TWI226927B - Herbal chip - Google Patents

Abstract

The present invention relates to an herbal chip comprising a plastic slide, a coating as a spacer on the plastic slide, and fractions or components obtained from herbs that are independently allocated in microarrays on the coating. The herbal chip is useful for screening for active ingredients contained in the herbs that have specific pharmacological or therapeutical functions.

Description

1226927 A7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs B7 V. Description of the Invention (1) Technical Field The present invention relates to the arrangement of herbal graded liquids or ingredients in a microarray on a plastic substrate. The plastic matrix with herbal grade liquid or ingredients is used as a platform for screening active ingredients with specific pharmacological or therapeutic effects in herbs. Background of the invention The use of herbs or their extracts or fractions for filling medicines has been known for many years. For example, the bark of willow has been used as an antipyretic and analgesic for 2000 years' and bark extract of Cinchona, Peru has been used since the 17th century to treat malaria. However, to date, only a few herbal active ingredients have been found to have specific pharmacological or therapeutic effects. For example, in the mid-20th century AD, salicypid (a salicyl alcohol glucoside) was identified as an active ingredient with antipyretic and analgesic activity in the bark of willows, and quinine was identified in Peru in 1802 Cinchona bark has active ingredients in the treatment of malaria. The discovery of active ingredients with specific pharmacological or therapeutic effects from herbs is time consuming and labor intensive. For example, after collecting more than 35,000 plant genera from all over the world and screening more than 110,000 samples, the antitumor drug taxol (taxol) was isolated from the bark of Taxus brevifolia. ). Generally, the general pattern of finding the active ingredients present in herbal medicines is to confirm the efficacy of the herbal medicines in the human body, and to use various physical and chemical methods to separate the active fractions of herbal medicines, and then isolate and purify them. Active ingredient. Isolate and purify herbs 7 paper sizes are applicable to Chinese National Standard (CNS) A4 specifications (210 X 297 public love) _ 4-(Please read the precautions on the back before filling this page)

Loading n n .f, n n -ϋ 1 an I n. 1226927 A7 B7 5. There are no general rules for the active ingredients contained in the description of the invention (2). Although based on pharmacology, the development of cell biology and molecular biology has made significant progress in understanding the cause of disease at the molecular or genetic level, and the technology of targetgun screening of herbal extracts has been developed with a view to It can directly hit to get the active ingredients that are in the herbal medicine, but isolating and purifying the active ingredients in the herbal medicine is still a labor-intensive and trial-and-error job, and its progress is still slow. This technology has a significant need to develop a novel method and / or tool for the rapid screening of active ingredients with specific pharmacological activity or therapeutic efficacy from herbs or their extracts or fractions. Techniques for immobilizing large biomolecules on a solid substrate (eg, nylon membrane) have been used in this technique, such as western blotting systems for immobilizing peptides or proteins, southern blotting systems for immobilizing DNA fragments, and The northern blotting method is used to immobilize RNA fragments and cause the immobilized molecules to react or react with the labeled probes, and then develop the resulting probe-labeled molecules. For example, the ligase immunosorbent assay (ELISA) involves immobilizing large biomolecules on a substrate, allowing the immobilized molecules to react or react with a marker, and subsequently coloring and developing the resulting labeled molecules. The E L I S A can be performed on a conventional 96-well microplate. Recently, High-density gndding technology has been used in this technology to detect whether there is a desired target in a biological sample (eg, DNA or protein), where the sample is pre-determined The arrangement is fixed on a solid substrate (eg, a glass slide) and hybridized with a labeled probe, followed by washing and development.丨 Loading (Please read the precautions on the back before filling this page)

i_l nn .1 J 、 · nnn ammmm fn an-The printed paper size of the employee's consumer cooperative of the Intellectual Property Bureau of the Ministry of Economy applies the Chinese National Standard (CNS) A4 (210 X 297 mm) -5- 1226927 Α7 Β7 Ministry of Economy Intellectual Property Bureau employee clothing cooperative printed clothes i. Description of invention (3) When the high-density column technology is applied, the biological sample is a homogeneous known DNA or protein library, and the labeled probe is not Homogeneous unknown or unidentified substance. The advantages of this high-density grid technology are that a very small volume of sample can be fixed to a small grid area on the surface of the substrate, and thousands of samples can be operated simultaneously. Further, a computer controlled 3-degree space robotic device and a single tip assembly (ie, a microarrayer) can be used to generate biological samples in a high-density array on the surface of the substrate. However, to date this high-density column technique has been used only for the analysis of large molecules, such as proteins and nucleic acids, where the sample immobilized on the surface of a solid substrate (for example, a glass slide) is homogeneous. This technique does not teach or suggest that heterogeneous samples containing large bioactive molecules (such as proteins or nucleic acids) or small bioactive molecules (such as secondary metabolites) can be immobilized on a solid substrate (eg, plastic slides) This technique also does not teach or suggest that homogeneous or heterogeneous samples of herbs or their extracts or fractions can be fixed on the surface of a solid substrate (eg, a plastic slide), especially if the sample is Microarrays are arranged. Further, this technique does not teach or suggest that by applying the high-density grid technology, the homogeneous or heterogeneous unknown sample disposed on the surface of a solid substrate (such as a 'plastic slide') and Homogeneous or heterogeneous known labeled probe action or reaction can be used for high throughput screening of biologically active molecules in herbs. Based on this, the present invention discloses a method using homogeneous or heterogeneous Novel platform for labeling probes to screen active ingredients in herbal medicines, including a homogeneous phase of herbal medicines immobilized on a solid substrate (eg, plastic slide) Uneven (Please read the back of the precautions to fill out this page)

Binding ---- Order -------- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) -6- 1226927 A7 B7 V. Description of the invention (4) Phase grading solution or Ingredients. SUMMARY OF THE INVENTION The present invention discloses a novel platform (called a herbal wafer), which includes a plastic slide, a coating of a spacer is filled on the plastic slide, and micro-arrays are separately arranged on the coating. Herbal grading solution or ingredients. The herbal wafer of the present invention is used for screening active ingredients with specific pharmacological activity or therapeutic effect in herbal medicine. The invention also discloses a method for producing the herbal medicine wafer. Furthermore, the present invention discloses a method for using the herbal medicine wafer to screen active ingredients contained in herbal medicine. Brief description of the drawings, Fig. 1 illustrates a perspective view of a plastic slide used to manufacture the herbal wafer of the present invention. FIG. 2A illustrates an example of the herbal medicine wafer of the present invention, in which the sample is gridded on the plastic slide shown in FIG. 1 with a 6 × 10 matrix. FIG. 2B shows the endogenous fluorescence of the column sample on the herbal medicine wafer shown in FIG. 2A before the hybridization reaction using the labeled probe. Figure 2C shows the herbal wafer shown in Figure 2A after hybridization using Cy 3 labeled TNF — a R (tumor necrosis factor alpha receptor) and Cy 5 labeled streptococcal avidin. The fluorescent image of the sample displayed in the upper column. Figure 3 shows two selections of green fluorescent (C y 3) signals (please read the precautions on the back before filling this page). Gmm§ I ϋ flu JV Xin-i_i nnnn I— n The paper size printed by the Bureau ’s Consumer Cooperatives applies the Chinese National Standard (CNS) A4 specification (210 x 297 mm ') -7- 1226927 Α7 Β7 V. Description of the invention (5) Product (Classification liquid 4 and 5 of Herbal C) It corresponds to the disposition points at positions B 7 and B 8 ′ and B 9 and B 10 on the herbal medicine wafer shown in FIG. 2A) for the competitive inhibition of TNF — a / TNF-a R binding. DETAILED DESCRIPTION The present invention discloses a novel platform (called a herbal chip), which is used to screen the active ingredients contained in herbs in a target-oriented manner to achieve high-efficiency results. The herbal medicine wafer of the present invention comprises a plastic slide, which is filled with a coating of a spacer on the plastic slide, and a herbal grading solution or component arranged or fixed on the coating in a microarray manner. In the herbal medicine wafer of the present invention, each herbal medicine graded solution or component is arranged in a microarray manner. The above is an undefined, homogeneous or heterogeneous, partially purified mixture derived from herbal medicine. . The graded solution or composition can be obtained by fractionating the extract of the herbal medicine by applying an instrument (for example, 'ΗPLC). Each configuration point on the herbal wafer may contain secondary metabolites of the herbal. The herbal wafer of the present invention uses a plastic slide instead of a conventional glass slide. The material of the plastic slide can be a homopolymer or a copolymer, which is prepared from one or more monomers, and the single system is selected from ethylene, vinyl halide, propylene, halopropylene, acrylate, methacrylate , Butadiene, acrylonitrile, orbornene or styrene, of which styrene polymers are suitable. The plastic slide also includes polycarbonate. The size of the plastic slide is equivalent to that of a microarrayer or a laser scanner. The grass of the invention (please read the notes on the back before filling out this page) | ------ 丨 丨 Order * ---- 丨 丨 丨 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the paper size is suitable for China National Standard (CNS) A4 specification (210 X 297 mm ') -8- 1226927 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (6) The advantage of using plastic slides for medicine chips is that they can be used Various chemicals are used to treat the surface of plastic slides, so that not only large molecules (such as proteins and DNA), but also small molecules (such as metabolites of herbs) can be fixed on the surface of plastic slides. This advantage is particularly important in contrast to the fact that conventional glass slides can only be used to immobilize large molecules such as proteins and DNA. Furthermore, the plastic slide can be easily molded into a desired shape, and it also has the advantage of low cost. In the preferred system of the present invention, the plastic slide is molded to have 2 grooves (as shown in FIG. 1), and the sample obtained from the herbal grade liquid or composition can be arranged in the groove. The surface of the groove, and then a solution containing a probe can be loaded onto the groove for a hybridization reaction. The depth of the two grooves may be the same or different, and is between less than 0.03 mm and as high as 0.5 mm. Further, as shown in FIG. 1, the relative 2 of each groove can be separately molded to support a glass cover, wherein the glass cover is used to prevent evaporation or loss of the contents loaded on the groove. Probe solution. In the herbal medicine wafer of the present invention, a polyfunctional aldehyde and a subsequent immersion in a precursor solution providing N Η 2 groups are used to previously treat the plastic slide, so that the generated plastic slide contains a surface with Reactive amine group. The precursor for providing the N Η 2 group may be organic or inorganic, and may be selected from the group consisting of Η Η 40Η, a primary amine, a secondary amine, or a tertiary amine, wherein the primary amine, the secondary amine, and the tertiary amine are aliphatic. And / or the aromatic portion can be used as an additional spacer. In the precursor of providing the N Η 2 group, it is suitable to directly provide the Η Η Η of the free Η 2 group. In the herbal wafer of the present invention, the coating on the plastic slide is made by (please read the precautions on the back before filling this page).

雠 — — I — — II # This paper size applies to China National Standard (CNS) A4 (210 X 297 mm ') _ 9-Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1226927 A7 B7 V. Description of the invention (7 ) Functional molecules (for example, polyfunctional epoxides) that act as spacers. The function of the multifunctional epoxide is to connect the ingredients contained in the herbal medicine to the pre-treated plastic slide. An active epoxy group at one end of the multifunctional epoxy is reacted with an amine group on the surface of the pre-treated plastic slide, and an active epoxy group at the other end of the multifunctional epoxy is adsorbed. Or react with the ingredients contained in herbs. In detail, molecules containing free radicals, hydrogen sulfide groups and / or amine groups in the herbal ingredients can form a covalent bond with the active epoxy group at the other end of the polyfunctional g-epoxide. Molecules can then be attached to the plastic slide. The polyfunctional epoxide suitably contains a chemical chain with a length of 6 to 24 carbon atoms, so that the ingredients contained in the herbal medicine will not be directly bound or adsorbed to the pre-treated plastic slide. In the herbal medicine wafer of the present invention, the bonding of each coated point to the coated plastic slide is firm, even after severe disengagement. In the present invention, not only macromolecules (such as proteins and DNA), but also small molecules (such as metabolites of herbs) can be fixed on the surface of the coated plastic slide in a homogeneous or heterogeneous manner. . The preparation of the herbal wafer of the present invention comprises the following steps: preparing a plastic slide (with grooves as appropriate), using a polyfunctional aldehyde and subsequently immersing in a precursor solution providing N Η 2 groups (suitably N Η 4) Η solution) to pre-treat the plastic slide, use multifunctional molecules (suitably polyfunctional epoxide) to coat the surface of the pre-treated plastic slide, and apply the plastic On the surface of the slide, by applying high-density grid technology, a microarray device is used to arrange and fix a large number of samples on the grid area in a microarray manner. Each sample point contains a uniform paper size. Applicable to China National Standard (CNS) A4 specification (210 X 297 public love).

1226927 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Α7 Β7 V. Description of invention (8) or heterogeneous herbal grading solution or ingredients. In the preparation of the herbal wafer of the present invention, integrating miniaturization technology can be used to increase the density of the grid sample on the coated plastic slide. The present invention also discloses a method for using the herbal wafer to pass the target A method for screening active ingredients contained in a herbal medicine in a guided manner, comprising the steps of: loading a solution containing a labeled probe onto the herbal wafer (eg, a groove of the herbal wafer) for hybridization reaction (wherein (By using a glass cover to cover each groove to prevent evaporation of the solution containing the labeled probe), and by means of an instrument (for example, a laser scanner) to visualize and identify that it can bind to the labeled probe Or reaction configuration points. The label of the connection probe may be a dye or a radioactive substance. The probes used in the present invention are based on known targets that are transformed into homogeneous or heterogeneous molecules based on a defined molecular mechanism, which may be, for example, antagonists such as selected cells, receptors, enzymes or proteins. Molecular compounds, competitive ligands, or antibodies. In the preferred system of the present invention, Cy 3 labeled tumor soil death factor alpha receptor (TNF — aR) and Cy 5 labeled streptococcal avidin (strepavidin) are used as probes to carry out Hybridization reaction. Therefore, for example, if a signal showing the combination of the component in the configuration point of the herbal wafer and the labeled TNF-αR is observed, it shows that there is at least one candidate compound in the component of the configuration point, which has a similar resistance to Biological activity of TNF-a R antibody. These candidate compounds can be used as antagonists to reduce the inflammatory response and therefore can be used to treat autoimmune diseases such as rheumatoid arthritis. This paper size applies to China National Standard (CNS) A4 specification (210 X 297 public love ') -11-(Please read the precautions on the back before filling this page) -------- Order ----- -II · 1226927 Printed by A7 B7, Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of Invention (9) The herbal wafer of the present invention can process and detect thousands of samples at the same time, so it can significantly increase the target-oriented screening of herbal medicines. Results and efficiency of biologically active ingredients. By using the herbal medicine wafer of the present invention, it is possible to quickly detect, isolate and identify any ingredient present in the herbal medicine with potential pharmacological activity or therapeutic effect. The present invention is further explained and illustrated by the following examples, but it must be understood that the scope of the present invention is not limited to the following examples. EXAMPLES Previous_Processing of plastic slides and preparation of coated plastic slides A molded plastic slide as shown in Figure 1 was prepared using a polymer of styrene, which contains 2 grooves. The size of the molded plastic slide is equivalent to that of a conventional glass slide used in a micro-array or laser scanner, wherein the depth of the groove is 0.05 mm. First, the molded plastic slide was immersed in a 0.4% glutaraldehyde solution (p Η 5 ♦ 0) at room temperature for 4 hours, and then rinsed with water and immersed in 3M NH4 at 60 ° C. H (pH l 1.0) for 4 hours. The resulting plastic slide was treated overnight at 37 ° C with 100 m Μ 1,4-butanediol-glycidyl ether (pH 1 1 · 〇). Finally, the plastic slide was rinsed with a 0.1 M NaHCO3 solution (pH 8.0) and allowed to dry. 1 Load the sample onto the coated plastic slide by using the microarray method and use the microarrayer BioGrid (purchased from BioRobotic, Cambridge). The paper size is in accordance with China National Standard (CNS) A4 (210 x 297). -12- (Please read the notes on the back before filling this page) "Shang -------- Order ---- # 1226927 A7 B7 V. Description of the invention (10) u κ) Place the sample to the above-mentioned coating Cover the plastic slide. First, the sample is dissolved and suspended on the 96-well trough microplate. The sample is continuously loaded from the 96-well trough microplate to the 96-well trough microplate using a 4-pin (0 · 2 〆m) tube. On the surface of the groove of the coated plastic slide, the herbal sample shown in Figure 2A is described as follows: The graded liquids of herbs A and B are obtained from different graded liquids of taraxacum mongoUcum. Crude extract of dandelion, crude extract of Herbal F series Hedyotis diffuse 'Grass Herbal C grades 1 to 9 are different and partially purified grades derived from cold throat, Herbal D is Lonicera japonica) crude extract 'Carthamus tinctorius' Liquid extract, Herbal medicine J is a purified ingredient obtained from safflower, crude extract of Herba Forsythia Forsythia (Forsythia suspensa), and Herbal I-type Paeonia (

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Paeonia lactiflora). After the placement points on the groove surface of the plastic slide were dried, the plastic slide was immersed in a 1 M T r s solution (ρ Η 8 · 0) at 37 ° C for 2 hours. A laser scanner (Axon, USA) was used to develop the generated plastic slide to confirm that all samples were adsorbed on the plastic slide (see FIG. 2B). The hybridization reaction and signal side measurement were performed using Cy 3 labeled tumor necrosis factor alpha receptor (TNF — α R) and Cy 5 labeled streptococcal avidin (strep a vi din) as probes. Hybridization reaction. In a hybridization solution (100 β 1 τ BS Τ buffer containing the two kinds of labeled probes, which contains 50 mM Tris, pH 7.3, 0.15 M NaCl, i paper standard (CNS) ) A4 size (210 X 297 mm). 13. "Installation (please read the precautions on the back # 1 to fill out this page). Order printed by the Intellectual Property Bureau Employee Consumer Cooperative of the Ministry of Economic Affairs. 1226927 A7 B7 V. Invention Description (11) And 0.02% Tween 2〇), two glass coverslips were used to cover the two grooves on the plastic slide. The plastic slide was allowed to stand at room temperature for 4 hours, followed by TBST buffer solution. Rinse 3 times and rinse 3 times with water. Finally, the plastic slide is dried at 37 ° C. The plastic slide is developed with a laser scanner (Axon, USA). The development results are shown in Figure 2C Recover the green fluorescent spot that inhibits the binding of TNF — a / TNF — α R on the development of Figure 2C. The herbal grading solution corresponding to the green fluorescent spot contains an active ingredient capable of binding to TNF-a R. Analysis of the biological activity of the sample showing a positive green fluorescent signal confirms that the fractions collected above In order to inhibit the biological activity of TNF-α / but Ν XF — a R binding, the analytical method described in the document Mancini et al., Biochemical Pharmacology 58: 851-859, 1999, in which the known component is "suramin" (Surami η) was used as a positive control group, and two samples showing green fluorescence (Herbal C's grades 4 and 5) were tested, which corresponded to FIG. 2 Α herbal wafers Β 7 and Β 8 and Β 9 and B 1 0)). The test results are shown in FIG. 3. "Install (please read the precautions on the back before filling this page)

1-— 1 mmm— immm J ， · 1 1 nn mMMmm n 印 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs-Lr · in 1 · txe _ This paper size applies to China National Standard (CNS) A4 (210 X 297 (Public) -14-

Claims (1)

1226927: ./ vv,, ..... A8 ：: ¾ B8 C8 D8 六 、 Applicable patent scope attachment: No. 90 1 1 3 695 Chinese patent application scope amendment (please read the precautions on the back before reading) (Fill in this page) Amended on January 12, 1993 1 · A herbal wafer for screening desired active ingredients in herbal medicine, comprising a plastic slide, a coating on the plastic slide, and a coating on the coating The microarrays are individually configured to be combined with the herbal grade liquid or ingredients on the plastic slide, wherein the coating comprises a polyfunctional aldehyde coupled to the plastic slide, and the polyfunctional aldehyde is coupled to provide at least one amine group. Compound, the compound capable of providing at least one amine group is bound to a polyfunctional epoxide, the polyfunctional epoxide contains at least 1 epoxy group capable of coupling with the amine group and at least 1 capable of grading with the herbal medicine Liquid or ingredient coupled epoxy. 2. If the herbal medicine chip of the scope of the patent application is No. 1, wherein the herbal grading solution or composition is homogeneous or heterogeneous. 3. If the herbal medicine wafer of item 1 of the patent application scope, wherein the herbal fraction or liquid is obtained by separating the herbal extract with high-performance liquid chromatography (ΗPLC). Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 4 · If any of the herbal medicine wafers in any of claims 1 to 3 of the patent application scope, wherein the herbal grade liquid or ingredient contains the secondary metabolite of the herbal medicine. 5. If the herbal wafer of item 1 of the patent application scope, wherein the material of the plastic slide is polycarbonate, or a homopolymer or copolymer prepared from one or more monomers, the single system is selected from ethylene, Vinyl halide, propylene, halopropylene, propionate, methacrylate, butadiene, acrylonitrile, orthobornene or styrene. The paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 1226927 Α8 Β8 C8 D8 6. Scope of patent application 6 · For example, the herbal wafer of the scope of patent application No. 5 in which the material of the plastic slide is a polymer of styrene. (Please read the precautions on the back before filling out this page) 7 · If the herbal medicine wafer in the scope of patent application item 1, the plastic slide contains 2 grooves. 8. The herbal medicine wafer according to item 1 of the application, wherein the polyfunctional aldehyde is glutaraldehyde. 9) The herbal medicine wafer according to item 1 of the patent application scope, wherein the compound providing an amine group is NΗ40Η. 10 • The herbal medicine wafer according to item 1 of the scope of patent application, wherein the epoxy group capable of coupling with the herbal grading solution or ingredients reacts with a free hydroxyl group, a hydrogen thio group or an amine group. 1 1 · The herbal wafer according to item 1 of the patent application, wherein the multifunctional epoxide contains a chemical chain of 6 to 24 carbon atoms. 1 2 · A method for manufacturing an herbal wafer of the scope of patent application, which includes the following steps: preparing a plastic slide, using a polyfunctional aldehyde and subsequently immersing in a solution of an amine-based compound to pre-treat the plastic slide The surface of the film is printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, and the surface of the pre-treated plastic slide is coated with a polyfunctional epoxide, and by using the polyfunctional epoxide with a herbal grade solution or ingredients The coupling method is to dispose the herbal grade liquid or ingredients on the coating of the plastic slide. 1 3 · The method according to item 12 of the scope of patent application, wherein the plastic slide contains 2 grooves, and the herbal grading solution or ingredients are arranged or fixed on the paper. The Chinese national standard (CNS) A4 specification is applicable. (210 X 297 mm) 1226927 A8 B8 C8 D8 _ 6. The surface of the groove in the patent scope. 14 · The method according to item 12 or 13 of the scope of application for a patent, wherein the herbal grading solution or component is arranged or fixed on the grid area in a microarray manner. 1 5 _ The method according to item 12 or 13 of the scope of patent application, wherein the herbal grading solution or composition is homogeneous or heterogeneous. 16. The method according to item 12 of the scope of patent application, wherein the polyfunctional aldehyde is glutaraldehyde. 17. The method according to item 12 of the scope of patent application, wherein the compound providing an amine group is NΗ40Η. 18. The method according to item 12 of the scope of patent application, wherein the polyfunctional epoxide contains at least one epoxy group at each terminal thereof. 19 · A method for screening active ingredients contained in herbal medicines by using the herbal medicine wafers in the scope of patent application, including the following steps: loading a solution containing a labeled probe onto the herbal wafers to perform a hybridization reaction, And develop and identify the herbal fraction or component that can react or bind with the labeled probe. 20 · The method according to item 19 of the scope of application for a patent, wherein the solution containing a standard wooden needle is homogeneous or heterogeneous. 2 1 · The method according to item 19 of the scope of patent application, wherein the mark is a dye or a radioactive substance. Packing-(Please read the «Notes on the back before filling out this page）, printed by the aT Consumer Intellectual Property Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs This paper is called ^^^^ -— 陆-^