CN105319332A

CN105319332A - Quality evaluation method of blood-fat-reducing traditional Chinese medicines on the basis of bio-titer

Info

Publication number: CN105319332A
Application number: CN201410315623.8A
Authority: CN
Inventors: 周亚伟
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-07-03
Filing date: 2014-07-03
Publication date: 2016-02-10

Abstract

The invention relates to a quality evaluation method of blood-fat-reducing traditional Chinese medicines on the basis of bio-titer, wherein the quality evaluation method employs the bio-titer as an evaluation index, and particularly, the bio-titer of reducing cholesterol and/or the bio-titer of reducing triglyceride is employed as the evaluation index. The bio-titer of reducing cholesterol includes the bio-titers of inhibiting absorption and synthesis of cholesterol or promoting metabolism or excretion of the cholesterol; and the bio-titer of reducing triglyceride includes the bio-titers of inhibiting absorption and synthesis of triglyceride or promoting excretion of the triglyceride. The quality evaluation method can be used as effective supplement of a traditional chemical detection method, can directly reacts clinical validity, and is a quality control and evaluation method which really satisfies traditional Chinese medicine theory, shows clinical application characters of traditional Chinese medicines and ensures safety and effect of the traditional Chinese medicines.

Description

A kind of reducing blood lipid class medicine quality evaluated method based on biological value

Technical field

The invention belongs to traditional Chinese medicine quality to control and assessment technique field, particularly a kind of medicine quality evaluated method based on estimation of biological potency, more particularly, relate to the hypolipidemic activity detection method of Chinese medicine and the biological quality evaluation method of antilipemic Chinese herbal medicine.

Background technology

Compare chemical drugs, traditional Chinese medicine ingredients is very complicated, and the modernization of its quality standard and internationalization are perplex the core that Chinese medicine develops always, and the key of the quality standard of Erecting and improving sets up science, reliable quality control and evaluation method.Present stage traditional Chinese medicine quality control with evaluate with the qualitative and quantitative analysis of chemical composition for Main Means.Along with deepening continuously of traditional Chinese medicine research, find that this pattern controls at traditional Chinese medicine quality and shown very large limitation gradually in appraisement system.Such as measure jamaicin and can not be confirmed to be the coptis or golden cypress, rely on separately their content how much accurately can not reflect the quality of quality of medicinal material.And for example single component curcumin content height, can not represent the quality of this taste quality of medicinal material of turmeric, more overallly can not reflect the compound preparation containing turmeric, the hypolipidemic activity height of such as Fat-reducing are blood-circulation promoting capsule.Using the content of indivedual or part index number composition as an index of quality standard, although can control the amount of index composition, but be only the amount of a certain or some composition controlled wherein, be difficult to the stability and the consistance that ensure all the components, more be difficult to the overall biological effect embodying Chinese medicine, ensure the consistance of its clinical efficacy.Therefore, propose biologically active to detect in recent years and be applied to traditional Chinese medicine quality control with appraisement system, and the assay method that the existing bibliographical information medicinal material associated biomolecules such as Radix Isatidis, rheum officinale, balloonflower root are tired also advises adding in quality of medicinal material standard biological activity determination item, to make up the defect of chemical detection means.Estimation of biological potency method is as a kind of quality analysis means based on biological activity determination, and it is expected to become one and really meets traditional Chinese medicine traditional theory, embodies Chinese medicine clinical practice feature, ensures Chinese medicine quality control and evaluation method safely and effectively.

But, current estimation of biological potency method is at the rarely seen report of the achievement in research of field of Chinese herbal, and the assay method of biological value for Reducing Blood Lipid by Chinese medicine medicine, or the quality assessment of the reducing blood lipid class Chinese medicine recorded based on biological value or detection method have not yet to see report.

Luo Rong have studied dried immature fruit of citron orange reducing blood lipid composition and effectiveness and biological effect thereof in its doctoral candidate's academic dissertation " dried immature fruit of citron orange reducing blood lipid composition and effectiveness biological effect study on evaluation way ", first demonstrate the dried immature fruit of citron orange with in vivo studies and there is fat biological activity, then the external biological effect index research dried immature fruit of citron orange relevant with high fat of blood and the reducing blood lipid effect of composition and effectiveness thereof is chosen, the external biological effect index that it adopts is Antioxidant Indexes, namely Ox-LDL is 1. adopted to damage HUVEC model, the impact that the research dried immature fruit of citron orange and composition and effectiveness thereof discharge HUVECICAM-1 developed by molecule and NO; 2. the HepG2 cell oxidative damage model adopting H2O2 and t-BOOH to induce, studies the dried immature fruit of citron orange and composition and effectiveness spills LDH, MDA content, T-AOC, Cu-Zn-SOD vigor, GSH-Px vigor, CAT effect of vigor; 3. utilize Flow Cytometry, the research dried immature fruit of citron orange and composition and effectiveness thereof are on the impact of MMP; 4. utilize single cell gel electrophoresis, the research dried immature fruit of citron orange is to the protective effect of the HepG2 cell DNA oxidative damage that H2O2 and t-BOOH induces.By the mensuration of reducing blood lipid external biological effect and in conjunction with HPLC method, determine the composition and effectiveness of dried immature fruit of citron orange reducing blood lipid: the scope being benchmark with aurantiin-aurantiamarin-poncirin-neohesperidin-naringenin-hesperetin (223.66:18.50:254.49:321.00:4.46:1), this composition and effectiveness and Fructus Aurantii Immaturus fat biological effect have equivalence, therefore, this composition and effectiveness can state the qualitative characteristics of dried immature fruit of citron orange reducing blood lipid clinical efficacy.Therefore the biological effect action effect standard of available dried immature fruit of citron orange composition and effectiveness, evaluate the inherent quality of Fructus Aurantii Immaturus, detect by the identical biological effect assay method of sample to be evaluated, and effect standard comparing therewith, calculate BER.This research is also using BER (BER) as the quality of metrics evaluation medicine, and 1 >=BERS >=0.9 and comparing and effect standard indifference through mathematics is qualified medicine; BERS>1 and to compare with effect standard indifference through mathematics be high-quality medicine; BERS<0.9 or compare variant for defective medicine with effect standard through mathematics.

Above-mentioned prior art builds its biological effect evaluation method by adopting Antioxidant Indexes method and ICAIU representation, and these external pharmacological models and reducing blood lipid disease there is no direct correlation, indication corresponding to antioxidation widely, can not direct correlation reducing blood lipid drug effect, therefore the method specificity of the prior art is not strong, needs to be found external model and the evaluation index of more formulated Hypolipidemic efficacy.

200910141969.X Chinese invention patent " a kind of Colestid quality evaluating method based on adjusting fat antiopxidant effect " discloses a kind of Colestid quality evaluating method based on adjusting fat antiopxidant effect.The evaluation index of the tune fat antioxidation biology effect that it adopts is: (1) Antioxidant Indexes: the HUVEC cell viability of OX-LDL induced damage, HUVEC nutrient solution NO, ET-1 content of OX-LDL induced damage, the HUVEC endothelial cell MDA content of OX-LDL induced damage, SOD vigor; (2) metabolite content index: BEL-7402 liver cell inner cholesterol content, TBA content inside and outside BEL-7402 liver cell.The same with prior art above, also adopt BER (BER) as the quality good or not of metrics evaluation Chinese medicine compound prescription in this patent, described BER is also by first determining composition and effectiveness, by the biological effect action effect standard of this composition and effectiveness, the identical biological effect assay method of sample to be evaluated is detected, and effect standard comparing therewith, the ratio of the biological effect of both calculating.Equally, Antioxidant Indexes and metabolite content index definitely, exclusively, intactly can not express the biologically active of reducing blood lipid.

Further, above-mentioned two parts of BERs described in the prior art all refer to the biological effect detecting sample and reference substance under identical condition, the ratio of the biological effect both then calculating.This BER can only the height of response sample and reference substance biological effect; The bioactive power of surveyed medicine relative to other similar drugs can not be reflected, feature and the intensity of this medicinal material or compound preparation Lipid-lowering activities in similar drug can not be evaluated.

Therefore, be necessary the quality evaluating method building a kind of reducing blood lipid class Chinese medicine based on estimation of biological potency, its evaluation index can the clinical efficacy of direct correlation reducing blood lipid, this evaluation method can comprehensive evaluation Reducing Blood Lipid by Chinese medicine group effect or its total quality, and this quality evaluating method also can be used for feature and the power of evaluating reducing blood lipid pharmacologically active between different medicine.

Summary of the invention

Existing blood lipid-lowering medicine quality control and evaluation method are main mainly with the qualitative and quantitative analysis of chemical index composition, but this chemical detection means cannot be associated with clinical efficacy, therefore, really effectively can not evaluate the quality of medicine.And the biological effect evaluation method of prior art by adopting Antioxidant Indexes method and ICAIU representation or metabolite content determination method to build fat-reducing medicament, but, these indexs and clinical Hypolipidemic efficacy there is no direct correlation, directly can not react medicine hypolipidemic activity power.For solving the defect existed in existing blood lipid-lowering medicine quality control and evaluation method, the invention provides a kind of blood lipid-lowering medicine quality evaluating method based on estimation of biological potency.With the extract of compound Chinese medicinal preparation or Chinese crude drug for test sample, the blood lipid-lowering medicine of determined curative effect product in contrast clinically, adopt ill vitro test method, use multiple biological standardization means, measure each medicine group (comprising test sample and the reference substance) absorption to cholesterol, synthesis, the absorption of metabolism and excretion and triglyceride, the biological effect of each application point of synthesis and metabolism, the biological value of each application point is recorded by the dose ratio of reactions such as calculating, evaluate from the group effect of many aspects to Chinese medicine, can be medication combined exploitation (such as with the reducing blood lipid class Drug combination of other mechanism of action, play better lipid-lowering effect by machine-processed complementation) or the medication of the clinical side of sending reference frame is provided, can also control with biological value and evaluate the inherent quality of test sample or active height.From different reducing blood lipid approach, this quality evaluating method reflects that described medicine fat biological is active strong and weak respectively, embody its group effect, with the validity direct correlation of clinical application, and can also compare hypolipidemic activity power and feature between different pharmaceutical.

For solving the deficiencies in the prior art, the invention provides a kind of reducing blood lipid class medicine quality evaluated method, it is using biological value as quality evaluation index.

Wherein, described quality evaluating method reduces cholesterol biological value and/or reduces triglyceride biological value as quality evaluation index.

Wherein, described reduction cholesterol biological value refers to the biological value suppressing cholesterol absorption, suppress cholesterol biosynthesis, promote cholesterol metabolic or promotion Cholesterol Excretion, evaluates with the height of measured biological value the power that Chinese medicine reduces hypocholesterolemic activity.

Wherein, described reduction triglyceride biological value refers to the biological value suppressing triglyceride to absorb, suppress triglyceride synthesis or promotion Triglyceride Metabolism in Patients, evaluates with the height of measured biological value the power that Chinese medicine reduces triglyceride activity.

In embodiments more of the present invention, the mensuration of described reduction cholesterol biological value detects the biological value of Chinese medicine to one or more link in the absorption of cholesterol, synthesis, metabolism, excretion four processes by experiment in vitro, evaluates with the height of measured biological value the power that Chinese medicine reduces hypocholesterolemic activity; The mensuration of described reduction triglyceride biological value detects the biological value of Chinese medicine to one or more link in the absorption of triglyceride, synthesis, metabolism three links by experiment in vitro, evaluates with the height of measured biological value the power that Chinese medicine reduces triglyceride activity.

In another embodiment, supplementing as above-mentioned quality evaluating method, particularly when medicine itself not plays the active component of effect for reducing blood fat, but the product produced after biological conversion is in vivo only active substance, this quality evaluating method also comprises makes described Chinese medicine after gut flora fermentation, obtain tunning, experiment in vitro is adopted to detect the absorption of this tunning to cholesterol, synthesis, metabolism and excretion and the absorption to triglyceride, the biological value of synthesis and metabolism links, quality evaluation index using the biological value of one or more link as lipopenicillinase class Chinese medicine.

In another embodiment, supplementing as above-mentioned quality evaluating method, particularly when medicine itself not plays the active component of effect for reducing blood fat, but the product produced after metabolism is in vivo only active substance, this quality evaluating method can also comprise makes described Chinese medicine obtain metabolic product under the effect of liver microsomes enzyme, experiment in vitro is adopted to detect the absorption of this metabolic product to cholesterol, synthesis, metabolism and excretion and the absorption to triglyceride, the biological value of synthesis and metabolism links, quality evaluation index using the biological value of one or more link as lipopenicillinase class Chinese medicine.

In some embodiments of the present invention, the reducing blood lipid class medicine quality evaluated method based on minor biological cycling provided, detect the biologically active of Chinese medicine to the absorption of cholesterol, synthesis, metabolism and excretion and each application point to the absorption of triglyceride, synthesis and metabolism by vitro test, to choose wherein biologically active test experience result be positive and dose-effect is the application point of correlativity, carry out estimation of biological potency, the index using the biological value of one or more application point as reducing blood lipid class medicine quality evaluated.

Particularly, two classes can be divided in order to the biological value characterizing hypolipidemic activity power of the present invention: reduce cholesterol biological value and reduce triglyceride biological value.

Wherein, reduce cholesterol biological value and comprise following four kinds:

A. the biological value of cholesterol intestinal absorption is suppressed;

B. cholesterol biosynthetic biological value is in vivo suppressed;

C. the biological value of cholesterol metabolic is promoted; And

D. the biological value of Cholesterol Excretion is promoted.

Reduce triglyceride biological value and comprise following three kinds:

E. the biological value of triglyceride intestinal absorption is suppressed;

F. triglyceride biosynthetic biological value is in vivo suppressed; And

G. the biological value of Triglyceride Metabolism in Patients is promoted.

More than for the height of 7 kinds of biological values in order to characterize hypolipidemic activity power can be used for evaluating or detecting or control traditional Chinese medicine quality.

In the specific embodiment of the present invention, described biological value can be any one in above-mentioned 7 kinds of biological values, also can be the combination in any of above-mentioned 7 kinds of biological values, the wherein combination of preferred above-mentioned 7 kinds of biological values, because it can by the absorption of complete detection drug on cholesterol, synthesis, metabolism and excretion and the absorption to triglyceride, the biological value of the links of synthesis and metabolism, active from its fat biological of many aspects comprehensive evaluation, reflect the reducing blood lipid effect of its entirety, and which application point to play effect for reducing blood fat by this quality evaluating method detection of drugs by, the blood fat reducing function feature of the different herbal species of comprehensive representation, quantize and characterize effect speciality of dissimilar blood lipid-lowering medicine, for the joint development or safer clinically of medicine, effectively, the side's of sending medication provides foundation exactly.

In some embodiment, Chinese medicine of the present invention can be Chinese medicine compound prescription, also can be single medicinal material material or medicine materical crude slice, can also be their extract.In some embodiments, described antilipemic Chinese herbal medicine is selected from one or more in following Chinese patent drug or Chinese crude drug contained by it or its medicine materical crude slice or extract: blood fat recovery capsule, stasis open capsule, Baolier capsules, gynostemma pentaphyllum total glycosides capsule, Fat-reducing are blood-circulation promoting capsule, lipopenicillinase channel activating soft capsule, fat must safe capsule, hypogastric lipid-lowering pill, red fragrant Qingzhi Granules.In an instantiation, described Chinese medicine is compound preparation Fat-reducing are blood-circulation promoting capsule, and its prescription is: cassia seed 100g, turmeric 1500g, rhizoma alismatis 2000g, pseudo-ginseng 500g and bermudagrass herb 2000g.In another instantiation, described Chinese medicine is Chinese crude drug turmeric.In another instantiation, described Chinese medicine is single preparations of ephedrine blood fat recovery capsule, is by the capsule of red colouring agent for food, also used as a Chinese medicine through being processed into.

Estimation of biological potency method of the present invention can be carry out according to " Chinese Pharmacopoeia " 2010 editions two annex XIV Bioassay-statistical methods, such as according to parallel line assay principle, carry out according to 22 methods in the parallel line analysis determination method specified under " Chinese Pharmacopoeia " 2010 editions two annex XIV Bioassay-statistical method items; Also can be adopt method in existing document, " research (II) of motherwort medicinal material biological assay method---oxytocin, motherwort dose-effect relationship and calibrating are suitable for the foundation of Effective pattern " " four-point method " shown in a literary composition that " biological assay of the biological detection method III-Herba Andrographitis of the antipyretic and antidotal type Chinese medicine " people such as " one point method " shown in a literary composition or Yang Minghua that the people such as such as Wang Shengmin deliver delivers; Other science, the feasible estimation of biological potency method that can quantize biological effect can also be adopted.Any restriction is not done, as long as can realize the object of the invention for which kind of estimation of biological potency method of employing and the present invention of statistical procedures method.

Exemplarily illustrate described in order to characterize the assay method of 7 kinds of biological values of hypolipidemic activity power, but should can not limiting the scope of the invention with this below.

The biological value of suppression cholesterol intestinal absorption of the present invention can measure as follows:

A. reference substance solution preparation: be reference substance with cholesterol absorption inhibitor, be dissolved in MEM nutrient solution, be made into the reference substance solution of respective concentration, filter, for subsequent use, described cholesterol absorption inhibitor is preferably Ezetimibe;

B. need testing solution preparation: with medicine to be measured for test sample, preferably, took the test sample powder of 10-100 mesh sieve, with appropriate solvent extraction, extract after filtration, according to the estimated value that test sample is tired, obtains need testing solution with distilled water diluting;

C. need testing solution and reference substance solution are diluted to the dosage group of variable concentrations with identical dose of distance, and agent distance is 0.5-1.0, such as, when being 0.6, and middle dosage=high dose * 0.6; Low dosage=middle dosage * 0.6;

D. data processing calculates with tiring: the cholesterol absorption inhibiting rate recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical cholesterol absorption inhibiting rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the bioactive power of Drug inhibition cholesterol absorption to be measured.

The biological value of suppression triglyceride intestinal absorption of the present invention can measure as follows:

A. reference substance solution preparation: taking pancreatic lipase inhibitors as reference substance, is solvent with deionized water, is made into the reference substance solution of respective concentration, filter, for subsequent use, described pancreatic lipase inhibitors is preferably Xenical or ATL-962;

D. data processing calculates with tiring: the pancreatic lipase activity inhibiting rate recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical pancreatic lipase activity inhibiting rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the bioactive power that Drug inhibition triglyceride to be measured absorbs.

Suppression cholesterol of the present invention in vivo biosynthetic biological value can measure as follows:

A. reference substance solution preparation: take HMG-CoA reductase inhibitor as reference substance, be solvent with reaction buffer, be made into the reference substance solution of respective concentration, filter, for subsequent use, described reaction buffer is such as by 70mM phosphate buffer, 2mMEDTA, 2mMDTT, 0.06%BSA and 0.15mMNADPH form, this pH of cushioning fluid is 7.0, and described HMG-CoA reductase inhibitor is preferably Simvastatin, general Liprevil;

D. data processing calculates with tiring: the HMG-CoA reductase maximum inhibition recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical HMG-CoA reductase maximum inhibition, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects Drug inhibition cholesterol to be measured biosynthetic bioactive power in vivo.

Suppression triglyceride of the present invention in vivo biosynthetic biological value can measure as follows:

A. reference substance solution preparation: with triglyceride synthetic inhibitor for reference substance, be dissolved in DMEM nutrient culture media, be made into the reference substance solution of respective concentration, filter, for subsequent use, described triglyceride synthetic inhibitor is preferably Acipimox;

B. need testing solution preparation: with medicine to be measured for test sample, preferably, took the test sample powder of 10-100 mesh sieve, with appropriate solvent extraction, extract after filtration, according to the estimated value that test sample is tired, obtains need testing solution with DMEM dilution;

D. data processing calculates with tiring: the free fatty acid recording the dosage group of the variable concentrations of test sample and reference substance respectively generates inhibiting rate, calculate the ratio that test sample and reference substance reach dosage when substantially identical free fatty acid generates inhibiting rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects the bioactive power of Drug inhibition lipolysis to be measured.

The biological value of promotion cholesterol metabolic of the present invention can measure as follows:

A. reference substance solution preparation: with LDL receptor (LDLR) activator for reference substance, be dissolved in DMEM nutrient culture media, be made into the reference substance solution of respective concentration, filter, for subsequent use, described LDL receptor (LDLR) activator is preferably jamaicin or Berberine hydrochloride;

D. data processing calculates with tiring: the LDLR rise rate recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical LDLR rise rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of cholesterol metabolic.

The biological value of promotion Triglyceride Metabolism in Patients of the present invention can measure as follows:

A. reference substance solution preparation: with lipoprotein lipase activator for reference substance, be dissolved in DMEM nutrient culture media, be made into the need testing solution of respective concentration, filter, for subsequent use, described lipoprotein lipase activator is preferably Bezafibrate, Clofibrate, Gemfibrozil, fenofibrate;

D. data processing calculates with tiring: the LPL enzymatic activity increment rate recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical LPL enzymatic activity increment rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of Triglyceride Metabolism in Patients.

The biological value of promotion Cholesterol Excretion of the present invention can measure as follows:

A. reference substance solution preparation: with bile acid absorption inhibitor for reference substance, be solvent by NaCl solution, be made into the need testing solution of respective concentration, filter, for subsequent use, described bile acid absorption inhibitor is preferably colesevelam, cholestyramine;

B. need testing solution preparation: with medicine to be measured for test sample, preferably, took the test sample powder of 10-100 mesh sieve, with appropriate solvent extraction, extract after filtration, according to the estimated value that test sample is tired, obtains need testing solution with NaCl solution dilution;

D. data processing calculates with tiring: the bile salt Percentage bound recording the dosage group of the variable concentrations of test sample and reference substance respectively, calculate the ratio of dosage when test sample and reference substance reach substantially identical bile salt Percentage bound, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of Cholesterol Excretion.

Beneficial effect of the present invention at least comprises:

1, reducing blood lipid class medicine quality evaluated method provided by the present invention is utilized to carry out traditional Chinese medicinal materials assortment qualification and quality evaluation, quality control and evaluation can also be carried out to Chinese medicine compound prescription or Chinese crude drug or its extract, characterize the inherent quality of reducing blood lipid class Chinese medicine with the biological value reducing cholesterol and/or the biological value that reduces triglyceride and activity strong and weak;

2, estimation of biological potency method sensitivity provided by the invention is high and reliable and stable, selected biological value index and hypolipidemic activity direct correlation, can Efficient Characterization fat biological active, the present invention efficiently solves the quality assessment of the blood lipid-lowering medicine based on estimation of biological potency, ensure that the qualitative, quantitative of blood lipid-lowering medicine uses, as supplementary and raising that is conventional and chemical detection, jointly can to check on its inherent quality from multiple angles, improve existing Quality Management Mode, and thinking and Technical Reference can be provided for the research of other medicine quality evaluated and control, economic and social benefit is huge,

3, the present invention is by the biological value of the absorption of complete detection drug on cholesterol, synthesis, metabolism and excretion and the links to the absorption of triglyceride, synthesis and metabolism, active from its fat biological of many aspects comprehensive evaluation, reflect the reducing blood lipid effect of its entirety, and detection of drugs which application point can play effect for reducing blood fat by, for medicine joint development or clinically safer, the side's of sending medication efficiently and accurately foundation is provided;

4, the present invention is by measuring the biological value of each application point, quantize the biological effect of each reducing blood lipid approach, structure biological effect is composed, clinically, doctor can need consciously according to biologically active direction and potency (property feature) side's of sending medication of the prompting of biological effect spectrum for the state of an illness, and the quantized value of biological value can become the scientific basis of clinical application.

5, the positive control drug used in fat biological titration method of the present invention is all that drug effect is clear and definite on this application point, be widely used clinically, the medicine of " mark post " property effect can be played, medicine to be measured is tired relative to the Relative biological of positive control drug can characterize its activity intensity in this similar drug, therefore, quality evaluating method of the present invention also can be used for feature and the intensity of evaluating fat biological activity between different medicine

6, the present invention not only may be used for the quality evaluating finished product or intermediate, and also can be used for the evaluation index of process optimization, the monitor and managment for production run is significant.

Embodiment

For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment, these embodiments only play illustrative effect, are not limited to range of application of the present invention, NM specific experiment method in the following example, conveniently experimental technique carries out usually.

Technological thought of the present invention is: first carry out biological activity determination experiment, namely detection of drugs is to the absorption of cholesterol, synthesis, metabolism and excretion and the biologically active to the absorption of triglyceride, synthesis and metabolism seven application points, choose that experimental result is positive and the application point that dose-effect is correlativity measures biological value further, described experimental result is that the positive refers to that test sample group is compared with negative control group, has significant difference statistically; Test positives control group result for positive for biological activity determination, and test sample group result is negative, then the biologically active testing result of this application point is judged to be feminine gender, and the application point of negative findings no longer carries out estimation of biological potency.Choose one or more biological activity determination experimental result for positive and dose-effect be correlativity application point measured by biological value as quality evaluation index, consider the cost that quality analysis detects and efficiency, preferably the biological value of 1 or 2 application point is as quality evaluation index, for the quality assessment of the quality control in traditional Chinese medicine quality evaluation, Chinese Traditional Medicine or final products.If the blood fat reducing function feature in order to fully understand medicine, for medication combined exploitation, such as with the reducing blood lipid class Drug combination of other mechanism of action, play better lipid-lowering effect by machine-processed complementation and scientific basis is provided, then preferably measure the positive and biological value of application point in dose-effect correlativity of each biological activity determination experimental result, by this can the blood fat reducing function feature of the different herbal species of comprehensive representation, quantize and characterize effect speciality of dissimilar blood lipid-lowering medicine, for the joint development of medicine provides foundation.

Biological value of the present invention is different from the definition of BER of the prior art (BER), and biological value of the present invention refers to the ratio of the dosage compared under the same conditions when test sample and reference substance (or standard items) produce identical or basic same reaction.

Specific embodiments of the invention have chosen Chinese patent drug Fat-reducing are blood-circulation promoting capsule, blood fat recovery capsule and Chinese crude drug turmeric.But method of the present invention is not limited to carry out quality assessment to these kinds, only exemplarily exemplify herein, not any type of restriction is done to the present invention, in fact, the medicine that any zoopery shows to have hypolipidemic activity all can adopt ill vitro test method of the present invention to measure its biological value relative to positive control medicine to carry out quality assessment, evaluate direction and the potency of its fat biological activity, the direction of described fat biological activity mainly refers to it plays effect for reducing blood fat by which approach, such as by suppressing cholesterol absorption, or by suppressing to synthesize in triglyceride body, described reducing blood lipid potency is exactly the biological value of corresponding positive contrast medicine on each application point.

Chinese patent drug Fat-reducing are blood-circulation promoting capsule used in the present invention (the accurate word Z20026429 of traditional Chinese medicines, Yunnan You Ke drugmaker), blood fat recovery capsule is (hereinafter referred to as Effects of Xuezhikang, the accurate word Z10950029 of traditional Chinese medicines, Beijing WBL Peking University Biotech Co., Ltd) all purchased from commercially available prod, two Chinese patent drugs use suitable dissolution with solvents, filtration respectively, filtrate is concentrated into 0.5g crude drug/ml, for subsequent use respectively as Fat-reducing are blood-circulation promoting capsule need testing solution, Effects of Xuezhikang need testing solution.

Turmeric medicinal material used in the present invention, also purchased from medicinal material market, is got dry medicinal material and is first crushed to 80 ~ 100 orders, then uses 10 times amount soak by water secondaries, each 2 hours, and liquid merges, and filter, filtrate is concentrated into 0.5g crude drug/ml, for subsequent use as turmeric need testing solution.

We have carried out associated biomolecule titration test respectively to compound Chinese patent medicine Fat-reducing are blood-circulation promoting capsule, folk prescription Chinese patent drug Effects of Xuezhikang and single medicinal material turmeric, now test findings are reported as follows:

Embodiment 1

First carry out biological activity determination to Fat-reducing are blood-circulation promoting capsule, turmeric, experimental result is as follows:

1, the bioactive mensuration of Fat-reducing are blood-circulation promoting capsule, turmeric suppression cholesterol absorption

Add the Fat-reducing are blood-circulation promoting capsule need testing solution of a certain amount of cholesterol and variable concentrations, turmeric need testing solution and Ezetimibe solution effects 24h in the Caco-2 cell (breaking up 17 days) of differentiation and maturation after, draw cell conditioned medium liquid, detect extracellular free cholesterol situation of change, evaluate the size of Fat-reducing are blood-circulation promoting capsule, the effect of turmeric suppression cholesterol absorption.Often group establishes 3 multiple holes, the blank group that be arranged in parallel, model group, cholesterol group and positive controls, in triplicate.Cholesterol absorption inhibiting rate is calculated as follows:

Cholesterol absorption inhibiting rate %=(administration group OD ₅₇₀-model group OD ₅₇₀)/cholesterol group OD ₅₇₀-model group OD ₅₇₀.

Specific experiment the results are shown in Table 1.

Table 1 Fat-reducing are blood-circulation promoting capsule, turmeric on cholesterol absorption inhibiting effect impact ( n=3)

2, the bioactive mensuration of Fat-reducing are blood-circulation promoting capsule, the absorption of turmeric suppression triglyceride

Triglyceride in food only has and can be absorbed by intestinal epithelial cell after pancreatic lipase digest and decompose is free fatty acid and monoglyceride.Based on this principle; in experiment with 96 orifice plates for carrier; by the drug response of pancreatic lipase, substrate triolein (principal ingredient is triglyceride) and variable concentrations; reaction conditions is: reaction buffer; the phosphate solution 25mM of pH7.5, pancreatic lipase concentration: 2mg/ml, substrate emulsion: every milliliter of 0.75mM natrium taurocholicum emulsifying soln 20 μ l triolein gained; temperature of reaction: 37 DEG C, the reaction time: 30min.

Experimental technique: under these experimental conditions, first be that the pancreatic lipase solution of 2mg/ml mixes with the Fat-reducing are blood-circulation promoting capsule of variable concentrations, turmeric need testing solution and Sai Keni positive reference substance solution respectively by concentration, add substrate triolein emulsion again and jointly hatch 30min, measure the growing amount of free fatty acid, detect variable concentrations test sample and reference substance to the inhibiting effect of pancreatic lipase activity with this, and then evaluate Fat-reducing are blood-circulation promoting capsule, turmeric to the inhibiting effect of triglyceride intestinal absorption.Be arranged in parallel enzyme solutions control group, Substrate controls group, blank group and standard control group, in triplicate.Specific experiment the results are shown in Table 2.

Data Processing in Experiment: the growing amount detecting enzymatic reaction free fatty acid, reflects pancreatic lipase activity with this, and calculates the inhibiting rate of medicine to pancreatic lipase.

Enzymatic activity is defined as: pH7.5, and under 37 DEG C of conditions, the enzyme dosage that catalyzing glycerol three ester per minute decomposes the fatty acid of generation 1 μm of ol is a unit of enzyme activity.

The computing formula of fatty acid growing amount is:

Wherein, OD _{mensuration group}represent administration group or the light absorption value of enzyme solutions control group under wavelength is 550nm; OD _{blank group}represent the light absorption value of blank product under wavelength is 550nm; OD _{standard items group}represent the light absorption value of standard items under wavelength is 550nm; Standard concentration is that now with the current, unit is mmol/mL according to the regulation preparation on " free fatty acid microdetermination kit #E1000 " instructions; V _srepresent sample volume, unit is mL.

Pancreatic lipase activity inhibiting rate computing formula is:

Table 2. variable concentrations Fat-reducing are blood-circulation promoting capsule, turmeric on the impact of pancreatic lipase ( n=3)

3, Fat-reducing are blood-circulation promoting capsule, turmeric suppress cholesterol biosynthetic bioactive mensuration in vivo

HMG-CoA reductase, HMG-CoA (substrate) and buffer system are jointly hatched, because HMG-CoA reductase catalysis HMG-CoA generates by NADPH hydrogen supply in the process of mevalonic acid (MVA), NADPH is at OD _339nmthere is the peak of light absorption value, by detecting the variable quantity of NADPH, judging whether medicine has the effect suppressing HMG-CoA reductase.

HMG-CoA reductase vitality test---spectrophotometric analysis

The absorbance value slip of 339nm is made to measure the activity of enzyme by monitoring due to the oxidation of NADPH.

First by before enzyme sample detection in 37 DEG C of water-bath 5min, each composition before adding cuvette in 25 DEG C of water-bath 5min, reaction buffer volume 1800 μ L is (containing pH7.0,70mM phosphate buffer, 2mMEDTA, 2mMDTT, 0.06%BSA, 0.15mMNADPH), add 100 μ LHMG-CoA reductases, after mixing, put into UV detector, detect OD in 5min _339nmreduction △ A1, obtain △ A1/5min; Add 100 μ L substrate HMG-CoA (final concentration is 100 μMs) and start reaction, detect OD in 5min _339nmreduction △ A2, draw △ A2/5min; Formula: E (U/L)=158.73/V _s× (△ A2/5-△ A1/5), and be provided with control group.

Experimental result calculates:

According to formula: E (U/L)=158.73/V _s× (△ A2/ △ T-△ A1/ △ T), calculates the enzyme activity of HMG-CoA reductase before and after administration, can obtain the inhibiting rate of medicine to HMG-CoA reductase activity by the change of enzyme activity after test sample effect:

Inhibition of enzyme activity rate=(E _{normal group}-E _{administration group})/E _{normal group}× 100%

Wherein, V _sfor sample volume, unit L; (△ A2/ △ T-△ A1/ △ T) represents enzyme reaction rate; In the present invention, △ T is such as that the implication that 5min, △ A1 and △ A2 refers to respectively is as implied above.

E _{normal group}represent Normal group, i.e. the enzyme activity of HMG-CoA reductase before administration; E _{administration group}represent administration group, i.e. the enzyme activity of HMG-CoA reductase after administration.Concrete outcome is in table 3.

Table 3 variable concentrations Fat-reducing are blood-circulation promoting capsule, turmeric on the impact of HMG-CoA reductase activity ( n=3)

4, Fat-reducing are blood-circulation promoting capsule, turmeric suppress triglyceride biosynthetic bioactive mensuration in vivo

Select murine preadipocyte cell 3T3-L1 cell to be experimental subjects, utilize TG synthetic inhibitor in-vitro screening model and positive drug Acipimox, MTT detects Fat-reducing are blood-circulation promoting capsule, turmeric to the maximal non-toxic concentration of 3T3-L1 cell.After the differentiation-inducing lipoblast of 3T3-L1 cell success, promote that TG resolves into free fatty acid (FFA) with isoprel and adenosine deaminase acting in conjunction in the successful 3T3-L1 cell of differentiation, variable concentrations test medicine Fat-reducing are blood-circulation promoting capsule is added on this basis in cell conditioned medium, turmeric effect 48h, cell controls group and positive controls are set simultaneously, positive drug is Acipimox, addition is 5mM, repeat 3 times respectively, detect the growing amount of each group of cells and supernatant free fatty acid, the power of Drug inhibition lipolysis ability is reflected with this, and calculate the inhibiting rate that medicine generates free fatty acid.

Free fatty acid growing amount computing formula is as follows:

Wherein, mensuration group OD _550nmrepresent administration group and cell controls group absorbance at 550 nm; Blank group OD _550nmrepresent blank group absorbance at 550 nm; Standard items group OD _550nmrepresent standard items absorbance at 550 nm; Standard concentration is that now with the current, unit is mmol/mL according to the regulation preparation on " free fatty acid microdetermination kit #E1000 " instructions; V _srepresent sample volume, unit is mL.

It is as follows that free fatty acid generates inhibiting rate computing formula:

Wherein, " cell controls group FFA measures " represents the growing amount of cells and supernatant free fatty acid before administration; The growing amount of cells and supernatant free fatty acid after " administration group FFA measures " expression administration.

After table 4. drug treating after 3T3-L1 cell each group FFA generate inhibiting rate ( n=3)

5, the bioactive mensuration of Fat-reducing are blood-circulation promoting capsule, turmeric promotion cholesterol metabolic

Hepatocellular carcinoma H22 is selected to be experimental subjects, utilize LDLR activator in-vitro screening model and positive drug Berberine hydrochloride, mtt assay detects Fat-reducing are blood-circulation promoting capsule to HepG2 cell maximal non-toxic concentration, three concentration are set on this basis, the tested Fat-reducing are blood-circulation promoting capsule of variable concentrations is added in cell conditioned medium, after turmeric and Berberine hydrochloride effect 72h, the total memebrane protein of cell surface is extracted by Membrane protein extraction kit, then the LDLR change in total memebrane protein is detected by LDL receptor ELISA detection kit, detect the expression of LDLR and calculate the rise rate of medicine to LDLR, the impact of Fat-reducing are blood-circulation promoting capsule on HepG2 cellular expression LDLR is reflected with this.

Wherein, mensuration group OD _450nmrepresent administration group and cell controls group absorbance at 450 nm; Blank group OD _450nmrepresent blank group absorbance at 450 nm; Standard items group OD _450nmrepresent standard items absorbance at 450 nm; The unit of standard concentration is mol/mL; V _sfor sample volume, unit is mL.

HepG2 cell LDLR rise rate after table 5. Fat-reducing are blood-circulation promoting capsule, turmeric effect 72h ( n=3)

6, the bioactive mensuration of Fat-reducing are blood-circulation promoting capsule, turmeric promotion Triglyceride Metabolism in Patients

Lipoprotein lipase (LPL) is the rate-limiting enzyme that plasma triglyceride decomposes, synthesize primarily of tissues such as fat, heart and skeletal muscle and secrete, triglyceride hydrolysis in catalysis blood is free fatty acid, increases the active triglyceride can effectively removed in circulation of LPL.Based on this principle, the 3T3-L1 cell that experiment adopts the Fat-reducing are blood-circulation promoting capsule of variable concentrations, turmeric acts on differentiation and maturation, mtt assay gropes medicine maximal non-toxic concentration, maximal non-toxic concentration basis is got three mass actions in the 3T3-L1 cell of differentiation and maturation, 3 porocytes of every mass action 96 orifice plate, establish non-administration cell controls group and blank group, positive controls simultaneously.Positive reference substance is Bezafibrate, and its concentration is after administration 36h, detects the activity of LPL in cells and supernatant, the change of LPL activity after observation administration.

LPL determination of activity is carried out as follows:

Get the 3T3-L1 cells and supernatant 100 μ l after drug effect, add in 96 orifice plates, then add equivalent substrate solution (by triolein, inactivated fetal bovine serum and reaction buffer formulated with 1:5:19), hatch 60min for 37 DEG C.The free fatty acid utilizing the extracting of free-fat acid detection kit to generate after hatching end, ultra-violet and visible spectrophotometer detects the OD value at 550nm wavelength place, detects the activity of enzyme.Experiment repetition 3 times, observes pharmacodynamic stability.

Utilize at 37 DEG C, the free fatty acid amount that under pH8.5 condition, hydrolysis triolein per hour generates quantizes enzymatic activity.By following formulae discovery enzymatic activity increment rate:

A: administration group free fatty acid growing amount B: cell controls group free fatty acid growing amount

Free fatty acid growing amount computing formula is as follows:

Wherein, pipe OD is measured _550nmrepresent administration group and cell controls group absorbance at 550 nm; Blank tube OD _550nmrepresent blank group absorbance at 550 nm; Standard pipe OD _550nmrepresent standard items absorbance at 550 nm; Standard pipe concentration refers to the concentration of standard items, and standard concentration is according to the regulation preparation on " free fatty acid microdetermination kit #E1000 " instructions, now with the current, is 1000 μm of ol/L here.

Table 6. variable concentrations Fat-reducing are blood-circulation promoting capsule, turmeric on the impact of LPL enzymatic activity ( n=3)

7, the bioactive mensuration of Fat-reducing are blood-circulation promoting capsule, turmeric promotion Cholesterol Excretion

At 37 DEG C, pH7.6, under the reaction system of 0.1MNaCl solution, by positive drug cholestyramine (5mg/ml) and variable concentrations Fat-reducing are blood-circulation promoting capsule (40mg/ml, 20mg/ml, 10mg/ml, 5mg/ml), turmeric solution (20mg/ml, 10mg/ml, 5mg/ml, 2.5mg/ml) jointly hatch 2h with taurocholate salt solusion respectively, through high speed centrifugation and precipitable after medicine conjucated bile acids salt, be arranged in parallel cholate control group, 3 repeated experiments respectively, by the surplus of taurocholate in detection reaction system centrifuged supernatant, the binding ability of medicine to cholate is reflected with this, and calculate the Percentage bound of medicine to cholate.Bile salt quantity measuring method is with reference to the assay method of cholate in 2005 editions " Chinese Pharmacopoeia " first middle calculus bovis factitiuses, and the computing formula of bile salt amount is:

Cholate Percentage bound computing formula is:

In this experiment, cholate control group cholate amount is 4.65mM; Administration group cholate amount is calculated formulae discovery by above-mentioned bile salt gauge and is obtained, and wherein, OD administration group refers under 605nm, the absorbance of administration group; OD blank group refers under 605nm, the absorbance of blank group; OD standard items group refers under 605nm, the absorbance of standard items group.

Table 7 variable concentrations Fat-reducing are blood-circulation promoting capsule, turmeric disappear on the impact of cholate Percentage bound ( n=3)

From table 1 to table 7, Fat-reducing are blood-circulation promoting capsule is to biosynthesizing, promotion Triglyceride Metabolism in Patients in suppression cholesterol intestinal absorption, suppression triglyceride intestinal absorption, suppression triglyceride body and promote that Cholesterol Excretion five application points all show biologically active, and the increase of bioactive intensity dosage and increasing, present good dose-effect relationship.Estimation of biological potency can be carried out further to these 5 application points, using the biological value of wherein one or more application point as quality evaluation index, in order to save the cost analyzed and detect, usually choose 1 or 2 kind of estimation of biological potency method as quality analysis detection means, for control or detection of drugs quality.

Turmeric to biosynthesizing in suppression cholesterol body, promote cholesterol metabolic and promote that Cholesterol Excretion 3 application points have biologically active that and the increase of bioactive intensity dosage and increasing presents good dose-effect relationship.Estimation of biological potency can be carried out further to these 3 application points.

Experimental result above it also illustrates turmeric simultaneously and plays Hypolipidemic efficacy mainly through reducing cholesterol, and the Fat-reducing are blood-circulation promoting capsule containing turmeric medicinal material then both can reduce triglyceride by reducing cholesterol, it passes through reasonable formula, balancedly regulate and improve cholesterol and triglyceride levels, reaching more reasonable, effective, lasting lipid-lowering effect.Further illustrate, single medicinal material turmeric can not represent the curative effect of whole prescription Fat-reducing are blood-circulation promoting capsule.Therefore, differentiate that the quality of compound preparation cannot really be monitored and evaluate to certain taste medicinal material with thin-layer method, similarly, also cannot effectively evaluate or control the quality of single medicinal material or compound preparation as index with the content of single index components.

Embodiment 2 Fat-reducing are blood-circulation promoting capsule suppresses the mensuration of the biological value of cholesterol intestinal absorption

A. reference substance solution preparation: Ezetimibe 3 (10mg), be dissolved in 5mLMEM nutrient solution, and repeatedly blow and beat mixing, ultrasonic 30min, gained liquid through coarse filter paper and 0.22M membrane filtration, collects last filtrate successively, is the Ezetimibe solution of concentration 6mg/ml, 4 DEG C of preservations, in contrast product solution for standby;

B. need testing solution preparation: prepare Fat-reducing are blood-circulation promoting capsule need testing solution by method as previously mentioned;

C. need testing solution and reference substance solution are diluted to the dosage group of variable concentrations with identical dose of distance, and agent distance is 0.5; Diluting solvent used is MEM nutrient culture media;

D. data processing calculates with tiring: the cholesterol absorption inhibiting rate recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical cholesterol absorption inhibiting rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects the bioactive power of Drug inhibition cholesterol absorption to be measured.

Table 8 Fat-reducing are blood-circulation promoting capsule on cholesterol absorption inhibiting effect impact ( n=3)

The biological value of setting Ezetimibe is 1U/g, then the relative potency of Fat-reducing are blood-circulation promoting capsule is 0.088U/g.

Embodiment 3 Fat-reducing are blood-circulation promoting capsule suppresses the mensuration of the biological value of triglyceride intestinal absorption

A. reference substance solution preparation: taking Xenical as reference substance, is solvent with deionized water, is made into the reference substance solution of respective concentration, filters, for subsequent use;

C. need testing solution and reference substance solution are diluted to the dosage group of variable concentrations with identical dose of distance, and agent distance is 0.5;

D. data processing calculates with tiring: the pancreatic lipase activity inhibiting rate recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical pancreatic lipase activity inhibiting rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects the bioactive power that Drug inhibition triglyceride to be measured absorbs.

Table 9 variable concentrations Fat-reducing are blood-circulation promoting capsule on the impact of pancreatic lipase ( n=3)

The biological value of setting Xenical is 1U/g, then the relative potency of Fat-reducing are blood-circulation promoting capsule is 0.3125U/g.

Embodiment 4 Fat-reducing are blood-circulation promoting capsule suppresses the mensuration of triglyceride biosynthetic biological value in vivo

A. reference substance solution preparation: be reference substance with Acipimox, be dissolved in DMEM nutrient culture media, be made into the need testing solution of respective concentration, filters, for subsequent use;

C. need testing solution and reference substance solution are diluted to the dosage group of variable concentrations with identical dose of distance, and agent distance is 0.7;

D. data processing calculates with tiring: the free fatty acid recording the dosage group gained of the variable concentrations of test sample and reference substance respectively generates inhibiting rate, relatively test sample and reference substance reach the ratio of dosage when substantially identical free fatty acid generates inhibiting rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects the bioactive power of Drug inhibition lipolysis to be measured.

After table 10 drug treating after 3T3-L1 cell each group FFA generate inhibiting rate ( n=3)

The biological value of setting Acipimox is 1U/g, then the relative potency of Fat-reducing are blood-circulation promoting capsule is 0.12U/g.

Embodiment 5 Fat-reducing are blood-circulation promoting capsule promotes the mensuration of the biological value of Triglyceride Metabolism in Patients

A. reference substance solution preparation: be reference substance with Bezafibrate, be dissolved in DMEM nutrient culture media, be made into the need testing solution of respective concentration, filters, for subsequent use;

B. need testing solution preparation: prepared by need testing solution: prepare Fat-reducing are blood-circulation promoting capsule need testing solution by method as previously mentioned; According to the estimated value that test sample is tired, obtain need testing solution with DMEM dilution;

D. data processing calculates with tiring: the LPL enzymatic activity increment rate recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical LPL enzymatic activity increment rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of Triglyceride Metabolism in Patients.

Table 11 variable concentrations Fat-reducing are blood-circulation promoting capsule on the impact of the active increment rate of LPL ( n=3)

The biological value of setting Bezafibrate is 1U/g, then the relative potency of Fat-reducing are blood-circulation promoting capsule is 0.047U/g.

Embodiment 6 Fat-reducing are blood-circulation promoting capsule promotes the mensuration of the biological value of Cholesterol Excretion

A. reference substance solution preparation: be reference substance with cholestyramine, be dissolved in NaCl solution, be made into the need testing solution of respective concentration, filters, for subsequent use;

B. need testing solution preparation: prepared by need testing solution: prepare Fat-reducing are blood-circulation promoting capsule need testing solution by method as previously mentioned; According to the estimated value that test sample is tired, obtain need testing solution with NaCl solution dilution;

D. data processing calculates with tiring: the bile salt Percentage bound recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical bile salt Percentage bound, using this ratio as the biological value relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of Cholesterol Excretion.

Table 12 Fat-reducing are blood-circulation promoting capsule disappear on the impact of cholate Percentage bound ( n=3)

The biological value of setting cholestyramine is 1U/g, then the relative potency of turmeric is 0.0625U/g.

Embodiment 7 turmeric suppresses the mensuration of cholesterol biosynthetic biological value in vivo

A. reference substance solution preparation: take Simvastatin as reference substance, be solvent with reaction buffer, be made into the reference substance solution of respective concentration, filter, for subsequent use, described reaction buffer is such as 70mM phosphate buffer, 2mMEDTA, 2mMDTT, 0.06%BSA, 0.15mMNADPH, this pH of cushioning fluid is 7.0;

B. need testing solution preparation: with medicine to be measured for test sample, took the test sample powder of 80-100 mesh sieve, with appropriate solvent extraction, extract after filtration, according to the estimated value that test sample is tired, obtains need testing solution with DMEM dilution;

D. data processing calculates with tiring: the HMG-CoA reductase maximum inhibition recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical HMG-CoA reductase maximum inhibition, using this ratio as the biological value relative to reference substance, the size of this ratio reflects Drug inhibition cholesterol to be measured biosynthetic bioactive power in vivo.

Table 13 variable concentrations turmeric on the impact of HMG-CoA reductase activity ( n=3)

The biological value of setting Simvastatin is 1U/g, then the relative potency of turmeric is 0.475U/g.

Embodiment 8 turmeric promotes the mensuration of the biological value of cholesterol metabolic

A. reference substance solution preparation: be reference substance with Berberine hydrochloride, be dissolved in DMEM nutrient culture media, be made into the reference substance solution of respective concentration, filters, for subsequent use;

C. need testing solution and reference substance solution are diluted to the dosage group of variable concentrations with identical dose of distance, and agent distance is 0.7; D. data processing calculates with tiring: the LDLR rise rate recording the dosage group gained of the variable concentrations of test sample and reference substance respectively, the ratio of dosage when relatively test sample and reference substance reach substantially identical LDLR rise rate, using this ratio as the biological value relative to reference substance, the size of this ratio reflects that medicine to be measured promotes the bioactive power of cholesterol metabolic.

Table 14 variable concentrations turmeric on the impact of average LDLR rise rate ( n=3)

The biological value of setting Berberine hydrochloride is 1U/g, then the relative potency of turmeric is 0.48U/g.

Embodiment 9 turmeric promotes the mensuration of the biological value of Cholesterol Excretion

B. need testing solution preparation: with medicine to be measured for test sample, took the test sample powder of 80-100 mesh sieve, with appropriate solvent extraction, extract after filtration, according to the estimated value that test sample is tired, obtains need testing solution with NaCl solution dilution;

Table 15 turmeric disappear on the impact of cholate Percentage bound ( n=3)

The biological value of setting cholestyramine is 1U/g, then the relative potency of turmeric is 0.125U/g.

In addition, drug quality evaluation method of tiring based on fat biological of the present invention is not really wanted to carry out estimation of biological potency one by one to 7 application points mentioned above, as drug production process or drug inspection means, promptness and cost are also very important Considerations, thus as one of preferred version of the present invention, the application point can choosing dose-effect relationship definite carries out estimation of biological potency, with this biological value and Chemical Evaluation index comprehensive evaluation drug quality.Below with the Chinese patent drug that lipopenicillinase drug effect is clear and definite: Effects of Xuezhikang is that example sets forth the present invention further.

Blood fat recovery capsule is by the capsule of red colouring agent for food, also used as a Chinese medicine through being processed into, the main chemical compositions of red colouring agent for food, also used as a Chinese medicine is Lovastatin, Lovastatin is the inhibitor of major rate-limiting enzyme Hydroxymethylglutaryl list acyl coenzyme A (HMG-CoA) reductase of body inner cholesterol synthesis, infers that Effects of Xuezhikang has accordingly and well suppresses biosynthetic effect in cholesterol body.Effects of Xuezhikang drug concentration is set as 1.5,0.75,0.375,0.188,0.094mg/ml, the method for living according to following detection enzyme, measures HMG-CoA reductase intensity of variation after administration, judges whether test sample has suppression

The effect of HMG-CoA reductase.

HMG-CoA reductase vitality test---spectrophotometric analysis

Experimental result calculates:

Wherein, V _sfor sample volume, unit L; (△ A2/ △ T-△ A1/ △ T) represents enzyme reaction rate; In the present invention, △ T elects 5min as, and the implication that △ A1 and △ A2 refers to respectively is as implied above.

E _{normal group}represent Normal group, both the enzyme activity of HMG-CoA reductase before administration; E _{administration group}represent administration group, i.e. the enzyme activity of HMG-CoA reductase after administration.

Utilize the method whether can have the effect of certain suppression HMG-CoA reductase activity by Preliminary detection test sample, and judge whether it exists good dose-effect relationship.

Experimental result is in table 16.

Table 16 positive drug Effects of Xuezhikang is to model preliminary experiment

Found by above-mentioned preliminary experiment, Effects of Xuezhikang is by suppressing HMG-CoA reductase active thus playing the effect reducing cholesterol levels, and dose-effect relationship is better.Therefore, the present invention chooses and suppresses the biological value that synthesizes in vivo of cholesterol as one of index evaluating Effects of Xuezhikang quality.

Experimental technique: by comparing reference substance Simvastatin with test sample Effects of Xuezhikang under identical condition, when reaching substantially identical HMG-CoA reductase inhibiting rate, the ratio of both dosage calculates the biological value of Effects of Xuezhikang suppression cholesterol biosynthesis.

Experimental result: shown in table 17.

Table 17 variable concentrations Simvastatin and Effects of Xuezhikang on the impact of HMG-CoA reductase activity ( n=3)

The biological value of setting Simvastatin is 1U/g, then the relative potency of Effects of Xuezhikang is 6.33U/g.This experimental result illustrates, with regard to the rejection ability to HMG-CoA reductase, Chinese patent drug Effects of Xuezhikang is stronger than chemical drugs Simvastatin, and this may be relevant with the synergy of principal ingredient Lovastatin and other compounds in red colouring agent for food, also used as a Chinese medicine.

Innovative point of the present invention is the quality determining method providing a kind of reducing blood lipid class Chinese medicine, and it is using biological value as evaluation index; Particularly, be that the biological value of the biological value and/or reduction triglyceride that reduce cholesterol is as evaluation index.Reducing cholesterol can be by suppressing the absorption of cholesterol, synthesis or promoting that one or more approach in the metabolism of cholesterol or excretion realizes; Reducing triglyceride can be by suppressing triglyceride to absorb, synthesizing or promote that one or more approach in triglyceride excretion realizes.Detect biological value as which kind of means of employing, this is not restricted in the present invention, as long as can to stablize, accurately and be easy to operation, meet the requirement that Pharmaceutical Analysis detects.

The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.

Claims

1. a reducing blood lipid class medicine quality evaluated method, is characterized in that, using biological value as quality evaluation index.

2. method according to claim 1, is characterized in that, to reduce cholesterol biological value and/or to reduce triglyceride biological value as quality evaluation index.

3. method according to claim 2, it is characterized in that, described reduction cholesterol biological value refers to the biological value suppressing cholesterol absorption, suppress cholesterol biosynthesis, promote cholesterol metabolic or promotion Cholesterol Excretion, evaluates with the height of measured biological value the power that Chinese medicine reduces hypocholesterolemic activity.

4. according to the method in claim 2 or 3, it is characterized in that, described reduction triglyceride biological value refers to the biological value suppressing triglyceride to absorb, suppress triglyceride synthesis or promotion Triglyceride Metabolism in Patients, evaluates with the height of measured biological value the power that Chinese medicine reduces triglyceride activity.

5. method according to claim 4, it is characterized in that, also comprise and make described Chinese medicine after gut flora fermentation, obtain tunning, adopt experiment in vitro to detect this tunning reduce cholesterol absorption, reduce cholesterol biosynthesis, promote cholesterol metabolic and promote Cholesterol Excretion and reduce the biological value that triglyceride absorbs, reduces triglyceride synthesis and promote Triglyceride Metabolism in Patients, the quality evaluation index using measured biological value as lipopenicillinase class Chinese medicine.

6. method according to claim 4, it is characterized in that, also comprise and make described Chinese medicine obtain metabolic product under the effect of liver microsomes enzyme, adopt experiment in vitro to detect this metabolic product reduce cholesterol absorption, reduce cholesterol biosynthesis, promote cholesterol metabolic and promote Cholesterol Excretion and reduce the biological value that triglyceride absorbs, reduces triglyceride synthesis and promote Triglyceride Metabolism in Patients, the quality evaluation index using measured biological value as lipopenicillinase class Chinese medicine.