JP4677601B2 - Flow injection analyzer and flow injection analysis method - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a technique for characterizing an enzyme reaction between an enzyme and an activity control component that controls the activity of the enzyme, and more particularly, an enzyme between an enzyme and an activity control agent using flow injection analysis. The present invention relates to a flow injection analysis apparatus and a flow injection analysis method that enable measurement of the nature of a reaction control function.

  Flow injection analysis is a widely used measurement method combined with a wide range of analysis techniques such as molecular weight analysis, mass spectrometry, and enzyme characterization, as well as chromatographic analysis. The principle of flow injection analysis is that the analyte is passed through a fixed layer using a transport medium (carrier) and separated using chemical and physical action by the fixed layer, or the analyte is injected, After mixing and reacting the reagent with the sample solution flow, for example, the refractive index, the ultraviolet-visible spectrum, the oxygen concentration, the electrochemical potential, and the mass spectrum are used to output the information to be analyzed.

  Particularly in recent years, the flow injection technique has been used as a method for tracing an enzyme reaction in combination with an immobilized enzyme technique. For example, in Japanese Patent Application Laid-Open No. 2002-257780 (Patent Document 1), by using an immobilized enzyme in which a peroxidase is immobilized in a buffer solution, a reduction in the hydrogen peroxide concentration due to the reaction between the test solution and hydrogen peroxide is performed. A measurement apparatus having a flow cell for measurement is disclosed. In JP-A-11-290096 (Patent Document 2), in order to measure the phosphate ion concentration, a mixture of a sample solution containing phosphate ions and a maltose-containing solution is immobilized with maltose phosphorylase or the like. A flow injection analysis method is disclosed in which a phosphate ion concentration is quantified by contacting with an immobilized enzyme and chemiluminescence in a flow cell. In Japanese Patent Laid-Open No. 7-198657 (Patent Document 3), a reaction unit including an immobilized enzyme reactor filled with an immobilized enzyme alone, and a detection unit for detecting a change resulting from a reaction of a specific component in a sample. A flow type measuring apparatus is disclosed that detects a peak position from a stored waveform, creates a calibration curve, and calculates the concentration of a specific component in an unknown sample. Furthermore, in JP-A-7-63725 (Patent Document 4), in order to monitor harmful substances in water, an immobilized enzyme membrane to which urease is immobilized and an immobilized microbial membrane to which nitrite-producing bacteria are immobilized are used. Thus, a method for monitoring harmful substances in water based on changes in the amount of dissolved oxygen is disclosed.

  As described above, the immobilized enzyme technique is combined with the flow injection analysis technique and used for analysis of various samples. On the other hand, enzymes are known to cause a continuous enzymatic reaction under certain conditions and to cause chemical reactions such as oxidation, reduction, and degradation of antibodies or target substances. It is also known that there are chemical components that inhibit enzyme function, so-called inhibitory components. JP-A-11-124330 (Patent Document 5) using the enzymes and inhibitory components described above provides a therapeutic agent for vascular diseases by combining fixed doses of angiotensin converting enzyme inhibitory component and calcium channel inhibitory component. We disclose what we can do.

  Japanese Patent Application Laid-Open No. 8-134090 (Patent Document 6) discloses galactokodiic acid, a method for producing the same, and a tyrosinase-inhibiting component containing the same, and discloses a drug that prevents blackening, that is, has a sunscreen effect. Has been. In addition, in Japanese Patent Laid-Open No. 2-163097 (Patent Document 7), an enzyme is allowed to act on a substrate immobilized on a carrier in an environment in which an inhibitory component coexists, under a flow condition, and its degradation product is measured. An activity measurement method is disclosed. As described above, it is known that a combination of an enzyme and an inhibitor component can provide a drug for controlling an important function in vivo. In addition, it is considered that there is a component that enhances the enzyme activity, and it has been desired to characterize the activity control component for the substrate-enzyme reaction.

  By the way, the inhibitory action between the enzyme and the inhibitory component is also expressed by a biochemical reaction, and the inhibitory ability of the inhibitory component and the recoverability of the enzyme activity, so-called activity control characteristics, may be different for a specific enzyme. This suggests that the efficacy of the drug activity and the effective period of the inhibitory component provided by the enzyme-activity control component combination may vary depending on the specific enzyme and the specific activity control component combination. Therefore, when using an inhibitory component having a high affinity or a low affinity for a specific enzyme, the concentration of the inhibitory component for obtaining a drug effect by a specific drug and the period until the next administration are different. It is also envisioned to have a significant impact on drug prescription and dosing cycle. Similarly, there may be an activity controlling component that activates the activity to the enzyme.

  Conventionally, the evaluation of the inhibition characteristics between the above-described enzyme-inhibiting components has been performed by a conventional batch test. FIG. 12 shows a conventionally known method for measuring enzyme inhibition characteristics. In many cases, an enzyme-reactive organism is quantified by adding an inhibitory component in vitro using, for example, absorptiometry or dissolved oxygen quantification (FIG. 12 (a)), or contains a specific enzyme. An inhibitory component is administered to the incubated cells, a biological reaction caused by a specific enzyme is generated in vivo, and the enzymatic reaction product formed in the cell is quantified (FIG. 12B). ). For these conventional techniques, see, for example, Yoko Funasaka, “Mechanism and Evaluation of Whitening Effect”, Fragrance Journal, Vol. 30 (9), pp. 67-72 (2002) (Non-Patent Document 1). be able to.

It can be said that the inhibition test by the batch test described above can accurately measure the enzyme inhibition characteristics when competitive inhibition does not occur. Here, considering that enzyme inhibition is performed based on a chemical reaction, a certain degree of chemical equilibrium is established in the inhibition reaction between the enzyme and the inhibitory component, and the inhibitory component exists in the vicinity of the enzyme. Therefore, it is considered that an environment where the substrate reacts competitively with the enzyme is actually generated. For this reason, although the inhibitory properties are high, the binding properties of the enzyme-inhibitory components are weak, so the function of the inhibitory components disappears early, or the inhibitory properties of the enzyme-inhibitory components are weak, but the inhibitory components around the enzyme It is conceivable that an inhibitory component that exhibits a predetermined inhibitory property over a long period of time is present due to the deterrence. In addition, as described above, there may be not only a substance that inhibits the reaction activity of the enzyme, but also an agent that enhances the reaction activity between the enzyme and the substrate. Therefore, by elucidating the above behavior in a drug that exerts a predetermined medicinal effect by a combination of enzyme and activity control component, it becomes possible to evaluate a new property of an inhibitory component or an activation component, and thus a novel drug It is expected that it will be possible to provide compositions and drug delivery systems.
JP 2002-257780 A JP 11-290096 A Japanese Patent Application Laid-Open No. 7-198657 JP-A-7-63725 JP-A-11-124330 JP-A-8-134090 Japanese Patent Laid-Open No. 2-163097 Funakosaka, "Mechanism and Evaluation of Whitening Effect", Fragrance Journal, Vol. 30 (9), pp. 67-72 (2002)

  The present invention has been made in view of the above problems, and the present invention is a novel that makes it possible to detect an enzyme reaction control property between an enzyme and an activity control agent using a flow injection analysis method. It is an object of the present invention to provide a flow injection analysis apparatus and a flow injection analysis method.

  The inventors of the present invention have made extensive studies in view of the above problems. As a result, a specific enzyme is supported on a carrier, and a sample and an activity control agent are alternately injected at predetermined intervals using a flow injection method. Thus, the inventors have found that it is possible to measure the properties between the enzyme and the activity control agent, and have reached the present invention.

  That is, the present invention introduces a sample to a column containing an immobilized enzyme carrying a specific enzyme. Thereafter, after a predetermined period when the peak due to the sample returns to the baseline, the inhibitory component, the inhibitory component or the activation component is injected. Further, after the predetermined period, the sample is injected again, and the peak due to the sample is detected. It was found that the peak obtained at this time changes the peak height corresponding to the chemical reactivity between the enzyme and the substrate with respect to the immobilized enzyme of the inhibitory component. That is, the present inventors show that the lower the peak height due to the injected substrate after the injection of the inhibitory component, etc., the inhibitory component can exist in a region where the enzyme-inhibitory component reaction can occur over a long period of time, In addition, when the peak height is high, the inhibitory component cannot be present in the region causing the enzyme-inhibitory component reaction, indicating that the enzyme activity is recovered early, and in the case of the activation component, the enzyme activity As a result, the present inventors have found that the formation can be detected.

That is, according to the present invention, a column that holds the immobilized enzyme and allows the sample to pass along with the transport medium;
An activity control component that controls the reaction activity between the sample and the immobilized enzyme in the transport medium, a first sample injection, an injection of the activity control component, a second sample injection, and an activity control component An injection device that injects alternately as a third sample injection after the time to measure the effect of the residue ;
A detection device for detecting a reaction under control with the immobilized enzyme corresponding to the second sample injection and the third sample injection, which is performed later than the activity control component;
A flow injection analyzer comprising: a system controller that calculates an activity characteristic of the activity control component by the second sample injection, and calculates a recoverability of the immobilized enzyme by the third sample injection. Can be provided.

  The sample in the present invention may be selected from the group comprising a whitening agent, an anti-pylori agent, an antisterolemia therapeutic agent, an anti-Utsu agent, an anti-HIV agent, an antifungal agent, and a diabetes therapeutic agent (insulin-independent). it can. The enzyme is an oxidoreductase, and the detection device can detect a dissolved oxygen concentration. The enzyme may be tyrosinase and the sample may be tyrosine.

According to the present invention, the activity control component for controlling the reaction activity between the sample in the transport medium and the immobilized enzyme held in the column by the injection device is used as the first sample injection, the activity control component, the second Alternately injecting as a third sample injection after a sample injection and a time to measure the effect of residual activity control component ;
Detecting a reaction under control with the immobilized enzyme corresponding to the second sample injection and the third sample injection performed by the detection device with a time delay from the activity control component;
Calculating an activity characteristic of the activity control component from the second sample injection by a system controller and calculating recoverability of the immobilized enzyme from the third sample injection;
A flow injection analysis method can be provided.

The step of alternately injecting the sample and the activity control component of the present invention includes the step of performing the second sample injection at the same concentration and in the same amount as the first sample injection. The time interval from the injection to the second sample injection can be 3 to 20 times the peak retention time of the sample. Further, the calculation of the activity characteristic is performed by calculating a ratio between a peak due to the first sample injection performed before the injection of the activity control component and a peak of the second sample injection injected after the injection of the activity control component. Can be used to calculate activity characteristics. The sample can be selected from the group comprising a whitening agent, an anti-pylori agent, an anti-sterolemia therapeutic agent, an anti-Utsu agent, an anti-HIV agent, an antifungal agent, and a diabetes therapeutic agent (insulin-independent). The enzyme is an oxidoreductase, and the detection device can detect a dissolved oxygen concentration. Tyrosinase can be used as the immobilized enzyme, and a drug containing tyrosine can be injected as the sample. The activity characteristic may be enzyme inhibition or enzyme activation, and the recoverability may be the recoverability of the immobilized enzyme from the influence of the activity control component.

  According to the present invention, it is possible to evaluate the activity control characteristics of an activity control agent and an enzyme in a substrate-enzyme reaction using flow injection analysis, enabling evaluation of an inhibitory component and an activation component, and a novel A novel flow injection analysis apparatus and a flow injection analysis method capable of providing novel information for drug design including various drug delivery methods can be provided.

  The present invention will be described below with reference to specific embodiments shown in the drawings, but the present invention is not limited to the embodiments described below.

FIG. 1 is a diagram showing a flow injection analyzer of the present invention (hereinafter simply referred to as an analyzer). The analyzer 10 of the present invention has a configuration generally similar to that of a high performance liquid chromatograph, and includes a liquid feed pump 12, a damper 14, injection valves 16 and 18, and a column 20. A buffer solution stored in a carrier reservoir 22 is supplied to the liquid feed pump 12. Any buffer known so far can be used as the buffer, for example, a 100 mM Tris-HCl buffer can be used. Furthermore, in the present invention, depending on the type of substrate used and the inhibitory or activating component, for example, MOPS-EDTA-sodium acetate buffer, acetic acid-sodium acetate buffer, 100 mM K-PO ₄ , 0.1 mM EDTA In addition to (pH 7.4), Tris-Ac buffer, Tris-Ac-ethanolamine buffer, a buffer contained in any commercially available buffer kit for standard amino acid analysis can be used. The buffer solution is generally sent as a carrier at a flow rate of 1.0 ml / min. The damper 14 is used for preventing the pulsating flow of the carrier sent by the liquid feeding pump 12, and any commercially available damper 14 can be used. In the specific embodiment of the present invention, the injection valves 16 and 18 may be a rotary valve with a loop, and an injection operation using a microsyringe may be possible. In another embodiment of the present invention, a programmed sample injection can be performed using an autosampler, as described below, instead of an injection valve.

  The column 20 holds an immobilized enzyme composed of an enzyme supported on a carrier, and biochemically interacts with the substrate and the inhibitory components injected from the injection valves 16 and 18 so that the amount of the substrate is dissolved. The amount of the substance to be detected such as oxygen is changed, and the substrate and the substance to be detected are moved to the detection device 22 together with the carrier. Examples of the enzyme that can be used in the present invention include tyrosinase, urease, HMG-CoA reductase, monoamine oxidase, reverse transcriptase, HIV protease, ergosterol synthase, glucan synthase, α-glucoxidase, maltose phosphorylase, alkaline phosphatase. , Amylase, protease and the like. However, in the present invention, any enzyme other than those described above with an appropriate inhibitory component or an enzyme with an appropriate enzyme activity evaluation method can be used without particular limitation. Examples of samples to which the present invention is applied include whitening agents, anti-pylori agents, anti-sterolemia therapeutic agents, anti-Utsu agents, anti-HIV agents, anti-fungal agents, and diabetes therapeutic agents (insulin-independent). However, like the enzyme described above, the present invention is not particularly limited.

  Furthermore, the components that can be used as an inhibitory component and an activating component in the present invention include kojic acid, ellagic acid, brabastadine, simvastatin, phenelzine, tranylcypromine, isocarboxazide, AZT (zidovudine), viramune, efavirenz, ritonavir, Examples include miconazole, itraquinazole, fluconazole, Micafungin, Caspofungin, acarbose, etc., but the present invention is not limited to these inhibitory components and activation components, and components obtained from herbal extracts and the like It can also be used.

  As described above, the carrier discharged from the column 20 sends the substance to be detected to the detection device 22 as the carrier moves, and the detection device 22 detects the detection target. In the present invention, the enzyme reaction can be detected directly by detecting the substrate or indirectly by detecting a specific component such as oxygen consumed in the substrate-enzyme reaction. Examples of the detection device that can be used in the present invention include a UV-VIS absorption spectrophotometer, a fluorescence spectrophotometer, a potentio / galvanostat, and an oxygen electrode. A recorder 24 is connected to the detection device 22 through an appropriate amplification device (not shown), and the voltage or current signal from the detection device 22 is plotted against time.

  In the more systemized embodiment of the present invention, the output of the recorder is sent to the system control unit 26 and displayed as digital-converted time-output data on a computer liquid crystal display, CRT, plasma display or the like. It can be set as the structure which can be represented to an apparatus. Furthermore, in the present invention, the operator can directly drive the analyzer and execute the analysis sequence of the present invention. In another embodiment of the present invention, a program for executing the analysis sequence of the present invention is included in the system controller 26, and the system controller 26 controls the autosampler 28 to automate the analysis sequence. You can also

  FIG. 2 is a partial cross-sectional view of a column 20 holding an immobilized enzyme that can be used in the present invention. The column 20 shown in FIG. 2 generally includes a thermostatic container 30 and an immobilized enzyme package 32 held in the thermostatic container 30. In the thermostatic container 30, water held at a constant temperature (about 30 ° C.) is circulated through the inlet 34 and the outlet 36, and the immobilized enzyme in the immobilized enzyme package 32 in the thermostatic container 30 is kept constant. The temperature is maintained. Further, the immobilized enzyme package 32 held in the thermostatic container 30 is packed fluidly tightly so that water does not leak and the carrier does not leak. More specifically, the immobilized enzyme package 32 is formed of an upstream carrier liquid passing portion 32a and a downstream filling portion 32b, and a filter 32c is provided upstream and downstream of the filling portion 32b. The carrier is prevented from flowing out.

  Inside the filling portion 32b, an enzyme supported on a carrier is filled together with the carrier, and can biochemically act on a substrate and an inhibitory component contained in the carrier supplied from the upstream side. Further, as shown in FIG. 2, the upstream of the constant temperature container 30 and the downstream side of the constant temperature container 30 prevent leakage of the constant temperature water, and the inflowing carrier passes through the immobilized enzyme package 32 and is discharged from the constant temperature container 30. As shown, packing structures or connectors 38a, 38b are provided upstream and downstream.

  The commercially available thing can be used for the immobilized enzyme which can be used in this invention, when a commercially available thing can be utilized. Further, the above-described immobilization treatment can be performed by immobilizing a specific enzyme on a carrier, and various immobilization methods can be used for immobilization of the enzyme. For example, as a method for immobilizing an enzyme, for example, a crosslinking method using glutaraldehyde or the like, a physical adsorption method using hydroxyapatite or the like, an ion binding method using an ion exchange resin, or a diazonium salt is used. Any immobilization method known in the art so far, such as a covalent bond method and a comprehensive method using alginic acid or a membrane, can be used and is not limited to a specific method.

  Further, in the present invention, various carriers such as silica, alumina, diatomaceous earth, and glass can be used as the carrier on which the enzyme is immobilized. Synthesized microporous glass can be used. The particle diameter of the carrier described above can be in the range of 100 mm to several mm, and the pore size can be in the range of about 10 nm to 200 nm.

  As the above-mentioned carrier, in addition to glass, electrodes made of platinum or gold that can be used for immobilization of enzymes, agar, reverse micelles, etc., corresponding to specific applications such as enzymes and use conditions, etc. Can be used. Although various treatments are possible when the enzyme is immobilized on the carrier, a predetermined amount of the carrier is treated with a silane coupling agent, a titanium coupling agent, or other metal-organic coupling agent to bind to an organic substance. A method in which an enzyme solution prepared using a buffer solution is added to a carrier and dried can be preferably used.

FIG. 3 is a diagram showing the analysis sequence in the present invention as a flowchart in the case of specifying the activity control characteristic between the enzyme and the inhibitory component. Note that the same analysis sequence can be used to measure the activation component or the activity characteristic of the activation component. Analysis sequence of the present invention starts from step S100, in step S102, in response to T ₀ after the analysis sequence is started has elapsed, in step S104, the first substrate injection are performed. This first substrate injection is an operation for giving a reference substrate peak height (A) in the analysis sequence. Next, in step S106, the analysis sequence proceeds to step S108 in response to reaching the time (T ₁ ) for injecting the inhibitory component, and injection of the inhibitory component is performed. After injection of the inhibitory components in step S108, in step S110, in response to whether reaches the time T _2, after a predetermined time interval (T _d), a second substrate injection row in step S112 Is called. This second substrate injection is the inhibition rate probe injection (B) for giving the inhibition rate of the inhibition component after _Td .

Thereafter, the time in the analysis sequence in step S114 is in response to a determination whether it is T _3, a third substrate injection is performed in step S116. This third substrate injection is a recovery probe injection (C) for measuring how much the influence of the inhibitory component remains. Thereafter, in step S118, the analysis sequence proceeds to step S120 in response to the determination that the sequence timeout time, which is the time at which the series of analysis is completed, and ends the analysis sequence. Note that the analysis sequence in FIG. 3 can be performed by an analysis person who determines the start of analysis and arrival of each time and controls the analysis apparatus. In the present invention, the analysis sequence includes a program for executing the analysis sequence. An analysis sequence can be automatically executed using a system controller and an autosampler.

FIG. 4 shows the data obtained according to the analysis sequence for evaluating the inhibitory activity shown in FIG. 3, the retention time is shown on the horizontal axis, and the vertical axis is the peak height (output current value of the oxygen electrode), so-called It is the figure shown as a chromatogram. First, at time T ₀ , a first substrate injection is performed, and a peak A is detected after a predetermined retention time (T _r ). In the present invention, the time from the injection of a component to the detection of the corresponding peak is referred to as the peak retention time (T _r ). Then, inhibitory components or activated components at time T ₁ is injected. Further, after that, a second substrate injection is performed after a predetermined delay time _Td , and the concentration of the detection target substance is observed as a peak B having an intensity different from that when no inhibitory component exists due to enzyme inhibition by the inhibitory component. . Moreover, when not an inhibitor but an activator is inject | poured, it will be observed as peak height shown with the broken line Ba with activation of enzyme activity. The chart shown in FIG. 4 gives a high peak (shallow dip) when the enzyme activity is high and a detection device and a detection substance so as to give a low peak (deep dip) when the enzyme activity is low. Corresponds to the selected embodiment. More specifically, the detection target is an oxygen molecule, an oxygen electrode is used as a detection device, and oxygen consumption by an enzyme reaction is reduced, that is, when enzyme activity is reduced and dissolved oxygen is increased, a relatively low peak is obtained. Is set to give.

Thereafter, a third substrate injection at T ₃ is performed, and a corresponding peak is observed as peak C. The intensity of the peak C is observed as a higher peak as the biochemical bond between the enzyme and the inhibitory component is weaker, and as the biochemical bond between the enzyme and the inhibitory component is higher as a smaller peak. When the above-mentioned peak intensities are respectively A, B, and C, the inhibition rate (Inhibition Ability) and the recovery rate (Reactivation Ratio) are given by the following equations (1) and (2), respectively.

ディレイタイムＴ_ｄは、活性の度合いおよび活性を制御する機序により応じて、例えば酵素および阻害成分の組み合わせにより適宜設定することができるものの、本発明の分析装置における基質ピークの検出までの時間（リテンションタイムＴ_ｒ）がインジェクションから数１０秒程度であることから、５ｍｉｎ〜３０ｍｉｎ程度の時間とすることができる。また、賦活成分または賦活成分の添加により酵素活性が高められる場合には、賦活率を、例えば賦活率＝(Ba/A-1) × 100により得ることができる。また、第２の基質インジェクションから、第３の基質インジェクションまでの時間間隔は、特に限定されるものではないが、分析シーケンスの全体の長さを考慮して、T_d程度とすることができる。

The delay time _Td can be set as appropriate depending on the degree of activity and the mechanism for controlling the activity, for example, by a combination of an enzyme and an inhibitory component, but the time until the detection of the substrate peak in the analyzer of the present invention ( Since the retention time T _r ) is about several tens of seconds from the injection, the time can be set to about 5 to 30 minutes. When the enzyme activity is increased by adding an activation component or an activation component, the activation rate can be obtained by, for example, activation rate = (Ba / A-1) × 100. The time interval from the second substrate injection to the third substrate injection is not particularly limited, but can be set to about _{Td in} consideration of the entire length of the analysis sequence.

In the present invention, it is preferable to set the alternate injection in the range of T _d / T _r = 3 to 20, where T _r is the retention time of the sample and T _d is the delay time. If the value of T _d / T _r is smaller than 3, it tends to be subject to baseline fluctuations such as air due to the previous sample injection, and tends to reduce the measurement accuracy. The value of T _d / T _r is set to 20 If it exceeds, the inhibitory component that is mainly competitive inhibition does not show sufficient inhibition, and the recovery rate value tends to decrease in accuracy. Particularly in certain embodiments of the invention, the value of T _d / T _r is in the range of about 4.5 to about 18 and is good for both inhibitory rate and recovery rate for many inhibitory components. It was found that results were obtained. FIG. 5 is a graph plotting the dependency of the inhibition rate on the delay time _Td in an embodiment in which tyrosinase is used as the enzyme and kojic acid is used as the inhibitory component. (2) indicates the case where the inhibitory component concentration is 0.5 mg / ml, and (2) indicates the inhibition rate (%) obtained when the inhibitory component concentration is 5 mg / l.

As shown in FIG. 5, it is shown that the inhibition rate decreases as the delay time _Td increases. This is because the inhibitory component flows out over time and does not exist near the enzyme. Further, when the concentration of the inhibitory component was high, there was a tendency that even when the delay time _Td was increased, the inhibition rate decreased, that is, the recovery of enzyme activity was saturated. This is thought to suggest that the inhibitory component may remain bound to the enzyme to some extent. That is, in the conventional batch method for measuring the inhibition rate, the inhibitory component is always present at a position where it can act biochemically on the enzyme. In addition to the inhibition reaction, the influence of competitive inhibition is also added, and it is considered that the inhibition rate is higher than actual. Therefore, in the present invention, even when the above-described competitive inhibition is the main or irreversible inhibition reaction, these are separated and accurately evaluated for competitive inhibition / irreversible inhibition of the enzyme-inhibiting component. It can be shown that Similarly, it is possible to evaluate the reaction mechanism of the enzyme-activating component.

  FIG. 6 is a diagram showing a system and software configuration of the analyzer according to the present invention. The analyzer 40 of the present invention shown in FIG. 6 includes an autosampler 42 and a column holder (or column oven) 44 that holds the immobilized enzyme at a constant temperature and holds the column. The autosampler 42 is controlled by a system control device 46 including a computer, which will be described later. When an analysis sequence is started by a command from the system control device 46, the substrate and the inhibitory component or activation are based on the command from the system control device 46. Perform component injection. Thereafter, when the substance to be detected reaches the detection device by the carrier, the detection device changes the output signal to enable data acquisition.

  Further, the system control unit 46 receives A / D converted digital data from the detection unit in response to the injection of the substrate and the inhibitory component by the autosampler 42, and stores it in a memory or the like in association with the sampling channel. Then, the peak search described above and the values of the inhibition rate and recovery rate are calculated. Further, in the analysis apparatus shown in FIG. 6, when the analysis sequence is completed, the time-peak intensity chart of the analysis sequence is displayed on the display device 46a with the sampling channel as the horizontal axis and the peak height as the vertical axis. be able to. In the embodiment shown in FIG. 6, a time-peak height graph, an inhibition rate, and a recovery rate are displayed on the display device 46a.

  The A / D conversion of the detection signal is performed based on a command from the system control device 46, and when there is sufficient space in the auto sampler 42 or the column holder 44, the A / D included in the auto sampler 42 or the column holder 44 is used. It may be executed and stored by a converter / memory, or the analog output from the detection device may be directly A / D converted by the system controller 46 and stored in the memory.

  Furthermore, the system control device 46 shown in FIG. 6 of the present invention includes a program for causing the analysis device to execute the analysis sequence of the present invention, and responds to input from a keyboard, mouse, stylus pen, or the like. The initial value setting, analysis start / end, and data analysis are enabled. Furthermore, in another system embodiment of the present invention, a plurality of enzyme columns may be prepared, evaluated in parallel, and collectively processed by the system control device 46, or multiple wells may be configured. Using a microtiter plate (for example, 96 wells), the enzyme is immobilized in the wells, and a condition similar to the carrier flow by the liquid feeding pump is generated by injecting and sucking the solution and reagent by the system controller 46. In addition, a large number of samples can be processed.

  Hereinafter, the analysis apparatus and the analysis method of the present invention will be described in more detail with examples applied to actual enzyme-inhibitory component systems and enzyme-activating component systems. The analysis apparatus and analysis method of the present invention were applied to a tyrosine-tyrosinase system, which is known to generate melanin in vivo, and the effect was examined.

  FIG. 7 shows a reaction mechanism in which melanin is formed from tyrosine in an in vivo reaction. As shown in FIG. 7, tyrosine in the living body is oxidized by tyrosinase in pigment cells existing in the vicinity of the skin tissue surface to generate L-3,4-dihydroxyphenylalanine (L-DOPA). The produced L-DOPA is further oxidized by tyrosinase to produce an intermediate product, dopaquinone. The produced dopaquinone is finally accumulated as melanin through a plurality of intermediate products such as leucodopachrome, dopachrome, 5,6-hydroxyindole, and indole-5,6-quinone. In FIG. 7, a compound surrounded by a thick frame is a substance to be detected that is detected by a batch method in many cases. Melanin is a causative substance that causes skin darkening such as sunburn or spots, and its production involves the substrate-enzyme reaction of tyrosine-tyrosinase. For this reason, it is known that an enzyme-inhibiting component that prevents the generation / accumulation of melanin is effective, and in view of the fact that it is used as a whitening agent, the effects of the analyzer and the analysis method of the present invention are verified. It is considered to be the most suitable biochemical reaction system.

A. (1) As a substrate substrate, commercially available L-tyrosine (manufactured by Sigma-Aldrich Japan Co., Ltd.) was used.
(2) Tyrosinase (derived from mushrooms, Sigma-Aldrich Japan, EC: 1.14.18.1) was used as the enzyme enzyme. The optimum pH is 6-7.
(3) As an enzyme immobilization treatment carrier, porous glass (CPG-10, mesh 200/400, pore diameter 24.2 nm, surface area 88.1 m ² / g, Funakoshi Chemical Co., Ltd.) was used. First, 3 g of microporous glass was weighed and 10% γ-aminopropyltriethoxysilane (γ-APTES) adjusted to pH 3.45 was added and reacted at 75 ° C. for 3 hours to fix the carrier. Alkylaminated. Thereafter, a 2.5% glutaraldehyde solution prepared using 10 mM phosphate buffer (pH 7.0) was added as a cross-linking agent to 1.0 g of the alkylaminated carrier, followed by drying. I let you. Thereafter, a tyrosinase solution prepared at 10 mg / ml on a 10 mM phosphate buffer was added for immobilization. Thereafter, the carrier on which the enzyme was immobilized was treated with a reducing agent (NaBH ₄ ).
(4) Creation of column The enzyme reduced after the immobilization was filled in an immobilized enzyme holder, and the immobilized enzyme holder was set in a thermostatic container to produce a column described in detail in FIG. The constant temperature container was maintained at about 20 ° C. by circulating constant temperature water.
(5) The liquid feeding system and the detection system analyzing apparatus were prepared by modifying a commercially available HPLC apparatus (liquid feeding pump PU-1580i) manufactured by JASCO Corporation so that the analysis method of the present invention can be applied. Specifically, a commercially available oxygen electrode (BO-P, manufactured by Biot Co., Ltd.) was used as the detection device, and was kept at room temperature. The output from the oxygen electrode is detected by an ammeter (HOKUTODENKO HA-104, manufactured by Hokuto Denko Co., Ltd.). The detected current is converted into a voltage, and a recorder (Rika Electric Co., Ltd., MULTI-PEN RECORDER (R-62MB) is used. )). The feeding conditions were a flow rate of 1 ml / min, a sample injection amount of 0.1 ml, and 50 mM phosphate buffer + 1 M NaCl (pH = 6.5) as a carrier.

B. Measurement of detection linearity of analyzer The sensitivity characteristics of the analyzer including the detector were examined using the analyzer described above. FIG. 8 shows a plot of the sensitivity characteristics of the device obtained according to the present invention with the substrate (tyrosine) concentration on the horizontal axis and the output current from the oxygen electrode on the vertical axis. As shown in FIG. 8, for the tyrosine substrate, linearity was confirmed in the range of approximately 0 to 2 mM. For this reason, in the present invention, the sample injection concentration was set to 1 mM, and examination was performed in a range where correction for the peak intensity due to the tyrosine concentration is not necessary.

C. Adjustment of inhibitory component As the inhibitory component, kojic acid (manufactured by Wako Pure Chemical Industries, Ltd.), which is known as an inhibitor, was used a herbal extract. Extraction of the medicinal component from the crude drug was performed by weighing 3 g of the crude drug and extracting the component with 30 ml of 50% ethanol solution for 2 days and then removing the ethanol by distillation under reduced pressure. Thereafter, freeze drying was performed to obtain a herbal extract as a powder. The obtained crude drug extract powder was dissolved in dimethyl sulfoxide and then adjusted with a carrier so as to have predetermined final concentrations (0.5 mg / ml and 5 mg / ml) to prepare an inhibitor component solution (dimethyl sulfoxide). The final concentration is 0.5% or less). FIG. 9 lists the inhibitory components used.

D. Measurement of inhibition rate and recovery rate (Experimental Example 1)
After the liquid feeding pump and oxygen electrode of the analyzer described in Section A were started and the baseline of the chart recorder was stabilized, 0.1 ml of 1 mM tyrosine was injected with a microsyringe. This was the first substrate injection. Thereafter, when the tail of the peak of the first substrate injection was stabilized, 0.1 ml of an inhibitory component of 0.5 mg / ml was injected with a microsyringe. After 6 min (T _d / T _r = 12) of this inhibitor component injection, the second substrate injection was performed under the same conditions as the first substrate injection. Further, after about 6 min, the third substrate injection was performed in the same manner.

FIG. 10 shows the obtained chromatogram. FIG. 10 shows calculation criteria for the values of T _r and T _d in the present invention. T _r is the time from the substrate injection until the peak of the substrate is observed, and in the embodiment shown in FIG. 10, T _r = 56 sec. The peak of the substrate ends at time T _end ≈3 min when the peak returns to the baseline. _Td can be defined as the interval from the injection of the activity control component to the second substrate injection, and the time interval until the third substrate injection can be obtained in the same manner.

  Three peaks A, B, and C shown in FIGS. 4 and 10 were obtained by the above-described four alternate injections. The inhibition rate and the recovery rate were calculated according to the formula (1) and the formula (2) using the peak height of the obtained peak.

(Experimental example 2)
Further, the inhibition rate and the recovery rate were calculated by conducting the same experiment as in Experiment 1 with the concentration of the inhibitory component being 5 mg / ml.

  The result is shown in FIG. In FIG. 11, the inhibition activity is judged using the inhibition rate, and the inhibition component having a high inhibition rate is indicated by ++, and hereinafter, the inhibition rate is indicated as ++, +, and − as the inhibition rate decreases. Further, the recovery rate was judged using the recovery rate, and indicated as ++ to ++, +, and − in order from the inhibitory component having the lowest recovery rate. Therefore, it can be said that the sum of the numbers of + is a component having high inhibitory activity including competitive inhibition. Table 1 shows the results of classifying the inhibitory components as A group to E group in descending order of the sum of the above +.

  As shown in Table 1, according to the present invention, crude drugs classified into Group A and Group B in Table 1 such as amacha and licorice powder are identified as inhibitory components having higher inhibitory activity against tyrosinase than kojic acid. We were able to. In addition, referring to FIG. 11, kojic acid, which is well known as an inhibitor, has high inhibitory activity but high recoverability. This indicates that the herbal extract components and kojic acid have different tyrosinase inhibition mechanisms. For this reason, kojic acid was classified into Group C in Table 1. The reason for this is that kojic acid loses its effect immediately, although it shows a high inhibitory ability immediately after administration. From this, it was shown that competitive inhibition is the main inhibition mechanism for inhibition of kojic acid on tyrosinase.

E. Comparative Experiment As a comparative experiment, the inhibitory activity against tyrosinase was measured by a batch test by coexisting with a solution obtained by mixing kojic acid and tyrosine with a phosphate buffer solution and monitoring dissolved oxygen using an oxygen electrode. The inhibition rate at this time is defined as (1−B / A) × 100, where A is the oxygen consumption amount in the absence of the inhibitory component, and B (A> B) is the oxygen consumption amount in the presence of the inhibitory component. Calculated as Tyrosinase was immobilized on an aminated PAN membrane (manufactured by Sakura Seiki Co., Ltd.) by a glutaraldehyde crosslinking method. The final concentration of tyrosine is 1 mM, and the concentration of kojic acid is 5 mg / ml, 0.5 mg / ml, 0.05 mg / ml, 5 × 10 ⁻³ mg / ml, 5 × 10 ⁻⁴ mg. / Ml. The results are shown in Table 2.

  As shown in Table 2, the batch test showed that the inhibition rate of kojic acid had a 100% inhibition rate even in a considerably low concentration region. On the other hand, in the result (flow (FIA)) obtained by the analyzer of the present invention, the inhibition rate of kojic acid was obtained as 77.5 (5 mg / ml) and 41.2 (0.5 mg / ml). It was shown that a sufficient inhibition rate was not obtained at a concentration lower than that.

  Although this result shows that kojic acid shows a high inhibition rate as shown in FIG. 11, considering the large recovery rate, the effect of competitive inhibition is overestimated in the batch test under conditions where competitive inhibition exists. It shows that it has been done. In addition, although the sample which shows activation activity was not obtained in the crude drug ingredient measured this time, if the same analysis sequence is used, it is possible for those skilled in the art to evaluate the activation activity of the enzyme-substrate reaction. It can be easily understood.

  As described above, according to the present invention, a novel flow injection analyzer and flow for elucidating the activity characteristics of the activity control component for the enzyme-substrate reaction, specifically, inhibition, recovery, activation, etc. An injection analysis method can be provided, which enables screening of new inhibitory components, generation of novel pharmaceutical compositions, and construction of drug delivery systems.

1 is a diagram showing a flow injection analyzer of the present invention (hereinafter simply referred to as an analyzer). The figure which showed the column 20 filled with the immobilized enzyme which can be used in this invention as a partial cross section. The figure which showed the analysis sequence in this invention as a flowchart. The figure which showed the data obtained according to the analysis sequence shown in FIG. 3 as what is called a chromatogram which showed the retention time on the horizontal axis. The figure which plotted delay time _Td dependence of the inhibition rate in embodiment which uses tyrosinase as an enzyme and made kojic acid an inhibitory component. The front view which showed the system configuration | structure of the analyzer of this invention. The figure which showed the reaction mechanism in which melanin is formed from tyrosine in in vivo reaction. The sensitivity characteristics of the device obtained according to the present invention are plotted with the substrate (tyrosine) concentration on the horizontal axis and the output current from the oxygen electrode on the vertical axis. The figure which showed the inhibitory component (herbal medicine extract and kojic acid) used in this invention. The figure which showed the chromatogram and each time reference | standard in the tyrosine-tyrosinase system obtained in this invention. The figure which showed the inhibition rate and recovery rate which were obtained by the flow injection analysis method of this invention. Schematic which showed the measuring method of the inhibition rate by the conventional batch test.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Analytical apparatus, 12 ... Liquid feed pump, 14 ... Damper, 16, 18 ... Injection valve, 20 ... Column, 22 ... Detection apparatus, 24 ... Recorder, 26 ... System controller, 28 ... Autosampler, 30 ... Constant temperature Container, 32 ... Immobilized enzyme package, 34 ... Constant temperature water inlet, 36 ... Constant temperature water outlet, 38a, 38b ... Packing structure, 40 ... Analyzer, 42 ... Autosampler, 44 ... Column holder, 46 ... System controller

Claims (11)

A column that holds the immobilized enzyme and through which the sample is passed along with the transport medium;
An activity control component that controls the reaction activity between the sample and the immobilized enzyme in the transport medium, a first sample injection, an injection of the activity control component, a second sample injection, and an activity control component An injection device that injects alternately as a third sample injection after the time to measure the effect of the residue ;
A detection device for detecting a reaction under control with the immobilized enzyme corresponding to the second sample injection and the third sample injection, which is performed later than the activity control component;
A flow injection analyzer comprising: a system controller that calculates an activity characteristic of the activity control component by the second sample injection and calculates a recoverability of the immobilized enzyme by the third sample injection .   The sample is selected from the group comprising a whitening agent, an anti-pylori agent, an anti-sterolemia therapeutic agent, an anti-Utsu agent, an anti-HIV agent, an antifungal agent, and a diabetes therapeutic agent (insulin-independent). The analyzer described in 1.   The analyzer according to claim 1, wherein the enzyme is an oxidoreductase, and the detection device detects a dissolved oxygen concentration.   The analyzer according to claim 3, wherein the enzyme is tyrosinase and the sample is tyrosine. An activity control component that controls the reaction activity between the sample in the transport medium and the immobilized enzyme held in the column by the injection device, the first sample injection, the activity control component, the second sample injection, and the activity Alternately injecting as a third sample injection after the time to measure the effect of residual control component ;
Detecting a reaction under control with the immobilized enzyme corresponding to the second sample injection and the third sample injection performed by the detection device with a time delay from the activity control component;
Calculating the activity characteristic of the activity control component from the second sample injection by a system controller and calculating the recoverability of the immobilized enzyme from the third sample injection . The step of alternately injecting the sample and the activity control component includes the step of performing the second sample injection at the same concentration and in the same amount as the first sample injection, and after the injection of the activity control component The analysis method according to claim 5, wherein a time interval until the second sample injection is 3 to 20 times a peak retention time of the sample. Further, the calculation of the activity characteristic is performed by calculating a ratio between a peak due to the first sample injection performed before the injection of the activity control component and a peak of the second sample injection injected after the injection of the activity control component. The analysis method according to claim 5, comprising calculating an activity characteristic using   The sample is selected from the group comprising a whitening agent, an anti-pylori agent, an anti-sterolemia therapeutic agent, an anti-Utsu agent, an anti-HIV agent, an anti-fungal agent, and a diabetes therapeutic agent (insulin-independent). The analysis method described in 1.   The analysis method according to claim 6, wherein the enzyme is an oxidoreductase, and the detection device detects a dissolved oxygen concentration.   The flow injection analysis method according to claim 9, wherein tyrosinase is used as the immobilized enzyme, and a drug containing tyrosine is injected as the sample. The analysis method according to claim 7, wherein the activity characteristic is enzyme inhibition or enzyme activation, and the recoverability is recoverability of the immobilized enzyme from the influence of the activity control component .

Images