TWI568746B - An oligopeptide for estimating free radical-producing ability and a method thereof - Google Patents

Description

Oligopeptides and methods for assessing free radical production ability

The present invention relates to an oligopeptide, particularly an oligopeptide for assessing the ability to generate free radicals. The invention further relates to a method of using the oligopeptide to assess the ability to generate free radicals.

In the metabolism of drugs, if a covalent bond homocleization occurs to form a free radical with unpaired valence electrons, it may be due to the high chemical activity of the free radicals. Body tissues and cells cause damage.

In order to prevent the harm caused to the organism by taking the drug, the first stage of screening in the cell experiment is generally carried out during the drug development process, and the drug candidate which causes the cell to generate free radicals is removed, however, when the candidate drug is In the case of a large number of cells, the experimentation of the cell will take a lot of time and money. In view of this, it is indeed necessary to provide a method capable of evaluating the free radical generating ability of the candidate drug.

In order to solve the above problems, the present invention provides an oligopeptide for evaluating a free radical generating ability comprising the amino acid sequence as shown in SEQ ID NO: 3.

Accordingly, the oligopeptide of the present invention for evaluating the ability to generate free radicals, which is easily oxidized and which can be oxidized to different oxidation states, can be used to evaluate the free radical generating ability of a sample to be tested. The efficacy of the invention.

Based on the same technical concept, the present invention further provides a method for evaluating free radicals. The method for producing capability comprises: providing a sample to be tested; mixing the sample to be tested and the oligopeptide as described above to obtain a mixture; and forming the mixture with a matrix to form a crystalline solid with a laser beam The oligopeptide in the mixture is ionized to form a gas phase ion, and the gas phase ion is detected by a mass-to-charge ratio analyzer to obtain an oligopeptide intensity value and at least an oxidized oligopeptide intensity value.

According to the method of the present invention for evaluating the ability of generating free radicals, the sample to be tested and the oligopeptide are transiently contacted, and the free radical generated by the sample to be tested is partially oxidized by the oligopeptide. The ratio between the oxidized oligopeptide and the oxidized oligopeptide is evaluated, and the free radical generating ability of the sample to be tested is evaluated as the efficacy of the present invention.

In the method for evaluating the ability of generating free radicals of the present invention, the mixture is irradiated with UV for 10 minutes before the mixture is formed into the crystalline solid with the substrate; the covalent bond can be caused to be split by UV irradiation. Occurring, the sample to be tested is accelerated to generate free radicals, and the time for oxidizing the oligopeptide is shortened, thereby achieving an effect of accelerating the reaction and shortening the time course.

S1‧‧‧ sample supply steps

S2‧‧‧oxidation step

S3‧‧‧ Analysis steps

Figure 1 is a flow chart of a method for evaluating free radical generating ability of this embodiment.

Figure 2: Test bar graph of oligopeptides with different amino acid sequences.

Figure 3: Test pattern of the method for evaluating free radical generating ability of this example.

Fig. 4a is a test bar graph of different concentrations of vitamin K3 before UV irradiation of the method for evaluating free radical generating ability of this example.

Figure 4b: Test bar graph of different concentrations of vitamin K3 after UV irradiation of the method for evaluating free radical generating ability of this example.

Figure 5a: Test bar graph of different types of non-steroidal anti-inflammatory drugs prior to UV irradiation of the method for assessing free radical production in this example.

Figure 5b: After UV irradiation of the method for evaluating free radical generating ability of this embodiment Test bar graph of different types of non-steroidal anti-inflammatory drugs.

Figure 6: Test bar graph of different types of paraben-treated cells of the method for evaluating free radical generating ability of this example.

The above and other objects, features and advantages of the present invention will become more <RTIgt; Referring to FIG. 1, a method for evaluating free radical generating ability according to an embodiment of the present invention comprises: a sample providing step S1, an oxidation step S2, and an analyzing step S3.

In detail, in the sample providing step S1, the sample to be tested may be any food or medicine that needs to be evaluated for its free radical generating ability, for example, a food or a medicine for which the safety of the living body needs to be evaluated, but only the present invention. Those of ordinary skill in the art will appreciate that there is no limitation thereto.

In the oxidation step S2, the sample to be tested may be mixed with an oligopeptide for evaluating the ability to generate free radicals to obtain a mixture comprising the amino acid sequence as shown in SEQ ID NO: 3. It is worth noting that the oligopeptide has a Cysteine residue which is easily oxidized, so that when the sample to be tested and the oligopeptide form the mixture, the free radical generated by the sample to be tested can be The cysteine residue is oxidized, and since the cysteine residue can be oxidized to an oxygen, two oxygen or three oxygen, the oligopeptide can have a different oxidation state after being oxidized.

In the oxidation step S2, the mixture can be further irradiated with UV for 10 minutes, which can promote the occurrence of covalent bond homolysis, accelerate the generation of free radicals in the sample to be tested, shorten the oxidation time of the oligopeptide, and achieve an accelerated reaction. , shorten the time course and other effects.

In the analysis step S3, a matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (matrix-assisted laser desorption/ionization-time of flight) may be selected. Mass spectrometer (MALDI-TOF MS) is used for analysis, but not limited to this. In detail, the mixture can be placed on a target plate of the matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, covering a matrix to form a matrix with the mixture. A crystalline solid that absorbs a laser beam having a wavelength of 330-360 nm. In this embodiment, the substrate may be α-cyanol-4-hydroxy-cinnamic acid (CHCA for short).

Then, the crystalline solid material is irradiated with a laser beam having a wavelength of 330 to 360 nm, so that the crystalline solid matter can be excited to form a gas phase ion, and the gas phase ion can be accelerated into a mass charge after being accelerated by the electric field. The ratio analyzer is configured to detect a mass-to-charge ratio (m/z) of the gas phase ions to obtain an oligopeptide intensity value and at least an oxidized oligopeptide intensity value. The mass-to-charge ratio analyzer may be selected from a time-of-flight analyzer (TOF analyzer), a quadrupole analyzer, and an ion trap mass-to-charge ratio analyzer. (ion trap analyzer, referred to as IT analyzer) and Fourier transform-ion cyclotron resonance (FT-ICR). In this embodiment, the mass-to-charge ratio analyzer is selected as a time-of-flight analyzer (TOF analyzer), which has a fast analysis speed and a matrix-assisted laser desorption free method. , can effectively save the time of sample analysis.

Accordingly, by comparing the oligopeptide intensity value with the at least oxidized oligopeptide intensity value, the free radical generating ability of the sample to be tested can be evaluated.

In order to confirm that the method for evaluating the free radical generating ability of the present embodiment can be used to evaluate the free radical generating ability of the sample to be tested, the following tests are carried out:

(A) Selection of amino acid sequence of oligopeptide

In the present experiment, the peptide shown in the first table was analyzed by the analysis step S3, whereby the most suitable oligopeptide was selected, and the results are shown in Fig. 2.

Table 1, amino acid sequence of the peptide of each group in the test

Referring to Fig. 2, among the oligopeptides of group A1 to group A5, the oligopeptide of group A3 has the strongest signal, and thus the oligopeptide of group A3 was used for subsequent experiments.

(B) Calculation of oligopeptide intensity values and oxidized oligopeptide intensity values

In this test, vitamin K3, which is known to generate free radicals, is taken as the sample to be tested, and the sample to be tested (concentration: 10 to 100 μg/mL) and the oligopeptide (50 to 100 μg/mL) are mixed to form the sample. After the mixture (equal volume mixing, total volume 2 μL), the mixture was irradiated with UV for 10 minutes, followed by analysis by MALDI-TOF MS, and the results were as shown in Fig. 2, except that the unoxidized oligopeptide was formed at position 775. In addition to a peak (m/z ^. [M+H] ^+. =775), oligopeptides with different oxidation states by vitamin K3 form a peak at positions 791, 807, and 823; The area of the peak formed by the position can obtain the intensity value of the oligopeptide, and the area of the peak formed at the positions of 791, 807, and 823 is the intensity value of the oxidized oligopeptide (for the convenience of subsequent explanation, the foregoing three positions are formed. The areas of the peaks are hereinafter referred to as the oxime oligopeptide strength value 〝, the quinone dioxy oligopeptide strength value 〞 and the 〝trioxy oligopeptide strength value 〞).

(C) Free radical production capacity of vitamin K3

In the present test, each group of samples to be tested shown in Table 2 is evaluated by the method described above, and the oxidation step S2 is separately performed under the conditions of not irradiating UV and irradiating UV, and the ratio is calculated by the following formula: ratio =monooxygen (dioxy or trioxo) oligopeptide intensity value / oligopeptide intensity value

Table 2, samples to be tested in each group of the test

Please refer to Figure 4a. Only in the case where the mixture is not irradiated with UV, only high concentrations of vitamin K3 (Group C3) can be detected to have free radical generating ability. Please refer to Figure 4b for details. In the case of UV irradiation of the mixture, different concentrations of vitamin K3 in the C1 to C3 groups can detect the ability to generate free radicals.

(D) Free radical production capacity of non-steroidal anti-inflammatory drugs

In this test, each group of samples to be tested shown in Table 3 is evaluated by the method described above, and the oxidation step S2 is performed separately under the conditions of not irradiating UV and irradiating UV, and the results are as shown in Figs. 5a, 5b, respectively. The figure shows.

Please refer to Figure 5a. In the case where the mixture is not irradiated with UV, only ibuprofen (Group D2) can be detected to have free radical generating ability. Please also refer to Figure 5b for UV irradiation. In the case of this mixture, both the ketoprofen of group D1 and the ibuprofen of group D2 were able to detect the ability to generate free radicals.

(E) The ability of parabens to promote the production of free radicals by cells

In this test, the paraben shown in Table 4 (100 μM) was added to human skin keratinocytes (HaCaT cell), and after 24 hours, the cell culture medium of each group was collected to a 3 kDa filter. The cell culture solution was filtered, and the filtered cell culture solution was further added to the oligopeptide (1 μL each) for 10 minutes at 37 ° C, and finally analyzed by MALDI-TOF MS. The results are shown in Fig. 6.

Referring to Figure 6, it is confirmed that the method of the present embodiment can also be applied to evaluate the ability of the sample to be tested to promote the production of free radicals by cells.

In summary, the method for evaluating the ability of generating free radicals of the present invention can temporarily contact the sample to be tested and the oligopeptide to partially oxidize the oligopeptide generated by the free radical generated by the sample to be tested. The ratio between the oxidized oligopeptide and the oxidized oligopeptide is evaluated, and the free radical generating ability of the sample to be tested is evaluated as the efficacy of the present invention.

Furthermore, the oligopeptide of the present invention for evaluating the ability to generate free radicals, which is easily oxidized and which can be oxidized to different oxidation states, can be used to evaluate the free radical generating ability of a sample to be tested. The efficacy of the invention.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

<110> Kaohsiung Medical University

<120> Oligopeptides and methods for evaluating free radical production ability

<130> PK14482

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<170> PatentIn version 3.5

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S1‧‧‧ sample supply steps

S2‧‧‧oxidation step

S3‧‧‧ Analysis steps

Claims (4)

An oligopeptide for evaluating the ability to generate free radicals, comprising the amino acid sequence as shown in SEQ ID NO: 3. A method for evaluating a free radical generating ability, comprising: providing a sample to be tested; mixing the sample to be tested and the oligopeptide as described in claim 1 to obtain a mixture; and causing the mixture to The matrix forms a crystalline solid, ionizing the oligopeptide in the mixture with a laser beam, forming the oligopeptide into a gas phase ion, and detecting the gas phase ion by a mass-to-charge ratio analyzer to obtain an oligopeptide Strength value, a single oxygen strength value, a double oxygen intensity value, and a trioxane strength value. A method for evaluating free radical generating ability as described in claim 2, wherein the substrate is α-cyano-4-hydroxycinnamic acid. A method for evaluating the ability to generate free radicals as described in claim 2, wherein the mixture is irradiated with UV for 10 minutes before the mixture is formed into the crystalline solid with the substrate.