Method for producing indigo leaves solution, and indigo leaves solution containing tryptanthrin

Abstract

To provide tryptanthrin-containing indigo leaves solution by various extract liquid.SOLUTION: In a method for producing indigo leaves solution, solution containing tryptanthrin is obtained from indigo leaves by using polyhydric alcohol solution as extract liquid. It is preferable that the polyhydric alcohol be glycerol. Further, it is also preferable that extraction by the extract liquid be performed by shaking extraction. Indigo leaves solution contains tryptanthrin obtained from indigo leaves by using polyhydric alcohol solution as extract liquid.

Description

Technical Field

[0001] The present invention relates to a method for producing a blue leaf solution by extracting components from blue leaves with a polyhydric alcohol such as glycerin water, and a blue leaf solution containing tryptanthrin.

Background Art

[0002] Conventionally, a method for producing a blue leaf solution containing tryptanthrin as described in Patent Document 1 has been known. This method for producing a blue leaf solution containing tryptanthrin involves bringing liquid limonene into contact with blue leaves for a certain period of time to obtain a solution containing tryptanthrin. The contact between the blue leaves and limonene is carried out by placing the blue leaves in the liquid limonene within 30 hours, and further, ascorbic acid is added to the liquid limonene.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the conventional method for producing a blue leaf solution containing tryptanthrin, limonene is used as the extract. Although there are various ways to use the blue leaf solution containing tryptanthrin obtained by this production method, depending on the intended use, it may not be preferable to use limonene. Therefore, a blue leaf solution containing tryptanthrin using a different extract has been desired as another option.

Means for Solving the Problems

[0005] The method for producing a blue leaf solution according to the present invention obtains a solution containing tryptanthrin from blue leaves using polyhydric alcohol water as an extract. The polyhydric alcohol is preferably glycerin. Further, the extraction with the extract is preferably performed by shaking extraction. The blue leaf solution of the present invention contains tryptanthrin obtained from blue leaves with polyhydric alcohol water as an extract.

Brief Description of the Drawings

[0006]

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Modes for Carrying Out the Invention

[0007] (Embodiment 1) The present invention extracts the components of blue with glycerin water.

[0008] Quantification of the content of blue glycerin extract and antibacterial tests were conducted as follows. [Sample Preparation and Extraction Method] Sample A. Add 12 mL of glycerol water (75: glycerol 75% solution) to 1 g of blue, and perform shaking extraction for 24 hours. Sample B. Add 12 mL of glycerol water (50: glycerol 50% solution) to 1 g of blue, and perform shaking extraction for 24 hours. Sample C. Add 12 mL of glycerol water (25: glycerol 25% solution) to 1 g of blue, and perform shaking extraction for 24 hours. Sample D. Add 12 mL of glycerol water (12.5: glycerol 12.5% solution) to 1 g of blue, and perform shaking extraction for 24 hours.

[0009] Samples A - D prepared in this way are shown in Figure 1. The shaking extraction was carried out at room temperature for 24 hours at 100 r.p.m. using an orbital shaker (Model OS - 200, Hangzhou Allshng instrument Co., Ltd., China).

[0010] [Quantification] The quantification of triptanthrin was carried out by diluting the extracts of Samples A - D 10 - fold and using HPLC (High - Performance Liquid Chromatography) under the following equipment and conditions. Measurement detector: Spectrophotometer detector SPD - 20A (Shimadzu Corporation) Liquid delivery pump: LC - 20AD (Shimadzu Corporation) Column: COSMOSIL 5PE - MS 4.6×250 mm (Nacalai Tesque, Inc.) Mobile phase: 40% acetonitrile Flow rate: 1.0 mL / min Detection wavelength: 254 nm

[0011] [Antibacterial test] Disk diffusion method (halo method) (1) Inoculate Staphylococcus aureus with a platinum loop into 3 mL of PBS (phosphate - buffered saline), and adjust to 2×10 5 cells / mL. (2) After immersing a sterilized cotton swab in the prepared bacterial suspension, rotate the agar medium about 60 degrees each time, and smear a total of 3 times to evenly distribute the inoculated bacteria. (3) Add 70 μL of blue glycerin extract to the disk. (4) Place the disk on the agar surface with forceps and gently press it from above. (5) Incubate at 37 °C in an incubator with the medium surface facing up. (6) After incubation, measure the diameter of the inhibition circle formed around the disk.

[0012] Liquid medium method (MIC method) (1) Dilute the cultured Staphylococcus aureus with PBS so that the O.D. 550nm is approximately 0.125, and further dilute it 10-fold with PBS to obtain the inoculation bacterial solution (about 10 7 CFU / mL). (2) Dispense 180 μL of Mueller-Hinton liquid medium containing blue glycerin extract into a 96-well plate. The final extract concentration was 10%, 5%, 2.5%, and 1.25%. (3) Dispense 10 μL of the inoculation bacterial solution into each well (about 10 5 CFU / well). (4) Incubate at 37 °C for 6 hours. (5) Add 10 μL of the color reagent to all wells and further incubate for 30 minutes to 1 hour. (6) Measure the absorbance at 450 nm using a microplate reader.

[0013] [Test results] First, the quantification of tryptanthrin in each sample was performed. The HPLC charts shown in Figures 2 to 5 below were quantified using a 10-fold dilution of each sample. In the figure, the horizontal axis is the detection time, indicating the time (RT: retention time) until a specific compound in the sample elutes from the column. The vertical axis shows the intensity of the detection signal obtained from the detector, represented by the height and area of each detection peak. Each peak indicates that different compounds were separated and detected, and the shape and position of the peak indicate the characteristics of the analyzed compound.

[0014] A. Glycerin (75) extract (see Figure 2) As shown in Table 1 below, in Sample A, a concentration of 10.22 μg / mL was obtained. This concentration is determined by converting the measured area and height into concentration using the calibration curve for the standard substance (the same applies hereinafter).

[0015] B. Glycerin (50) Extract (see Figure 3) As shown in Table 1 below, in Sample B, a concentration of 10.21 μg / mL was obtained.

[0016] C. Glycerin (25) Extract (see Figure 4) As shown in Table 1 below, in Sample C, a concentration of 8.24 μg / mL was obtained.

[0017] D. Glycerin (12.5) Extract (see Figure 5) As shown in Table 1 below, in Sample D, a concentration of 7.41 μg / mL was obtained.

[0018] The quantified concentrations (μg / mL) are summarized as follows.

Table 1

[0019] Figure 6 is a diagram showing the results of the antibacterial test by the disk diffusion method. It was found that in each of Samples A to D, inhibition zones of a certain size were formed, and the growth of Staphylococcus aureus around the disks was inhibited. The sizes of the inhibition zones are as shown in Table 2. For comparison, disks of only glycerin water were tested at each concentration, but no inhibition zones were formed at any concentration. In this figure, the samples (lower row) and the comparative samples (upper row) are glycerin (75), glycerin (50), glycerin (25), and glycerin (12.5) from left to right.

[0020]

Table 2

[0021] Figure 7 shows the state of growth inhibition by the antibacterial test using the liquid medium method. Table 3 below shows the content of the samples for each plate used in the test.

[0022] [Table 3]

[0023] Figure 8 shows the results for each concentration by the antibacterial test using the liquid medium method. In this figure, the results of the final extract concentration, 10%, 5%, 2.5%, and 1.25% are shown for each concentration of glycerol water, and the percentage (% of control) indicating how much it is inhibited with the sterile medium as 100% is shown.

[0024] [Evaluation of Test Results] As a result of conducting tests by changing the glycerol concentration during the extraction of indigo, the extraction amount of triptanthrin, which is the main component of indigo, increased in a glycerol concentration-dependent manner by HPLC. In addition, as a result of examining its antibacterial activity by the disk diffusion method and the liquid medium method, an antibacterial action was clearly recognized by the disk diffusion method as compared with glycerol water alone, and the action was concentration-dependent. On the other hand, by the liquid medium method, significant antibacterial activity was recognized with glycerol (75) and glycerol (50). The viable cell count decreased by 1.15% with the addition of 10% of the glycerol (75) extract shown in Sample A and by 26.78% with the addition of 5%. A decrease in the viable cell count of 5.93% at 10% and 28.45% at 5% was also recognized with the glycerol (50) extract shown in Sample B, and it was revealed that the growth of bacteria was significantly inhibited.

[0025] Furthermore, the antibacterial test against Escherichia coli was also carried out in the same manner as above. Samples A and B described above were used for the test.

[0026] Liquid Medium Method (MIC Method) (1) Dilute the cultured Escherichia coli with PBS so that the O.D. at 550 nm is about 0.125, and further dilute it 10-fold with PBS to obtain the inoculum bacterial solution (about 107 CFU / mL). (2)Dispense 180 μL of Mueller-Hinton liquid medium containing blue glycerin extract into a 96-well plate. The final extract concentrations were 10%, 5%, 2.5%, and 1.25%. (3)Dispense 10 μL of the inoculum bacterial solution into each well (about 105 CFU / well). (4)Incubate at 30 °C for 6 hours. (5)Add 10 μL of the chromogenic reagent to all wells and incubate for an additional 30 minutes to 1 hour. (6)Measure the absorbance at 450 nm using a microplate reader.

[0027] Figure 9 is a diagram showing the results for each concentration in the antibacterial test by the above liquid medium method. In this figure, the results for the final extract concentrations of 10%, 5%, 2.5%, and 1.25% are shown for each concentration of glycerin water, and the percentage of inhibition (% of control) is shown with the sterile medium as 100%.

[0028] As a result of this test, although growth inhibitory effects on Escherichia coli were observed for glycerin (75) and glycerin (50) alone, a clear antibacterial effect was confirmed for the blue glycerin extract compared to these alone. Growth of Escherichia coli was inhibited by 96.40% with 10% of the glycerin (75) extract of sample A and by 91.26% with 10% of the glycerin (50) extract of sample B.

[0029] [Polygonum tinctorium] The Polygonum tinctorium that can be used in the present invention is a blue-containing plant such as Polygonum tinctorium (Polygonaceae), Polygonum meyerianum (Polygonaceae), Isatis tinctoria (Brassicaceae), Strobilanthes cusia (Acanthaceae), Indigofera tinctoria (Fabaceae), etc. In particular, Polygonum tinctorium, an annual plant belonging to the Polygonaceae family, is preferable for use because it is easily available and rich in the specific component tryptanthrin.

[0030] In addition, the indigo plant used in the present invention is not particularly limited in terms of its origin or cultivation method, and may be a naturally occurring indigo plant, a cultivated one, or a mutant strain obtained by breeding these by a conventional method. Further, the indigo plant used in the present invention may be a culture obtained by tissue culture, callus culture, cell culture, or the like. When using the plant body as a raw material for extraction, a part or all of the plant body can be used. Also, the plant body may be in a state containing moisture, a frozen state, a dried state, or a mixture thereof. From the viewpoint of ease of handling, it is desirable to use the dried state.

[0031] In the heat drying step, only the indigo leaves are collected from the indigo plant harvested as described above and heat dried. For heat drying, the indigo leaves are placed in the drying chamber of a drying device. Then, while blowing high-temperature air heated by a burner, the indigo leaves are stirred. The air volume is set to such an extent that the indigo leaves fly in the drying chamber so that the air hits the entire indigo leaves uniformly.

[0032] In the above test, the extraction of tryptanthrin was carried out with glycerin water, but the following polyhydric alcohols can also be used as the extraction liquid instead of glycerin water. 1,3-Butylene glycol (1,3-butanediol): A dihydric alcohol having two alcohol groups, mainly used as a solvent for food flavors and a humectant in cosmetics and the like. Ethylene glycol (ethane-1,2-diol): A dihydric alcohol having two alcohol groups, mainly used as a coolant and an antifreeze. Propylene glycol (propane-1,2-diol): A dihydric alcohol having two alcohol groups, used in various fields such as foods, pharmaceuticals, and cosmetics. Erythritol: A tetrahydric alcohol having four alcohol groups, which may be produced naturally and is used as a substitute for sugar. Xylitol: A pentahydric alcohol having five alcohol groups, which has an effect of preventing the formation of dental calculus and is used in sweeteners and oral care products. Sorbitol: A hexavalent alcohol with six alcohol groups, which is used in foods, pharmaceuticals, cosmetics, etc.

Claims

1. A method for producing a blue leaf solution, characterized by obtaining a solution containing tryptanthrin from blue leaves using a polyhydric alcohol solution as an extractant.

2. The method for producing a blue leaf solution according to Claim 1, wherein the polyhydric alcohol is glycerin.

3. The method for producing a blue leaf solution according to Claim 1 or 2, wherein the extraction with the extractant is performed by shaking extraction.

4. A blue leaf solution containing tryptanthrin obtained from blue leaves using a polyhydric alcohol solution as an extractant.

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