CN104435095A - Common rabdosia leaf total-terpene active part and extraction method, application and composition of active part - Google Patents
Common rabdosia leaf total-terpene active part and extraction method, application and composition of active part Download PDFInfo
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Abstract
The invention discloses a common rabdosia leaf total-terpene active part and an extraction method, application and a composition of the active part. The common rabdosia leaf total-terpene active part disclosed by the invention contains 0.02-0.5wt% of oleanolic acid and 0.05-1wt% of ursolic acid. A main component of a medicine is processed into a proper pharmaceutic preparation for treating immune liver injury diseases; the common rabdosia leaf total-terpene active part is capable of improving liver states and does not have side effects; pharmacodynamic experiments prove that the curative effect of the common rabdosia leaf total-terpene active part is obvious; because medicinal raw materials can be easily obtained, industrialization is easily realized; the common rabdosia leaf total-terpene active part can be prepared into various dosage forms according to requirements; a more convenient, more active and better quality-controllability modern traditional Chinese medicine is provided clinically; patients can have more benefits; and therefore, enormous social benefits are generated. The invention further discloses the extraction method of the common rabdosia leaf total-terpene active part, application for resisting immune liver injury diseases, and the pharmaceutical composition containing the common rabdosia leaf total-terpene active part.
Description
Technical field
The present invention relates to Chinese medicine Isodon amethystoides (Benth.) C.Y.Wuet Hsuen and extract field, be specifically related to extract in a kind of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site, the therefrom king of medicine Chinese date method of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site and the application of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site in anti-immune liver damage disease, there is the pharmaceutical composition of the total terpene effective site of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
Background technology
Viral hepatitis is a kind of whole world commonly encountered diseases, and China belongs to popular serious area, and the health of the people in the serious threat of all kinds viral hepatitis, and western countries are maximum with hepatitis C, China's Major Epidemic hepatitis B.In China, hepatitis b virus carrier accounts for 10% of population, accounts for more than 1/3 of whole world carrier.Existing hepatitis B patient is about 3,000 ten thousand.The primary disease course of disease is delayed, and easily changes chronic active hepatitis, hepatitis interstitialis chronica and hepatocarcinoma into.China changes hepatocarcinoma into because suffering from hepatitis B and causes the dead and reach more than 160,000 people every year, causes the dead if add because of acute hepatic failure and hepatitis interstitialis chronica, more, and therefore, the treatment of chronic hepatitis just becomes a significant problem in the urgent need to address.
The domestic and international types of drugs for chronic hepatitis treatment is a lot of at present, be summed up, substantially can be divided three classes: (one) antiviral agents, mainly contain interferon and several ucleosides analog, interferon is about 30-50% to therapeutic effect that is B-mode and hepatitis C, comparison hepatitis B seems to the effect of hepatitis C slightly good, but discontinue medication most patients recurrence in latter a year, and expenses for medicine is quite expensive, import interferon tens thousand of unit of client need RMB, domestic interferon patient's expenses for medicine is also more than 10,000 yuan, interferon needs intramuscular injection, the side reaction of influenza-like symptom is had after some patients's medication.Some ucleosides analog not as interferon to the therapeutic effect of hepatitis, also exists after discontinuing medication and occurs rebound rebound phenomenon, and drug price is also quite expensive, and some patients has side reaction.For improving curative effect, abroad someone tries out interferon and some nucleoside antiviral agent and merges and use, and result curative effect has no raising.(2) improve liver function medicine, also known as anti-hepatocyte injury medicine, as bifendate, Fructus Schisandrae Chinensis preparation and some Chinese herbal medicine, glycyrrhizin is first succeeded in developing by Japan, and silymarin is German product.This kind of medicine is to reduction serum transaminase, and jaundice eliminating cellulitis has certain effect, and the two cruel effect of the biphenyl wherein developed voluntarily by China is better than other medicines of the same type.But this kind of medicine is after stopping using, most patient also occurs that namely knock-on recurs, and turns out cloudy like inefficacy to the chronic index of hepatitis virus, and expenses for medicine is comparatively cheap, and side reaction is few.(3) immunomodulator, as transfer factor, interleukin II, thymosin etc., be mainly derived from non-specific immunomodulator, mostly being used in can not the patient of application of interference extract for treating, or as drug combination, Treatment chronic Hepatitis B, HBeAg negative conversion rate is at about 20-30%, the definite curative effect of this type of Drug therapy hepatitis need more checking, and price is also quite expensive.As can be seen from the above, there is no the satisfied medicine controlling chronic viral hepatitis at present.
Chronic viral hepatitis is hepatitis B particularly, there is due to hepatitis virus the biological heredity materials such as DNA, the RNA that can be fitted together in human liver cell, the hereditary material of host cell is utilized to copy, above-mentioned molecular biosciences, causes the searching of antiviral agent to be the difficult problem that the world today faces.In addition, nearly all viral generation, not only depends on virus, also depends on the individual factors of people.Current research finds, the response of body self epidemic disease has very important significance in the development mechanism of viral hepatitis, hepatitis B virus is mainly caused by immunoreation hepatocyte wound, the hepatocellular apoptosis of hepatites virus infections induction simultaneously or necrosis are all with fragmentation or the damage phenomenon of diffusivity degraded, therefore except antiviral, promoting that the hepatocellular reparation of damage is with the immunoregulation medicament looking for specific aim stronger, also will be one of the direction of such disease therapeuticing medicine research.
From Chinese herbal medicine, research and develop hepatic is a field having prospect.External existing successful precedent, the glycyrrhizin class medicine developed from Radix Glycyrrhizae as Japan and Europe the silymarin developed, oneself becomes liver-protecting product important in international natural drug market.Also there are many natural medicinal formulations in current China clinically for the treatment of hepatitis, but from clinical efficacy, kind sales volume and added value, Chinese herbal and crude drugs preparations is not preponderated.Since China's " new drug approval method " enforcement in 1985, the national new Chinese medicine of approved about 1000, wherein can be used for the new Chinese medicine about 70 for the treatment of hepatitis, and most of anti-hepatitis new Chinese medicine is three classes, four classes, illustrate for refractory diseases such as chronic hepatitiss, Chinese medicine has certain unique curative effect, also illustrate simultaneously, there is low-level repetition phenomenon in anti-hepatitis new drug development, due to bioactive components and the mechanism of action indefinite, do not have the modern times of high-efficiency low-toxicity anti-hepatitis Chinese medicine to emerge so far.
Labiatae Rabdosia contains various active material, more than 70 new diterpene-kind compounds of 25 kinds in the Rabdosia that Cheng Peiyuan etc. have studied to China have made summary, find that wherein considerable part compound has cell toxicant, antitumor and antibacterial activity, and discussion has been done to diterpene-kind compound anti-tumor activity gene and structure activity relationship in Rabdosia plant.In pharmacodynamics, the bibliographical information protection of Amethystoidin A to carbon tetrachloride hepatic injury, sends out it and has obvious liver protection effect.Bibliographical information Rabdosia plant Folium solani verbascifolii Rterifdia Haraa compound preparation treatment hepatitis B has obvious curative effects clinically.
Liver is the major organs producing cytokine, and be again one of first target organs of cytokine, the balance of cytokine network is not normal is hepatic diseases, as hepatocyte injury, the Fibrotic principle pathological mechanism of hepatocyte that chronic viral hepatitis causes.Recent study the most widely transcription regulatory factor NF-κ B is the most important transcription regulatory factor regulating pro-inflammatory cytokines, cell surface receptor and adhesion factor to express.In the inflammation damnification of liver, NF-κ B by regulation and control pro-inflammatory cytokines (IL-6, TNF-α), chemotactic factor and cell adhesion factor transcribe the acute and chronic inflammation participating in liver directly.
Research in recent years shows, tumor necrosis factor-alpha (TNF-α) plays very important effect in inflammation damage.Rear hour of ConA injection 2; TNF-α has been peak value; give anti-tnf-alpha serum in advance; serum TNF-cc level declines; and protect liver damage wound completely; in view of the ability of macrophage synthesis TNF-α is greater than T lymphocyte, after can inferring T lymphocyte activation, stimulating expression of macrophage discharges TNF-α, may play a significant role in this pathological process.
Isodon amethystoides (Benth.) C.Y.Wuet Hsuen Rabdosia amethy stoides (Benth) Hara system Labiatae Rabdosia plant, the mountain area of our province primary growth northern band in Suzhou is Wild Medicinal.For a long time, the local masses have and take the tradition that Isodon amethystoides (Benth.) C.Y.Wuet Hsuen soup carrys out disease therapy, and spread the saying of " be skin ulcer be not skin ulcer, three bowls of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen soup ".Among the peoplely be used for the treatment of acute infectious hepatitis, there is antibacterial, antiinflammatory and antitumor action etc.Isodon amethystoides (Benth.) C.Y.Wuet Hsuen contains a lot of chemical composition; as Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extract IG-1 and H-1, H-9; Isodon amethystoides (Benth.) C.Y.Wuet Hsuen IG-1 and A prime; Isodon amethystoides (Benth.) C.Y.Wuet Hsuen B prime, Diterpenes, triterpenes, total terpene, flavone and glucoside etc., wherein total ter penoids has significant protective effect to the immune hepatic injury that concanavalin A, Con A (ConA) is induced.
In the pathogenesis of viral hepatitis, the hepatocyte immunologic injury that the lymphocytic activation of T causes is the direct factor causing liver function injury.The experimental liver damaged (as bacteria lipopolysaccharide, D aminogalactose) applied for many years the hepatic injury that mediates, how can not reflect this pathological characteristic well, not to be suitable for the evaluating drug effect having the immunomodulator of liver protection effect or affect immune hepatinica.Induce the hepatic injury of mice specificity from successful Application concanavalin A, Con As such as Tiegs in 1992, its pathogenic characteristic and our pathomechanism of human viral's hepatitis known today have many similarities.It is generally acknowledged, in liver, immunoreation is one of important mechanisms causing viral hepatic injury, such as, in the pathogenic process of hepatitis B, causes the direct factor of hepatocyte injury and destruction to be the cell immune response that T cell mediates.The foundation of the liver injury model brought out with amynologic mechanism, the research for hepatic injury opens new approach, significant to the control of research viral hepatitis.Therefore, we select Immune liver injury to carry out the research of the total terpene liver protection effect of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.But the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen and be not easy extract, this serves certain obstruction to the application of the total terpene of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, be unfavorable for the total terpene of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen medicine promote.
Summary of the invention
The invention discloses the method for extracting this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site in a kind of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site, therefrom king of medicine Chinese date and the application of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site in anti-immune hepatic injury, there is the pharmaceutical composition of the total terpene effective site of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.The present invention have studied the preliminary molecular mechanisms of action of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene liver protection effect, for the medicine total for Isodon amethystoides (Benth.) C.Y.Wuet Hsuen terpene being developed to control hepatic injury provides theoretical foundation.The present invention extracts the total ter penoids of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, isolation and purification, and based on the pathological characteristic of human viral's hepatitis immunologic derangement, set up the model of immunological liver injury, observe the impact on immunological liver injury in mice serum enzyme and histopathology of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene, the total terpene of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is on the impact of immune hepatic injury critical inflammatory medium T NF-α and IFN-γ protein level, and the regulatory mechanism research to TNF-α upstream signaling molecule consideration convey record molecule NF-κ B, inquire in immunologic liver injury process, the preliminary molecular mechanisms of action of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene liver protection effect, for the medicine total for Isodon amethystoides (Benth.) C.Y.Wuet Hsuen terpene being developed to control hepatic injury provides theoretical foundation.
The present invention is achieved in that the total terpene effective site of a kind of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, and it contains oleanolic acid and ursolic acid, by raw material weight percentage ratio composition containing the oleanolic acid of 0.02% to 0.5%, the ursolic acid of 0.05% to 1%.By the percentage by weight of the total terpene effective site of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, content of oleanolic acid accounts for 0.8% to 8%, and ursolic acid content accounts for 1.0% to 12%.
The present invention also provides a kind of method extracting total terpene effective site in therefrom king of medicine Chinese date, and it comprises the following steps:
(1) get clean Isodon amethystoides (Benth.) C.Y.Wuet Hsuen to pulverize, add 15 to 35 times of weight, 70% to 95% ethanol, 50 to 80 DEG C of supersound extraction 40 to 100min, extracting liquid filtering, filtrate is with hydrochloric acid adjust pH=1-3, and to filtrate reflux hydrolysis 20 to 40min, the water adding 0.5-1.0 times of volume in hydrolyzed solution fully mixes, again with isopyknic chloroform extraction several times, combining extraction liquid;
(2) by the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extract concentrating under reduced pressure of gained, carry out post separation, with water and ethanol elution, collect respective streams part, lyophilizing, obtains the total terpene effective site of above-mentioned Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As the further improvement of such scheme, this detached dowel is macroporous resin column, and the macroporous adsorbent resin of this macroporous resin column is at least one in D101, AB-8, HPD100A, HPD400, HPD600, HPD750.Preferably, adsorption time is after 6 to 10 hours, carries out eluting with 1 to 4BV/h flow velocity, first with the washing post removing water-solubility impurity of 3 to 6BV, then use 3 to 6BV40% to 60% ethanol elution, merge ethanol elution, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As the further improvement of such scheme, this detached dowel is polyamide column, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 25-40, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As the further improvement of such scheme, this detached dowel is sephadex column, and this sephadex column is selected from G-10, G-25, G-50, LH-20.Preferably, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 20-50, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, and lyophilizing, obtains the effective portion of the total terpene of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As the further improvement of such scheme, this detached dowel is reverse C18, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 10-20, an eluting 2-4 column volume, starts to collect stream part and collects corresponding stream part 5-11 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As the further improvement of such scheme, this detached dowel is C8 post, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 20-50, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
The present invention also provides the application of a kind of above-mentioned Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site in anti-immune hepatic injury.Particularly in the application for the treatment of viral hepatitis, autoimmune hepatitis, hepatic necrosis, fatty liver, cholestasis, hepatic fibrosis, liver cirrhosis and hepatocarcinoma etc.
The present invention also provides a kind of pharmaceutical composition for the treatment of immune liver damage disease, comprises the total terpene effective site of a kind of above-mentioned Isodon amethystoides (Benth.) C.Y.Wuet Hsuen as main component in this pharmaceutical composition.
Beneficial effect of the present invention is as follows:
(1) a kind of total terpenoid effective site extracted from Chinese crude drug Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is provided, suitable pharmaceutical preparation is become as the immune liver damage disease for the treatment of using the main component production and processing of medicine, liver state can be improved, have no side effect, prove that it is evident in efficacy through pharmacodynamics test;
(2) the medicine material source in the present invention is easy to get, be easy to industrialization, various dosage form can be made as required, for clinically providing convenient, more effectively, the more controlled modern Chinese medicine of quality, for patient brings more interests, thus produce huge social benefit;
(3) the immune liver damage disease that medicine of the present invention is treated mainly comprises: viral hepatitis, autoimmune hepatitis, hepatic necrosis, fatty liver, cholestasis, hepatic fibrosis, liver cirrhosis and hepatocarcinoma etc.
Accompanying drawing explanation
Fig. 1 is the chromatogram that oleanolic acid and ursolic acid reference substance measure.
Fig. 2 is oleanolic acid and the ursolic acid chromatogram of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extracting solution sample determination.
Fig. 3 oleanolic acid canonical plotting.
Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D, Fig. 4 E, Fig. 4 F be the total terpene various dose of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen to the HE colored graph of the protective effect of hepatic injury: Fig. 4 A Normal group; Fig. 4 B Normal group+Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site high dose group; Fig. 4 C model group; Fig. 4 D Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site low dose group; dosage group in Fig. 4 E Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site, Fig. 4 F Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site high dose group.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen contains oleanolic acid and ursolic acid, by raw material weight percentage ratio composition containing the oleanolic acid of 0.02% to 0.5%, the ursolic acid of 0.05% to 1%.The molecular formula of oleanolic acid and ursolic acid is as follows respectively:
Extract the method for total terpene effective site in Chinese medicine Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, concrete steps are as follows.
(1) get clean Isodon amethystoides (Benth.) C.Y.Wuet Hsuen to pulverize, put into ultrasonic tank, add the ethanol of 15 to 35 times of weight 70% to 95%, 50 to 80 DEG C of supersound extraction 40 to 100min, extracting liquid filtering, filtrate is with hydrochloric acid adjust pH=1-3, to filtrate reflux hydrolysis 20 to 40min, the water adding 0.5-1.0 times of volume in hydrolyzed solution fully mixes, then with isopyknic chloroform extraction 1-3 time, combining extraction liquid.
(2) by the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extract concentrating under reduced pressure of gained to proper volume, carry out post separation, detached dowel is selected from macroporous resin column, polyamide column, sephadex column, oppositely C18 and C8 post, with the ethanol elution of water and variable concentrations, collect respective streams part, lyophilizing, obtains the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
Wherein, 1, when using macroporous adsorptive resins to be separated, preferred macroporous adsorbent resin is D101, AB-8, HPD100A, HPD400, HPD600, one or more macroporous adsorbent resin in HPD750, maximum applied sample amount is 1 to 4BV resin demand, pH is 3 to 6, resin blade diameter length ratio is 1 ︰ 6 to 1 ︰ 15, adsorption rate is 1 to 4BV/h, adsorption time is after 6 to 10 hours, eluting is carried out with 1 to 4BV/h flow velocity, first with the washing post removing water-solubility impurity of 3 to 6BV, use 3 to 6BV 40% to 60% ethanol elution again, merge ethanol elution, lyophilizing, obtain the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen,
2, when using polyamide column to be separated, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 25-40, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, and lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen;
3, when using sephadex column to be separated, preferred polydextran gel is selected from G-10, G-25, G-50, LH-20, with water elution, the weight ratio of extracting solution and filler is 1 ︰ 20-50, after being preferably 1 ︰ 25-40, an eluting 1-3 column volume, start to collect stream part 2-6 column volume, lyophilizing, obtains the effective portion of the total terpene of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen;
4, when using anti-phase C18 post to be separated, with water elution, the weight ratio of extracting solution and filler is 1 ︰ 10-20, after an eluting 2-4 column volume, start to collect stream part and collect corresponding stream part 5-11 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen;
5, when using anti-phase C8 post to be separated, with water elution, the weight ratio of extracting solution and filler is 1 ︰ 20-50, after an eluting 1-3 column volume, preferred elution volume is 2 column volumes, starts to collect stream part 2-6 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
As follows to the determination method of oleanolic acid and ursolic acid in above-mentioned gained Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extracting solution.
Experimental agents: oleanolic acid and ursolic acid reference substance, be all purchased from Nat'l Pharmaceutical & Biological Products Control Institute's chromatographic condition and system suitability test: Shim-pack vp-ODS C
18chromatographic column (250mm × 4.6mm, 5 μm).Mobile phase methanol-water (90: 10); Determined wavelength: 210nm; Flow velocity: 0.8mL/min; Column temperature: 25 DEG C.Theoretical cam curve calculates with oleanolic acid and ursolic acid and is all not less than 4000.
Reference substance solution: precision takes oleanolic acid and ursolic acid reference substance 5.8mg, 2.4mg that 105 DEG C are dried to constant weight, be settled to 10mL with dissolve with methanol, be made into the solution that concentration is 0.58mg/mL, 0.24mg/mL, cross 0.45 μm of filter membrane, be reference substance stock solution, for subsequent use.
Need testing solution: it is appropriate that precision pipettes Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extract, evaporate to dryness, and residue adds dissolve with methanol and is settled in 10mL measuring bottle, shakes up, 0.45 μm of microporous filter membrane filters, and obtains need testing solution.
Sample size measures: accurate absorption reference substance and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, the results are shown in Fig. 1-2, reference substance is consistent with the retention time of oleanolic acid in sample chromatogram figure and ursolic acid, and good separation, reaches the related request of separating degree.
As a result, in Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extracting solution, content of oleanolic acid accounts for 0.02% to 0.5% of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen medical material, and ursolic acid content accounts for 0.05% to 1% of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen medical material.
As follows to the determination method of ter penoids in above-mentioned gained Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site.
Experimental agents: oleanolic acid reference substance, be purchased from Nat'l Pharmaceutical & Biological Products Control Institute's reference substance solution: precision takes the oleanolic acid reference substance 5.0mg that 105 DEG C are dried to constant weight, 25mL is settled to dissolve with methanol, be made into the solution that concentration is 0.20mg/mL, be reference substance stock solution, for subsequent use.
Need testing solution: it is appropriate that precision pipettes Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site lyophilized powder, adds dissolve with methanol and be settled in 10mL measuring bottle, shaking up, obtain need testing solution.
Standard curve is set up: accurate absorption oleanolic acid reference substance solution (0.20mg/mL) 0.2 respectively, 0.4,0.6,0.8,1.0mL is in different tool plug test tubes and number, water-bath volatilizes, add the 5% vanillin-glacial acetic acid solution 0.4mL of brand-new, perchloric acid 1.6mL, by methanol constant volume to 5mL, in 70 DEG C of heating in water bath after jolting mixing, frozen water cools.To accompany, reagent does blank, and spectral scan result shows, and has maximum absorption band at 517nm place, selects 517nm place to measure absorption value.With absorbance A for vertical coordinate, reference substance concentration C is that abscissa returns, and obtains equation A=0.8125C+0.0043, r=0.9996 (see Fig. 3).Oleanolic acid is good in 0.008 ~ 0.04mg/mL scope internal linear relation.
Total terpene content in determined by ultraviolet spectrophotometry Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site: the appropriate reference substance of accurate absorption and sample solution respectively, heating in water bath solution makes solution to be measured volatilize, add the 5% vanillin-glacial acetic acid solution 0.4mL just made subsequently, perchloric acid 1.6mL, and by methanol constant volume to 5mL, rocked by solution to be put in 70 DEG C of water-baths evenly and heat, frozen water cools.Retinue reagent is done blank, measures its absorption value at 517nm place.
As a result, total terpene content in Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site lyophilized powder (calculating with oleanolic acid) is for 52%-70%.
Oleanolic acid and ursolic acid content in high effective liquid chromatography for measuring Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site: measure by the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen of above-mentioned high-efficient liquid phase chromatogram condition to purification total terpene effective site lyophilized powder, in the total terpene effective site of result Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, content of oleanolic acid accounts for 0.8% to 8% of lyophilized powder, and ursolic acid content accounts for 1.0% to 12% of lyophilized powder.
The pharmacodynamics test of the anti-immune hepatic injury of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site proves, it carries out in the steps below:
1 experiment material
1.1 laboratory animal
Male ICR mouse, body weight 20 scholar 2g, Yangzhou University's medical experiment animal center provides.
1.2 Experimental agents
Concanavalin A (ConcanavalinA, ConA): Sigma Products; Pyrrole alkane dithiocar-bamate (Pyrrolidinedithioearbamate, PDTC), Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site, is provided by Suzhou University's Pharmaceutical Biotechnology institute.
2 experimental techniques
2.1ConA causes immune liver injury model preparation method
With reference to related documents, ConA 18mg/kg mouse tail vein injection is prepared liver injury model.And the amount of required ConA is calculated according to body weight, then diluting with aseptic PBS is 0.3mL volume injection, eyeball blood sampling is plucked after injecting latter 8 hours, the centrifugal 10min of blood 3000rpm/min, get serum ultra cold storage freezer frozen in order to next step detect needed for, measure the activity of ALT, AST, and put to death mice in the 8th hour, get liver and detect corresponding index.
2.2 animal packet design
After ICR mice adaptability feeds three days, be divided into 6 groups at random, often organize 10.(1) Normal group: every day gavage equal volume normal saline, once a day, continuous 5 days.(2) immune liver injury model group: every day gavage equal volume physiological saline solution, once a day, continuous 5 days, after the 5th day gavage, half an hour, tail vein injection ConA 18mg/kg was to cause immune liver injury model.(3) PDTC treatment group: every day gavage equal volume normal saline, and in the 5th day at ConA by first 1 hour of above-mentioned dosage injection, ConA and PDTC dilution is equally all 0.3mL volume by PDTC120mg/kg lumbar injection.(4) administration of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site is by high dose 10mg/kg every day, middle dosage 3mg/kg, low dosage 1mg/kg gavage mice, continuous gavage 5 days, 1 hour tail vein injection ConA 18mg/kg after the 5th day gavage.
2.3 Testing index
2.3.1 serum enzyme inspection
2.3.1.1 reagent: glutamate pyruvate transaminase detection kit (ALT, reitman-frankel method), purchased from Shanghai Ke Xin biotech company; Glutamic oxaloacetic transaminase, GOT detection kit (AST, reitman-frankel method), purchased from Shanghai Ke Xin biotech company.
2.3.2 histopathologic examination
2.3.2.1 reagent: 10% neutral formalin (by 40% formaldehyde 10mL, the PBS of pH=7.4 is formulated); Hematoxylin (element): hematoxylin 1 gram, 8.8 grams, aluminum sulfate, dehydrated alcohol 10mL, distilled water 200mL.First use distilled water heating for dissolving aluminum sulfate, then pour into the Lignum Sappan seminal fluid being dissolved in dehydrated alcohol in the aluminum sulfate liquid that dissolved, boil 1 minute, slightly cool, slowly add sodium iodate 0.1 gram, dye liquor becomes aubergine, with gauze lid bottleneck, after using front filter paper filtering, every 100mL adds glacial acetic acid 5mL; Yihong liquid: after ethanol being dissolved in the grinding of 1 gram, Yihong with Glass rod, instillation acetic acid makes in the pasty state, and addend ml distilled water filters, dried in an oven by the sediment leached, dissolve in 95% ethanol 200mL.
2.3.3 the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is on the impact of serum levels of inflammatory cytokines TNF-α, IFN-γ
Experiment grouping is the same with administration, plucks eyeball and gets blood, centrifugally leave and take serum after modeling administration 8h.ELISA method measures the expression of TNF-α, IFN-γ (pg/mL).
2.3.4 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site on the impact of the expression of hepatic tissue NF-κ B p65 experiment grouping and administration the same, put to death mice after modeling administration 8h, cut open the belly and get liver.Western-blot method measures the expression of murine liver tissue NF-κ B p65.
3 statistical dispositions
The data obtained all with
represent, adopt SPSS11.0 statistical software analytical data, adopt one factor analysis of variance: adopt when variance is neat during LSD method (least significant difference method) heterogeneity of variance and adopt Dunnett T3 method.The horizontal α of variance analysis test of homogeneity
1=0.05, level of significance test α
2=0.05.
4 testing results
4.1ALT and AS checks: after ConA injects 8 hours, Serum ALT and AST concentration significantly raise, and compared with Normal group, difference has significance (p<0.01), proves modeling success.PDTC is as NF-kB activity inhibitor, the damage that ConA causes mouse liver can be resisted, obviously can reduce Serum ALT and AST level, and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site heavy dose group also significantly can reduce the level of Serum ALT and AST, both all have significant difference (p<0.01) compared with model control group.And small dose group is compared with model group, there is certain significance (p<0.05).The results are shown in Table 1.
Table 1 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site is on the impact (n=10) of immune hepatic injury mice serum ALT and AST
Note: compared with Normal group,
#p<0.05;
##p<0.01; Compared with model control group,
*p<0.05;
*p<0.01
4.2 pathological examination results
4.2.1 visual results: model group liver enlargement, there is the ecthyma gangrenosa differed in size on surface.Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site group, that positive drug control group liver changes comparatively model group is light.Normal group liver shows no obvious abnormalities.
4.2.2 light microscopy checking: this experiment evaluates Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site respectively to the improvement effect of blood lipid under the principle following the classification of hepatic injury pathological grade in hepatocellular degeneration and hepatic necrosis two.HE coloration result is shown in Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D, Fig. 4 E, Fig. 4 F, adopt 6 groups, Fig. 4 A is Normal group, Fig. 4 B is Normal group+Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site high dose group, Fig. 4 C is model group, and Fig. 4 D is Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site low dose group, and Fig. 4 E is dosage group in model group+Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site, Fig. 4 F is model group+Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site high dose group, and their histological scores is in table 2.
Hepatocellular degeneration aspect, χ
2=55.835, p=0.000; Hepatic necrosis aspect, χ
2=40.731, p=0.000; Show that each group of curative effect exists significant difference.Compared with normal group, especially significantly (p < 0.05/5), Animal Model is successful in the pathological changes such as the degeneration of model group liver and necrosis.In hepatocellular degeneration and hepatic necrosis, each administration group improves the Be very effective of liver degeneration, and has certain dose dependent.Microscopic observation the results are shown in Table 2.
Table 2 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site is on the impact (n=10) of the liver morphology of each group of mice
Note:
#p < 0.05/5,
*p < 0.001, compares with model group.
The level of 4.3 each group peripheral blood TNF-αs, IFN-γ
As shown in table 3, compare with normal group, model group mouse peripheral blood TNF-α, IFN-γ level all significantly raise (p<0.05).Compare with model group, matched group, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site various dose group all can obviously reduce mouse peripheral blood TNF-α, IFN-γ level, there is significant difference (p<0.05).
Table 3 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site to the comparison of each group of mice serum TNF-α, IFN-γ level (
)
Note: compare with normal group,
##p<0.01,
#p<0.05, compares with model
*p < 0.01,
*p < 0.05.
4.4 the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is on the impact of the expression of hepatic tissue NF-κ B p65
Western-blot method measures the expression of murine liver tissue NF-κ B p65.Statistical result, in table 4, is compared with normal group, and the level of model group NF-κ B p65 obviously raises, and there were significant differences for statistics (p<0.05); Compare with model group, the level of matched group and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site various dose group NF-κ B p65 has significant change, and there were significant differences for statistics (p<0.05).Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site various dose group obviously can reduce the expression of immunological liver injury in mice hepatic tissue NF-κ B p65.
Table 4 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen effective site on the impact of the expression of each group of hepatic tissue NF-κ B p65 (
)
Note: compare with normal group,
##p<0.01,
#p<0.05, compares with model
*p < 0.01,
*p < 0.05.
The present invention further discloses the application of pharmaceutical composition in the immune liver damage disease of preparation treatment that the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site containing oleanolic acid and ursolic acid forms, particularly in the application for the treatment of viral hepatitis, autoimmune hepatitis, hepatic necrosis, fatty liver, cholestasis, hepatic fibrosis, liver cirrhosis and hepatocarcinoma etc.
The present invention extracts the total ter penoids of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, isolation and purification, and based on the pathological characteristic of human viral's hepatitis immunologic derangement, set up the model of immunological liver injury, observe the impact on immunological liver injury in mice serum enzyme and histopathology of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene, the total terpene of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is on the impact of immune hepatic injury critical inflammatory medium T NF-α and IFN-γ expression, and the regulatory mechanism research to TNF-α and IFN-γ upstream signaling molecule consideration convey record molecule NF-κ B, inquire in immunologic liver injury process, the preliminary molecular mechanisms of action of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene liver protection effect, for the medicine total for Isodon amethystoides (Benth.) C.Y.Wuet Hsuen terpene being developed to control hepatic injury provides theoretical foundation.
Effect of the present invention:
(1) a kind of total terpenoid effective site extracted from Chinese crude drug Isodon amethystoides (Benth.) C.Y.Wuet Hsuen is provided, suitable pharmaceutical preparation is become as the immune liver damage disease for the treatment of using the main component production and processing of medicine, liver state can be improved, have no side effect, prove that it is evident in efficacy through pharmacodynamics test;
(2) the medicine material source in the present invention is easy to get, be easy to industrialization, various dosage form can be made as required, for clinically providing convenient, more effectively, the more controlled modern Chinese medicine of quality, for patient brings more interests, thus produce huge social benefit;
(3) the immune liver damage disease that medicine of the present invention is treated mainly comprises: viral hepatitis, autoimmune hepatitis, hepatic necrosis, fatty liver, cholestasis, hepatic fibrosis, liver cirrhosis and hepatocarcinoma etc.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, is characterized in that: it contains oleanolic acid and ursolic acid, by raw material weight percentage ratio composition containing the oleanolic acid of 0.02% to 0.5%, the ursolic acid of 0.05% to 1%.
2. the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, is characterized in that: it contains oleanolic acid and ursolic acid, and by the percentage by weight of the total terpene effective site of this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen, content of oleanolic acid accounts for 0.8% to 8%, and ursolic acid content accounts for 1.0% to 12%.
3. therefrom extract a method for total terpene effective site in king of medicine Chinese date, it is characterized in that, it comprises the following steps:
(1) get clean Isodon amethystoides (Benth.) C.Y.Wuet Hsuen to pulverize, add 15 to 35 times of weight, 70% to 95% ethanol, 50 to 80 DEG C of supersound extraction 40 to 100min, extracting liquid filtering, filtrate is with hydrochloric acid adjust pH=1-3, and to filtrate reflux hydrolysis 20 to 40min, the water adding 0.5-1.0 times of volume in hydrolyzed solution fully mixes, again with isopyknic chloroform extraction several times, combining extraction liquid;
(2) by the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen extract concentrating under reduced pressure of gained, carry out post separation, with water and ethanol elution, collect respective streams part, lyophilizing, obtains the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen as claimed in claim 1 or 2.
4. in therefrom king of medicine Chinese date as claimed in claim 3, extract the method for total terpene effective site, it is characterized in that, this detached dowel is macroporous resin column, the macroporous adsorbent resin of this macroporous resin column is at least one in D101, AB-8, HPD100A, HPD400, HPD600, HPD750, adsorption time is after 6 to 10 hours, eluting is carried out with 1 to 4BV/h flow velocity, first with the washing post removing water-solubility impurity of 3 to 6BV, use 3 to 6BV40% to 60% ethanol elution again, merge ethanol elution, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
5. in therefrom king of medicine Chinese date as claimed in claim 3, extract the method for total terpene effective site, it is characterized in that, this detached dowel is polyamide column, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 25-40, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
6. in therefrom king of medicine Chinese date as claimed in claim 3, extract the method for total terpene effective site, it is characterized in that, this detached dowel is sephadex column, this sephadex column is selected from G-10, G-25, G-50, LH-20, and with water elution, the weight ratio of extracting solution and filler is 1 ︰ 20-50, after an eluting 1-3 column volume, start to collect stream part 2-6 column volume, lyophilizing, obtains the effective portion of the total terpene of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
7. in therefrom king of medicine Chinese date as claimed in claim 3, extract the method for total terpene effective site, it is characterized in that, this detached dowel is reverse C18, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 10-20, an eluting 2-4 column volume, starts to collect stream part and collects corresponding stream part 5-11 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
8. in therefrom king of medicine Chinese date as claimed in claim 3, extract the method for total terpene effective site, it is characterized in that, this detached dowel is C8 post, with water elution, the weight ratio of extracting solution and filler is after 1 ︰ 20-50, an eluting 1-3 column volume, starts to collect stream part 2-6 column volume, lyophilizing, obtains the total terpene effective site of purity >=50% Isodon amethystoides (Benth.) C.Y.Wuet Hsuen.
9. the application of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen as claimed in claim 1 or 2 total terpene effective site in anti-immune hepatic injury.
10. treat the pharmaceutical composition of immune liver damage disease for one kind, it is characterized in that: comprise the total terpene effective site of a kind of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen as main component in this pharmaceutical composition, this Isodon amethystoides (Benth.) C.Y.Wuet Hsuen total terpene effective site is the total terpene effective site of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen as claimed in claim 1 or 2.
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| CN105218616A (en) * | 2015-10-23 | 2016-01-06 | 山东省果树研究所 | A kind of method extracting ursolic acid and Oleanolic Acid from Blueberry |
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| CN106581146A (en) * | 2017-01-11 | 2017-04-26 | 安徽科技学院 | Isodon amethystoides heat-clearing bacteriostatic granule and production method thereof |
| CN108432816A (en) * | 2018-03-14 | 2018-08-24 | 淮北师范大学 | The extracting method and active constituent of common rabdosia leaf active constituent are in application agriculturally |
| CN108432816B (en) * | 2018-03-14 | 2020-07-28 | 淮北师范大学 | Extraction method of active ingredients of Wangzao date and application of active ingredients in agriculture |
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