Methods for evaluating antidepressant effects

Abstract

The present invention aims to provide a method for evaluating the serotonin reuptake inhibitory effect of a composition containing valerian extract. [Solution] The present invention provides a method for evaluating antidepressant effects, which involves quantitatively analyzing one or more components contained in at least a part of a plant of the genus Valerian or a processed product thereof, and evaluating the antidepressant effect of the test substance by referring to the results of the quantitative analysis and criteria for antidepressant effect.

Description

Technical Field

[0001] The present invention relates to a method for evaluating antidepressant effects.

Background Art

[0002] Valeriana fauriei Briq. has been traditionally used as a raw material for women's medicines in Japan and is formulated into pharmaceuticals for menopause, menstrual pain, blood circulation disorders, irregular menstruation, cold sensitivity, etc., expecting an anxiolytic effect. It is also used as a flavoring agent for foods such as confectioneries.

[0003] Valeriana officinalis has been used as a folk medicine in Europe, expecting a sedative effect since ancient times, and has a relatively high level of recognition in Japan. Therefore, it is also formulated into herbal tea, bath agents, cosmetics, and health food supplements.

[0004] Patent Document 1 describes that a composition containing an NSAID and a Valeriana extract can more greatly improve pain caused by stress or trauma and the release from muscle tension. Patent Document 2 describes that a processed Valeriana product in combination with acetaminophen, ethenzamide, and allylisopropylacetylurea exhibits an improved effect on pain-induced insomnia (sleep maintenance disorder). Patent Document 3 describes that a combination of a Valeriana extract and any one of ascorbic acid, glutamic acid, and ondj extract can exert an analgesic effect and enhance a sedative effect.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] Patent documents 1-3 do not describe the relationship between the components of valerian extract and their respective physiological activities, nor do they describe or suggest any method for evaluating activity. Since valerian is a natural product, it is expected that its activity will vary depending on the extraction conditions and raw materials. Furthermore, if there is a method for selecting extracts that exhibit higher activity, it is hoped that the development of antidepressants and antidepressant food compositions will be advanced.

[0007] The present invention aims to provide a method for evaluating the serotonin reuptake inhibitory effect of a composition containing valerian extract. [Means for solving the problem]

[0008] The present invention provides the following [1] to

[10] . [1] A method for evaluating the antidepressant effect of a test substance, which involves quantitatively analyzing one or more components contained in at least a part of a plant of the genus Valerian or a processed product thereof, and evaluating the antidepressant effect of the test substance by referring to the results of the quantitative analysis and the criteria for antidepressant effect. [2] The method according to [1], wherein the genus Valeriana is one or more species selected from Valeriana, European Valeriana, Indian Valeriana, climbing Valeriana, and lisian Valeriana. [3] The method according to [1] or [2], wherein one or more components include at least one component that is involved in serotonin reuptake inhibition. [4] The method according to any one of the following [1] to [3], wherein one or more components are selected from the group consisting of kesyl glycol diacetate, canoconyl acetate, α-kesyl acetate, and canocol. [5] Quantitative analysis is performed using liquid chromatography-mass spectrometry (LC-MS / MS) and / or high-performance liquid chromatography (HPLC) as described in any one of the following [1] to [4]. [6] The method according to any one of items [1] to [5], wherein one or more components are kesyl glycol diacetate and canoconyl acetate, and the method comprises calculating a predicted value of relative serotonin uptake from the concentrations obtained by quantitative analysis using the following formula 1. (Formula 1) Predicted relative serotonin uptake (%) = -440 × log[KNA concentration (μM)] - 327 × log[KGD concentration (μM)] + 122 × log[KNA concentration (μM)] × log[KGD concentration (μM)] + 1259 [7] The method described in [6], wherein the antidepressant effect is determined to be high if the predicted value of relative serotonin uptake calculated by Equation 1 is 130% or less. A method for selecting candidate active ingredients for an antidepressant or antidepressant food composition, comprising evaluating one or more components contained in at least a part of a valerian plant or a processed product thereof, which is the test substance, by the method described in any one of items [8], [1], to [7]. A method for producing an antidepressant, comprising using at least a portion of a plant of the genus Valerian selected by the method described in [9][8] or a processed product thereof as an active ingredient. A method for producing an antidepressant food composition, using at least a portion of a plant of the genus Valerian selected by the method described in

[10] [8] or a processed product thereof as an active ingredient. [Effects of the Invention]

[0009] According to the present invention, a method for accurately and easily evaluating serotonin reuptake inhibitory effects is provided, and it is expected to contribute to the evaluation, selection, and development of serotonin reuptake inhibitors containing components of plants of the genus Valerian. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the amount of serotonin uptake from valerian ethanol extract. [Figure 2] Figure 2 shows the amount of serotonin uptake when (a) only kessyl glycol diacetate (KGD) is added, or (b) when KGD and a fraction of the ethanol extract of canokoso are mixed and added, respectively. [Figure 3] Figure 3 shows the amount of serotonin uptake of (a) canokonyl acetate (KNA), (b) canocol, or (c) α-kessyl acetate (α-KA). [Figure 4] Figure 4 shows the correlation between the measured serotonin uptake amount and the predicted value of Samples 1 to 13 containing KNA, canocol, α-KA, and KGD in various mixing ratios in cell experiments. The numbers in the graph indicate the sample numbers (Table 1). [Figure 5] Figure 5 shows the serotonin uptake inhibitory effect when KNA, KGD, canocol, and α-KA are mixed. [Figure 6] Figure 6 is a graph showing the correlation between the serotonin uptake amount calculated from the model formula based on the KNA and KGD concentrations of the canokoso extract obtained by various extraction methods and the serotonin uptake amount measured by cell experiments. The numbers in the graph indicate the sample numbers (Table 2). [Figure 7] Figure 7 is a graph showing the correlation between the serotonin uptake amount calculated from the model formula based on the KNA and KGD concentrations of the canokoso extract of various commercial products and the serotonin uptake amount measured by cell experiments. The numbers in the graph indicate the sample numbers (Table 4). [Mode for Carrying Out the Invention]

[0011] [1. Evaluation Method for Antidepressant Effect] [Test Substance] In the evaluation method of the present invention, the test substance is at least a part of a plant of the genus canokoso or a processed product thereof.

[0012] Examples of plants of the genus Valeriana include, for example, Valeriana fauriei Briq., Valeriana officinalis, Valeriana jatamansi (Valeriana wallichii), Valeriana flaccidissima, and Valeriana phu (Valeriana edulis). The plant body of the Valeriana genus plants may be a part thereof, for example, a part including roots, rhizomes, leaves, preferably a part including roots and / or rhizomes. Examples of processed products include those obtained by subjecting the plant body or a part thereof of the Valeriana genus plants to processing treatments such as crushing treatment, drying treatment, heat treatment, extraction treatment, solid-liquid separation treatment, concentration treatment, granulation, and / or powdering treatment (preferably including drying treatment, crushing treatment, and extraction treatment). More specifically, for example, processed products obtained by drying and pulverizing the raw plant body (all or part) of Valeriana fauriei (Valeriana fauriei powder: it may be either coarse powder or fine powder), processed products obtained by drying and grinding the raw plant body or a part thereof of the Valeriana genus plants (ground product), and extracts of the Valeriana genus plants. Examples of extracts of the Valeriana genus plants include, for example, extracts obtained by extracting the raw plant body or a part thereof, dried product, or dried crushed product of the Valeriana genus plants with a solvent, dried products thereof (dried extracts), and further powdered dried extracts thereof. Preferably, they are processed products of the roots and rhizomes of the Valeriana genus plants, more preferably dried extracts obtained by drying and crushing a part including the roots and rhizomes of the Valeriana genus plants and then extracting with a solvent. Examples of extraction solvents include alcohols such as ethanol, mixed solvents of alcohol and water, and hexane, hydrous alcohol (preferably, alcohol 30% by mass or more, 40% by mass or more, or 50% by mass or more), and hexane. The extraction may be either cold soaking or ultrasonic extraction. Also, the extraction time can be determined according to the extraction conditions. For example, in the case of cold soaking, it is 3 to 60 hours, and in the case of ultrasonic extraction, it is 15 minutes to 3 hours.

[0013] [Subject of quantitative analysis][[ID=[5]] The target of quantitative analysis is one or more components contained in the test substance. Preferably, the components include those involved in serotonin reuptake inhibition. Such components can be selected by analyzing the components contained in the test substance and confirming the activity of candidate substances. Examples of components involved in serotonin reuptake inhibition include canokol (formula (1)), kessyl glycol diacetate (KGD: formula (2)), canokonyl acetate (KNA: formula (3)), and α-kessyl acetate (α-KA: formula (4)), with KNA and KGD being preferred.

[0014] [ka]

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] [Methods of quantitative analysis] Quantitative analysis can be performed by methods such as mass spectrometry and high-performance liquid chromatography. Specifically, examples include GC-MS, LC-MS, LC-MS / MS, FAB-MS, EI-MS, CI-MS, FD-MS, MALDI-MS, ESI-MS, HPLC-MS, FT-ICR-MS, CE-MS, ICP-MS, Py-MS, and TOF-MS.

[0019] [Evaluation of the antidepressant effect of the test substance] The evaluation of antidepressant effects can be performed by referring to the results of quantitative analysis and the criteria for antidepressant effects. The criteria for antidepressant effects are preferably statistical regression equations with the content of specific components in the test substance as variables. This allows for quantitative determination of antidepressant effects (e.g., serotonin reuptake inhibition) by substituting the content of the relevant component into the regression equation. The regression equation can be calculated based on populations in which the composition of each component of the test substance has been altered (e.g., compositions with increased or decreased amounts of specific components). The calculation of the regression equation can be performed using statistical analysis software.

[0020] For example, when the target components are KGD and KNA, the regression equation shown below is Equation 1. When using Equation 1, the concentration (content) of KGD and KNA in the test substance can be substituted into Equation 1 to calculate the predicted value of relative serotonin uptake.

[0021] (Formula 1) Predicted relative serotonin uptake (%) = -440 × log[KNA concentration (μM)] - 327 × log[KGD concentration (μM)] + 122 × log[KNA concentration (μM)] × log[KGD concentration (μM)] + 1259

[0022] If the predicted value calculated using this formula is 130% or less, it can be determined that the substance has a high reuptake inhibitory effect, i.e., a high antidepressant effect.

[0023] [2. Application of methods for evaluating antidepressant effects] The above method for evaluating antidepressant activity can be applied to the selection of candidate active ingredients for antidepressants or antidepressant food compositions. For example, by quantitatively analyzing at least a portion of a Valerian plant or its processed product (e.g., Valerian extract) that is being considered as an antidepressant or antidepressant food composition, and substituting specific concentrations into a pre-prepared regression equation, its antidepressant activity can be accurately predicted without actually measuring it. If the predicted value is above a certain level, it can be used as an active ingredient in actual antidepressants or antidepressant food compositions. [Examples]

[0024] The present invention will be described in detail below with reference to examples. The following examples are merely illustrative of the present invention and are not limited to these examples. Generally, "Kanokosou" is a polysemous term that refers to the plant genus, the plant itself, and the crude drug. However, in the following examples, the crude drug "Kanokosou," that is, the roots and rhizomes of the plant "Kanokosou," were used.

[0025] Reference Example 1 [Serotonin uptake effect of valerian extract] A sample (chopped) of Valerian from Mie Prefecture was immersed in three times the amount of 99.5% ethanol at room temperature for 48 hours. This was repeated twice to obtain an ethanol extract of Valerian (ethanol cold immersion: Sample 1 in Table 1 below). Human Embryonic Kidney cells 293 (HEK293 cells) transiently expressing recombinant human serotonin transporter (hSERT) were then mixed with the above-mentioned ethanol extract of Valerian (ethanol cold immersion: the amount used shown in Figure 1) and a radioactive isotope ( 3 Serotonin (10 nM) labeled with H) was dissolved in Krebs-Ringer's-Hepes (KRH) buffer supplemented with 5% acacia gum and 0.015% olive oil, added, and incubated at 37°C for 10 minutes. After aspirating the buffer, cells were lysed with 0.1 M NaOH / 0.5% SDS solution, neutralized with 0.1 M HCl, and radioactivity was measured using a liquid scintillation counter to determine serotonin uptake. Serotonin uptake was measured similarly except that KGD alone (amount shown in Figure 2(a)) or KGD and valerian ethanol fraction (hexane layer of ethanol extract fractionated by open column: amount shown in Figure 2(b)) was added instead of valerian ethanol extract (Figures 2(a) and (b)). Serotonin uptake was measured similarly for mock cells and control HEK293-hSERT cells, except that they were treated with a buffer without the sample. Sertraline (0.6 μM) was used as a positive control.

[0026] The introduction of hSERT increased serotonin uptake, but the addition of valerian ethanol extract decreased serotonin uptake in a concentration-dependent manner (Figure 1). Furthermore, KGD, the main component of valerian, does not have serotonin uptake inhibitory activity on its own (Figure 2a), but its activity was enhanced when combined with a fraction of valerian (Figure 2b).

[0027] Further fractionation of the valerian fraction by open column and HPLC yielded KNA, the novel compound canocol, α-KA, and KGD (Equations 1-4). From the results of the fractions and individual compounds, all of these compounds showed concentration-dependent serotonin uptake inhibitory activity (Figures 2 and 3).

[0028] Example 1 [Investigation of the mixing ratio of each component] In Reference Example 1, the addition of KGD increased the serotonin uptake inhibitory effect of the valerian fraction, suggesting that compounds in valerian may interact with KGD to exert their activity. Therefore, based on a Definitive Screening Design (DSD), the activity of 13 combinations of the four activity-related compounds, mixed in amounts actually contained in valerian extract, or twice or half of those amounts (Table 1), was evaluated, and their interactions were examined. Valerian ethanol extract (extraction method: manufactured by the same method as in Example 2 (ethanol cold maceration); 40 mg / ml as valerian) contained KNA, kanokol, α-KA, and KGD at 260, 90, 70, and 1200 μM, respectively, and Sample 13 is a mixture of the four compounds in these amounts.

[0029] [Table 1]

[0030] The serotonin uptake levels in each individual study were shown as a percentage of the control group (a group of hSERT-introduced HEK293 cells without sample addition), and further shown as a relative amount to the value for sample 13. Regression analysis revealed that the serotonin uptake inhibitory effect of Valerian is due to the interaction between KNA and KGD, and the following regression equation was obtained to predict the interaction.

[0031] (Regression equation) Relative serotonin uptake (%) = -440 × log[KNA concentration (μM)] - 327 × log[KGD concentration (μM)] + 122 × log[KNA concentration (μM)] × log[KGD concentration (μM)] + 1259

[0032] The predicted and actual values ​​of serotonin uptake using this regression equation are r 2 A correlation of 0.79 was observed (Figure 4), allowing for the estimation of the interaction of activity from the concentrations of KNA and KGD. However, since this regression equation was obtained under conditions where canocol and α-KA were also present, the activity was evaluated by sequentially mixing each compound in amounts found in valerian. As a result, the activity was enhanced by mixing KNA and KGD, supporting the regression equation. Furthermore, the activity was enhanced by the addition of canocol (Figure 5).

[0033] Example 2 [Examination of extraction conditions] Various valerian extracts (Samples 1-6) were prepared using valerian grown in Mie Prefecture, with different extraction solvents and methods (Table 2).

[0034] [Table 2]

[0035] These samples were analyzed by LC-MS under the following conditions. Equipment used: HPLC (DIONEX Ultimate 3000) MS / MS(TSQ QUANTUM ACCESS MAX)system(Thermo Fisher Scientific, MA, USA) Column: Mightysil RP-18 GP II, 3.0 id × 150 mm, 3 μm (Kanto Chemical) Mobile phase: 0.1% formic acid (A), acetonitrile (B) 55% B (0-13 min), increase from 55% to 80% (13-25 min) Flow rate: 0.5ml / min Column temperature: 40℃ Detection fragments and detection time for each compound KGD:219.30→201.20m / z, 5.5 minutes Α-KA:221.21→203.29m / z, 10.2 minutes KNA:322.13→281.13m / z, 11.4 minutes kanokol:205.31→149.33m / z, 15.4 minutes

[0036] As a result, the presence of KNA, canocol, α-KA, and KGD was confirmed under all conditions (Table 3).

[0037] [Table 3]

[0038] Furthermore, serotonin uptake was measured for each sample. Specifically, human embryonic kidney cells 293 (HEK293 cells) transiently expressing recombinant human serotonin transporter (hSERT) were subjected to samples 1-6 (valerian crude drug concentration: 40 mg / mL) and radioactive isotopes ( 3 After adding serotonin (10 nM) labeled with H), the mixture was incubated at 37°C for 10 minutes, and the amount of serotonin uptake was measured.

[0039] When the activity estimated using this method was compared with the actual activity measurement results based on the measured component amounts in each sample, a high correlation was obtained between the estimated value and the measured value (r 2 =0.95) (Figure 6).

[0040] These results demonstrate that the serotonin reuptake inhibitory activity of valerian extract can be estimated using the method of the present invention, and that it is useful for investigating extraction conditions for valerian extract that have high serotonin reuptake inhibitory activity.

[0041] Example 3 [Investigation of the activity of the raw materials] The serotonin uptake inhibitory activity of extracts made from commercially available valerian was investigated. Valerian extract (valerian concentration: 40 mg / mL) was prepared using commercially available samples (dried roots) 1-4 shown in Table 4, under common extraction conditions (cold immersion in ethanol, repeated twice for 48 hours at room temperature).

[0042] [Table 4]

[0043] [Table 5] The weight shown is the weight per gram of sample.

[0044] As a result, the presence of KNA, α-KA, and KGD was confirmed in all samples (Table 5). When the activity estimated by this method was compared with the actual activity measurement results based on the measured component amounts, a high correlation was obtained between the estimated values ​​and the measured values ​​(r 2 =0.95) (Figure 7).

[0045] From this, it is clear that the method of the present invention is useful for selecting and examining valerian raw materials and valerian varieties that have high serotonin uptake inhibitory activity.

Claims

1. A method for evaluating antidepressant effects, which involves quantitatively analyzing one or more components contained in at least a part of a plant of the genus Valerian, which is the test substance, or in a processed product thereof, and evaluating the antidepressant effect of the test substance by referring to the results of the quantitative analysis and criteria for antidepressant effect.

2. The method according to claim 1, wherein the plant of the genus Valeriana is one or more species selected from Valeriana fauriei, Valeriana verna, Valeriana japonica, Valeriana sylvestris, and Valeriana lisianthus.

3. The method according to claim 1 or 2, wherein one or more components include at least one component that is involved in inhibiting serotonin reuptake.

4. The method according to claim 1 or 2, wherein one or more components are selected from the group consisting of kesyl glycol diacetate, canoconyl acetate, α-kesyl acetate, and canocol.

5. The method according to claim 1 or 2, wherein quantitative analysis is performed using liquid chromatography-mass spectrometry (LC-MS / MS) and / or high-performance liquid chromatography (HPLC).

6. The method according to claim 1 or 2, wherein one or more components are kesyl glycol diacetate and canoconyl acetate, and the method includes calculating a predicted value of relative serotonin uptake from the concentrations obtained by quantitative analysis using the following formula 1. (Formula 1) Relative serotonin uptake (%) = -440 × log [concentration of canoconyl acetate (μM)] - 327 × log [concentration of kesyl glycol diacetate (μM)] + 122 × log [concentration of canoconyl acetate (μM)] × log [concentration of kesyl glycol diacetate (μM)] + 1259

7. The method according to claim 6, wherein the antidepressant effect is determined to be high if the predicted value of the relative serotonin uptake calculated by formula 1 is 130% or less.

8. A method for selecting candidate active ingredients for an antidepressant or antidepressant food composition, comprising evaluating one or more components contained in at least a part of a plant of the genus Valerian, which is a test substance, or a processed product thereof, by the method described in claim 1 or 2.

9. A method for producing an antidepressant, comprising using at least a portion of a plant of the genus Valerian selected by the method of claim 8, or a processed product thereof, as an active ingredient.

10. A method for producing an antidepressant food composition, using at least a portion of a plant of the genus Valerian selected by the method of claim 8, or a processed product thereof, as an active ingredient.

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