research, found that this monomer pharmacological action is clear, has the inhibition hepatitis B replication, protecting liver, lowering enzymes, promoting the function of the gallbladder to alleviate jaundice, effects such as immunomodulatory, toxic side effect is extremely low, safe to use, can be the new Chinese medicine that vast hepatitis B patient provides a kind of high-efficiency low-toxicity.
Summary of the invention
The object of the present invention is to provide the pharmaceutical dosage form of the preparation method and the treatment hepatitis B thereof of kutkin I.
The present invention further provides the Chinese medicine composition that is used for the treatment of hepatitis B that contains kutkin I.
Kutkin I (Picroside I) belongs to the iridoid glycoside compounds.This compound is white unformed powder, soluble in water and methyl alcohol, fusing point 100-102 ℃.Molecular formula is C 24H 28O 11, structural formula is as follows:
Figure S2007101643215D00041
The invention provides the preparation method of kutkin I with industrial value.Its separation purification method is quick, simple, practical, and production cost is lower, and suitability for industrialized production is highly advantageous to.
The method of kutkin I extraction provided by the invention, separation, purifying is as follows:
(1) extracts: dry rhizome or its pulverizing product of getting Tibet picrorhiza rhizome (Picrorhiza scrophulariiflora Pennell) and India's Rhizoma Picrorhizae (Picrorhiza kurroa Royle ex Benth), water, ethanol, acetone equal solvent or its two kinds, the extraction of multiple mixed solvent, extracting solution is being evaporated to dried (or organic solvent-free) below 70 ℃, add water to suitable volume, the centrifugal precipitation of removing gets supernatant liquor;
(2) separate: supernatant liquor is by the good adsorption column of conventional processing, and water and Different concentrations of alcohol wash-out are respectively collected and contained the thin layer inspection and know the elutriant that kutkin I is arranged, and concentrating under reduced pressure, vacuum-drying below 70 ℃ are pulverized, the kutkin I crude product;
(3) purifying: with kutkin I crude product anhydrous alcohol solution, filter, filtrate is admixed silica gel, water-bath volatilizes solvent, loads on the silicagel column top, with the two mixed solvent wash-out of ethyl acetate, acetone or its, collect effluent liquid, drying under reduced pressure, pulverize the kutkin I sample.
Used Rhizoma Picrorhizae medicinal material can be the medicinal material of pure natural in the said extracted process, also can be the medicinal material of artificial propagation.The Rhizoma Picrorhizae medicinal material of employing artificial growth can guarantee the steady sources of medicinal material, and is quality controllable, and can reduce production costs.
Used Rhizoma Picrorhizae medicinal material can be ground into the granularity of 10～30 order sizes in the said extracted process, can help the extraction of effective constituent.
Used solvent can be water, ethanol, acetone in the said extracted process, both can use separately, also can merge with two or more solvent and use.The extracting method that adopts can use backflow, diacolation or apparatus,Soxhlet's to extract.
The filler of used filling adsorption column can be the resin of nonpolar or low-pole in the above-mentioned sepn process, as styrene type (comprising vinyl toluene type, ethyl styrene type), vinyl cyanide type resin etc., specifically can adopt D-101 type, ZTC type, AB-8 type etc.
Optimal conditions in said extracted, separation, the purge process can pass through L 9(3 4) orthogonal table carries out the quadrature screening experiment, is index with the content and the rate of transform of kutkin I, filters out optimum experimental condition.
Kutkin I content is more than 95% in the kutkin I sample that makes according to the method described above, and the rate of transform is more than 55%.The extraction result is stable, and good reproducibility can be used for large-scale production.
Kutkin I of the present invention during disease, can use separately when being used for the treatment of hepatitis B, and the form of pharmaceutical composition that also can be by containing kutkin I is used.
Pharmaceutical composition provided by the invention can be by method known in the art preparation, and can be by oral, hypogloeeis, through administrations such as skin, muscle or subcutaneous, mucocutaneous, urethra, vagina or veins.
The invention provides with the kutkin I is activeconstituents, is used for the treatment of the pharmaceutical preparation and the related dose forms of hepatitis B.This pharmaceutical preparation is to be the effective active composition with above-mentioned kutkin I, and has comprised acceptable other auxiliary material components on the pharmaceutics, and wherein said kutkin I content surpasses 90%, preferably is higher than 95%.Pharmaceutical preparation of the present invention comprises oral preparation and non-intestinal drug delivery agent, and wherein oral preparation comprises capsule, oral liquid, chewing agent, pill, tablet, granule etc., and non-intestinal drug delivery agent comprises injection liquid formulation and freeze-dried powder injection type etc.Pharmaceutical preparation of the present invention also comprises local administration preparation, comprises creme, ointment, patch, sprays etc.Formulation of the present invention is not limited to this.
Available auxiliary type agent can be conventional filling agents such as starch, dextrin or cyclodextrin, sucrose, stearate when the preparation oral preparations.Can prepare by methods such as aseptic spraying drying, low-temperature vacuum drying, lyophilizes at the preparation lyophilized injectable powder.Each preparation later stage preparation technology and equipment all belong to the routine techniques of pharmacy field, the present invention does not limit this, so will not describe in detail at this.
Creativeness of the present invention is to prove the effect that kutkin I has the obvious treatment hepatitis B by serial experiment.In order to prove that kutkin I has therapeutic action to hepatitis B, comprise therapeutic action acute, chronic hepatitis B, kutkin I and related dose forms that the contriver uses the above method to make, adopted different animal models, carried out a large amount of experimentation on animalies by oral and injecting pathway, experimental result shows: (1) causes the liver of Rats with Acute Liver Injury and mouse that tangible prevention protection and therapeutic action are arranged to tetracol phenixin, D-amido semi-lactosi and paracetamol; (2) cause the liver of chronic hepatic injury rat that tangible prevention protection and therapeutic action are arranged to autoallergic and tetracol phenixin; (3) can obviously increase bilirubin direct content in the normal rat bile, tangible choleretic effect is promptly arranged; (4) can increase how bilirubin direct (TBIL), gpt (ALT) content in ester (ANIT) the icterogenicity liver injury mice serum of isothiocyanic acid-1-, promptly how ester causes mouse jaundice liver damage that the obvious treatment effect is arranged to isothiocyanic acid-1-; (5) can obviously increase normal mouse macrophage phagocytic index, non-specific immune function is had tangible enhancement; (6) can obviously be increased in the normal mouse immune organ weight, promote the generation of hemolysin, have the effect that strengthens humoral immune function; (7) the duck hepatitis B there is the obvious treatment effect; (8) can obviously reduce hepatitis B virus surface antigen and the antigenic content of e in the 2.2.15 cell culture supernatant.
Main pharmacodynamics experimental technique of the present invention and result are as follows:
Test an injection kutkin I and D-Gal is caused the therapeutic action of chmice acute liver injury
Observe the injection kutkin I causes the chmice acute liver injury to D-Gal (D-Galn) provide protection by the variation that detects mice serum ALT, AST.Get Kunming mouse by body weight be divided at random blank group, model control group, Glycyrrhizic acid,diammonium salt group (50mg/kg), injection Rhizoma Picrorhizae sweet-I little (5mg/kg), in (10mg/kg), big (15mg/kg) three dosage groups, except that the blank group, all the other D-Gals of respectively organizing equal abdominal injection 1000mg/kg cause the chmice acute liver injury, each administration group 1h after modeling, 11h and 21h press 0.1mL/10g volume intravenous administration, behind the modeling 22h, pluck eyeball and get blood survey Serum ALT, AST value, and calculate the liver coefficient.The results are shown in following table.
Table 1 injection kutkin I to D-Gal cause acute liver damage mice serum ALT, AST influence (
Figure S2007101643215D00071
)
Group Dosage (mg/kg) n ALT(U/L) AST(U/L)
The blank group - 12 49.65±16.58 128.42±36.55
Model control group - 12 427.42±198.78△△△ 499.34±331.53△△△
The Glycyrrhizic acid,diammonium salt group 50 12 189.31±100.99** 279.61±109.70*
Annotate the body kutkin I
Small dose group 5 12 253.94±103.23* 321.86±103.22
Middle dosage group 10 12 231.25±145.44* 280.86±56.21*
Heavy dose of group 15 12 200.87±100.97** 249.67±78.42*
Annotate: compare * P＜0.05, * * P＜0.01 with model control group; Compare △ △ △ P＜0.001 with the blank group.
Table 2 injection kutkin I to D-Gal cause acute liver damage mouse liver coefficient influence (
Figure S2007101643215D00081
)
Group Dosage (mg/kg) n Liver coefficient (%)
The blank group - 12 4.76±0.38
Model control group - 12 5.21±0.53△
The Glycyrrhizic acid,diammonium salt group 50 12 5.47±0.45
The injection kutkin I
Small dose group 5 12 5.13±0.33
Middle dosage group 10 12 5.02±0.61
Heavy dose of group 15 12 4.77±0.53
Annotate: compare △ P＜0.05 with the blank group.
Above-mentioned experimental result shows: the injection kutkin I is little, in, big three dosage groups compare with model control group, ALT, AST value all have remarkable reduction (P＜0.05 or P＜0.01).
Conclusion: the injection kutkin I has the obvious treatment effect to the chmice acute liver injury that D-Gal brings out.
Test two injection kutkin Is to CCl 4Cause the prevention provide protection of rat chronic liver injury
Observe the injection kutkin I to CCl 4Cause the prevention provide protection of rat chronic liver injury.Get Wistar rat male and female half and half, according to body weight be divided at random blank group, model control group, Glycyrrhizic acid,diammonium salt group (30mg/kg), injection kutkin I little (3.5mg/kg), in (7mg/kg) and big (10mg/kg) dosage group.The equal tail vein injection administration of each administration group, every day 1 time, 4 weeks of successive administration.After the administration 7 days, except that blank group rat, the equal subcutaneous injection 25%CCl of all the other each rats 4Peanut oil solution 2mL/kg 2 times weekly, in totally 3 weeks, causes the chronic hepatic injury model.24 hours abdominal aortic bloods after the last administration are measured Serum ALT, AST, TP, ALB, MDA, SOD, GSH content and liver starch, liver oxyproline.The results are shown in following table.
Table 3 injection kutkin I is to CCl 4Cause rat chronic liver injury serum AST, ALT influence (
Figure S2007101643215D00091
)
Group Dosage mg/kg n AST(U/L) ALT(U/L)
The blank group - 12 135.22±37.10 29.71±7.72
Model control group - 12 700.73±154.75 △△△ 284.24±2 08.94 △△△
The Glycyrrhizic acid,diammonium salt group 30 12 310.10±118.52 * 123.84±111.60 *
The injection kutkin I
Small dose group 3.5 14 293.82±159.68 *** 103.40±49.39 **
Middle dosage group 7 14 183.42±87.72 *** 63.24±30.28 ***
Heavy dose of group 10 14 199.11±75.71 *** 62.82±25.09 ***
Annotate: compare with model control group *P＜0.05, *P＜0.01, * *P＜0.001; Compare with the blank group △ △ △ P＜0.001.
Table 4 injection kutkin I is to CCl 4Cause the influence that the rat chronic liver decreases TP, ALB (
Figure S2007101643215D00092
)
Group Dosage (mg/kg) n TP(g/dl) ALB(g/dl)
The blank group - 12 66.77±3.21 40.60±4.20
Model control group - 12 66.64±3.40 36.07±2.61 △△
The Glycyrrhizic acid,diammonium salt group 30 12 64.37±2.58 38.52±1.78 *
The injection kutkin I
Small dose group 3.5 14 66.52±5.11 40.12±4.24 *
Middle dosage group 7 14 67.12±5.43 41.20±5.20 **
Heavy dose of group 10 14 66.48±4.56 44.74±3.76 ***
Annotate: compare with model control group *P＜0.05, *P＜0.01, * *P＜0.001; Compare with the blank group △ △P＜0.01.
Table 5 injection kutkin I is to CCl 4Cause the liver starch and the liver oxyproline of rat chronic liver injury influence ( )
Group Dosage (mg/kg) n Liver starch (the mg/g liver is heavy) Liver oxyproline (μ g/g)
The blank group - 12 9.72±1.17 191.2±51.1
Model control group - 12 4.78±2.85 △△△ 553.3±112.5 △△△
The Glycyrrhizic acid,diammonium salt group 30 12 6.59±1.46 * 32 0.6±115.4 ***
The injection kutkin I
Small dose group 3.5 14 6.59±1.46 519.71±156.62
Middle dosage group 7 14 6.73±1.49 ** 339.7±119.4 **
Heavy dose of group 10 14 7.41±1.84 ** 281.3±108.7 ***
Annotate: compare with model control group *P＜0.05, *P＜0.01; Compare with the blank group △ △ △P＜0.001.
Table 6 injection kutkin I is to CCl 4Cause MDA, SOD, GSH content in the rat chronic liver injury serum influence (
Figure S2007101643215D00102
)
Group Dosage mg/kg n MDA (nmol/mL) SOD (nU/mL) GSH (mg/L)
The blank group - 12 4.04±1.20 151.98±11.27 130.63±23.97
Model control group - 12 9.06±1.04 △△△ 161.77±28.29 △ 107.22±27.62 △
The Glycyrrhizic acid,diammonium salt group 30 12 6.82±1.48 *** 137.07±19.69 143.52±30.12 **
The injection kutkin I
Small dose group 3.5 14 6.91±2.03 ** 138.62±15.93 122.57±22.30
Middle dosage group 7 14 6.80±1.40 *** 138.93±19.94 137.81±19.49 **
Heavy dose of group 10 14 4.30±1.65 *** 155.00±10.80 ** 144.68±32.69 **
Annotate: compare with model control group *P＜0.05, *P＜0.01, * *P＜0.001; Compare with the blank group △P＜0.05, △ △ △P＜0.001.
Above-mentioned experimental result shows: the injection kutkin I can obviously reduce Serum ALT, AST, MDA and liver oxyproline, and increased SOD, GSH, TP, ALB and glycogen initial value have significant difference (P＜0.05, P＜0.01 or P＜0.001).
Conclusion: the injection kutkin I is to CCl 4Cause the rat chronic liver injury tangible prevention provide protection is arranged.
Test three kutkin Is and tetracol phenixin is caused the therapeutic action of rat chronic liver injury
Observe the kutkin I oral administration causes the rat chronic liver injury to tetracol phenixin therapeutic action.Get the Wistar rat and adopt tetracol phenixin (CCl 4) cause chronic hepatic injury after, be divided at random model control group, Biphenylylmethylcarbinol group (150mg/kg), kutkin I little (15mg/kg), in (30mg/kg), big (60mg/kg) dosage group, and set up blank group parallel running.Each organizes continuous gastric infusion, 4 weeks of successive administration.Blank group and model control group give equal-volume physiological saline.After the last administration 24 hours, carry out abdominal aortic blood after each treated animal anesthesia, separation of serum is surveyed Serum ALT, AST, TP, ALB value, and is got liver and do the pathological tissue inspection, mensuration liver oxyproline and liver glycogen content.The results are shown in following table.
Table 7 kutkin I to the influence of chronic hepatic injury rat blood serum ALT, AST, A/G (
Figure S2007101643215D00111
)
Group Dosage n ALT AST A/G
mg/kg (U/L) (U/L)
The blank group 12 40.85±6.35 149.17±20.51 1.1866±0.18
Model control group 18 562.52±85.64 △△△ 843.78±28.46 △△△ 0.8876±0.21 △△△
The Biphenylylmethylcarbinol group 150 14 340.90±74.41 *** 511.35±111.61 *** 1.0962±0.15 **
Kutkin I
Small dose group 15 14 468.25±74.80 ** 768.12±117.13 1.0361±0.24
Middle dosage group 30 14 436.46±67.03 *** 742.39±108.63 * 1.0486±0.16 *
Heavy dose of group 60 14 389.25±86.24 *** 583.88±129.37 *** 1.0860±0.20 *
Annotate: compare with the blank group, △ △ △P＜0.001; Compare with model control group, *P＜0.05 *P＜0.01 * *P＜0.001.
Table 8 kutkin I to the influence of chronic hepatic injury rats'liver glycogen, hydroxyproline content and liver coefficient ( )
Group Dosage (mg/kg) n Liver glycogen (mg/g) Oxyproline (μ g/g) Liver coefficient (%)
The blank group 12 21.07±141 8.42±4.65 2.682±0.25
Model control group 18 7.14±2.30 △△△ 19.74±7.55 △△ 5.859±1.34 △△△
The Biphenylylmethylcarbinol group 150 14 14.53±3.87 *** 12.03±9.59 * 4.072±0.75 ***
Kutkin I
Small dose group 15 14 9.67±3.14 * 13.90±8.40 * 4.982±1.12
Middle dosage group 30 14 12.14±5.65 ** 13.37±8.66 * 4.671±1.51 *
Heavy dose of group 60 14 14.12±4.69 *** 11.52±5.30 ** 3.721±1.74 ***
Annotate: compare with the blank group, △ △P＜0.01 △ △ △P＜0.001; Compare with model control group, *P＜0.05 *P＜0.01 * *P＜0.001.
Above-mentioned experimental result shows: kutkin I is little, in, heavy dose of group and model control group relatively, ALT, liver oxyproline obviously reduce (P＜0.05), liver glycogen obviously raise (P＜0.05); In the kutkin I, heavy dose of group obviously reduces with model control group comparison AST, liver coefficient, A/G ratio obviously raise (P＜0.05).Pathological examination results points out each administration group and model control group relatively not to have significant difference.
Conclusion: kutkin I has the obvious treatment effect to rat chronic liver injury due to the tetracol phenixin.
Test of the influence of four injection kutkin Is to normal macrophage phagocytosis of mice
Observe of the influence of injection kutkin I to normal macrophage phagocytosis of mice.Divide blank group, endoxan group (50mg/kg), LEVAMISOLE HCL group (30mmg/kg), injection kutkin I small dose group (5mg/kg), middle dosage group (10mg/kg), heavy dose of group (15mg/kg) at random with the ICR mouse, totally 6 groups, the endoxan group is pressed the 0.02mL/10g intraperitoneal injection every day, the LEVAMISOLE HCL group is pressed the 0.2mL/10g gastric infusion, each dosage group of injection kutkin I is all by 0.1mL/10g volume tail vein injection relative medicine, blank group tail vein injection equal-volume physiological saline, once a day, continuous 7 days.24h after the last administration carries out carbon and cleans up experiment, calculates phagocytic index.Get Mouse Liver simultaneously and spleen is weighed, calculate organ coefficient.The results are shown in following table:
Table 9 injection kutkin I to the influence of normal mouse macrophage phagocytic index ( )
Group Dosage mg/kg n Phagocytic index
The blank group - 14 0.0383±0.0069
The endoxan group 50 14 0.0286±0.0114△
The LEVAMISOLE HCL group 30 14 0.0478±0.0069△△
The injection kutkin I
Small dose group 5 14 0.0431±0.0121
Middle dosage group 10 14 0.0456±0.0081△
Heavy dose of group 15 14 0.0470±0.0120△
Annotate: compare △ P＜0.05 △ △ P＜0.01 with the blank group
Table 10 injection kutkin I to the influence of laboratory animal organ coefficient (
Figure S2007101643215D00141
)
Group Dosage mg/kg n Liver coefficient (%) Spleen coefficient (%)
The blank group - 14 5.5087±0.4529 0.5031±0.0975
The endoxan group 50 14 5.4878±0.6011 0.2210±0.0553△
The LEVAMISOLE HCL group 30 14 5.5092±0.4461 0.5165±0.0711
The injection kutkin I
Small dose group 5 14 5.3290±0.2976 0.5348±0.3772
Middle dosage group 10 14 5.2614±0.5175 0.4874±0.0928
Heavy dose of group 15 14 5.3466±0.3689 0.5027±0.0922
Annotate: compare △ △ △ P＜0.001 with the blank group
Above-mentioned experimental result shows: in the injection kutkin I, heavy dose of group and blank group relatively, phagocytic index all rises to some extent, learns by statistics to handle difference and all have significance (P＜0.05), organ coefficient does not then have considerable change (P＞0.05).
Conclusion: the injection kutkin I has tangible enhancement to non-specific immune function.
Test of the influence of five injection kutkin Is to normal mouse humoral immune function
By observing the injection kutkin I, detect its influence to humoral immune function to the influence that the normal mouse hemolysin generates.ICR kind mouse is divided into 6 groups at random by body weight, it is the blank group, LEVAMISOLE HCL (30mg/kg) group, endoxan group (20mg/kg) group, injection kutkin I little (5mg/kg), in (10mg/kg), big (15mg/kg) dosage group, wherein except that the LEVAMISOLE HCL group was pressed the 0.2mL/10g gastric infusion, each was organized all by 0.1mL/10g tail vein injection relative medicine, continuous 8 days.Administration second day, each organizes mouse peritoneal injection CRBC suspension 0.2mL, and continuous 7 days, 1h after the last immunity got blood and carries out hemolysin O D pH-value determination pH, got thymus gland, organ coefficient is weighed, calculated to spleen.The results are shown in following table.
Table 11 injection kutkin I to the influence of normal mouse immune organ (
Figure S2007101643215D00151
)
Group Dosage mg/kg n Thymus gland coefficient (%) Spleen coefficient (%)
The blank group 14 0.37±0.09 0.62±0.15
The endoxan group 20 14 0.10±0.03△△△ 0.19±0.04△△△
The LEVAMISOLE HCL group 30 14 0.52±0.57 0.57±0.15
The injection kutkin I
Small dose group 5 14 0.41±0.06 0.71±0.19
Middle dosage group 10 14 0.38±0.06 0.63±0.07
Heavy dose of group 15 14 0.40±0.03 0.90±0.29△△
Annotate: compare △ △ P＜0.01, △ △ △ P＜0.001 with the blank group.
Table 12 injection kutkin I to the influence of normal mouse hemolysin content ( )
Group Dosage (mg/kg) n Hemolysin O D value
The blank group - 14 1.83±0.01
The endoxan group 20 14 0.16±0.02△△△
The LEVAMISOLE HCL group 30 14 1.84±0.02△
The injection kutkin I
Small dose group 5 14 1.88±0.12
Middle dosage group 10 14 2.04±0.32△
Heavy dose of group 15 14 1.8 6±0.01△△△
Annotate: compare △ P＜0.05 △ △ P＜0.01 △ △ △ P＜0.001 with the blank group
Above-mentioned experimental result shows: with the blank group relatively in the injection kutkin I, heavy dose of group hemolysin O D value all obviously raises, learning processing difference by statistics all has significance (P＜0.05, P＜0.001).Obviously raise with the heavy dose of group of blank group comparison injection kutkin I spleen coefficient, learning processing difference by statistics has highly significant (P＜0.01).
Conclusion: the injection kutkin I has the effect that strengthens the normal mouse humoral immune function.
Test the choleretic effect of six injection kutkin Is to normal rat
By observing of the influence of injection kutkin I, the choleretic effect of research injection kutkin I to bilirubin direct content in choleresis and the bile.Method is got 50 of Wistar male rats, body weight 220～240g, according to body weight be divided at random blank group, positive drug phenylpropyl alcohol capsule and pill group (250mg/kg), injection kutkin I little (3.5mg/kg), in (7mg/kg) and big (10mg/kg) dosage group, totally 5 groups.Open the abdominal cavity behind the rat anesthesia, make common bile duct intubated and drained bile, collect the preceding 0h of medicine～0.5h bile, then respectively from sublingual vein administration 1mL/100g, collect 0h～0.5h after the administration, 0.5h～1.0h, 1.0h～1.5h, 1.5h～2.0h, 2.0h～2.5h, the bile flow of 2.5h～3.0h is also measured bilirubin direct content.The results are shown in following table.
Table 13 injection kutkin I to the influence of rat bile flow (
Figure S2007101643215D00161
)
Group Dosage mg/kg n 0h～0.5h (mL) before the medicine 1.0h～1.5h behind the medicine (mL) 0.5h～1.0h behind the medicine (mL) 1.0h～1.5h behind the medicine (mL)
The blank group - 10 0.31±0.10 0.31±0.10 0.25±0.06 0.26±0.09
Positive controls 250 10 0.32±0.08 0.40±0.05 △** 0.43±0.06 △△** 0.45±0.05 △△△***
Annotate the body kutkin I
Small dose group 3.5 10 0.30±0.08 0.39±0.08 △* 0.40±0.09 △*** 0.41±0.10 △**
Middle dosage group 7 10 0.32±0.10 0.43±0.13 * 0.43±0.07 △*** 0.46±0.06 △△***
Heavy dose of group 10 10 0.33±0.09 0.43±0.10 △** 0.46±0.07 △△*** 0.49±0.05 △△△***
Group Dosage mg/kg n 1.5h～2.0h behind the medicine (mL) 2.0h～2.5h behind the medicine (mL) 2.5h～3.0h behind the medicine (mL)
The blank group - 10 0.25±0.04 0.23±0.06 0.23±0.05
Positive controls 250 10 0.43±0.07 △*** 0.40±0.09 △** 0.32±0.09
Annotate the body kutkin I
Small dose group 3.5 10 0.34±0.04 ** 0.32±0.09 0.33±0.05 *
Middle dosage group 7 10 0.45±0.08 △*** 0.41±0.05 △*** 0.36±0.07 **
Heavy dose of group 10 10 0.45±0.06 △△*** 0.42±0.05 △*** 0.39±0.08 ***
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with the blank group; With comparison △ P＜0.05 before the self administration, △ △ P＜0.01, △ △ △ P＜0.001.
Table 14 injection kutkin I to the influence of rat bile bilirubin direct (
Figure S2007101643215D00171
)
Group Dosage mg/kg n 0h～0.5h (μ mol/L) before the medicine 0h～0.5h behind the medicine (μ mol/L) 0.5h～1.0h behind the medicine (μ mol/L) 1.0h～1.5h behind the medicine (μ mol/L)
The blank group 10 75.75± 13.03 77.43± 15.58 80.82± 15.83 81.57± 20.07
Phenylpropyl alcohol capsule and pill group 250 10 74.92± 16.07 106.52± 17.05***△△△ 125.65± 17.20***△△△ 142.69± 13.95***△△△
The injection kutkin I
Small dose group 3.5 10 77.44± 17.87 93.60± 21.59 111.02± 23.54**△△ 118.54± 24.24**△△△
Middle dosage group 7 10 75.31± 17.20 106.47± 31.87*△ 116.14± 29.45**△△△ 129.59± 28.17***△△△
Heavy dose of group 10 11 73.92± 16.25 101.77± 26.36*△△ 126.93± 28.33***△△△ 134.34± 27.50***△△△
Group Dosage mg/kg n 1.5h～2.0h behind the medicine (μ mol/L) 2.0h～2.5h behind the medicine (μ mol/L) 2.5h～3.0h behind the medicine (μ mol/L)
The blank group - 10 78.56± 16.09 72.54± 18.70 71.09± 14.72
Phenylpropyl alcohol capsule and pill group 250 10 115.12± 14.92***△△△ 104.22± 15.07***△△△ 100.80± 15.59***△△
The injection kutkin I
Small dose group 3.5 10 115.59± 29.14**△△ 98.77± 23.36**△ 90.77± 15.29**
Middle dosage group 7 10 124.93± 24.57***△△△ 103.48± 23.51**△△ 99.37± 24.37**△
Heavy dose of group 10 11 120.77± 26.49***△△△ 112.42± 22.97***△△△ 92.41± 18.65**△
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with the blank group; With comparison △ P＜0.05 before the self administration, △ △ P＜0.01, △ △ △ P＜0.001.
Above-mentioned experimental result shows: each dosage group of injection kutkin I is compared with the blank group, and each time period bile flow all has rising in various degree, and can obviously increase the bilirubin direct content (P＜0.05, P＜0.01, P＜0.001) in the bile.
Conclusion: the injection kutkin I has tangible choleretic effect.
Test seven injection kutkin Is to isothiocyanic acid-1-how ester cause the therapeutic action of mouse jaundice liver damage
Observe of the therapeutic action of injection kutkin I by the variation that detects mice serum total bilirubin (TBIL), conjugative bilirubin (DBIL), gpt (ALT), glutamic-oxal(o)acetic transaminase (AST) to the jaundice liver damage.Kunming mouse by body weight be divided at random blank group, model control group, positive mattress Cape jasmine Huang (200mg/kg), injection kutkin I little (5mg/kg), in totally 6 groups of (10mg/kg), big (15mg/kg) dosage groups, 12 every group.Except that the blank group, all the other each groups all gavage ANIT (100mg/kg) peanut oil solution by 0.1mL/10g and cause chmice acute jaundice model, and 6h, 24h, 47h are administered three times after the modeling.After the last administration 1 hour (be after the modeling 48 hours), each treated animal is plucked eyeball and is got blood, and centrifugal serum is surveyed TBIL, DBIL, ALT, AST.Get liver, the calculating organ coefficient of weighing.The results are shown in following table.
Table 15 injection kutkin I to ANIT cause mouse jaundice liver damage serum T BIL and DBIL influence (
Figure S2007101643215D00191
)
Group Dosage mg/kg n Serum T BIL (μ mol/l) Serum DBIL (μ mol/l)
The blank group 12 9.23±2.61 7.43±2.57
Model control group 12 140.91±33.85 △△△ 98.65±29.51 △△△
The Yinzhihuang Injection group 200 12 100.20±14.37 ** 82.30±11.31
The injection kutkin I
Small dose group 5 12 108.46±35.89 85.80±24.73
Middle dosage group 10 12 111.01±34.63 * 85.35±20.37
Heavy dose of group 15 12 114.05±18.58 * 90.46±16.41
Annotate: compare with model control group *P＜0.05, *P＜0.01; Compare with the blank group △ △ △P＜0.001.
Table 16 injection kutkin I to ANIT cause mouse jaundice liver damage Serum ALT and AST influence (
Figure S2007101643215D00192
)
Group Dosage mg/kg n Serum ALT (U/L) Serum AST (U/L)
The blank group - 12 46.83±13.99 143.2 5±37.56
Model control group - 12 887.65±243.47 △△△ 871.16±119.45 △△△
The Yinzhihuang Injection group 200 12 618.72±268.33 * 742.56±166.10 *
The injection kutkin I
Small dose group 5 12 786.58±255.43 832.58±147.15
Middle dosage group 10 12 688.59±215.03 * 807.50±207.15
Heavy dose of group 15 12 697.91±154.22 * 767.23±149.16
Annotate: compare with model control group *P＜0.05; Compare with the blank group △ △ △P＜0.001.
Above-mentioned experimental result shows: in the injection kutkin I, heavy dose of group and model control group relatively, serum T BIL, ALT value all obviously reduce, and learn processing by statistics and have significant difference (P＜0.05).All the other each indexs and model control group compare, no significant difference (P＞0.05).
Conclusion: the injection kutkin I has the obvious treatment effect to mouse jaundice liver damage.
Test of the influence of eight injection kutkin Is to the duck hepatitis B
Observe of the influence of injection kutkin I to duck hepatitis B virus (DHBV).Experiment is established 6 groups, normal control group, model group, positive drug group (lamivudine 20mg/kg), injection kutkin I little (10mg/kg), in (20mg/kg), big (40mg/kg) dosage group, except that the normal control group, all the other each groups all adopt sheldrake hepatitis B model, intravenous route is adopted in administration, detects (T before the administration respectively 0), the 5th day (T after the administration 5), the 10th day (T 10) and drug withdrawal after the 3rd day (P 3) content of DHBV-DNA and preceding s surface antigen (DHBpresAg) in the duck serum.Experiment repeats three batches.The results are shown in following table.
Table 17 injection kutkin I (HD-I) is to the influence of duck serum DHBV-DNA content
Batch Group Dosage mg/kg Duck number Join clear DHBV-DNA average optical density value (± s)
T 0 T 5 T 10 P 3
1 Model group 12 0.327±0.053 0.26±0.071 0.307±0.065 0.323±0.062
3TC 20 13 0.381±0.093 0.168±0.077***△△ 0.284±0.043*** 0.307±0.070**
HD-I (little) 10 10 0.365±0.108 0.273±0.060** 0.30±0.060 0.319±0.046
HDI (in) 20 9 0.376±0.069 0.256±0.055** 0.314±0.054 0.298±0.075**
HD-I (greatly) 40 10 0.361±0.071 0.208±0.055*** 0.231±0.064***△ 0.189±0.052***△△△
Model group 11 0.658±0.048 0.673±0.075 0.678±0.083 0.67±0.055
3TC 20 14 0.608±0.108 0.229±0.120**△△△ 0.444±0.109***△△△ 0.460±0.113***△△△
2 HD-I (little) 10 12 0.609±0.071 0.588±0.093△ 0.399±0.082***△△△ 0.478±0.123**△△△
HD-I (in) 20 13 0.640±0.090 0.465±0.070***△△△ 0.524±0.117***△△ 0.521±0.107***△△△
HD-I (greatly) 40 14 0.641±0.056 0.455±0.114***△△△ 0.434±0.138***△△△ 0.299±0.133***△△△
Model group 10 0.303±0.077 0.286±0.078 0.293±0.056 0.300±0.068
3TC 20 13 0.304±0.144 0.162±0.037**△△△ 0.211±0.052△△ 0.222±0.080△
3 HD-I (little) 10 12 0.308±0.059 0.308±0.070 0.334±0.063 0.293±0.109
HD-I (in) 20 11 0.269±0.134 0.21±0.153 0.205±0.022△△ 0.167±0.056**△△
HD-I (greatly) 40 11 0.378±0.138 0.328±0.138 0.235±0.024**△△ 0.235±0.069**△
Annotate: *: t check in pairs: administration group different time (T 5, T 10, P 3) (T before duck serum DHBV-DNA average optical density value and the administration not 0) average optical density value relatively.*P＜0.05，**P＜0.01，***P＜0.001。
△: t check between group: administration group different time (T 0, T 5, T 10, P 3) duck serum DHBV-DNA average optical density value and model group (T 0, T 5, T 10, P 3) average optical density value relatively.△P＜0.05，△△P＜0.01，△△△P＜0.001
Table 19 injection kutkin I (HD-I) is to the influence of duck serum DHBpresAg level
Batch dosage duck count duck serum DHBpresAg average optical density value (
Figure S2007101643215D00211
)
Group
Inferior
(mg/kg) (only) T 0T 5T 10P 3
Model group 12 1.392 ± 0.156 1.267 ± 0.266 1.326 ± 0.148 1.341 ± 0.191
3TC 20 13 1.401±0.142 0.986±0.202***△△ 1.207±0.058* 1.190±0.125***△
1 HD-I (little), 10 10 1.482 ± 0.143 1.105 ± 0.329***, 1.196 ± 0.263**, 1.186 ± 0.258***
HD-I (in) 20 9 1.400 ± 0.120 0.977 ± 0.264** △, 1.187 ± 0.256*, 1.060 ± 0.281** △
HD-I (greatly) 40 10 1.429 ± 0.260 0.111 ± 0.421 1.101 ± 0.441*, 1.061 ± 0.425*
Model group 11 0.450 ± 0.068 0.391 ± 0.072 0.392 ± 0.059 0.397 ± 0.093
3TC 20 14 0.434±0.077 0.400±0.093 0.349±0.088** 0.320±0.0.092***△
2 HD-I (little), 10 12 0.507 ± 0.077 0.449 ± 0.106 0.448 ± 0.129 0.361 ± 0.124**
HD-I (in) 20 13 0.501 ± 0.058 0.420 ± 0.075*, 0.392 ± 0.044***, 0.396 ± 0.079**
HD-I (greatly) 40 14 0.493 ± 0.066 0.435 ± 0.059*, 0.369 ± 0.060*** 0.337 ± 0.111***
3 model group 10 0.741 ± 0.079 0.732 ± 0.093 0.741 ± 0.059 0.704 ± 0.112
3TC 20 13 0.687±0.184 0.485±0.194***△△ 0.575±0.171**△△ 0.615±0.124*
HD-I (little) 10 12 0.764 ± 0.056 0.597 ± 0.156*** △ 0.614 ± 0.111*** △ △ 0.607 ± 0.116*** △
HD-I (in) 20 11 0.727 ± 0.050 0.519 ± 0.021*** △ △ △, 0.636 ± 0.106* △, 0.557 ± 0.121*** △ △
Figure A20071016432100241
Annotate: *: t check in pairs: administration different time (T 5, T 10, P 3) (T before duck serum DHBpresAg OD value and the administration not 0) the OD value relatively.*P＜0.05，**P＜0.01，***P＜0.001。
△: t check between group: administration group different time (T0, T5, T10, P3) duck serum DHBpresAg OD value and model group (T 0, T 5, T 10, P 3) the OD value relatively.△P＜0.05，△△P＜0.01，△△△P＜0.001。
Above-mentioned experimental result shows: to DHBV-DNA assay result in the duck serum be: in the three batches of experiments injection kutkin I little, in, before the 3rd day duck serum DHBV-DNA average optical density value and the administration significance decline (P＜0.001 or P＜0.01) is arranged relatively after the 5th day, the 10th day and the drug withdrawal after the administration of heavy dose of group; The injection kutkin I is little, in, after the administration of heavy dose of group the 5th day, the 10th day with drug withdrawal after the 3rd day serum DHBV-DNA level respectively with corresponding model group administration after the 5th day, the 10th day with drug withdrawal after compared in the 3rd day and all to have significance to descend (P＜0.001 or P＜0.01 or P＜0.05), and have certain dose-effect relationship.To DHBpresAg assay result in the duck serum be: in the three batches of experiments injection kutkin I little, in, before the 3rd day duck serum DHBpresAg OD value and the administration significance decline (P＜0.001 or P＜0.01 or P＜0.05) is arranged relatively after the 5th day, the 10th day and the drug withdrawal after the administration of heavy dose of group; The injection kutkin I is little, in, after the administration of heavy dose of group the 5th day, the 10th day with drug withdrawal after the 3rd day serum DHBpresAg level respectively with corresponding model group administration after the 5th day, the 10th day with drug withdrawal after compared in the 3rd day significance descend (P＜0.001 or P＜0.01 or P＜0.05) all arranged.
Conclusion: in the injection kutkin I body dhbv dna is had than the obvious suppression effect.
Test nine injection kutkin Is to the inhibiting in vitro study of hepatitis B virus
Whether observation in vitro injection kutkin I is inhibited to hepatitis B virus (HBV).Detect the toxicity of injection kutkin I with mtt assay to the 2.2.15 cell; Detect the content that different concns injection kutkin I acts on behind the 2.2.15 cell different time HBV related antigen-surface antigen (HBsAg) and e antigen (HBeAg) in the cells and supernatant, the half toxic concentration (TC of calculating medicine pair cell with the ELISA method 50) and to antigenic half-inhibition concentration (IC 50), with TC 50And IC 50Ratio (being therapeutic index) estimate the validity and the security of medicine, TI＞2 are safe and effective.The normal control group is set simultaneously, blank group, and positive drug lamivudine (3TC) group parallel running.The results are shown in following table.
Table 20 injection kutkin I is to the therapeutic index of HbsAg and HBsAg
Medicine TI to HBsAg TI to HBsAg
Batch 1 2 3 1 2 3
The injection kutkin I 2.0 5.0 1.9 4.8 5.2 4.5
3TC 5.2 5.4 3.3 - 1.7 -
Annotate: criterion is: TI＞2 are for effective; 1≤TI≤2 are poisonous effective; TI＜1 is a toxic action.
The explanation of above-mentioned experimental result: the three batches of experiment injection kutkin Is are respectively 2.97 and 4.83 to the mean value of HBsAg and HBeAg therapeutic index; The positive drug lamivudine is 4.83 to the therapeutic index of HBsAg, and HBeAg is not had restraining effect.
Conclusion: the injection kutkin I is external to have the obvious suppression effect to HBV.
The pharmacodynamic experiment result shows that kutkin I not only has protecting liver, lowering enzymes, promoting the function of the gallbladder to alleviate jaundice and immunoregulation effect preferably, but also has significant anti-HBV effect.
In order to study the toxic action that kutkin I may exist body, estimate the drug safety that utilizes the Chinese medicine medicine that this monomer is prepared into, the inventor has carried out the general toxicity experiment and specific toxicity is tested.
One, general toxicity experiment
1. studies on acute toxicity
Experimental result shows the LD of the injection type intravenous administration of kutkin I to mouse 50Value is 1040mg/kg, and intravenous administration is to the LD of rat 50Value is 171.4mg/kg, and intraperitoneal injection is to the LD of rat 50Be 552mg/kg.Medicine does not have obvious acute toxicity damage to vital tissue and the internal organs of animal.
2. long term toxicity research
(1) kutkin I injection type intraperitoneal injection is to the long term toxicity research of rat
Establish 4 groups altogether, be respectively solvent control group (equal-volume physiological saline), the little 40mg/ (kgd) of kutkin I injection type, middle 150mg/ (kgd) and big three dosage groups of 300mg/ (kgd) are equivalent to 5 times, 21 times, 42 times of pharmacodynamics effective dose respectively.With general performance, body weight, food ration, hematology and serum biochemistry index, routine urinalysis, marrow classification, organ weights, organ coefficient before and after the animals administer serves as to detect index, and be aided with the pathological examination that animal is respectively organized internal organs, the toxicity situation of comprehensive evaluation medicine.The result shows that the heavy dose of treated animal body weight gain of kutkin I injection type is slow, and all the other every indexs are all normal.In, every detection index of small dose group animal also shows no obvious abnormalities.The result of histopathologic examination shows that three dosage treated animals are respectively organized the internal organs no abnormality seen.Be safe dose in can thinking below the dosage.
(2) kutkin I injection type intravenous drip administration is to the long term toxicity research of Beagle
Establish 4 groups altogether, be respectively solvent control group (equal-volume physiological saline), the little 20mg/ of kutkin I injection type (kgd), middle 77.46mg/ (kgd) and big three dosage groups of 150mg/ (kgd).Be equivalent to 10,40 and 70 times of pharmacodynamics effective dose respectively.Observation index is the same.The result shows that the every detection index of large, medium and small dosage treated animal is no abnormality seen also.The pathology detection results suggest, three each vital tissue internal organs Non Apparent Abnormalities of dosage treated animal.
Comprehensive above-mentioned two long term toxicity results of study, we think that injection kutkin I toxicity is lower, safety range is wide, considerably beyond the scope of pharmacodynamics effective dose.
Two, specific toxicity experiment
1. the mutagenesis of kutkin I research
Studied kutkin I potential mutagenicity in vitro and in vivo respectively by " Salmonella typhimurium reverse mutation experiment ", " Chinese hamster lung fibroblast (CHL) chromosome aberration experiment " and " mouse bone marrow cells is had a liking for the polychromatocyte micronucleus and formed experiment ".The result shows, no matter kutkin I in external still body, does not all have significantly direct or indirect mutagenesis.
2. the Study on Teratogenic of kutkin I
Sensitive period of teratogenesis reproductive toxicity test by kutkin I is observed kutkin I at rat sensitive period of teratogenesis successive administration, but whether the teratogenesis fetal hair is given birth to.The result shows that each dosage of kutkin I does not all have tangible teratogenesis.
In sum, the kutkin I toxic side effect is less, pharmacological action is clear and definite, it not only has protecting liver, lowering enzymes, promoting the function of the gallbladder to alleviate jaundice and immunoregulation effect preferably, but also has significant anti-HBV effect, this is one of creative place of the present invention, is the not available characteristics of domestic like product.As injection, this drug effect is rapid, and acute hepatitis is had good therapeutic action in addition.As oral preparation, its major advantage is less than toxicity such as Western medicine such as lamivudine, aciclovirs, should take for a long time.The above-mentioned kutkin I that experimental results show that is a kind of active compound that hepatitis B comprises the acute or chronic hepatitis B disease for the treatment of, and the pharmaceutical composition that contains kutkin I is the good medicine for the treatment of the hepatitis B disease simply.
Rhizoma Picrorhizae can adopt the method for artificial growth to breed at present, has guaranteed the steady sources of medicinal material, and is quality controllable, and a large amount of plantation can reduce production costs with exploitation.In view of China is hepatitis B big country, the medicine that kutkin I is developed to the treatment hepatitis B will have than wide market application prospect.
Specific embodiment
The present invention is further elaborated with following embodiment, but the present invention is not limited to the content that the following example comprises.
The preparation method of embodiment 1 kutkin I
Get Rhizoma Picrorhizae medicinal material 3000g (kutkin I content 3.19% in the medicinal material), be ground into the medicinal powder of 10 mesh sieves.70% alcohol reflux with 12 times of medicinal material bulking values (v/w), it is 1.18～1.20 concentrated solution that the gained percolate is condensed into relative density at 60 ℃, the gained concentrated solution adds the entry dilution, centrifugal, the gained supernatant liquor passes through D-101 macroporous adsorptive resins (resin path: high=1: 10 with the speed of 1.2L/Kg.h, resin demand is 2.5 times of medicinal material weight), after treating that soup flows into resin fully, use the water of 20 times of medicinal material bulking values (v/w) successively, 32 times of medicinal material bulking values (v/w), 15% ethanol elution impurity, use 20 times of medicinal material bulking values (v/w), 25% ethanol elution kutkin I then, collect elutriant, 60 ℃ of concentrating under reduced pressure, vacuum-drying, pulverize, with dehydrated alcohol medicinal powder is dissolved again, filter, filtrate is admixed silica gel, and 60 ℃ of water-baths volatilize solvent, loads on (silica gel footpath: high=1: 10, silicagel column top, the silica gel consumption is 1 times of medicinal material weight), with 10 times of medicinal material bulking values (V/W) eluent ethyl acetate, collect effluent liquid, 60 ℃ of drying under reduced pressure, pulverize kutkin I sample 56.85g, kutkin I content 95.24%, kutkin I weight 54.14g, the kutkin I rate of transform 5 6.5 7%.(annotate: the volume number of one hour effluent liquid of " L/Kg.h " expression per kilogram medicinal material, down together.)
The preparation method of embodiment 2 kutkin Is
Get Rhizoma Picrorhizae medicinal material 84kg (kutkin I content 3.19% in the medicinal material), be ground into the medicinal powder of 10 mesh sieves.70% ethanol speed diacolation with 8 times of medicinal material bulking values (v/w), it is 1.18～1.20 concentrated solution that the gained percolate is condensed into relative density at 60 ℃, gained concentrated solution thin up, centrifugal, the gained supernatant liquor is by ZTC type resin column, after treating that soup flows into resin fully, use the water of 18 times of medicinal material bulking values (v/w) successively, 25 times of medicinal material bulking values (v/w), 15% ethanol elution impurity is used 25 times of medicinal material bulking values (v/w), 25% ethanol elution then, collect elutriant, 60 ℃ of concentrating under reduced pressure, vacuum-drying is pulverized, use 1 times of medicinal material bulking value (v/w) dehydrated alcohol that medicinal powder is dissolved again, filter, filtrate is admixed silica gel, and 60 ℃ of water-baths volatilize solvent, load on the silicagel column top, with 10 times of medicinal material bulking values (V/W) acetone wash-out, collect effluent liquid, 60 ℃ of drying under reduced pressure, pulverize kutkin I sample 1.55kg, kutkin I content 95.30%, kutkin I weight 1.48kg, the kutkin I rate of transform 55.12%.
[embodiment 3] injection kutkin I preparation technology
Prescription: kutkin I 60.0g, make 1000.
Method for making: get kutkin I 60.0g, add the about 2800mL of water for injection, 65 ℃ of water-baths make dissolving, add total amount of preparation 2.0 ‰ gacs (6.0g), stir half an hour, filter, add water to 3000mL on filter, continue with 0.45 μ m, 0.22 μ m millipore filtration classified filtering, filtrate quantitatively is sub-packed in the 10mL cillin bottle with every bottle of 3mL, lyophilize, recharge high-purity nitrogen, jump a queue, roll lid, packing, promptly.
[embodiment 4] kutkin I tablet producing technology
Prescription: make 1000 altogether, each component and content are as follows:
Coptis glycosides I 30g
Starch 180g
Microcrystalline Cellulose 60g
Lactose 30g
Magnesium Stearate 1.5g
Method for making: get the kutkin I sample, wherein kutkin I content surpasses 90%, and it was pulverized 80 mesh sieves, mixes with starch, Microcrystalline Cellulose, lactose etc., with 8% starch slurry system softwood, drying, whole grain adds Magnesium Stearate, mixing, compressing tablet, quality inspection, packing, promptly.

Claims (4)

1. the preparation method of a kutkin I is characterized in that:
(1) extracts: dry rhizome or its pulverizing product of getting Tibet picrorhiza rhizome (Picrorhiza scrophulariiflora Pennell) and India's Rhizoma Picrorhizae (Picrorhiza kurroa Royle ex Benth), water, ethanol, acetone solvent or its two kinds, the extraction of multiple mixed solvent, extracting solution be evaporated to below 70 ℃ dried, add water to suitable volume, the centrifugal precipitation of removing gets supernatant liquor;
(2) separate: by the good adsorption column of conventional processing, water and Different concentrations of alcohol wash-out are respectively collected and are contained the thin layer inspection and know the elutriant that kutkin I is arranged with the supernatant liquor of gained, concentrating under reduced pressure, vacuum-drying, pulverizing below 70 ℃, the kutkin I crude product;
(3) purifying: with kutkin I crude product anhydrous alcohol solution, filter, filtrate is admixed silica gel, water-bath volatilizes solvent, loads on the silicagel column top, with the two mixed solvent wash-out of ethyl acetate, acetone or its, collect effluent liquid, drying under reduced pressure, pulverize the kutkin I sample, kutkin I content surpasses 90%.
2. preparation method according to claim 1 is characterized in that extracting method is backflow, diacolation or apparatus,Soxhlet's extracting method.
3. preparation method according to claim 1, the filler that it is characterized in that filling adsorption column used in the sepn process is nonpolar or the resin of low-pole, as styrene type (comprising vinyl toluene type, ethyl styrene type), vinyl cyanide type resin.
4. preparation method according to claim 3 is characterized in that resin used in the sepn process is D-101 type, ZTC type, AB-8 type.
CN2007101643215A 2004-12-15 2004-12-15 Method for preparing picroside I Expired - Fee Related CN101186627B (en)

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CN102920831A (en) * 2012-11-20 2013-02-13 济南康众医药科技开发有限公司 Preparation method of rhizoma picrorhizae total saponins
CN103601769A (en) * 2013-11-27 2014-02-26 扬子江药业集团有限公司 Preparation process for extracting, separating and purifying picroside I and picroside II from Picrorrhiza Kurrooa Royleex Benth
CN104402950A (en) * 2014-12-29 2015-03-11 济南东源生物医药技术有限公司 Preparation method of kutkin I crystal
CN104693253A (en) * 2015-03-30 2015-06-10 济南东源生物医药技术有限公司 Industrial preparation method for high-purity 6'-O-trans-cinnamyl catalpol
CN104892699A (en) * 2015-05-05 2015-09-09 济南东源生物医药技术有限公司 Method for preparing high-purity 6'-O-trans-cinnamylcatalpol
CN105017355A (en) * 2015-07-21 2015-11-04 济南同生生物医药科技有限公司 6'-O-trans-cinnamoylcatalpol normal phase pressured column chromatography preparation method
CN105198941A (en) * 2015-09-21 2015-12-30 济南同生生物医药科技有限公司 Aluminum oxide pressurized column chromatography preparation of 6'-O-trans cinnamyl catalpa alcohol
CN106749451A (en) * 2017-03-27 2017-05-31 山东康裕生物科技有限公司 It is a kind of to extract the method for separating picroside Ⅰ and picroside Ⅱ
CN112168834A (en) * 2020-10-12 2021-01-05 上海中医药大学 Application of picroside I in preparation of medicine for preventing and treating hepatic fibrosis

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CN102920831B (en) * 2012-11-20 2014-07-23 济南康众医药科技开发有限公司 Preparation method of rhizoma picrorhizae total saponins
CN102920831A (en) * 2012-11-20 2013-02-13 济南康众医药科技开发有限公司 Preparation method of rhizoma picrorhizae total saponins
CN103601769A (en) * 2013-11-27 2014-02-26 扬子江药业集团有限公司 Preparation process for extracting, separating and purifying picroside I and picroside II from Picrorrhiza Kurrooa Royleex Benth
CN103601769B (en) * 2013-11-27 2015-08-19 扬子江药业集团有限公司 A kind of preparation technology of extraction separation and purification picroside Ⅰ and Picroside II from Rhizoma Picrorhizae
CN104402950B (en) * 2014-12-29 2017-06-16 济南东源生物医药技术有限公司 A kind of picroside Ⅰ crystallization preparation method
CN104402950A (en) * 2014-12-29 2015-03-11 济南东源生物医药技术有限公司 Preparation method of kutkin I crystal
CN104693253A (en) * 2015-03-30 2015-06-10 济南东源生物医药技术有限公司 Industrial preparation method for high-purity 6'-O-trans-cinnamyl catalpol
CN104892699A (en) * 2015-05-05 2015-09-09 济南东源生物医药技术有限公司 Method for preparing high-purity 6'-O-trans-cinnamylcatalpol
CN105017355A (en) * 2015-07-21 2015-11-04 济南同生生物医药科技有限公司 6'-O-trans-cinnamoylcatalpol normal phase pressured column chromatography preparation method
CN105017355B (en) * 2015-07-21 2017-10-24 济南同生生物医药科技有限公司 The positive pressurized column chromatography preparation method of the trans cinnamyl Catalpols of 6 ' O
CN105198941A (en) * 2015-09-21 2015-12-30 济南同生生物医药科技有限公司 Aluminum oxide pressurized column chromatography preparation of 6'-O-trans cinnamyl catalpa alcohol
CN106749451A (en) * 2017-03-27 2017-05-31 山东康裕生物科技有限公司 It is a kind of to extract the method for separating picroside Ⅰ and picroside Ⅱ
CN106749451B (en) * 2017-03-27 2019-04-05 山东康裕生物科技有限公司 A method of extracting separation picroside Ⅰ and picroside Ⅱ
CN112168834A (en) * 2020-10-12 2021-01-05 上海中医药大学 Application of picroside I in preparation of medicine for preventing and treating hepatic fibrosis

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