CN1317974A - Method for identifying and confirming consistent bio-functionality of natural compsns. - Google Patents

Method for identifying and confirming consistent bio-functionality of natural compsns. Download PDF

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CN1317974A
CN1317974A CN 99810878 CN99810878A CN1317974A CN 1317974 A CN1317974 A CN 1317974A CN 99810878 CN99810878 CN 99810878 CN 99810878 A CN99810878 A CN 99810878A CN 1317974 A CN1317974 A CN 1317974A
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composition
tonic
regimen
prepared product
digestion
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B·兰德斯
G·维洛克
J·A·里尼格尔
Z·弗兰克
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PARACALSIAN Inc
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PARACALSIAN Inc
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Abstract

The present invention provides methods for evaluating the biofunctionality of natural products. Since natural products typically comprise a variety of active components, both identified and unidentified, and normal dosage is by ingestion, methods of the invention simulate normal physiological digestion and absorption from the digestive tract. Methods are disclosed which provide for evaluation of biofunctionality and quality assurance testing for natural compositions such as herbs, herbal extracts and formulations derived therefrom, even though active components and their specific activities may remain unidentified.

Description

Identify and confirm the method for natural composition consistent bio functionality
The application requires the royalty interest of U.S. Provisional Application series number 60/092,686 and U.S. Provisional Application series number 60/094,360, and the former submitted on July 14th, 1998, and the latter submitted on July 28th, 1998.
Invention field
The present invention relates to the active method of a kind of evaluation natural prodcuts biological function, and the natural prodcuts that can be used for this method, this natural prodcuts are raw material form and the final products that can be consumed.
Background of invention
Usually be not considered to medicine as the natural prodcuts of regimen tonic, yet consumer usually expects natural prodcuts and has many identical characteristics to the medicine expection.The medicament (medicine) that is designed to the disease particular treatment is by Food and Drug Administration (FDA) management, and medicament is made up of single active component or limited definite mixture of active principles.In case selects and prepared a kind of medical compounds, and it is specified press the FDA criterion, prove that the disease for institute's desire treatment is safe and effective, only need this product of analysis so that guarantee the proper content of this reactive compound.For doctor and consumer, its curative effect is decided by the history of this chemical compound clinical effectiveness of proof then.
Being different from medicament, usually is a difficult problem for the evaluation and the standardization of natural composition.Its composition may be difficult to measure, and usually is too many so that can not make it standardization with the statistics confidence level.In addition, how the chemical composition of natural prodcuts grows, gathers in the crops and processes if will depending on also where its material reaches, and environmental condition such as nutriment and disease and pest.And active component may be unknown here, also may be unfavorable for its activity for a kind of this standardization of type compound, also may be unknown for the proper proportion of inferring effective various active composition perhaps.
Proving conclusively under the environment of technology based on traditional qualitative analysis of standardization natural material, development having formed natural prodcuts and other food therapy tonic market.As mentioned above, thisly guarantee that quality and conforming strategy do not guarantee by claiming that on Product labelling this series products of promoting has consistent biological agent.For example, a kind of popular product that is called as Herba Hyperici Monogyni, the quality of its hint is based on the standardization to single chemical substance (hypericin), and this chemical substance may not cause the effect that causes Herba Hyperici Monogyni to be sold.
In the U.S., because " health of regimen tonic and education regulations " permission (DSHEA), consumer is the non-specific statement of nutrition supply and general health-care effect to the impression of natural product, for example " antioxidant maintenance cell integrity ".Such statement connotation is to inform consumer about the potential effect in keeping normal body structure and function, and not as need " federal food drug and cosmetic act, medicine and cosmetics regulations " (FFDCA) under the control direct statement to the treatment of specific health problem.
Because for the statement of these products progressively upgrading that becoming, and correspondingly also be subjected to more double check of consumer and administrative organization, so more and more importantly, set up the method for quality conclusive evidence, so that these statements have abundant foundation, and mass product that its statement effect is not provided and the product zone of having done like this separated.
When the effect of its statement is provided, set up and use the biologic test method that designs for assessment natural prodcuts effect, a kind of method of estimating some quality of materials and content is provided, and the chemical constituent quantity that this material contains can have tangible change in different prepared products.In addition, different with the medicament that is designed traditionally by single binding mode performance specificity and curative effect, natural prodcuts tend to have a plurality of site of actions or multiple binding mode, when they are merged, can produce the effect of expection.
The objective of the invention is to be devoted to solve above-mentioned about guaranteeing and the problem of the reliable level of the corresponding biological activity of natural prodcuts function.
Summary of the invention
The present invention uses the quality of the complex mixture of a kind of functional technical measurement and evaluation form right compositions feature tomorrow.
The invention relates to a kind of method of estimating natural composition or extract Biofunctional, the method comprises makes natural composition stand mimic digestion, mimic absorption, perhaps stand this two kinds of effects, so that obtain a kind of prepared product of this natural composition, determine then to exist in this prepared product or do not have one or more compositions or biological activity, and make and exist in this prepared product or do not exist these one or more compositions or biological activity to be associated with the Biofunctional of this natural composition or extract.Mimic digestion and mimic absorption can be one after the other to carry out or carry out simultaneously.
Mimic digestion can be mimic peptic digestion or mimic intestinal digestion, perhaps can be the combining form of the two.Mimic digestion is carried out in acidic gastric juice, preferred pH is about 1.2, and wherein also may contain have active digestive enzyme under acid condition, perhaps carries out in neutral intestinal juice, preferred pH is about 7.4, wherein also may contain activated digestive enzyme under neutrallty condition.
Mimic absorption comprises in order to simulate some compositions of bioavaliability or bioactive transmembrane transport.In preferred embodiments, this film is the Caco-2 cell of monolayer.A target of the present invention is that for any given natural composition provides a kind of detection system, wherein this system is special in order to assess the sort of compositions associated biomolecule functionality.The invention provides several detection methods, be used to assess the biological functionality that natural composition is expected in vivo, and therefore can assess this natural composition to treating the effectiveness of a certain selectivity pathological state, perhaps as the people is consumed the regimen tonic, the effectiveness of the 26S Proteasome Structure and Function that is used to keep fit.Effectively detection method can also comprise can be clearly or mensuration may be relevant with natural composition the detection method of ill effect.
The present invention is also further at the regimen tonic that contains natural composition, this tonic have basically by batch the composition concordance, can assess described composition concordance by a kind of method, the method comprises makes the sample of this regimen tonic or natural composition stand mimic digestion, mimic absorption or this two kinds of effects, so that obtain the prepared product of this sample, determine then to have one or more compositions in this prepared product, and the existence of one or more compositions is associated with the composition concordance.
So-called composition concordance is meant at least a required active component or bioactive consistent with basically a batch biological functionality.
Another aspect of the present invention is a mensuration system, is used for natural composition is set up standard, and the quality and the concordance that are used to guarantee natural food therapy tonic.It or not the active component of only attempting to identify any given complicated natural composition that is consumed with quantitative assay, but design this detection system, and depend on special special detection method so that corresponding natural prodcuts are guaranteed its effect and concordance so that assess the whole biological functionality of the sort of compositions that is consumed.
The present invention is also further at a kind of method, be used to prepare and comprise the regimen tonic that has basically by batch conforming natural composition, can assess the concordance of this composition by a kind of method, the method comprises that the sample that makes this natural composition stands mimic digestion, mimic absorption, perhaps these two kinds of effects, so that obtain the prepared product of this sample, determine then in this prepared product, to have or do not exist a certain or several compositions, and select to contain the prepared product of these one or more compositions.
The invention still further relates to a kind of bioactive novel method of Semen Ginkgo compositions that is used to estimate, be included in and make a kind of neurocyte stand one or more when said composition exists effectively to reduce the material or the condition of these neurocyte activity, and measure the activity of this neurocyte.
Another aspect of the present invention is a kind of bioactive novel method of Saw Palmetto Berries compositions that is used to estimate, comprise by a kind of said composition, 5-hydroxy tryptamine of containing is provided, the not combined androgen receptor of compound HSP90 and the mixture that androgen is replied components D NA, and detect the formation that androgen receptor-androgen is replied components D NA complex, detect androgen is replied the inhibitory action that receptor is transformed into DNA combining form.
The accompanying drawing summary
Fig. 1 show sample preparation method is to the influence of Echinacea purpurea Moench (Echinacea) with reference to product macrophage activation ability.The TNF-α mean+SD that data representation is produced by the RAW264.7 macrophage system, this cell are used to from not 20 μ g/ml Echinacea purpurea Moench (Echinacea purpurea) the medical herbs processing of same preparation method.E-DMSO is the capsular inclusions of reference product that is dissolved in dimethyl sulfoxine (DMSO) solvent; E-50%ETOH is the reference product capsule contents that is dissolved in 50% ethanol/water solution; E-digestion preparation is the reference product capsule that is subjected to simulating the preparation of digestion scheme.Placebo is the placebo soft capsule that is provided by R.P.SchererNA.
Fig. 2 shows that Echinacea purpurea Moench stands to simulate the dose-response of the prepared product of digestion to the activating macrophage ability with reference to product, data represented three meansigma methodss that repeat culture hole.Standard deviation is less than 5%.
Fig. 3 is presented at after the simulation digestion, compares the ability of 7 Echinacea purpurea Moench material sample activating macrophages with two reference standards and a placebo.Data represented three mean+SD that repeat culture hole.
After the digestion of Fig. 4 display simulation, compare the ability (placebo has been done calibration) of 11 Echinacea purpurea Moench outturn sample activating macrophages with reference standard.Data represented three mean+SD that repeat culture hole.
Fig. 5 shows for the result of the test of Echinacea purpurea Moench with reference to product and placebo sample determination functional living being effective degree.By with Caco-2 cell monolayer incubation do simulation absorb before and measure the activity of activating macrophage afterwards.Cylinder is represented the mean+SD in three repeated trials holes.
Fig. 6 shows that 5 batches of Echinacea purpurea Moenchs are with reference to the variation between product batch.The mean+SD in data represented three replication holes.
Detailed Description Of The Invention
To describe in detail the present invention with the specific representative embodiment of the present invention now, it should be understood that and only attempt with these embodiments property embodiment as an illustration, and the present invention be not limited.
For in this disclosed especially representative embodiment, the noun natural composition is used to mean the composition that is considered to can be used as the regimen tonic as stipulating in DSHEA, be used for for whole body health, keeps normal 26S Proteasome Structure and Function. Natural composition preferably derives from plant, and more preferably is to derive from herbaceous plant, still, also can be the source of non-plant, for example from the composition of animal and other non-plant organism, for example, algae, fungi, bacterium etc. By its final form processing, natural composition comprises the selected part in its source itself and source. Vegetable composition and draft composition can comprise for example stem, flower, fruit, root etc. Packaged leaves of plants complete or that pulverized, what for example be used for making tea is exactly natural composition. Natural composition comprises the extract that comes from the source thing, for example by solvent extraction from source thing isolated composition. Solvent can comprise water, ethanol, methyl alcohol, acetone etc. Each those of ordinary skill of this area is known the example for the preparation of other solvent of extract. The natural composition material by the source thing of modified that can also comprise controlling oneself can be by means of other process, and for example modification or digestion enzymatic or chemistry are carried out this processing. Natural composition is to comprise all or part of of a Plants, draft, animal or other living organism, and can without or through processing with physics, enzymatic or chemical method processing. Natural composition also comprises the composition that has added other material. Other material is for example to be added into to preserve or the prepared product of preparing natural composition, comprises for example anticorrisive agent, filler, adhesive, lubricant etc. Natural prodcuts are comprised of natural composition, are in can use immediately or edible form.
Different from the pharmaceutical composition that usually comprises the single-activity compound, natural composition is by the compositions of mixtures of lateral reactivity thing and interaction composition. It is the main composition of being responsible for producing required biological agent that a kind of like this mixture may only contain two kinds or three kinds identified, but generally all contains many kinds. In addition, when when being produced such as the mixture of the extract of the material in viable organism, found or extract from naturally occurring material, natural composition comprises many supplementary elements usually, they may be existing with different amounts relatively on a small quantity individually, but they combine the considerable part that may consist of whole natural composition. These additional submembers may producing needed biological agent and preventing in the adverse side effect, contribute to the effect of mainly wanting active component synergistically.
Although well-knownly be, may can remarkably be affected by other material of simultaneously administration the bioavaliability of bioactivator, therefore and affect its effect, but natural composition is different from the medicament composition of simultaneously administration, especially when natural composition be from naturally occurring material, to be extracted in some way, and caused forming when not only containing main component but also containing the composition of submember mixture.
Biological functionality as used herein means the ability that integral body that composition causes the state of living organism or feature changes. For the composition of natural origin, biological functionality means when the ability that is used as medicine or regimen tonic unhealthful or comfortable some aspect when edible. Usually determine natural composition to healthy and comfortable whole effect by means of clinical testing, measure the effect of the statement purposes of the healthy state of being correlated with for the treatment of or disease in the test. The effect that natural composition is stated can comprise, but be not limited to, when being in the state of high stress, strengthen immune system, and promotion wound healing (purple coneflower), keep prostate health (saw palmetto), promote the circulation of tip and brain blood and improve [Dan (ginkgo (Ginkgo biloba)), improve liver function and prevent liver damage (silymarin (Silymarin)), and circulation stimulus and antemetic (race (Ginger root)). Usually do not do clinical testing by the method for expection, because expense is too high. And some effects that cause for natural composition are as keeping fit and comfortable, and prevent disease or defective mode, can't measure by means of existing clinical testing.
The biologically active of composition means in the biochemical reaction that external test is tested or it is measured, and composition affects the ability of a cell or one group of cell particular organisms feature. This feature including, but not limited to, the change of visible phenotypic, the change of gene expression style (being the change of specific mRNA and protein or polypeptide level), the molecule secretion that can affect other physiology course changes (cell factor for example, interleukin, histamine), perhaps cell to be measured to the change (being that cell is to the responsiveness of outside stimulus) of the chemical agent sensitiveness of known action. In order to reach required biological function effect, can be evaluated like this for the effect of special natural composition, namely by measuring some relevant specific biologically active of the sort of special biological function effect.
Because the natural composition that is ingested generally all comprises the mixture of lateral reactivity composition, and because depend on the interior validity (being bioavaliability) of the actual body of every kind of composition of natural composition, bioactive scope may have suitable change, so importantly the assessment of biological functionality should comprise some reliable prediction experiments, predict that what biologically active of this natural composition will occur in vivo. Therefore, in case the present invention is based on digestion and/or the absorption that this natural composition has stood simulation, how it shows in the several functions test, provides one to be used for predicting reliably the in vivo unique system of effect of natural composition. Carry out these tests with the form of specific selection bioassary method, the closely-related biologically active of biological function effect is caused by natural composition in external proof and body, and often determines by clinical testing, carries out subsequently mechanics study. And, for reliable result is provided, proved that these detection methods have the specificity of replying and stable performance.
Also adopted several sample preparation methods except given natural prodcuts are selected the detection method, so as to reach with human body in active maximum possible interrelated. This sample preparation method may comprise the digestion model of using simulation. The digestion of simulation can comprise the peptic digest of simulation, the intestinal digestion of simulation, the oral digestion of simulation, perhaps their combining form. Particularly in order to assess the bioavaliability of natural composition active component, detection method of the present invention can adopt the simulation natural composition by the sample preparation method of each link of alimentary canal, extends to colon from the oral cavity.
As pointed in this paper other places, may have important effect by alimentary canal for the bioactive ingredients of given natural composition. May reduce or destroyed by the digestion process activity. On the other hand, those processes may cause release or the activation from the inactive precursor thing of natural composition. Preparation that can the designing natural extract makes it to protect otherwise some compositions of digested destruction under one's belt. For example, pharmaceutical field is known, can use in the sour environment of stomach relatively undissolved, and the preparation that in the neutral environment of intestines, is easy to dissolve.
Therefore the digestion of simulation means a kind of method, is designed to simulate natural composition by the situation of each sections of alimentary canal. Digestion can be peptic digest, intestinal digestion or the two combination. In order to reach this purpose, the present invention generally adopts the gastric juice of simulation or the intestinal juice of simulation, or the saliva of simulation. Therefore, make the composition of natural composition be subject to various processing, comprise by the degraded inactivation, activate and select by modifying, thereby some composition may be absorbed on one's own initiative, and other compositions spread passively or are not absorbed fully.
Peptic digestion generally means to make determined material is stood to have the effect that is lower than about 2.0pH liquid, and this liquid also contains at activated digestive enzyme of acid pH such as pepsin.The peptic digestion effect can also include the stirring that helps liquid and material mixing, can simulate generation wriggling under one's belt like this.Preferred acidity scope is about pH1.0-pH2.0, more preferably about pH1.2, and the mimic gastric juice of the present invention can contain gastric acid, most preferably pepsin.Most preferred gastric juice is prepared according to the method for American Pharmacopeia (USP) the 23rd volume, and contains pepsin.Natural composition is contacted, preferably at about 37 ℃ at about 25～40 ℃ with this gastric juice.Peptic digestion continues one period that can effectively digest this natural composition or extract.Digestion can be carried out about 2 minutes-Yue 10 hours, preferably was that digestion was carried out about 30 minutes-3 hours.Most preferably be that digestion continues 2 hours.
Intestinal digestion be included in mixture when leaving stomach and entering intestinal in and by the acidity of digestion mixture.The mimic intestinal juice of the present invention is at about pH6.0-pH8.0, preferably at about pH6.5-pH7.5.This liquid can also contain from pancreas or the excretory digestive enzyme of enteral wall.The example of this kind of enzyme has pancreatinum, trypsin, chymase, carboxypeptidase, elastoser, lipase and amino peptidase.The intestinal digestion effect occurs in neutral pH, preferably also includes the stirring that helps liquid and material mixing, stirs to be similar to the mode that takes place in intestinal.Intestinal digestion is carried out at about pH6.5-pH8.0.More preferably intestinal digestion is to carry out at pH7.2-pH7.6.Most preferably, intestinal digestion is to carry out at pH7.4.One period that can digest this natural composition or extract is effectively carried out in intestinal digestion.Digestion can be about 2 minutes-10 hours.Preferably digestion is about 30 minutes-3 hours.Most preferably digestion is to continue about 2 hours.
In another embodiment of the invention, mimic absorption comprises one or more compositions of crossing over biomembrane transhipment natural composition.Gastral Absorption occurs in the epithelium layer that forms the digestive tract liner.For example, aminoacid and monosaccharide are absorbed by active transport and stride across epithelial membrane and enter blood capillary.Epithelial membrane also allows to pass through some material by means of diffusion, and can get rid of some other material by means of similar transporting mechanism.Gastral Absorption occurs in its whole length, comprises intestinal absorption, and stomach absorbs, and the Sublingual absorbs and oral mucosa absorbs.The present invention can adopt a kind of mimic absorbing model, and its simulation is crossed over the cell that is positioned at the digestive tract inwall from digestive tract environment transhipment natural composition composition, enters body fluid and tissue.In a representative embodiment of the present invention, use cell monolayer simulation physiological absorption.This cell monolayer is based upon a kind of permeability surface, all can contact culture medium with two surfaces that cause this monolayer, and this cell monolayer forms a barrier, and the culture medium of these monolayer two sides is mechanically separated.This cell monolayer is realized transport function, simulates gastral absorption.Therefore, mimic absorption comprises the base side from the single composition of the top side of cell monolayer transhipment natural composition to cell monolayer.
In some cases, this when one embodiment of the invention comprise when using the simulation Digestion compatible with mimic absorption (for example, the condition of simulation digestion is to being used to simulate the cell monolayer or the not infringement of other film of absorption), mimic digestion and mimic absorption can be carried out simultaneously.In other cases, incompatible when the condition of simulation digestion with the condition that simulation absorbs, then need in order step to carry out.The step of carrying out means simultaneously to a certain degree overlapping in the carrying out of digestion step and absorption step.Do not need a kind of digestion of composition and absorb simultaneously to take place.
Comprise the Caco-2 cell according to the present invention in order to simulate the representative cell that Absorption can be used for setting up monolayer.Spendable other cell includes, but are not limited to HT29 human colon cancer cell and Madin-Darby dog kidney (MDCK) cell.When being coated over a kind of surface, make material flow to base side, otherwise too, this cell can constitute the biomembrane that can be used to simulate physiological absorption effect and bioavaliability from the top side.So-called biomembrane means a kind of structure, for example a kind of cell monolayer, and it forms a kind of physical barrier, and some molecule can be crossed over this barrier by transhipment, depends on molecular property.The alternative of cell monolayer may be used to simulation certainly and absorb.Usually alternative contain can active transport biological structure, the digestive organ of alternative including, but not limited to obtaining, and by being planted in cell in the synthetic medium at the organ that reconstitutes or the film of external generation from laboratory animal.No matter mimic Digestion comprises that natural composition composition that simulation to whether changes by digestion process is from digestive tract to body fluid with any process of each link of physiological absorption of tissue.May carry out in very wide time range for a kind of prepared product simulation Absorption, and depend on the character of prepared product, biological activity of being measured and biotic component are crossed over it and absorbed film.Mimic absorption can continue about 5 minutes-48 hours.More preferably embodiment comprises lasting 15 minutes-about 4 hours time range.In the most preferred embodiment, mimic being absorbed in about 2 hours finished.
By another kind of method, animal model can be used to simulate digestion and the absorption to natural composition.Give the feeding animal natural composition, determine bioavaliability by test then, for example measure and have or do not exist certain composition or certain specific biological activity in serum or the blood.Can find out this biological activity to animal by monitoring physiology or behavior change.
In order to measure the activity of natural composition, have virtually no restriction for the type that can be used for external test method of the present invention.Seeking this class algoscopy generally can seek and prove the clinical research that positive clinical effect is arranged for the natural prodcuts of being estimated from for example literature search.Also can retrieve the document of the research of describing the biomechanism relevant then with clinical efficacy.Useful especially research is the research that those particular compositions for the natural prodcuts that are successfully used to clinical trial are done bright or hinted the mechanism of action.And then every kind of mechanism of action is carefully estimated the scope of natural prodcuts functionality.Use master pattern known in the art and can determine the proper range of functionality.Press down and to select and to estimate the sample of natural prodcuts according to the functionality scope of having established.Can select one or more vitro bioassay methods to be used for this natural composition, can be according to following these bioassary methods of one or several Standard Selection: this bioassary method and relation at the seen clinical effect of people; Repeatability; Expense-effect; And/or as the effect of quality conclusive evidence instrument.
Be used for determining whether exist other method of certain composition to include, but are not limited in the natural composition, by means of physics, definite method of chemistry or immunologic assay method or test.When this biological activity was needed just, then situation can be, at the biological response that demonstrates for a long time a certain active component, perhaps a certain active component builds up to effective level in biosystem after repeatedly giving this prepared product.In this case, the alternative method of mensuration active component is particularly useful.Even a kind of a certain special composition of natural composition identified, the bioavaliability of this composition need not determined by means of the relevant algoscopy of biological activity therewith yet after simulation digestion and/or simulation absorbed.As long as by making this natural composition stand mimic Digestion and/or mimic Absorption, just can use any easy method to determine whether it is present in the prepared product.Measuring the existence of a certain composition can carry out simultaneously with mimic digestion step and/or absorption step, perhaps carries out after them.Measure simultaneously and mean that mensuration and digestion or absorption step have to a certain degree overlapping, still do not need to take place simultaneously.
In another aspect of this invention, provide a kind of natural prodcuts that can be used as the regimen tonic, it comprises a kind of natural composition that obtains from a collection of natural composition, and wherein the representative sample of this batch is estimated its biological functionality with a kind of method.This natural prodcuts can be different from natural prodcuts in the past, and wherein particularly when comparing with natural prodcuts in the past, natural prodcuts of the present invention have stronger biological function or form concordance.For example,, the biological function concordance of this collection is compared with many samples in the past, consequently can carry out the statistics inference and established this bigger conforming evidence by collecting many samples of packing respectively of natural prodcuts of the present invention.
The present invention can be used for determining the biological functionality of natural composition, and guarantees that natural prodcuts such as regimen tonic have consistent biological functionality.This for example comprises, any undressed or vegetable material of having processed, and the concentrate of the compositions of the extract of various standardizations or extract and whole medical herbs, and be not limited to natural prodcuts as the regimen tonic.Can be used for primary and processed natural composition by what the inventive method provided equally to quality and conforming conclusive evidence, for example those are used for the feedstock composition of production regimen tonic.Therefore, the present invention includes and have basically, assess by above-mentioned any method in this its concordance by batch conforming natural prodcuts.
For every kind of natural composition, determined functional character that it is unique and in order randomly to measure the algoscopy of these features.At the stage of recognition, repeat these mensuration with clinical certified product, so that determine admissible biological functionality scope.In case determined its admissible biological functionality scope for a certain product, this measures rule and can be used for measuring each independent batch.The target of this mensuration is to determine that every batch has all stably kept required feature, as in originally determined feature of affirmation stage.
Analytical method of the present invention applies to extensively multiple natural prepared product.The invention is characterized in it is to select to be used to measure bioactive algoscopy according to biomechanism, this biomechanism is likely the reason of this natural prepared product biological functionality.This may mean usually only selects two kinds or three kinds of mechanism to be used for biological activity determination.
For some natural prodcuts and compositions, desired is to guarantee that certain composition or bioactive level are higher than a certain minima.For some other natural prodcuts, can require to guarantee that certain composition or biological activity are within a certain scope.In some cases, legal product should be in standard composition or active about 50%.Preferably this product should be in standard composition or active 75%.Most preferably, these products should be in standard composition or active 90%.
Can measure and checking with any algoscopy any product, depend on the purposes that this product is sold.For example, can have the draft product of more than one purposes by diverse ways or standard test, this depends on the purposes of its expection.Similarly, it is desirable to measure different product with identical desired use by identical method.
Another feature of the present invention is, can select bioactive scope, reach can the required biological functionality of known generation scope so that may carry out clinical trial more easily, spend more effective.Before being used for clinical trial, material is made prescreen, make it to establish a measurement range, can cause reducing in this scope and have the grouping number that higher degree is replied.
In one embodiment of the invention, the biological functionality of the natural composition that contains Echinacea purpurea Moench or its extract is assessed.Laboratory research shows that the composition of Echinacea purpurea Moench has immunostimulatory activity (Stimpel etc., 1984 to Mus and people's mononuclear cell; Wagner etc., 1988; Luettig etc., 1988; Roesler etc., 1991; Burger etc., 1997; See etc., 1997).Echinacea purpurea Moench-activated macrophage shows stronger propagation, and cytokine production increases, the phagocyte increased activity, and the ability of killing tumor cell and antibacterial and fungal pathogens strengthens (Stimpel etc., 1984; Coeugniet etc., 1987; Wagner etc., 1988; Leuttig etc., 1989).When natural composition of the present invention contains Echinacea purpurea Moench or Echinacea purpurea Moench extract, biological activity test can comprise the immunostimulatory activity of measuring prepared product.Immunostimulatory activity means the ability of stimulating immune system cytoactive.This cell comprises the B cell, T cell and macrophage etc.This class cell of one type may be activated by this stimulation, and perhaps situation can be, causes immunocyte to interact to a kind of stimulation of cell type, from and cause and activate other cell type.Activation is including, but not limited to, inducing cell propagation, and secrete cytokines or other signal transmit molecule, and molecule such as the nitrogen oxide or the peroxide of oxygen are carried in secretion.In certain embodiments of the present invention, immunostimulatory activity comprises the ability that phagocyte such as macrophage is provided stimulation.Preferred macrophage is RAW264.7, and is the generation of tumor necrosis factor to the preferred bioassay standard of immunostimulatory activity.Activate this class cell and can also activate other cell type.
According to the present invention, active or to form conforming mensuration be by at first making this natural composition stand mimic digestion and/or mimic Absorption to the Echinacea purpurea Moench compositions, subsequently the prepared product that generates is carried out biological activity test, measure it lymphocyte activity or cytophagic influence.The content of Echinacea purpurea Moench compositions can have significant change, can measure biological activity by means of the method for embodiment 1.For example, the content that has stood the Echinacea purpurea Moench of simulation digestion can be about 10mg-5000mg.More preferably content is 100mg-2500mg, and most preferred content is about 750mg.In the present invention's embodiment in this respect, be to measure the Echinacea purpurea Moench compositions, the immunostimulatory activity of extract or prepared product by the generation of the monitoring responsive cell factor.For example, detect the activation (Leutting etc., 1989) of macrophage by the generation of measuring TNF-α.Being used to stimulate the content of the Echinacea purpurea Moench compositions that TNF-α produces can be the about 5 μ g/ml of about 0.1 μ g/ml-, even more preferably about 0.5 μ g/ml-2.5 μ g/ml, and 1 μ g/ml most preferably.Other the material of replying comprises il-1, interleukin-6, interleukin-8, nitrogen oxide, reactive oxygen species and arachidonic acid metabolite.The commercially available reagent box that is used to detect these materials is easy to obtain.Can monitor its cytophagy or killing to macrophage to antibacterial.In addition, macrophage activation and cytokine produce the immune cell responses that promotes to form other, as cytotoxicity (ADCC) (Delfino etc., 1991 of NK cell (NK) cytoactive and antibody-dependent cell; See etc., 1997).
Alternatively, can measure of the effect of certain compositions by the propagation and the vigor that detect cell to immunostimulatory activity.The activation of macrophage and production of cytokines help development and activate immunocyte such as NK cell and lymphocyte (See etc., 1997 of other type; Delfino etc., 1991).The polysaccharide in Echinacea purpurea Moench-source does not bring out the lymphocytic propagation (Luettig etc., 1989) that exsomatizes basically, still, because (Coeugniet etc., 1987) stimulate peripheral blood mononuclear cell (macrophage in external (See etc., 1997) and body, NK cell, and lymphocyte; PBMCs), at external certain lymphopoiesis and NK cell-stimulating of having taken place.In one embodiment of the invention, with the Echinacea purpurea Moench compositions-treated PBMCs that had prepared by method of the present invention, and the number of mensuration living cells.The ultimate density of the Echinacea purpurea Moench prepared product that uses can be about 0.1 μ g/ml-5 μ g/ml, about 2.5 μ g/ml of 0.5 μ g/ml-even more preferably, and 1 μ g/ml most preferably.Comprise monitoring metabolism dyestuff 3-(4,5-dimethylthiazole-2-yl)-2, the conversion of 5-hexichol tetrazolium bromide (MTT) for detecting the preferred also method easy to implement of viable count purpose.Can also use other metabolism dyestuff of knowing.The method of mensuration vigor also includes, but are not limited to, and to labeled nucleotide or aminoacid (for example measures 3The H-thymidine) absorption and by means of give birth to luminescence method measure the ATP level (for example use the ViaLight test kit, LumiTech company, Nottingham, U.K).The method that is used to measure the macrophage-stimulating effect is not limited to and detects TNF-α and measure cell proliferation.Other method includes, but are not limited to detect to prostaglandin, other cytokine such as IL-1 β, IL-1 α, and nitrogen oxide, and oxygen base such as superoxides and peroxide is synthetic.Well known in the art is to measure cytokine and the biological activity that is produced by cultured cell.For example, the ELISA test kit of mensuration cytokine and cell proliferation detecting kit all are easy to obtain.
In another embodiment of the invention, measured the biological functionality of the natural composition that contains Semen Ginkgo or its extract.Nearest research has been described Semen Ginkgo extrac (GBE) to suffering from following dissimilar disease patients' beneficial effect: dementia, and mood change is with aging and old and feeble relevant recognition function obstacle, and attention and hypomnesis, mental disorder, depressed and anxiety (Warot etc., 1991; Hofferberth, 1989).Laboratory research also shows in the body, and the aging animal that gives Semen Ginkgo extrac is compared with control mice less degenerative structural change (Barkats etc., 1994) is arranged in Hippocampus.GBE contains four kinds of different plant chemical ingredients usually, comprises flavonoid glycoside, terpenoid, lotus anthocyanidin and organic acid.Shown that flavonoid can remove free radical and play an antioxidant action.It is believed that the free-radical oxidation reaction is relevant with ageing process, and infer it is the main cause of intellectual deterioration.The oxidation tense situation works in the pathogenesis of several nerve degeneration sexual disorders.Shown that ginkgo biloba flavonoid can prevent cytolipin peroxidation and cell death (Joyeux etc., 1995; Robak etc., 1988), prevent the neurocyte that causes by the oxidation tense situation downright bad and apoptosis (Oyama etc., 1996; Ni etc., 1996), and can reduce oxidoreduction metabolism (Oyama etc., 1994) in the neurocyte of immobilized and load calcium ion.In one embodiment of the invention, be the activity of measuring the Semen Ginkgo compositions as follows: at first make this natural composition stand mimic digestion and/or mimic absorption, measure the biological activity that forms prepared product subsequently, remove the ability of free radical so that determine it.Can be according to the ginkgo biloba content that the method for embodiment 1 is digested at about 5mg-1000mg.More preferably this content is 25mg-500mg, and most preferred this content is about 180mg.In one embodiment, be the removing of observing free radical by the decolouring of monitoring free radical reagent.Obviously, each those of ordinary skill of this area also can use many other mensuration their exist and the oxidant and the method for inactivation, measure the Semen Ginkgo compositions after simulation digestion and/or absorption.By being woven into, the Semen Ginkgo compositions can measure the ability that it removes free radical in the solution that contains free radical.Preferred free radical can be by means of for example absorbance detection, and the change by solution absorbance detects the removing to it.According to the free radical for the treatment of cancellation, the content of required Semen Ginkgo can change, and the concentration of Semen Ginkgo can be about 1 μ g/ml-500 μ g/ml, even more preferably 5 μ g/ml-200 μ g/ml, most preferably 10 μ g/ml-100 μ g/ml.
Pressing another aspect of the present invention, is the biological functionality that detects the natural composition that contains Semen Ginkgo or its extract with its ability that suppresses platelet activating factor (PAF).The terpenoid component of Semen Ginkgo contains ginkgolide, and wherein ginkgolide B is the strong inhibitor of platelet activating factor (PAF) (Nunez etc., 1986; Smith etc., 1996).Also known PAF can activate the cell (Braquet etc., 1987) that participates in inflammation, regulates the immunne response (Prescott etc., 1990) of cell, changes hemorheology and vascular permeability (Kinn etc., 1998).For neurological, PAF participates in the incident (Baker, 1995) after shock reaction and the ischemia, and PAF also can reduce big cerebral blood flow (Kinn etc., 1998), regulates activity (Bazan etc., 1995 of synapse; Yue etc., 1994), in brain development, play an important role, and be associated with some CNS obstacle (Hattori, 1994; Smith etc., 1996).Shown that in experimental model ginkgolide B can suppress the overwhelming majority of these functions of PAF.In one embodiment of the invention, be to be used for assessing inhibitory action to platelet activation by after mimic digestion and/or Absorption, measuring Semen Ginkgo compositions anticoagulant.Each technical staff of this area understands, also has many other approach can assess with the platelet relevant active inhibitory action that interacts and all can be used for the present invention.Can detect fixed hematoblastic activation of instrumentation and inhibition thereof with a kind of gathering.The method of producing enrichment platelet blood plasma is well known in the art.The mixture of a kind of agonist that comprises platelet, brings out platelet activation (platelet activating factor) and Semen Ginkgo compositions is provided.Agonist can be the PAF of racemization.Preferred agonist is L-PAF.The concentration of this agonist can be about 0.001 μ M-0.1 μ M, preferably is 0.003 μ M-0.05 μ M, and most preferably about 0.006 μ M.Can select a concentration range for determined Semen Ginkgo compositions.Can use the Ginkgolide of a standard, compare to the inhibitory action of platelet activation with Semen Ginkgo compositions and prepared product.The concentration of standard Ginkgolide can be at about 0.05 μ M-10 μ M, even more preferably 0.25 μ M-2.5 μ M, most preferably 1.0 μ M.
Also have in another embodiment of the present invention, tested Semen Ginkgo neuroprotective cell and avoided the toxic biological activity relevant with some material and growth conditions.Based on to Alzheimer ' s onste and the latest developments be familiar with and the Semen Ginkgo that it is believed that beneficial effect to age related discernment obstacle, we have set up the detection method of each step of analog neuron degenerative process, and this detection method can be used to assess the biological activity of Semen Ginkgo compositions.Known excitatory amino acid (glutamic acid) and β-starch peptide (Janssens etc., 1995 that the virose material of neurocyte and condition comprised high concentration; Behl etc., 1994).Serum starvation also can impel the neuronal apoptosis of cultivation.The neuroprotective algoscopy that comprises a kind of novelty in one embodiment of the invention, neurocyte are cultured under the known toxicity condition.When existing and do not have Semen Ginkgo, make neurocyte stand their combining form of above-mentioned conditioned disjunction.Determine the reduction of Semen Ginkgo compositions or prevent toxic ability by the vigor of measuring cell.It is following multiple to have proved that the avertible injury of Semen Ginkgo comprises: the apoptosis that (1) serum starvation brings out; (2) exitotoxicity that brings out of glutamic acid; (3) the ischemic cytotoxicity state of simulation; (4) cytotoxicity of non-polymeric β-starch peptide; (5) cytotoxicity of fibril β-starch peptide; The apoptosis that brings out when (6) high concentration glutamic acid exists; (7) apoptosis that β-the starch peptide brings out when existing; (8) cytotoxicity of exitotoxicity damaged condition thereupon after the simulation ischemia; The cytotoxicity of β when (9) having the Fe ion-starch peptide; The cytotoxicity of β when (10) high concentration glutamic acid exists-starch peptide; The apoptosis that brings out when (11) having β-starch peptide and high concentration glutamic acid.Is conspicuous to virose other condition of neurocyte and their combining form for those of ordinary skill in the art, also can be used to assess the biological activity of Semen Ginkgo compositions.In neuroprotective test of the present invention, a kind of mixture is provided, it comprises neurocyte, a kind of cytotoxic substance, its content is enough to reduce the vigor of this cell, and the Semen Ginkgo prepared product.Cytotoxic substance can be a glutamic acid, exists with the concentration of about 1mM-50mM, and preferably be the about 20mM of 2.5mM-, most preferably 10mM.Cytotoxic substance can also be β-starch peptide, exists with the concentration of about 0.05 μ M-10 μ M, and preferably be the about 1.0 μ M of 0.2 μ M-, most preferably be 0.5 μ M.Test extract and compositions determine that they suppress Cytotoxic degree.
The present invention also can be used for assessing the biological functionality of the compositions that contains Herba Hyperici Monogyni or its extract.Proved that Herba Hyperici Monogyni is to slightly to moderate depressive patients effectively (Chatterjee etc., 1998a; Laakmann etc., 1998), and almost be free from side effects (Vorbach etc., 1997; Linde etc., 1996).Though its model of action is not still known, the indication of vitro bioassay method, it is main specific activity (Muller etc., 1997) that the 5-HT rephotography is gone into the inhibitory action of going into the dopamine rephotography.The inhibitory action that the 5-HT rephotography is gone into is many model of action that synthetic resisting-down is generally confirmed.
For Herba Hyperici Monogyni, some other model of action also is possible.Embodiment of the present invention are not limited to measure the inhibitory action that the 5-HT rephotography is gone into, and for example may comprise, measure inhibitory action (Bladt etc., 1994 to monoamine oxidase, MAO; Thiede etc., 1994) or GABA is incorporated into inhibitory action (Baureithel etc., 1997 of GABA receptor; Cott, 1997).
In one embodiment of the invention, make the compositions that contains Herba Hyperici Monogyni stand mimic digestion and/or mimic Absorption, and the prepared product that forms is tested the inhibition activity that the 5-HT rephotography is gone into.Bioactive test can comprise provides one to contain the Herba Hyperici Monogyni prepared product, synaptosome prepared product and the mixture that is selected from the neurotransmitter of dopamine and 5-HT, and detect of the absorption of synaptosome prepared product to neurotransmitter.As described in this paper other places, can by for example in 0.32 ice-cold M sucrose liquid the homogenization cerebral tissue prepare synaptosome.In the process of preparation synaptosome, the weight of weighing cerebral tissue approx, add sucrose liquid amount can be about 1 times of volume-50 times volume, preferably be about 5 times of volume-15 times volumes, and most preferably be about 9 times of volumes.The amount that is used for measuring the synaptosome prepared product of cultivation can be about 5 μ l-1ml, preferably is 20 μ l-250 μ l, and 50 μ l most preferably.Neurotransmitter can be dopamine or 5-HT, and the concentration that exists in extract can be about 2nM-500nM, preferably is 5nM-100nM, most preferably is about 50nM.The amount that can be used to measure by the method for embodiment 1 bioactive Herba Hyperici Monogyni compositions can have suitable change.For example, the amount that stands to simulate the Herba Hyperici Monogyni of Digestion can be about 10mg-5000mg.More preferably amount is 100mg-2500mg, and most preferred amount is about 900mg.Measure the short-cut method of taking in and to use radiolabeled neurotransmitter.Make things convenient for the example of the neurotransmitter of labelling to have 14C-5-HT, 3H-5-HT, 3The H-dopamine.The time that rephotography goes into to measure can be about 15 seconds-15 minutes, even more preferably 1-10 minute, and most preferably 5 minutes.
Find that at this inhibition activity that Herba Hyperici Monogyni prepared product still keeps the 5-HT rephotography to go into after simulation digestion remains at low levels although compare with the material of beginning.It seems that active forfeiture occur in the peptic digestion process, rather than in the intestinal absorption process.This point has been given prominence to a key character of the present invention, can simulate the bioavaliability from the different preparations of a certain natural prodcuts exactly.For example, can stop natural food therapy tonic dissolving to enter the preparation of intestinal environment by gastric environment, will improve the bioavaliability of acid instability active component significantly until it.Therefore, in another embodiment of the present invention, can measure the bioactive forfeiture of natural prodcuts preparation by means of mimic digestion and Absorption, and select according to this active preservation.
In another embodiment, the present invention can be used for assessing the activity of the compositions that contains Saw Palmetto Berries or its extract.Saw Palmetto Berries is the most common by forming from the fruit extract of America Saw Palmetto Berries (Serenoarepens) as the regimen tonic.In Europe, Saw Palmetto Berries is that a kind of being widely accepted with extensive clinical research is used for medicine with the relevant symptom of benign prostatic hyperplasia (BPH).Although the definite etiology of BPH is not thrown a flood of light on as yet, but, as if the metabolism of androgen steroid and effect, and by 5 testosterone is changed into dihydrotestosterone (5-hydroxy tryptamine) and with the development of BPH very strong relation (Strauch etc., 1994 are arranged; Isaacs etc., 1983).Making the additional factor of BPH aggravation, is that some are by the leukocyte of infiltration and mediator of inflammation such as Prostaglandins and Leukotrienes (Robinnette, 1988 of the local growth factor such as basic fibroblast growth factor (bFGF) generation; Theyer etc., 1992).
In order to illustrate clinical effectiveness, the described external activity of Saw Palmetto Berries is comprised: inhibitory action (Strauch etc., 1994 of 5 to patient's BPH report; Delos etc., 1994), androgen is incorporated into the inhibition of androgen receptor and activates androgen-responsiveness reporter gene (Sultan etc., 1984; Ravenna etc., 1996), anti-estrogen activity (Di Silverio etc., 1992), Role in Plant Signal Transduction (Vacher etc., 1995 of obstruction prolactin antagonist and fibroblast growth factor; Paubert-Braquet etc., 1998), and the enzyme (cyclo-oxygenase and lipoxygenase) (Paubert-Braquet etc., 1997) that suppresses to produce the inflammation Prostaglandins and Leukotrienes.Experiment has shown that Saw Palmetto Berries can stop the prostate growth of androgen mediation in the body of neuter, and the pharmacokinetics labelling experiment shows that the main component (for example linoleic acid, oleic acid) of Saw Palmetto Berries is distributed in prostate (Chevalier etc., 1997; Paubert-Braquet etc., 1996).
But, these researchs do not obtain in vivo may the mechanism of action about Saw Palmetto Berries any concluding data.For example, Proscar reduces the level of circulation 5-hydroxy tryptamine and the level of prostate specific antigen (PSA) significantly, and PSA is gene outcome (Strauch etc., 1994 that androgen is regulated; Rhodes etc., 1993).In some research, use the patient of Saw Palmetto Berries to show that as one man the level of these materials does not have significance,statistical to reduce (Casarosa etc., 1988 in the serum; Braeckman, 1994).On the contrary, recently some studies show that the extract that gives Saw Palmetto Berries has reduced the intraprostatic 5-hydroxy tryptamine of patient BPH really, and hint is the mechanism of action (Di Silverio etc., 1998) of Saw Palmetto Berries to the inhibition of 5 in the prostate.
In embodiments of the invention, to the composition measuring that contains Saw Palmetto Berries and extract thereof they suppress the ability of 5s.In specific embodiment, be to assess biological activity by measuring the metabolic inhibition of one or more 5 substrates in the mixture (for example testosterone and/or Progesterone), this mixture comprises the 5 from rat liver microsome, NADPH and this 5 substrate and Saw Palmetto Berries compositions.The concentration of Saw Palmetto Berries compositions is about 5 μ g/ml-500 μ g/ml, preferably is at 10 μ g/ml-100 μ g/ml, most preferably is about 50 μ g/ml.When preparing microsome with equal-volume, as given in embodiment 5, the MC amount that is present in this determination test can be about 2 μ l/200 μ l, preferably is 5 μ l-50 μ l, most preferably is 20 μ l.The 5 substrate that exists is about 5 μ g/ml-500 μ g/ml, preferably is 20 μ g/ml-200 μ g/ml.Most preferably, the concentration of testosterone is 25 μ g/ml, and the concentration of Progesterone is 20 μ g/ml.Begin reaction by adding NADPH, add NADPH ultimate density be about 20 μ g/ml-2mg/ml, even more preferably 50 μ g/ml-1mg/ml, most preferably 490 μ g/ml.Incubation under about 30 ℃-40 ℃, even more preferably 35 ℃-39 ℃, most preferably at about 37 ℃, the incubation persistent period is about 5 minutes-2 hours, preferably is 30 minutes-90 minutes, most preferably is about 1 hour.
As discussing at this, possible binding mode is to suppress androgen receptor by SPE to be transformed into DNA combining form.Found that SPE suppresses aromatic hydrocarbon receptor and is transformed into DNA combining form.Suppress the discovery of aromatic hydrocarbon receptor transformation and the commentary of this document criticism property according to present SPE, can think that at this SPE is noncompetitive, the inhibitor that nonspecific HSP90-changes in conjunction with hormone receptor.Use noun " nonspecific " to be meant that SPE has the aromatic hydrocarbon receptor of inhibition, androgen receptor, and other receptor protein that may can stand similar transformation in addition is transformed into the ability of DNA combining form.Although aromatic hydrocarbon receptor is not the part of androgen or estrogen receptor family, directly be not included in hormone signal transduction on yet, but these all receptors all use similar mechanism to keep not coordinate ability and are used to activate into coordinate DNA-combining form, and all have their bonded with it corresponding DNA response elements of determining.Demonstrated the effect of the anti-estrogen of SPE, it can prevent nuclear estrogen receptor (Di Silverio etc., 9192; Bombardelli and Morazzoni, 1997).Discovery is 20 μ g/ml for the SPE IC50 that is measured.This with the SPE that finds to enzyme inhibition activity in identical scope.
In a kind of like this determination test, in cytosol or nuclear extract, androgen response element DNA is diluted, the not coordination androgen receptor that is incorporated into HSP90 derives from this extract, be diluted to the concentration of about 0.01nM-1000nM, 0.1nM-10nM even more preferably, most preferably about 1.0nM.Depend on the detection method that is adopted, can be with androgen response element biotinylation.Prepare microsome as mentioned above.Employed amount can preferably at 100 μ l-1000 μ l, most preferably be about 500 μ l between about 30 μ l-3000 μ l in determination test.The testosterone that exists preferably at 1 μ g/ml-100 μ g/ml, most preferably is about 50 μ g/ml at about 0.5 μ g/ml-200 μ g/ml.Then Saw Palmetto Berries material and NADPH are joined in this mixture.Every kind concentration can be about 1.0 μ g/ml-1000 μ g/ml, preferably is 10.0 μ g/ml-200 μ g/ml, most preferably is about 100 μ g/ml.Can adopt detecting based on aromatic hydrocarbon receptor and the similar condition of response element.It is well known in the art being used to detect the method that complex forms between complex such as androgen response element DNA and the androgen receptor.For example, these methods can be based on immunologic, depend on a kind of element to the bonded method of solid phase carrier.
Therefore, the embodiment that the present invention tests Saw Palmetto Berries compositions or extract comprises the formation that a kind of mixed liquor is provided and detects androgen receptor-male response element complex, this mixed liquor comprises the Saw Palmetto Berries prepared product, 5, cofactor NADPH, testosterone, with the compound not coordination of HSP90 androgen receptor, and androgen response element DNA.Alternatively, this test can comprise the formation that a kind of mixed liquor is provided and detects aromatic hydrocarbon receptor-aromatic hydrocarbons response element DNA complex, and this mixed liquor comprises the Saw Palmetto Berries prepared product, with the compound not coordination of HSP90 aromatic hydrocarbon receptor, and aromatic hydrocarbons response element DNA.
Another aspect of the present invention provides a kind of novel method of testing, can monitor the inhibitory action of the SPE antagonism-androgen of androgen receptor transformation.This method of testing provides to measure simultaneously because 5 suppresses 5-hydroxy tryptamine and has formed the active contribution of antagonism-androgen.
The useful especially aspect of this novel test method is that it provides serenoa repens extract and commodity and the bioactive a kind of measurement of the compositions that contains Saw Palmetto Berries by simulation Digestion of the present invention and Absorption preparation.
What often follow that BPH exists is the chronic inflammatory disease that hormone brings out, and this inflammation is to cause (Theyer etc., 1992) because inflammatory macrophage and neutrophil's infiltration enter prostate.Known prostaglandin such as prostaglandin E 2(PGE 2) and leukotriene (leukotriene B4, generation LTB4) they are mediator of inflammation (Robinette, 1988 that produced by these cells; Theyer etc., 1992).Research is in the past reported, and serenoa repens extract is at vitro inhibition cyclo-oxygenase (COX), and report, concentrates on prostatic serenoa repens extract composition and also suppresses this fermentoid (Pubert-Braquet etc., 1997; Chaudry etc., 1994).In addition, a research shows, PGE in the urine 2Concentration reduces with urine stream and improves be associated (Rolland etc., 1981).The embodiment of therefore, embodiment of the present invention comprise assessment Saw Palmetto Berries compositions or extract anti--inflammatory activity.Related to the research hint that the inflammation immunocyte soaks in the past, the secretion of mediator of inflammation (for example cyclo-oxygenase and the macrophage derived cell factor) plays a major role in the disease that Saw Palmetto Berries makes it to improve.And, there is the pharmacokinetics data sheet to understand the Saw Palmetto Berries bioavaliability of target site in vivo, also support anti--inflammatory effect mechanism (Chaudry etc., 1994 of Saw Palmetto Berries; Plosker etc., 1996).As discussed above, can measure the activation of macrophage with number of ways.In preferred embodiments, be by measuring prostaglandin E 2(PGF 2) inhibition that produces assesses the biologically active level of Saw Palmetto Berries compositions or extract.
In one embodiment of the invention, the biological activity and the composition concordance of ginseng composition or extract are estimated.Radix Ginseng has been used about 2000 in traditional Chinese medicine, be used for stamina intensifying and improve replying tired and emergency rating.Though still unclear to its mechanism of action, observed its effect, relate to central nervous system's (memory, study and behavior, D ' Angelo etc., 1986), neuroendocrine function (Hiai etc., 1979), immunologic function (Scaglione etc., 1996), antioxidation (Kim etc., 1996), carbohydrate and lipid metabolism (Wink etc., 1993), and cardiovascular system (Rimar etc., 1996; Fukuda etc., 1995).
Think that Radix Ginseng acts on hypothalamus, cause the release corticotropin, it acts on hypophysis again and discharges ACTH, and ACTH acts on the adrenal gland and discharges corticosteroid.The release action of corticosteroid (Corticotrosteroids) and thyroliberin (ACTH) causes adaptation (Fulder, 1980 to stress state in brain and health; Fulder, 1981).Radix Ginseng is also by combination with activate glucocorticoid receptor (GR), mineralcorticoid receptor and progesterone receptor and show potential synergism (Pearce etc., 1982; Lee etc., 1997).
The activity relevant with Radix Ginseng is considered to because the ginsenoside, shown it can in conjunction with and activate glucocorticoid receptor (GR), and be incorporated into mineralocorticoid and progesterone receptor.The most generally the activity of Yan Jiu glucocorticoid comprises that regulating nitrogen oxide closes the ketone isozyme, particularly suppresses inducibility nitric oxide synthase (iNOS) (Di Rosa etc., 1990; Park etc., 1996).Stress/inflammatory conditions under the inhibition expressed of the iNOS of glucocorticoid receptor (GR) mediation, to blood flow, keep myocardial contraction and useful immunocompetence all has facilitation, and T and bone-marrow-derived lymphocyte are expanded, raise cytokine receptor (Radomski etc., 1990 on the immunocyte; Hawrylowicz etc., 1994).
Embodiment of the present invention can appraiser's conopsea extraction and compositions stimulate the biological activity that produces with inhibited oxidation nitrogen of inducing of corticosteroid.In preferred embodiments, with Radix Ginseng extract to animals administer and measure the level of corticosterone in the blood plasma.In another embodiment, to the activated cultivation macrophage of IFN-γ, test the ability that Radix Ginseng extract inhibited oxidation nitrogen produces.
Following examples are described the present invention in detail, show how some special representative embodiment carries out, its material, and instrument and equipment and method step, they should be understood that it only is illustrative.Particularly do not plan the present invention is limited to the method for enumerating especially at this material, condition, procedure parameter and instrument and equipment etc.
Various publications have been quoted, patent and patent application in the application's full text.These publications, the instruction of patent and patent application and disclosure spy are all introduced this and are applied for reference, so that describe the situation with the relevant field of the present invention more fully.
Be appreciated that and expect that those skilled in the art may do some changes to inventive principle disclosed herein, so these modification will comprise within the scope of the present invention.Embodiments of the invention
Embodiment 1 is in order to assess simulation digestion and the absorption of biological functionality to natural composition:
By after mimic digestion and/or absorption process, material being measured, improved the natural composition definite method of back that be eaten to its effect with bioassary method.Simulate the stringency that these physiological process have increased the assessment biological functionality, its result can indicate possible in vivo activity better.Because medical herbs and natural extract all are compound materials, Digestion can increase or reduce absorption and biological activity subsequently.Mimic digestion:
Final preparation compositions (tablet, capsule, hard gelatin capsule etc.) is added in the 15ml simulated gastric fluid of the method preparation of rolling up by USP the 23rd.This gastric juice has about pH1.2, contains pepsin, therefore can simulate the digestive environments of stomach.Then with this content 37 ℃ with 250 RPM joltings 2 hours in case the dissolving detected materials.
After this incubation, by the NaOH solution that adds 0.5ml 2.2N formed mixture is adjusted to about pH7.4, add 2 times of spissated simulated intestinal fluids that equal-volume (15.5ml) contains 2 times of dilution pancreatin then.At 37 ℃ with 250RPM jolting culture bottle 2 hours.The solution of Xing Chenging is similar to the physiology intestinal juice that contains pancreatin like this, and the detected materials edible by the oral cavity is exposed to wherein.With this mixture quick freezing, be stored in-80 ℃ and treat further test then.The assessment of mimic absorption and bioavaliability:
Except the effect of the digestive environments that may simulated by said process, the functionality of compositions depends on that also it withdraws from digestive tract and enter the ability that circulation reaches site of action.Application colon carcinoma cell line Caco-2 has proved the good method at extracorporeal evaluate Absorption and bioavaliability, and this cell line can be differentiated to form the monolayer (S.Yee, 1997) of enteric epithelium sample when applying with high density.The Caco-2 cell of one deck differentiation on flat board can allow material to flow to base side from the top side, on the contrary too, can be used for testing absorption and bioavaliability to detected materials.The feature of this modular system has been described well and show with the body that the animal and human is carried out in bioavaliability research consistent (Yee, 1997; Taylor etc., 1997).
Will be available from American type culture collection (ATCC, Rockviie, MD) Caco-2 cell is kept at the improved Eagle ' of Dulbecco ' s s culture medium (DMEM, Gilbco, Grand Island is housed, NY) in the T-75 culture bottle, this culture medium contain 10% hyclone (Gemini Bio-Products, Calabasas, CA) and penicillin-streptomycin solution (Sigma of 100 units/ml, St.Louis, MO).In order to carry out bioavaliability assessment, with cell be coated in the Transwell filter membrane (Corning-Costar, Cambridge, MA) on, in case after converging, keep again treating in about 21 days that it is differentiated to form enteric epithelium sample monolayer.
The convenient substituting product of Caco-2 cell be BIOCOAT  intestinal environmental system (BectonDickenson, Franklin Lakes, NJ).This test kit can provide a cellular layer that converges in about 3 days.
Detected materials can be dissolved in DMSO or 50% ethanol, perhaps may comprise the sample that aliquot is come self simulation digestion scheme (see above).Will from digestion scheme diluted sample, perhaps at 50 ℃ of incubations so that the heat inactivation pancreatin.This step is used to remove pancreatin, and this enzyme may be poisoned cell, and therefore may destroy the integrity of Caco-22 monolayer.Then testing sample is added to the top side of cell, again at 37 ℃ of incubations.The temperature of incubation can be lower.The BIOCOAT system can make detection at room temperature carry out, and the incubation time can be from about 10 minutes-2 hours or longer.With 30,60 and 90 minutes after the detected materials administration, be used for biological functionality mensuration from top side and substrate side room collection aliquot sample.When using the cell line to be grown in and to cultivate in the same culture medium of Caco-2 monolayer to carry out biological function when measuring, then can use the cell culture hole that base side at this monolayer contains target cell line to be measured to carry out this program.In case Absorption is finished, can remove the Caco-2 cell monolayer that this shifts hole and support.Affirmation to simulation digestion scheme:
In order to confirm above-described simulation digestion program, implemented two types analytical test.The concordance of simulation digestion program has been determined in first kind of analytical test.Table 1 shows in the different experiments of carrying out in three days, different vegetable materials is simulated the pH that digests after finishing.In second kind of analytical test (table 2), use the biological activity that is used to assess two kinds of different vegetable materials from material at least three kinds of different Digestion of parent material of same batch.It is active to use Echinacea purpurea Moench macrophage activation test (embodiment 2) or Semen Ginkgo free radical scavenging test (embodiment 3) assessment.Be set forth in data show in table 1 and the table 2 relative standard deviation (CV), the indication variability shows that the active CV of simulation Digestion and biological function is less than 10%.
The concordance of table 1-simulation Digestion
Product/sample PH the 1st day PH the 2nd day PH the 3rd day
Herba Hyperici Monogyni ????6.76 ????6.65 ????6.83
Echinacea purpurea Moench ????6.85 ????6.66 ????6.88
Semen Ginkgo ????6.85 ????6.75 ????6.88
Radix Ginseng ????7.02 ????6.80 ????6.98
Saw Palmetto Berries ????6.98 ????6.80 ????7.03
The bioactive concordance of table 2-
Sample Reply ????CV
Echinacea purpurea Moench (digestion 1) ????1027pg/ml?TNF-α ????9.2％
Echinacea purpurea Moench (digestion 2) ????1165pg/ml?TNF-α
Echinacea purpurea Moench (digestion 3) ????977pg/ml?TNF-α
Semen Ginkgo (digestion 1) ????EC 50=44.60μg/ml ????7.7％
Semen Ginkgo (digestion 2) ????EC 50=43.21μg/ml
Semen Ginkgo (digestion 3) ????EC 50=50.01μg/ml
Semen Ginkgo (digestion 4) ????EC 50=42.20μg/ml
The bioactive external test method of 2 pairs of Echinacea purpurea Moench prepared products of embodiment:
Based on the ability that they stimulate macrophage to produce TNF-α and stimulate the cell proliferation of PBMCs, estimated Echinacea purpurea Moench extract and the compositions of using above-mentioned simulation digestion and absorbing model preparation with in vitro tests.Macrophage produces the algoscopy of TNF-α:
With 1 * 10 6The density of individual cell/ml places the culture plate in 24-or 96-hole, incubation 24 hours with RAW 264.7 macrophages.Though finding that this macrophage ties up to can produce a small amount of TNF-α when placing culture plate, the TNF-alpha content in 24 hours incubation process in the culture medium can reduce.The Echinacea purpurea Moench compositions of variable concentrations is added in the RAW264.7 cell, measures the concentration of TNF-α.The Echinacea purpurea Moench compositions is dissolved standard substance, product and the prepared product that passes through to simulate digestion or simulation digestion and simulate the absorption preparation.(Endogen, Woburn MA) measure the level of TNF-α by ELISA, and by relatively carrying out quantitatively with reorganization TNF-α standard substance to use mice TNF-α ELISA test kit.Production ratio is:
A major advantage of simulation Digestion is to natural composition simulation Digestion.Second advantage is to preserve the biological activity of relevant composition.By the capsule 's content that contains Powdered Echinacea purpurea Moench product being prepared with common solvent (dimethyl sulfoxide or 50% ethanol) extracted overnight with by the simulation Digestion.Fig. 1 shows the comparison that macrophage that the different prepared products by placebo and Echinacea purpurea Moench powder-product bring out produces TNF-α.Echinacea purpurea Moench product by the preparation of simulation Digestion has kept remarkable a large amount of TNF-α stimulating activity, and is not higher than the activity of placebo by the product proof of two kinds of common extracting method preparations.And as showing among Fig. 2, the macrophage activation ability is a dose dependent, shows the specificity of effect.The comparison of Echinacea purpurea Moench raw material and product:
After simulation digestion, measure the macrophage activation activity of multiple Echinacea purpurea Moench raw material and product.As shown in Fig. 3, compare when same standard material and placebo, have only 2 batches of proofs can induce macrophage to produce TNF-α significantly in 7 batches of raw materials.To relatively being presented among Fig. 4 of obtainable Echinacea purpurea Moench outturn sample.In 11 tested products, have only 3 with standard material relatively prove can activating macrophage.Bioavaliability:
The standardized product of digestion and the top side that placebo puts on the Caco-2 cell monolayer will be simulated.After 2 hours, from then on the top side of monolayer and base side are taken out sample, dilute 10 times, measure the functional activity of TNF-α.The activity of Fig. 5 demonstration mensuration before simulating absorption and after the simulation absorption to standardized product and placebo sample.Cylinder is represented the mean+SD in three replication holes.
In order to assess the permeability of Caco-2 monolayer, when having measured t=0 in the substrate of top side and t=2 hour the time in base side substrate the content of TNF-α.By deduct the numerical value of placebo from the numerical value that standardized product is obtained, removed any effect that produces cytokine by the Caco-2 cell.38,945 TNF of unit have been added to the tip side of this monolayer.After the incubation 2 hours, be measured to 30,720 TNF of unit in base side.Evaluate proximate permeability (P by the following calculating of Yee (1997) then App): P App=(F *V D)/(SA *M D), at this F=active unit/per second (4.27), V D=top side volume (0.5ml), SA=film surface area (1.13cm 2), and the total units in the volume of MD=top side.According to other research, this value of calculation, P App=48 * 10 -6, be equivalent to about 70% or higher (Yee, 1997) that this material prediction is absorbed.Affirmation to product:
By mimic Digestion measure 5 batches of Echinacea purpurea Moench products criticize with criticize between concordance.In comparable scope, induce for 4 batches that describe among Fig. 6 and produce TNF-α in the macrophage.Induce significantly less activity for the 5th batch.In order to test conclusive evidence, these comparison tests are combined with other research, so that establish the biological functionality claimed range that a certifiable batch products must be fit to.With the test method determination batch sample of conclusive evidence like this, so that ensure the quality of products.The product that only is suitable for the biological functionality proper range just is identified.The inducing action of on cell proliferation:
With Echinacea purpurea Moench sample treatment peripheral blood lymphocytes (PBMCs, Clonetics, San Diego CA) by mimic harmonization of the stomach intestinal digestion effect preparation.Melt a bottle cell (about 5 * 10 7Individual cell), and be suspended in the lymphocyte growth culture medium that is preheated to 37 ℃ (LGM-3, Clonetics) in.100 μ l cell suspension are assigned in each hole of 96 hole microcomponent culture plates.With testing sample, the Cebo-Caps of digestion, Echinacea purpurea Moench standard material and concanavalin A (Con A, positive control) are prepared into 10 times of concentration in the LGM-3 culture medium, and to adding detected materials 10 μ l separately in the hole.With about 72 hours of cell incubation, the application cell titration TMProliferation assay test kit (Promega, Madison, WI) propagation/vigor of the assessment of the conversion by metabolism dyestuff MTT cell.The 1ml MTT dye solution that provides is added in the 5ml LGM-3 liquid, every hole is added 50 μ l MTT/LGM-3 culture medium.After 2 hours, add 100 μ l termination/solubilising liquid at 37 ℃ of incubations, measure optical density at 570nm with BioTEK ELISA culture plate analyzer.Table 3 shows the dose-response relationship between Echinacea purpurea Moench concentration and the cell proliferation.Table 4 shows the dose-response relationship between positive control Compound C on A and the cell proliferation.The numerical value of Echinacea purpurea Moench or each concentration of Con A is represented the meansigma methods in 8 holes.With single tail Student ' S T-test (one-tailed Student ' s T-test) Cebo-Caps is reacted computational statistics significance (P).The cell proliferation summit is in the Echinacea purpurea Moench concentration of about 1 μ g/ml digestion.Con A has also produced dose dependent response, has also produced high-caliber stimulation at 1 μ g/ml, therefore the dosage of the later experiment of selected conduct.
The dose response of table 3-Echinacea purpurea Moench standard material and PBMC propagation
Echinacea purpurea Moench concentration (μ g/ml) Average OD 570 Standard deviation ????％CV The P value
????5 ????0.590 ????0.022 ????3.73 ????＜0.01
????1 ????0.668 ????0.036 ????5.40 ????＜0.01
????0.5 ????0.597 ????0.086 ????14.37 ????＜0.01
????0.1 ????0.560 ????0.099 ????17.74 ????＜0.01
Placebo ????0.425 ????0.050 ????11.72
Concanavalin A (1 μ g/ml) ????0.790 ????0.035 ????4.39 ????＜0.01
The dose response of table 4-concanavalin A and PBMC propagation
Concanavalin A concentration (μ g/ml) Average OD 570 Standard deviation ????％CV The P value
????10 ????0.777 ????0.024 ????3.05 ????＜0.01
????5 ????0.714 ????0.018 ????2.58 ????＜0.01
????1 ????0.716 ????0.013 ????1.88 ????＜0.01
????0.5 ????0.463 ????0.027 ????5.9 ????＜0.01
Placebo (1 μ g/ml) ????0.415 ????0.029 ????7.01 ?????-
Table 5 show to use makes the PBMC proliferation assay from the cell of same donor, the repeatability between the degree of variation of test bay and Echinacea purpurea Moench standard substance batch.Table 6 proof all has significant breeder reaction from the PBMCs of three different donors.For every group of test, use 1 μ g/ml to measure multiple 8 holes 3 days or more time through the Echinacea purpurea Moench or the Con A of digestion.Echinacea purpurea Moench of all batches and Con A compare with placebo all significant difference (P＜0.01).
Repeatability (the OD of table 5-PBMC breeder reaction 570Measure)
Sample The 1st day The 2nd day The 3rd day The 4th day 3/4 day meansigma methods
Placebo ????0.479 ?0.425 ?0.458 ?0.469 ?0.458
Echinacea purpurea Moench standard substance lot number 1 ????0.579 ?0.654 ?0.665 ?0.664 ?0.641
Echinacea purpurea Moench standard substance lot number 2 ????- ?0.631 ?0.669 ?0.665 ?0.652
Echinacea purpurea Moench standard substance lot number 3 ????0.542 ?0.629 ?0.692 ?0.641 ?0.626
Echinacea purpurea Moench standard substance lot number 4 ????0.594 ?0.649 ?0.689 ?0.650 ?0.643
?Con?A ????0.784 ?0.790 ?0.762 ?0.722 ?0.765
Table 6-is from the breeder reaction (OD of different donor PBMCs 570Measure)
Sample The PBMC of No. 1 donor The PBMC of No. 1 donor The PBMC of No. 1 donor The meansigma methods of donor
Placebo ????0.407 ????0.295 ????0.361 ????0.355
Echinacea purpurea Moench standard substance lot number 1 ????0.613 ????0.623 ????0.527 ????0.588
Echinacea purpurea Moench standard substance lot number 2 ????0.572 ????0.59l ????0.553 ????0.587
Echinacea purpurea Moench standard substance lot number 3 ????0.585 ????0.577 ????0.473 ????0.545
Echinacea purpurea Moench standard substance lot number 4 ????0.606 ????0.612 ????0.497 ????0.572
?Con?A ????0.761 ????0.904 ????0.735 ????0.800
The bioactive external test method of 3 pairs of Semen Ginkgo prepared products of embodiment:
The test method determination of setting up for Semen Ginkgo the direct antioxidant activity of this herbal products, suppress the ability that platelet activating factor is incorporated into its receptor, and the ability of Semen Ginkgo prevention nerve cell death in the mitochondrion injury or after being exposed to amyloid-beta peptide.Detect the external test method of free radical scavenging activity:
(St.Louis MO) is dissolved in the 500ml ethanol for DPPH, Sigma, is stored in-80 ℃ by aliquot with 167mg hexichol picrylhydrazyl.By 20ml 1M Tris-HCl (pH7.4) being mixed into 880ml of 50% ethanol/water solution, formation determination buffer (BS).Test is carried out in 96 hole microtitration plates, at first sample to be determined is diluted to the highest test concentrations in the BS buffer, in BS this sample is made 2 times of serial dilutions subsequently.Then each hole is added 100 μ lDPPH solution, with this titer plate incubation 30 minutes at room temperature.Measure optical density at 540nm.
Table 7 shows the mean percentage of variable concentrations Semen Ginkgo composition removing free radical.Said composition comprises the Semen Ginkgo extrac of standardized not digestion process, has stood the extract of the same standardized of simulation Digestion, and three different Semen Ginkgo products that also stood the simulation Digestion.The result shows that the free radical scavenging activity of Semen Ginkgo is subjected to the influence of Digestion.In addition, according to the EC that calculates.Value, obviously, the Semen Ginkgo product of the different trade marks of three " equivalence " standardized extract (24% flavonoid, 6% terpenoid) of having mixed Semen Ginkgo has significantly different antioxidants and renders a service.
The free radical (%) that table 7-removes
Extract concentrations (μ g/ml) Standardized Semen Ginkgo extrac (dimethyl sulfoxine) Standardized Semen Ginkgo extrac (digestion) Product A (digestion) Product B (digestion) Products C (digestion)
????200 ????100 ????100 ????100 ????100 ????100
????100 ????100 ????100 ????98.1 ????100 ????79.7
????50 ????97.9 ????77.3 ????59.5 ????79.2 ????45.7
????25 ????58.2 ????43 ????34.2 ????45.7 ????27.4
????12.5 ????33.4 ????24.6 ????19.4 ????24.0 ????14.5
????6.25 ????18.1 ????13.0 ????11.2 ????13.7 ????7.9
????3.12 ????9.0 ????6.2 ????5.7 ????5.3 ????4.5
????EC50 ????(μg/ml) ????12.9 ????25.2 ????40.2 ????22.72 ????62.8
External test method to the platelet activating factor inhibition:
From the rabbit arteria auricularis blood sampling of fasting, this rabbit was not used medicine and immunity inoculation at least two weeks.Do venipuncture with butterfly valve conduit (No. 21 * 3/4 inch syringe needle, 3  inch conduits).Take out 1ml blood and discard, take out the 30ml whole blood then and directly enter in the syringe that contains 3ml 3.2% sodium citrate.The upset syringe makes the blood mixing several times lightly.The blood sample that demonstrates haemolysis or fibrin formation sign discards.Shake definite do not occur " eddy current " gently when lifting facing to light, the hematoblastic blood plasma (PRP) that is rich in that demonstrates platelet activation sign (form change) is also discarded.The preparation of PRP:
The 30ml anticoagulation is transferred in the aseptic plastic centrifuge tube of two 15ml, by in room temperature, isolates PRP 3 times, each centrifugal interval 2-3 minute with 650 * g continuous centrifugal in 30 minutes.The PRP that obtains after each centrifugal is transferred in the aseptic plastic pipe of 15ml, adds a cover and at room temperature place 30 minutes, analyze platelet function then.Whether assessment PRP activates (see above), if be activated then discard.Contain the few blood plasma of platelet (PPP) by 2000 * g centrifugal remaining cell precipitation component acquisition in 10 minutes at room temperature.PPP is transferred in another aseptic plastic pipe.
From PRP, take out 10 μ l aliquot and make platelet count.This aliquot of dilution PRP in the isoosmotic cell counting liquid of 20ml counts then earlier.Regulate hematoblastic number to 3000 among the PRP, 000/ μ l with the few blood plasma of platelet (PPP) that contains from body.The mensuration of platelet aggregation:
With Chrono-Log platelet aggregation meter, 37 ℃ and stir with the speed of 850 rpm under carry out the platelet aggregation optical detecting.During each mensuration, get 450 μ l PRP and be added in the gathering cuvette that has Teflon  stirring rod, cuvette is put into the measuring cell of assembling meter.To contain 450 μ l from body PPP, the cuvette of stirring rod and 50 μ l 0.15M NaCl (being preheated to 37 ℃) is put into and is assembled the position of meter with reference to the chamber.Use baseline setting pattern and determine baseline.The record distance is with respect to the most a large sum of deflection (apart from mm) of the PPP baseline of PRP on chart.Under 37 ℃ of stirrings,, add 5 μ l inhibitor or the extracts be dissolved in DMSO with the Hamilton microsyringe subsequently, perhaps add other excipient, make it and platelet response 1 minute, add 5 μ l platelet agonists then PRP preheating 1 minute.After adding agonist or tester, record aggregate response curve 3 minutes.Preferred agonist is L-α-phosphatidylcholine, and (L-PAF Sigma), finds it than DL-α-phosphatidylcholine to β-acetyl-γ-O-cetyl, and β-acetyl-γ-O-cetyl (raceme PAF) has significantly higher activity.Press the hematoblastic aggregation of following mensuration:
100% platelet aggregation effect is represented in the light transmission of PRP liquid behind the adding saturated level PAF.0% platelet aggregation effect is represented in the light transmission that adds excipient (for example DMSO) back contrast PPP liquid.For the effect of other solution composition, carry out the directional light transmission measurement by PPP solution and correct light transmission values similar processing.In order to determine the range of linearity of this method of testing, carried out containing the assaying reaction of variable concentrations L-PAF.In order to measure the inhibitory action of Semen Ginkgo extrac, selected to be in the L-PAF concentration in this test range of linearity.Determine hematoblastic aggregation according to following equation: the percent of platelet aggregation effect=100 * T o/ T Max, at this T oBe light transmissive value added in the assaying reaction, T MaxIt is the light transmission value added that does not contain the control reaction of Semen Ginkgo extrac.
There is linear relationship in discovery between the concentration of L-PAF and the platelet aggregation effect under L-PAF low dosage (＜0.1 μ M).The concentration that is higher than 0.1 μ M has been brought out maximum platelet aggregation effect.Table 8 shows, for L-PAF, uses the linear dose-response and the EC of different preparations of platelets acquisitions 50Value is highly repeatably.
In order to confirm this method of testing, with ginkgolide B and not the Semen Ginkgo control material of digestion process obtained measurement result.Table 9 shows the dose-response analysis of the anticoagulant effect that ginkgolide B brings out L-PAF.Made dose-response curve in 4 different PAF concentration representing platelet activation effect varying level (percent of maximum reaction).Expect as the competitive inhibitor that PAF-brought out aggregation, for ginkgolide B, its IC 50It is the degree that depends on platelet activation.Show for the data that linear regression coefficient obtained,, have the linear response (based on correlation coefficient r) of anticoagulant effect at 0.02 μ M and the PAF that is higher than this concentration.Therefore, (0.02 μ M) is used for further test with this PAF concentration, so that obtain to have the maximum sensitivity of linear response.
Table 8-is for the dose-response relationship of different preparations of platelets
Experiment numbers Rabbit number Platelet activation effect (the EC of L-PAF 50) Correlation coefficient (r)
????1 ????7037 ????0.008 ????0.95
????2 ????7037 ????0.006 ????＞0.98
????3 ????7037 ????0.006 ????＞0.98
????4 ????8052 ????0.006 ????＞0.98
????5 ????7261 ????0.006 ????＞0.98
????6 ????7036 ????0.006 ????＞0.98
Table 9-ginkgolide B is to the inhibitory action of platelet aggregation
The concentration of L-PAF (μ M) The percent of maximum platelet aggregation effect IC to ginkgolide B calculating 50????(μM) The dose-response correlation coefficient (r) of ginkgolide B linearity
????0.01 ????63％ ????0.2 ????0.90
????0.02 ????87％ ????0.65 ????＞0.99
????0.05 ????90％ ????3.3 ????＞0.99
????0.075 ????96％ ????6.4 ????＞0.99
Table 10 shows for ginkgolide B IC 50The repeatability of measuring is that selected 0.02 μ M L-PAF concentration was measured at different three days.For the not interior on the same day IC that measures 50, the r value approaches complete match (r=1).This proof for same preparations of platelets, can be determined to pinpoint accuracy the activity of inhibitor.
Table 10-ginkgolide B is to the inhibitory action (0.02 μ M L-PAF) of platelet aggregation
The experiment number The percent of maximum platelet aggregation effect IC for ginkgolide B calculating 50(μM?) The correlation coefficient of dose-response (r)
????1 ????87％ ????0.65 ????＞0.99
????2 ????89％ ????1.0 ????＞0.99
????3 ????89％ ????1.1 ????＞0.99
Average difference CV ????0.92 ????0.24 ????26％
Though identical PAF concentration not necessarily causes the platelet aggregation effect of par when using different preparations of platelets,, when with Semen Ginkgo standard substance activity being object of reference when measuring, improved the accuracy and reliability that obtains.With selected PAF concentration, three irrelevant preparations of platelets have been measured the IC of Semen Ginkgo with reference to material and ginkgolide B standard substance 50Value.Finding in the scope of being measured has linear relationship (r＞0.99, data is unlisted) between Semen Ginkgo concentration and the anticoagulant.And Semen Ginkgo is with respect to activity (the ginkgolide B IC of ginkgolide B 50/ Semen Ginkgo IC 50) irrelevant in selected PAF concentration and preparations of platelets.Therefore, can be with the relative activity (IC of Semen Ginkgo detected materials 50The IC of/Semen Ginkgo standard substance 50) to be used for comparison Semen Ginkgo prepared product active to the functional inhibition of platelet aggregation effect.Table 12 be presented between same batch the different prepared products and different batches with reference to accessible repeatability between the material.The affirmation of product:
For the required minimum activity of product validation depends on accuracy and the degree of accuracy that is used to measure active method of testing, from identical data repeatability with reference to the different prepared products of material, and different batches is with reference to the variability between the material.Measuring the standard deviation lower (2.7%, table 11) that this method of testing shows in the relative activity for identical with reference to material.As be displayed in Table 12, the variability that in same batch, causes by the sample preparation thing and batch between variability also lower.These considerations make it to select one must be satisfied with the activity level that a certain product reaches affirmation.In case be determined, this method of testing can be used for measuring Semen Ginkgo compositions and the extract that has prepared by simulation digestion and/or Absorption.
Table 11-is with respect to the ginkgolide B standard substance, and Semen Ginkgo is with reference to the inhibitory action of product material to platelet aggregation
The experiment number The active percent of ginkgolide B standard substance
????1 ????2.2
????2 ????2.1
????3 ????2.1
Average difference CV ????2.1 ????0.06 ????2.7％
Table 12-is with respect to the ginkgolide B standard substance, and Semen Ginkgo is with reference to the inhibitory action of product material to platelet aggregation
The Semen Ginkgo prepared product The active percent of ginkgolide B standard substance
Lot number
1, sample preparation thing 1 ????2.2
Lot number 1, sample preparation thing 2 ????2.4
Lot number 1, sample preparation thing 3 ????2.1
Lot number 1, sample preparation thing 4 ????2.2
Lot number 2 ????2.1
Lot number 3 ????2.2
Average difference CV ????2.20 ????0.11 ????5.0％
The external test method of neuroprotective:
Make neuronal precursor (people or inhuman) stand the condition of neurodegenerative process in the analogue body.Make these constrained optimizations, so that proof is with reference to the protective effect of product Semen Ginkgo material.When existing and not existing, the metabolic activity of processed cell is compared with the metabolic activity of processed cell not with reference to product medical herbs material.
Beta amyloid peptide (25-35) is to buy from Bachem Bioscience company.This peptide is dissolved in DMSO, becomes the ultimate density of 1mM, at N 2Be distributed into aliquot in the gas, be kept at-30 ℃.A fresh aliquot is used in each experiment, and is diluted to required concentration with culture medium before just will using.Glutamic acid available from Sigma (St.Louis, MO).In the pure water of 18M Ω, make the stock solution of 10mM, and filtration sterilization.Be kept at-30 ℃ with aliquot, before using, dilute with culture medium.
(Rockville MD) has obtained PC-12 precursor neuronal cell line (rat adrenal gland pheochromocytoma), cultivates containing 10% hyclone (FBS) and 5% hot deactivation horse serum, and exists in the DMEM culture medium of 1% penicillin/streptomycin solution from ATCC.Heat-inactivated horse serum be available from Sigma (St.Louis, MO).Other tissue culture's reagent is available from Gibco.Make the tissue culture superficial growth of cell at the glue primordial covering.According to the explanation of manufacturer prepare the fibril form collagen (the original collagen of cell, Cohesion Technologies company, Palo Alto, CA), and be used for the bag be organized culture bottle and 96 well culture plates, form a thin layer.By grinding cell is taken off from culture plate.With pressure-vaccum cell mass is smashed, is filtered this cell suspension by cellular filter so that obtain uniform cell quantity, the experiment before 24 hours with 10 4The cell density repopulating cell in/hole.With reference product Semen Ginkgo material pretreatment cell, this material is dissolved among the DMSO with the concentration of 10mg/ml, is diluted to required concentration with culture medium before just will using.
Table 13 shows glutamic acid, and beta amyloid peptide and the combination of the two are to the cytotoxicity of PC12 cell.The most significant decline of activity (percent of residual living cells) occurs in 48-72 hour.By 96 hours,, make cell number begin to increase because the breeding of remaining living cells is duplicated.
The neurocyte toxicity of table 13-glutamic acid and beta amyloid peptide: cell activity (%)
Time (hour) 10mM glutamic acid 0.5 μ M beta amyloid peptide 10mM glutamic acid+0.5 μ M beta amyloid peptide
????48 ????71 ????47 ????33
????72 ????59 ????33 ????18
????96 ????50 ????60 ????33
Table 14 shows the dose-response relationship with reference to product Semen Ginkgo material that is added in the culture medium, and the glutamic acid that causes, and the cytotoxicity of beta amyloid peptide and this two compositions reduces.In processing back 48 hours (tables 14) and back 72 hours (table 15) mensuration of processing cell activity.In all cases, add reference product material and all caused the activity increase.
Table 14-Semen Ginkgo with reference to the product material to the toxic protective effect of neurocyte: the activity (%) of handling back 48 hours PC12 cells
Semen Ginkgo concentration (μ g/ml) 10mM glutamic acid 0.5 μ M beta amyloid peptide 10mM glutamic acid+0.5 μ M beta amyloid peptide
????0 ????75 ????43 ????35
????25 ????84 ????51 ????43
????50 ????92 ????55 ????52
????100 ????102 ????66 ????61
Table 15-Semen Ginkgo with reference to the product material to the toxic protective effect of neurocyte: PC12 cell activity (%): handled back 72 hours
Semen Ginkgo concentration (μ g/ml) 10mM glutamic acid 0.5 μ M beta amyloid peptide 10mM glutamic acid+0.5 μ M beta amyloid peptide
????0 ????64 ????40 ????25
????25 ????70 ????51 ????37
????50 ????79 ????54 ????43
????100 ????86 ????65 ????52
Also studied the ability that is subjected to the serum starvation cell of beta amyloid peptide stimulation with reference to product silver product material protection.Table 16 demonstration is further reduced by the activity that serum starvation causes with two concentration cultured cells of beta amyloid peptide.May extend into the cell of serum starvation with reference to the protective effect of product Semen Ginkgo material.This method of testing is used to measure its neuroprotective effect by the Semen Ginkgo prepared product that simulation digests and/or Absorption forms.
Table 16-Semen Ginkgo is with reference to the protective effect of product material to neurocyte toxicity and serum starvation: PC12 cell activity (%)
Semen Ginkgo extrac concentration (μ g/ml) Serum 0.5 μ M β-4 amyloid 1.0 μ M beta amyloid peptide
48 hours ????0 ????- ????41 ????41
????50 ????- ????69 ????58
????0 ????+ ????49 ????47
????50 ????+ ????80 ????66
72 hours ????0 ????- ????35 ????36
????50 ????- ????50 ????49
????0 ????+ ????47 ????53
????50 ????+ ????80 ????77
The bioactive external test method of embodiment 4 Herba Hyperici Monogyni prepared products:
Use extract and compositions that above-mentioned mimic digestion method prepares Herba Hyperici Monogyni (Hypericum perforatum).In order to reduce the degraded of active component, peptic digestion and intestinal digestion effect were respectively handled 1 hour.Experiment in vitro is estimated formed prepared product and is suppressed 3The H-dopamine and 3The ability of H-5-HT (5-hydroxy tryptamine) reuptake in the rat layer synaptosome.This illustrative method of testing usually with resist-the down binding mode is relevant, and is and relevant with the possible mechanism of action of Herba Hyperici Monogyni preparation.The synaptosome preparation:
Preparing rat brain synaptosomes according to the method for Gray and Whittaker (1962) from striatum is used for 3The H-dopamine uptake is measured, and perhaps is used for from the full brain preparation of removing cerebellum 3H-5-HT absorbs mensuration.Briefly, with the male Sprague-Dawley rat sacrificed by decapitation, take out brain rapidly.Striatum or the full brain that removes cerebellum are weighed, in the ice-cold 0.32M sucrose liquid of 9 times of volumes, make homogenate with Potter-Elvejhem homogenizer.With this homogenate 0-4 ℃ with 1000g centrifugal 10 minutes.Topple over and supernatant and be used for picked-up experiment. 3The H-dopamine and 3The picked-up of H-5-HT:
Press (1986) described method mensuration picked-ups such as Bonnet.37 ℃ of rough synaptosome prepared products of incubation 50 μ l aliquot in 1-2.5ml incubation culture medium, incubation culture medium composed as follows: 109mM NaCl, 3.55mM KCl, 2.4mM CaCl 2, 0.61mM MgSO 4, 1.1mM KH 2PO 4, 25mM NaHCO 3, the 5.4mM glucose, the 0.025mM nialamide, pH7.4, and contain 20nM 3H-dopamine or 20nM 3H-5-HT (uses preceding 30 minutes with 95%O 2, 5%CO 2To this culture medium ventilation).Adopt 5 minutes incubation time.By stopping picked-up, filter by Whatman GF/B glass fibre filter vacuum decompression immediately with the ice-cold culture medium dilution of 1.5ml thereupon.Wash this filter 2 times with the ice-cold culture medium of 3ml, spend the night in drying at room temperature.This filter was being added continuously the 0.5ml distilled water lasting 30 minutes, and after 3ml 1 and 5ml Ready Safe  (Beckman) the scintillation solution cocktail, measuring with LS 6500 TA scintillation counters (Beckman) 3The H-radioactivity. 3The H-dopamine and 3The picked-up of the ratio of H-5-HT is defined in total picked-up of 37 ℃ poor with 4 ℃ of picked-ups.With GBR 12783 and imipramine conduct respectively 3The H-dopamine and 3The reference compound of H-5-HT picked-up test.The stock solution that will prepare in DMSO (10mg/ml) centrifugal 10 minutes with 17000g, its supernatant are used for test.Sample is 4 ℃ of maximum storages about 5 days in amber bottle.In the incubation culture medium, done further dilution.
Table 17 shows with reference to product Herba Hyperici Monogyni compositions and has stood simple peptic digestion or stood the compositions of harmonization of the stomach intestinal digestion effect, to the inhibitory action of 5-HT reuptake.The radioactivity weighted mean value of 4 parts of duplicate readings of each sample of data representation, and the active Student ' of the contrast significance s T-test statistics that contrast is mated is analyzed (P).Prove that all Herba Hyperici Monogyni samples have significant 5-HT reuptake inhibition.But, be dissolved in DMSO in compare with reference to the Herba Hyperici Monogyni product, simulated the Herba Hyperici Monogyni product of peptic digestion effect, its inhibitory action has reduced significantly.It is active that mimic intestinal digestion effect does not further reduce inhibition significantly.
The inhibitory action that table 17-Herba Hyperici Monogyni is gone into the 5-hydroxy tryptamine rephotography
Sample Average counter/minute P value to suitable contrast
Solvent blank ????31,529
Be dissolved in the reference product Herba Hyperici Monogyni of DMSO ????13,312 ????P＜0.01
Mimic gastric juice (SGF) ????29,429
With reference to product Herba Hyperici Monogynis (～88 μ g/ml are in SGF) ????24,597 ????P＜0.01
Mimic gastric juice/mimic intestinal juice (SGF/SIF) ????31,810
With reference to product Herba Hyperici Monogynis (～88 μ g/ml are in SGF/SIF) ????26,225 ????P＜0.01
Table 18 shows by the dose-response relationship of the Herba Hyperici Monogyni of digestion process with reference to product and the reduction of 5-HT reuptake.The radioactivity weighted mean value of 4 parts of duplicate readings of each sample of data representation, and the active Student ' of contrast significance s T-test statistics analysis (P) to being mated.Also can carry out similar test to dopamine reuptake inhibition.
Table 18-Herba Hyperici Monogyni suppresses the dose response that the 5-hydroxy tryptamine rephotography is gone into
Sample Average counter/minute P value to suitable contrast
Solvent blank (DMSO) ????32,184 ??????-
Be dissolved in the reference product Herba Hyperici Monogyni of DMSO ????16,728 ????＜0.01
Mimic gastric juice/mimic intestinal juice (SGF/SIF) ????37,785 ??????-
With reference to product Herba Hyperici Monogynis (SGF/SIF) (400 μ g/ml) ????23,104 ????＜0.01
With reference to product Herba Hyperici Monogynis (SGF/SIF) (200 μ g/ml) ????28,860 ????＜0.01
With reference to product Herba Hyperici Monogynis (SGF/SIF) (100 μ g/ml) ????34,924 ????0.08
The bioactive external test method of 5 pairs of Saw Palmetto Berries prepared products of embodiment:
5 or cyclo-oxygenase suppress algoscopy and can be used for measuring with above-mentioned simulation digestion and the Saw Palmetto Berries compositions of absorption pattern preparation and the activity of other serenoa repens extract and commodity.Preparation rat liver microsomes and 5:
Use CO 2Gas anesthesia male Sprague-Dawley rat (the Hilltop Animal Lab., Scottdale, PA).Remove the connective tissue of parcel liver, pour into buffer (HepespH7.2,1.5mM EDTA, 1.0mM DTT and 10% glycerol) perfusion liver 2 times with HEDG.Take out liver then, in the HEDG buffer, make liver homogenate (every gram liver wet weights 5ml buffer).With this homogenate 4 ℃ with 9000 * g centrifugal 20 minutes.Then with the supernatant that forms 4 ℃ with 100000 * g centrifugal 1 hour.With the microsome precipitation resuspending that obtains in HEDG and make it evenly (knocking 3 times) with the speed of 100rpm, the equivalent packing, quick freezing also is stored in-80 ℃.By detecting the active algoscopy that suppresses of the metabolic 5 of androgen:
For each Saw Palmetto Berries sample to be measured, in the kaliumphosphate buffer of 20mM pH7.2, this material is diluted to required concentration (50 μ g/ml serenoa repens extract).Add 20 μ l microsomes (enzyme) in succession, testosterone (25 μ g/ml ultimate density) and Progesterone (20 μ g/ml, ultimate density).Then reactant mixture is added the 5 substrate.Start enzymatic reaction by adding NADPH (490 μ g/ml ultimate density), 37 ℃ of incubations 1 hour.Prepare sample by the ether extraction substrate then and be used for the HPLC analysis.With the peak area integration of testosterone and Progesterone, compare with the standard substance of testosterone and Progesterone, according to remaining the not cubage inhibitory action of metabolism substrate.
Begin to find that the 5 activity is digested the inhibition of buffer and Cebo-Caps largely.But, as shown in Table 19, digestion product boiled 2 minutes or, reduced the non-specific inhibitory action of those compositions effectively, and the inhibitory action that is included in the Saw Palmetto Berries prepared product keeps uninfluenced 5 to 50 ℃ of heating 5 minutes.The average percentage that when data representation in the table 19 exists 50 μ g/ml Saw Palmetto Berries prepared products and 5 to be total to incubation the testosterone metabolism is suppressed.
These data show, after simulation digestion, even by boil or heat make the intestinal enzyme deactivation afterwards the Saw Palmetto Berries material still have activity.Lost active (data are unlisted) by the filtering sample of 0.2 μ syringe filter.
Behind the material that table 19-heat treated is digested to the inhibitory action of 5
Sample Handle Suppress percent
Saw Palmetto Berries is with reference to product ?????- ????59％
Saw Palmetto Berries is with reference to product Boil ????42％
Saw Palmetto Berries is with reference to product ????50℃ ????55％
Placebo Boil ????0％
Placebo ????50℃ ????10％
By detecting the algoscopy that 5-hydroxy tryptamine generates and the transformation of androgen receptor suppresses 5:
Single multidigit point algoscopy can be at the synergistic activity between in-vitro simulated 5-hydroxy tryptamine generation inhibition and the inhibition of 5-hydroxy tryptamine transformation androgen receptor.Following mixture is used to measure biological activity: reorganization 5 or comprise the microsome of 5, cofactor NADPH, the substrate testosterone of limiting concentration, the brain liquid or the nuclear extract that comprise the not coordination androgen receptor composition that is incorporated into HSP90, and the biotinylation androgen response element DNA of limiting concentration.The composition that can add other when needing to above-named composition.
Add the Saw Palmetto Berries compositions, comprise the positive control of the similar preparation sample that lacks the Saw Palmetto Berries compositions and the negative control that not only lacked testosterone but also lacked the Saw Palmetto Berries compositions stimulates this mixed liquor of aliquot [the Saw Palmetto Berries compositions is ... ].This algoscopy can detect by the catalytic inhibitory action that generates 5-hydroxy tryptamine from testosterone of 5.5-hydroxy tryptamine is incorporated into androgen receptor, and changes this receptor, causes androgen receptor can be incorporated into androgen response element DNA.Therefore, generate by suppressing 5-hydroxy tryptamine, Saw Palmetto Berries can suppress androgen receptor and be transformed into the form that can be incorporated into androgen response element DNA.Whether changed in order to detect androgen receptor, made this mixed liquor and the neutral Avidin that is incorporated into the ELISA check-out console, Avidin or streptavidin contact, biotinylated androgen response element DNA is immobilized in this mixed liquor thereby make.If the androgen receptor in this mixed liquor has been changed by 5-hydroxy tryptamine, then can and therefore be immobilized with androgen response element DNA formation complex.The unconjugated material of flush away.This ELISA check-out console is contacted with anti--androgen receptor antibody, cause the androgen response element-androgen receptor complex of this antibodies on check-out console.Use multiple technologies known in the art and can detect this antibody and complex.For example, available enzyme or fluorescent marker, perhaps other detectable this antibody of part labelling.By preferable methods, be that the anti-antibody that makes second antibody promptly be coupled to alkali phosphatase is incorporated into antibody and the complex that is connected on the check-out console.Use the enzyme catalysis of para-nitro-pheneye phosphate (PNPP) detection of alkaline phosphatase, and in ELISA check-out console analyzer, do colorimetric determination.Contain the activity of the reaction deduction negative control of Saw Palmetto Berries, the activity divided by positive control deduction negative control obtains the inhibiting percent of serenoa repens extract, is used to assess IC with the several dosage in this algoscopy scope 50To PGF 2Produce inhibiting algoscopy:
Measuring the Saw Palmetto Berries compositions suppresses to produce prostaglandin E by the macrophage that stimulates with gamma interferon (IFN-γ) 2(PGE 2) ability.
Be supplemented with 2mM L-glutaminate (Gibco), 10% hyclone (Gemini, Carlsbad, CA) and the DMEM of 1% penicillin/streptomycin liquid (Gibco, Long Island, NY) in training grow RAW 264.7 macrophages (ATCC, Rockville, MD).At cell density is 10 6Make passage during individual cell/ml, be used for all experiments with this identical cell number.The RAW264.7 cell inoculation is entered 24 hole tissue culturing plates (1ml/ hole).When the Saw Palmetto Berries prepared product that adds and do not add by simulation Digestion preparation in as embodiment 1, hatch cell to be measured, and add 10 units/ml IFN-γ (Gibco) stimulation simultaneously and induce PGE 2Produce.(St.Louis MO) is used as the positive control of the method to known synthesizing Cox-2 inhibitors indole first mycin for Indomethacin, Sigma.By using PGE 2The quantitative ELISA test kit (OxfordBiomedical Research, Oxford, MI), in conjunction with the concentration known PGE that enters tissue culture medium (TCM) by dilution 2The standard curve of making is measured PGE in the tissue culture medium (TCM) 2The PGE that level comes quantitative assay to produce 2Amount.
Table 20 show three concentration by the Saw Palmetto Berries of digestion process with reference to product material or indole first mycin with by the RAW 264.7 macrophages generation PGE that stimulates with IFN-γ 2Dose-response relationship.Next we have assessed the repeatability between Saw Palmetto Berries standard substance batch.Make three batches of 20 identical μ g/ml Saw Palmetto Berries standard substance stand mimic Digestion, and measuring (table 21) on the same day.
The reference product Saw Palmetto Berries of table 20-indole first mycin and digestion process is to PGE 2The inhibitory action that produces
Indole first mycin concentration (μ g/ml) The reaction of indole first mycin (inhibitory action percent) Saw Palmetto Berries concentration (μ g/ml) The reaction of Saw Palmetto Berries (inhibitory action percent)
????0.01 ????74.8 ????40 ????73.8
????0.001 ????53.9 ????20 ????47.7
????0.0001 ????41.8 ????10 ????18.8
Table 21-three batches of Saw Palmetto Berries with reference to the product prepared product to PGE 2The inhibitory action that produces
Sample PGE 2Produce the percent that suppresses Standard deviation ????％CV
Lot number
1 Saw Palmetto Berries (20 μ g/ml) ????48.1 ??????- ????-
Lot number 2 Saw Palmetto Berries (20 μ g/ml) ????48.9 ??????- ????-
Lot number 3 Saw Palmetto Berries (20 μ g/ml) ????57.5 ??????- ????-
The reaction meansigma methods ????51.5 ????5.2 ????10.2
The bioactive external test method of 6 pairs of Radix Ginseng prepared products of embodiment:
Bioactive algoscopy is based on the inducing action of corticosteroid and the glucocorticoid regulating action to the nitric oxide synthase isozyme.Algoscopy to the corticosterone inducing action:
Will by Hilltop Lab Animal (Scottdale, PA) the Swiss mice of Gong Geiing (20-25 gram) is fed in having the rebasing Merlon cage of wood shavings, 72 ℃, 12 hours illumination/dark cycle.Animal is supplied with rodent, arbitrarily drinking-water.Corticosterone I 125(Costa Mesa CA) provides radioimmunoassay (RIA) test kit, uses according to the explanation of manufacturer by ICN Biomedicals.The gen-seng that is used to set up and confirms this assay method is to comprise the product of the Radix Ginseng extract (G115 ) of clinical assays and proof.
The preparation testing sample is to the concentration of 60mg/ml in phosphate-buffered saline (PBS), perhaps stand the processing of simulation digestion scheme as described in example 1 above, the content of employed Radix Ginseng detected materials that different is has produced the Radix Ginseng extract of ultimate density 50mg/ml.5 animals of each processed group, the dosage with per 100 gram the weight of animals 50mg Radix Ginseng extracts gives detected materials or the contrast of PBS excipient by peritoneal injection.Take blood plasma in 1 hour after the administration, use the level that quantitative RIA method is measured corticosterone.Check the credit that takes statistics to analyse with Students T-.Table 22 shows the typical consequence that obtains according to these programs.Significance,statistical (P) is to be benchmark with the saline control.This digital proof, its corticosterone activity holds out against mimic Digestion, has function in vivo.
The generation of the inductive corticosteroid of table 22-Radix Ginseng
Testing sample The corticosteroid that produces The P value
Contrast ????62.7 ?????-
The Radix Ginseng for preparing among the PBS ????427.3 ????0.002
The Radix Ginseng of digestion process ????450.8 ????＜0.001
The algoscopy that inhibited oxidation nitrogen produces:
With the RAW264.7 macrophage (ATCC, Rockville is MD) at DMEM (Gibco, Long Island, NY) the middle cultivation is supplemented with 2mM L-glutaminate (Gibco) among this DMEM, (Gemini, Carlsbad is CA) with 1% penicillin/streptomycin liquid for 10% hyclone.Make cell surpass 10 6The cell density of individual cell/ml is used for all experiments with this identical cell number.The RAW264.7 cell inoculation is entered 96 hole tissue culturing plates (100 μ l/ hole).When the Radix Ginseng prepared product that adds and do not add by simulation Digestion preparation in as embodiment 1, cultivate cell to be measured, and add 10 units/ml IFN-γ [source] stimulation simultaneously and induce the nitrogen oxide generation.(Sigma, St.Louis MO) are used as positive control to Progesterone.(Miller etc., 1996).By using Griess reagent (Sigma, St.Louis, MO) (Miller etc., 1996), in conjunction with measuring the standard curve that nitrite is made by contain known μ M in tissue culture medium (TCM), the side-product nitrite of measuring nitrogen oxide in the tissue culture medium (TCM) comes the nitrogen oxide amount of quantitative assay generation.
Table 23 show three various dose by the dose-response relationship between the Radix Ginseng material of digestion process or Progesterone contrast and the nitrogen oxide generation inhibitory action.In order to confirm that this activity is not because direct free radical scavenging, also measured Radix Ginseng prepared product through digestion process by the method for describing among the embodiment 3.Demonstrating up to the dosage of 400 μ g/ml does not have direct free radical scavenging activity, and the reduction that shows nitric oxide levels very may be because the inhibitory action that nitrogen oxide is produced.
The inhibition that table 23-Progesterone and Radix Ginseng produce nitrogen oxide Effect
Progesterone concentrations (μ g/ml) Suppress percent Radix Ginseng extract concentration (μ g/ml) Suppress percent
????100 ????96.7 ????400 ????87.6
????50 ????79.0 ????200 ????31.7
????25 ????33.3 ????100 ????21.6
Table 24 show for 200 μ g/ml set concentration by the Radix Ginseng of digestion process with reference to product material, nitrogen oxide produced inhibiting batch between repeatability.5 batches that are measured have shown some active variability, but this generally variability less than 10% (based on relative standard deviation, %CV).
The inhibitory action that table 24-Radix Ginseng produces nitrogen oxide with reference to product
Sample Reaction (μ M) Suppress percent Standard deviation ????％CV
Placebo ????27.0 ?????- ????0.71 ????2.6
Radix Ginseng lot number 1 ?????- ????51.8 ????0.75 ????1.4
Radix Ginseng lot number 2 ?????- ????51.9 ????0.42 ????0.8
Radix Ginseng lot number 3 ?????- ????46.2 ????0.90 ????1.9
Radix Ginseng lot number 4 ?????- ????42.8 ????0.79 ????1.8
Radix Ginseng lot number 5 ?????- ????43.5 ????1.46 ????3.4
Totally ?????- ????47.2 ????4.15 ????8.8
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Claims (100)

1. method of assessing natural composition or extract biological functionality comprises:
Make this natural composition stand mimic Digestion, mimic Absorption or this two kinds of effects are so that obtain the prepared product of this natural composition;
Measure and whether have a certain or several compositions or biological activity in this prepared product; And
Make in this prepared product these one or more compositions or bioactive existence or do not exist and be associated with the biological functionality of this natural composition or extract.
2. method of assessing natural composition or extract biological functionality comprises:
Make this natural composition stand mimic Digestion, mimic Absorption or the effect of the two are so that obtain the prepared product of this natural composition;
Measure and whether have a certain or several compositions in this prepared product; And
Make the existence of these one or more compositions or do not exist and be associated with the biological functionality of this natural composition or extract.
3. method of assessing natural composition or extract biological functionality comprises:
Make this natural composition stand mimic Digestion, mimic Absorption or the effect of the two are so that obtain the prepared product of this natural composition;
Be determined at and whether have a certain or several biological activitys in this prepared product; And
Make interior one or more the bioactive existence of this prepared product or do not exist and be associated with the biological function of this natural composition or extract.
4. the method for claim 1 comprises making described natural composition stand mimic digestion and mimic absorption.
5. the method for claim 4, wherein mimic digestion and mimic absorption are to carry out in succession.
6. the method for claim 4, wherein mimic digestion and mimic absorption are to carry out simultaneously.
7. the process of claim 1 wherein that mimic digestion comprises peptic digestion or intestinal digestion.
8. the process of claim 1 wherein that mimic digestion comprises peptic digestion and intestinal digestion.
9. the method for claim 7, peptic digestion wherein comprises makes natural composition stand mimic effective a period of time of gastric juice continuous action, so that digest this natural composition or extract.
10. the method for claim 9, simulated gastric fluid wherein comprises at least a gastric enzyme.
11. the method for claim 9, simulated gastric fluid wherein comprise pepsin and have the pH of about 1.0-2.0.
12. the method for claim 9, simulated gastric fluid wherein comprise pepsin and the pH with about 1.2.
13. comprising, the method for claim 7, intestinal digestion wherein make natural composition stand mimic effective a period of time of intestinal juice continuous action, so that digest this natural composition or extract.
14. the method for claim 13, simulated intestinal fluid wherein comprise at least a pancreas enzyme that is selected from pancreatinum, trypsin, chymase, carboxypeptidase and elastoser.
15. absorbing, the simulation that the process of claim 1 wherein comprises that one or more compositions of transporting said composition cross over a kind of biomembrane.
16. the simulation that the process of claim 1 wherein absorbs and to comprise one or more compositions of said composition are transported to base side from a kind of top side of cell monolayer.
17. the method for claim 16, cell monolayer wherein comprises the Caco-2 cell.
18. the natural composition that the process of claim 1 wherein is to obtain from plant origin.
19. the natural composition that the process of claim 1 wherein is to obtain from the herbaceous plant source.
20. the natural composition that the process of claim 1 wherein is to obtain from animal origin.
21. the natural composition that the process of claim 1 wherein comprises Echinacea purpurea Moench or its extract.
22. the method for claim 21 wherein is to determine whether to exist biological activity by the immunostimulatory activity of measuring this prepared product.
23. the method for claim 22 also further comprises:
This prepared product and T are provided lymphocytic mixture; And
Measure the content of the tumor necrosis factor that exists in this mixture after time at predetermined incubation.
24. the method for claim 22 also further comprises:
The mixture of this prepared product and a macrophage system is provided, and
Measure the content of the tumor necrosis factor that exists in this mixture after time at predetermined incubation.
25. the method for claim 24, macrophage system wherein is RAW264.7.
26. the method for claim 21 wherein is to determine whether to exist biological activity by the propagation of measuring peripheral blood lymphocytes (PBMCs).
27. the method for claim 26, wherein the mensuration to propagation comprises the transformation of measuring metabolism dyestuff MTT.
28. the natural composition that the process of claim 1 wherein comprises Herba Hyperici Monogyni or its extract.
29. the method for claim 28 wherein is to measure as follows whether to have biological activity:
Provide one to comprise this prepared product, synaptosome prepared product and be selected from dopamine and the mixture of the neurotransmitter of 5-hydroxy tryptamine; And
Detect synaptosome prepared product in this mixture to the picked-up of this neurotransmitter.
30. the natural composition that the process of claim 1 wherein comprises Saw Palmetto Berries or its extract.
31. the method for claim 30 wherein is to measure biological activity as follows:
A mixture that comprises this prepared product, microsome, cofactor NADPH and one or more substrates is provided; And
Detect the metabolism of these one or more substrates.
32. the method for claim 31, one or more 5 substrates wherein are testosterone and Progesterone.
33. the method for claim 30 wherein is to measure its biological activity as follows:
Provide a kind of comprise this prepared product, 5, cofactor NADPH, testosterone, with the mixture of HSP90 compound not coordination androgen receptor and androgen response element DNA; And
Detect the formation of androgen receptor in this mixture-androgen response element complex.
34. the method for claim 33, androgen receptor wherein-androgen response element complex is immobilized on a kind of solid phase carrier.
35. the method for claim 33 wherein is by colorimetry or this androgen receptor of fluoroscopic examination-androgen response element complex.
36. the method for claim 30 wherein is to measure its biological activity as follows:
A kind of mixture that comprises this prepared product, not coordinate aromatic hydrocarbon receptor and aromatic hydrocarbons response element DNA is provided; And
Detect the formation of aromatic hydrocarbon receptor in this mixture-aromatic hydrocarbons response element DNA complex.
37. the method for claim 36, androgen receptor wherein-androgen response element complex is immobilized on a kind of solid phase carrier.
38. the method for claim 36 wherein is by colorimetry or this androgen receptor of fluoroscopic examination-androgen response element complex.
39. the method for claim 30 wherein is to measure biological activity as follows:
A kind of mixture that comprises this prepared product, macrophage and gamma interferon (IFN-γ) is provided; And
Detect the activation of this macrophage.
40. the method for claim 39, wherein the mensuration to this macrophage activation comprises the detection prostaglandin E 2(PGE 2) or the generation of nitrogen oxide.
41. the natural composition that the process of claim 1 wherein comprises Semen Ginkgo or its extract.
42. the method for claim 41 wherein is to determine its biological activity by measuring free radical scavenging activity.
43. the method for claim 41 wherein is to determine biological activity by measuring the amount that platelet activating factor is suppressed.
44. the method for claim 41 wherein is by measuring neurovirulent inhibitory action to be determined its biological activity.
45. the method for claim 44 wherein comprises the inhibiting mensuration of neurotoxicity:
When having this prepared product, make neurocyte stand effectively to reduce the material of neurocyte activity or the effect of condition; And
Measure the activity of this neurocyte.
46. the natural composition that the process of claim 1 wherein comprises Radix Ginseng or its extract.
47. the method for claim 46 wherein is to determine biological activity as follows:
Give this prepared product to subjects; And
Measure the amount that corticosterone produces in this subjects.
48. the method for claim 46 wherein is to determine biological activity as follows:
A mixture that comprises this prepared product, macrophage and gamma interferon (IFN-γ) is provided; And
The inhibitory action amount that mensuration produces nitrogen oxide in this macrophage.
49. one kind comprise have basically by batch the regimen tonic of the conforming natural composition of composition, the method assessment of this composition concordance by comprising the steps:
Make the sample of this regimen tonic or this natural composition stand mimic digestion, mimic absorption or this two kinds of effects are so that obtain the prepared product of this sample;
Determine to exist in this prepared product a certain or several compositions; And
Make the existence and composition concordance associated of these one or more compositions.
50. one kind comprise have basically by batch the regimen tonic of the conforming natural composition of composition, the method assessment of this composition concordance by comprising the steps:
Make the sample of this regimen tonic or this natural composition stand mimic digestion, mimic absorption or this two kinds of effects are so that obtain the prepared product of this sample;
Determine to exist in this prepared product a certain or several compositions; And
Make the existence and composition concordance associated of these one or more compositions.
51. one kind comprise have basically by batch the regimen tonic of the conforming natural composition of composition, the method assessment of this composition concordance by comprising the steps:
Make the sample of this regimen tonic or natural composition stand mimic digestion, mimic absorption or this two kinds of effects are so that obtain the prepared product of this sample;
Determine to exist in this prepared product a certain or several biological activitys; And
Make this one or more bioactive existence and form the concordance associated.
52. the regimen tonic of claim 49 wherein makes this regimen tonic or natural composition stand mimic digestion and mimic absorption.
53. the regimen tonic of claim 52, wherein simulation digestion and simulation absorption are to carry out in succession.
54. the regimen tonic of claim 52, wherein mimic digestion and mimic absorption are to carry out simultaneously.
55. the regimen tonic of claim 49, simulation digestion wherein comprises peptic digestion or intestinal digestion.
56. the regimen tonic of claim 49, simulation digestion wherein comprises peptic digestion and intestinal digestion.
57. comprising, the regimen tonic of claim 55, peptic digestion wherein make this regimen tonic or natural composition stand effective a period of time of simulated gastric fluid continuous action, so that digest this natural composition or extract.
58. the regimen tonic of claim 57, simulated gastric fluid wherein comprises gastric enzyme.
59. the regimen tonic of claim 57, simulated gastric fluid wherein comprise pepsin and have the pH of about 1.0-2.0.
60. the regimen tonic of claim 57, simulated gastric fluid wherein comprise pepsin and the pH with about 1.2.
61. comprising, the regimen tonic of claim 55, intestinal digestion wherein make this regimen tonic or natural composition stand mimic effective a period of time of intestinal juice continuous action, so that digest this natural composition.
62. the regimen tonic of claim 61, simulated intestinal fluid wherein comprise at least a pancreas enzyme that is selected from pancreatin, trypsin, chymase, carboxypeptidase and elastoser.
63. absorbing, the regimen tonic of claim 49, wherein simulation comprise that one or more compositions of transporting said composition cross over a kind of biomembrane.
64. the regimen tonic of claim 49, wherein simulation absorb and comprise one or more compositions of said composition are transported to base side from a kind of top side of cell monolayer.
65. the regimen tonic of claim 64, cell monolayer wherein comprises the Caco-2 cell.
66. the regimen tonic of claim 49, natural composition wherein are to obtain from plant origin.
67. the regimen tonic of claim 49, natural composition wherein are to obtain from the herbaceous plant source.
68. the regimen tonic of claim 49, natural composition wherein are to obtain from animal origin.
69. the regimen tonic of claim 49, natural composition wherein comprise Echinacea purpurea Moench or its extract.
70. the regimen tonic of claim 69, wherein determined its immunostimulatory activity of this prepared product.
71. the regimen tonic of claim 70 wherein is to determine its composition concordance as follows:
This prepared product and T are provided lymphocytic mixture;
Measure the content of the tumor necrosis factor that exists in this mixture after the predetermined incubation time; And
Content and composition concordance associated with this tumor necrosis factor.
72. the regimen tonic of claim 70 wherein is to determine its composition concordance as follows:
The mixture that comprises this prepared product and macrophage system is provided;
Measure the content of the tumor necrosis factor that exists in this mixture after time at predetermined incubation; And
Content and composition concordance associated with this tumor necrosis factor.
73. the regimen tonic of claim 72, macrophage system wherein is RAW264.7.
74. the regimen tonic of claim 69 wherein is to determine its composition concordance as follows:
The mixture that comprises this prepared product and peripheral blood lymphocytes (PBMCs) is provided;
Measure the propagation of this PBMCs in this mixture; And
Propagation and composition concordance associated with this PBMCs.
75. the regimen tonic of claim 74, wherein the mensuration to propagation comprises the transformation of measuring metabolism dyestuff MTT.
76. the regimen tonic of claim 49, natural composition wherein comprise Herba Hyperici Monogyni or its extract.
77. the regimen tonic of claim 76 wherein is to determine its composition concordance as follows:
Provide one to comprise this prepared product, synaptosome prepared product and be selected from dopamine and the mixture of the neurotransmitter of 5-HT;
Detect of the picked-up of synaptosome prepared product to this neurotransmitter; And
With this intake and composition concordance associated.
78. the regimen tonic of claim 49, natural composition wherein comprise Saw Palmetto Berries or its extract.
79. the regimen tonic of claim 78 wherein is to determine its composition concordance as follows:
A mixture that comprises this prepared product, microsome, cofactor NADPH and one or more 5 substrates is provided;
Detect the metabolism of these one or more substrates; And
Metabolism and composition concordance associated with this substrate.
80. the regimen tonic of claim 79, one or more 5 substrates wherein are testosterone and Progesterone.
81. the regimen tonic of claim 78 wherein is to determine its composition concordance as follows:
Provide a kind of comprise this prepared product, 5, cofactor NADPH, testosterone, with the mixture of HSP90 compound not coordination androgen receptor and androgen response element DNA;
Detect the formation of androgen receptor-androgen response element complex; And
Formation and composition concordance associated with this complex.
82. the regimen tonic of claim 81, androgen receptor wherein-androgen response element complex is immobilized on a kind of solid phase carrier.
83. the regimen tonic of claim 81 wherein is by colorimetry or this androgen receptor of fluoroscopic examination-androgen response element complex.
84. the regimen tonic of claim 78 wherein is to determine its composition concordance as follows:
A kind of mixture that comprises this prepared product, not coordinate aromatic hydrocarbon receptor and aromatic hydrocarbons response element DNA is provided;
Detect the formation of aromatic hydrocarbon receptor-aromatic hydrocarbons response element DNA complex; And
Formation and composition concordance associated with this complex.
85. the regimen tonic of claim 84, androgen receptor wherein-androgen response element complex is immobilized on a kind of solid phase carrier.
86. the regimen tonic of claim 84 wherein is by colorimetry or this androgen receptor of fluoroscopic examination-androgen response element complex.
87. the regimen tonic of claim 78 wherein is to determine its composition concordance as follows:
A kind of mixture that comprises this prepared product, macrophage and gamma interferon (IFN-γ) is provided;
Detect the activation of this macrophage; And
Activation and composition concordance associated with this macrophage.
88. the method for claim 87, wherein the mensuration to this macrophage activation comprises the detection prostaglandin E 2Or the generation of nitrogen oxide.
89. the regimen tonic of claim 49, natural composition wherein comprise Semen Ginkgo or its extract.
90. the regimen tonic of claim 89 wherein is to determine to form concordance by the removing activity of observation free radical with this activity and composition concordance associated.
91. the regimen tonic of claim 89 wherein is to the inhibition of platelet activating factor with will now suppress and form the concordance associated and determine the composition concordance by observation.
92. the regimen tonic of claim 89 wherein is to determine to form concordance by observation to neurovirulent inhibitory action with this inhibitory action and composition concordance associated.
93. the regimen tonic of claim 92 wherein is to determine to form concordance as follows:
When having this prepared product to exist, make neurocyte stand effectively to reduce the material of neurocyte activity or the effect of condition;
Measure the activity of this neurocyte; And
Activity and composition concordance associated with neurocyte.
94. the regimen tonic of claim 49, natural composition wherein comprise Radix Ginseng or its extract.
95. the regimen tonic of claim 94 wherein is to determine its composition concordance as follows:
With this prepared product to the subjects administration;
Measure the generation of corticosterone in this subjects; And
Generation and composition concordance associated with corticosterone.
96. the regimen tonic of claim 94 wherein is to determine its composition concordance as follows:
A mixture that comprises this prepared product, macrophage and gamma interferon (IFN-γ) is provided; And
The inhibitory action that mensuration produces nitrogen oxide in this macrophage; And
The inhibitory action that will produce nitrogen oxide with form the concordance associated.
97. a method for preparing the regimen tonic that comprises natural composition, this regimen tonic have basically by batch concordance, the method assessment of this composition concordance by comprising the steps:
Make the sample of this natural composition stand mimic digestion, mimic absorption or this two kinds of effects are so that obtain the prepared product of this sample;
Determine whether there are a certain or several compositions in this prepared product; And
Selection comprises the prepared product of these one or more compositions.
98. the bioactive method of assessment Semen Ginkgo compositions comprises:
When having said composition to exist, make neurocyte stand effectively to reduce one or more materials of this neurocyte activity or the effect of condition; And
Measure the activity of this neurocyte.
99. the method for claim 98, one or more materials wherein are beta amyloid peptide or glutamic acid.
100. an assessment Saw Palmetto Berries compositions bioactive method comprises that detection replys the inhibitory action that receptor is transformed into DNA combining form to androgen, this detection method comprises:
Provide one comprise said composition, 5-hydroxy tryptamine, with the mixture of HSP90 compound not coordination androgen receptor and androgen response element DNA; And
Detect the formation of androgen receptor-androgen response element DNA complex.
CN 99810878 1998-07-14 1999-07-14 Method for identifying and confirming consistent bio-functionality of natural compsns. Pending CN1317974A (en)

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US20080293159A1 (en) * 2007-05-24 2008-11-27 Murray Goulburn Co-Operative Co. Ltd. Validation Process
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CN101968480B (en) * 2009-10-27 2013-09-11 闽南师范大学 Simulation and detection method for content of microelements in medicine capable of being absorbed by human body
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CN102680654B (en) * 2011-12-19 2014-10-15 河南科技大学 Method for building model for collectively evaluating bioavailability and toxicity of cadmium accumulation in food for human body

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