Method for retaining estradiol

Abstract

The purpose of the present invention is to provide a method for retaining estradiol, which is an estrogen agonist having an estrogenic effect, in pomegranate seeds.　This method for retaining estradiol contained in pomegranate seeds is characterized by comprising: a step for extracting a solution, using an extraction solvent, from a pulverized sample obtained by pulverizing pomegranate seeds; a step for centrifuging the solution; and a step for drying the supernatant obtained by the centrifugation under reduced pressure. A preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention is characterized in that the extraction solvent is ethanol.

Description

Method for Retention of Estradiol

[0001] The present invention relates to a method for retaining estradiol, which retains estradiol from pomegranate seeds.

[0002] Pomegranate fruit has long been known to be beneficial to the human body and is commonly used in food and pharmaceuticals. It is considered that estrogens contained in pomegranate fruit are largely involved in such beneficial effects. In particular, estrogens are said to be useful for promoting skin health, preventing and improving Alzheimer's disease, and the like. These estrogens are female hormones synthesized and secreted in the human body, and estradiol, estriol, and estrone are three representative types thereof.

[0003] In recent years, based on the aforementioned beneficial effects, attempts have been made to extract and utilize estrogens from pomegranate fruit. As an extraction method thereof, for example, there is a method for obtaining a powdered pomegranate fruit component. Specifically, juice squeezed from the fruit is dried, or a pomegranate solution is extracted using an extraction solvent from powder obtained by crushing the fruit and removing moisture, and the extraction solvent is removed from the pomegranate solution.

[0004] For example, cosmetics that efficiently exhibit cosmetic effects by percutaneously absorbing female hormones, which are active ingredients contained in pomegranate fruit, are known (see, for example, Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2001-131053

[0006] However, in any of the extraction methods including the above-mentioned patent documents, extraction and retention of estrogen from discarded pomegranate seeds, rather than fruit, have not been achieved.

[0007] In addition, at present, in most cases, pomegranate seeds are basically treated as waste and are not effectively utilized, and the effective utilization of waste is being reconsidered.

[0008] On the other hand, hormones, including estrogen, are molecules released into the bloodstream from hormone-producing organs that exert biochemical effects at receptors on all cells in the body. Hormones control and regulate various functions such as metabolism, reproduction, growth, aging, and immunity. Even small amounts of hormones can have a significant effect. While optimal hormone secretion levels maintain youth and health, it is believed that a decrease in hormones is related to age-related physical ailments and decreased vitality.

[0009] Therefore, the present invention aims to provide a method for retaining estradiol, an estrogenic substance having estrogenic effects, in pomegranate seeds.

[0010] In order to achieve the above objective, the inventors of this invention diligently studied ways to effectively utilize pomegranate seeds, and as a result, arrived at the present invention.

[0011] In other words, the present invention provides a method for retaining estradiol contained in pomegranate seeds, comprising the steps of: extracting a solution from a pulverized sample obtained by crushing pomegranate seeds using an extraction solvent; centrifuging the solution; and drying the supernatant obtained by centrifuging under reduced pressure.

[0012] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the extraction solvent is characterized by being ethanol.

[0013] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the centrifugation conditions are characterized by being 1500 to 3000 rpm.

[0014] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the estradiol is characterized in that it is 17α-estradiol or 17β-estradiol.

[0015] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the supernatant is characterized by being dried under reduced pressure using a rotary evaporator or a vacuum dryer.

[0016] The present invention provides a method for retaining estradiol in pomegranate seeds, which has the advantage of enabling the effective utilization of pomegranate seeds that were conventionally treated as waste. Furthermore, the present invention provides a method for retaining estradiol in pomegranate seeds, which has the advantage of enabling the effective retention of estradiol, which would otherwise be considered a contaminant when mixed into cosmetics, health foods, or pharmaceuticals, in pomegranate seed extract.

[0017] The present invention provides a method for retaining estradiol contained in pomegranate seeds, comprising the steps of: extracting a solution from a pulverized sample obtained by crushing pomegranate seeds using an extraction solvent; centrifuging the solution; and drying the supernatant obtained by centrifuging under reduced pressure. The solution extracted from the pulverized sample obtained by crushing pomegranate seeds using an extraction solvent is pomegranate seed extract, which is an extract derived from pomegranate seeds.

[0018] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the estradiol is characterized in that it is 17α-estradiol or 17β-estradiol.

[0019] Estradiol has estrogen-like effects and exhibits the following properties: 1. It acts on estrogen receptors, thus influencing hormone-related pathways. 2. It affects reproductive function, influencing the regulation of the menstrual cycle and follicular development. 3. It regulates bone density, thus contributing to the prevention of osteoporosis. 4. It affects vascular function, promoting vasodilation and contributing to lower blood pressure and reduced arteriosclerosis. 5. It affects cholesterol, contributing to an increase in HDL and a decrease in LDL in the blood. 6. It has neuroprotective and cognitive protective effects, thus contributing to the prevention of neurodegenerative diseases such as Alzheimer's disease. 7. Regarding skin health, it promotes the synthesis of collagen and elastin. 8. It has anti-cancer effects and influences the immune system, thus providing a preventive effect against breast and ovarian cancer.

[0020] Therefore, a method for actively retaining estradiol, a useful component, in pomegranate seeds is desired. Surprisingly, according to the present invention, after investigating extraction methods, it was found that elastodiol can be effectively retained in pomegranate seed extract.

[0021] Here, we will first describe one example of a method for preparing pomegranate seed extract. First, prepare the pomegranate seeds. Wash and dry the pomegranate seeds as needed. It is preferable to dry them thoroughly, in order to ensure uniform grinding later.

[0022] Next, the pomegranate seeds are crushed. The crushing method is not particularly limited, and known crushing machines such as ball mills, hammer mills, roller mills, rod mills, sample mills, stamp mills, disintegrators, mortars, and blenders with cooling devices can be used. However, since the heat generated during crushing may cause decomposition of the pomegranate seed composition, the crushing time should be set to a few seconds and repeated more than ten times.

[0023] Next, the pomegranate seeds are crushed to obtain a pulverized product, which can then be immersed in various solvents. In a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the extraction solvent is ethanol, from the viewpoint of more effectively retaining estradiol.

[0024] When extracting the aforementioned solution, the pulverized sample and extraction solvent can be packed into a centrifuge tube. However, an extraction tube may also be used when extracting the aforementioned solution. In this case, the solution can be transferred to a centrifuge tube after extraction. The extraction solvent is, for example, ethanol, and if 50 g of pulverized sample is used, approximately 250 to 500 ml can be packed into the centrifuge tube. However, the packing amount can be appropriately changed depending on the amount of pulverized sample packed into the centrifuge tube, etc.

[0025] After the pulverized sample and extraction solvent have been packed into the centrifuge tube, a grinding blade may be inserted and the blade rotated at high speed around its axis. This allows the pulverized sample in the centrifuge tube to be ground and dispersed, and the solution to be extracted.

[0026] In this invention, immersion can be carried out under gentle stirring. Various solutions are obtained by immersing the pulverized material in various solvents. Stirring is performed on each solution according to its state, and in some cases the solution may be left to stand. When stirring is performed, there are no particular limitations, but stirring can be continued for 10 to 48 hours, preferably about 1 day (24 hours).

[0027] Pomegranate seed extract can then be obtained by separating the supernatant, but in a preferred embodiment of the present invention, the step of centrifuging the solution may be included.

[0028] The extracted solution can be centrifuged to separate the pulverized sample from the solution using a centrifuge. For example, the centrifugation conditions can be set to a rotation speed of 1500 to 3000 rpm, preferably 3000 rpm, and a rotation time of 15 to 25 minutes, for example, about 20 minutes. However, the centrifugation conditions may be appropriately adjusted depending on the volume of the solution, etc.

[0029] This centrifugation process ensures that the solution is reliably separated from the pulverized sample due to the difference in density. Only the supernatant of the separated solution can be transferred to a so-called pear-shaped flask or similar container. The amount of supernatant transferred can be adjusted as appropriate, for example, depending on the volume of the solution.

[0030] Furthermore, in a preferred embodiment of the method for retaining estradiol contained in pomegranate seeds according to the present invention, the supernatant is characterized by being dried under reduced pressure using a rotary evaporator or a vacuum dryer.

[0031] Furthermore, the supernatant can be evaporated to dryness if necessary. Evaporation to dryness can be carried out using an evaporator on a warm bath at 20°C to 60°C, preferably 37°C to 40°C. By evaporating to dryness, the pomegranate seed extract can be stored for a long period of time.

[0032] Furthermore, while conventional methods sometimes use separating agents in various extraction processes, the present invention's method for extracting estradiol allows for the simple and efficient elimination of the supernatant obtained by centrifuging with an evaporator or the like, without the use of filter media or filter paper.

[0033] An embodiment of the present invention will be described below, but the present invention is not limited to the embodiment described below.

[0034] Example 1: The following experiment was conducted to investigate a method for retaining estradiol, the active ingredient in pomegranate seeds. PSE-S and PSE-I were prepared as pomegranate seed extracts.

[0035] First, PSE-S was prepared as follows. PSE-S is an extract obtained by extracting from the seeds of Punica granatum L. with ethanol. 100g of crushed pomegranate seeds were mixed with 1000g of ethanol and extracted. After filtering the extract, the solvent was removed by distillation, and a 1,3-butylene glycol solution was added. The mixture was left to stand in the refrigerator for 1 to 10 days, and then filtered to obtain PSE-S. When incorporating it into cosmetics, etc., a two-filtration process was included in the manufacturing method to prevent turbidity and other issues, and the filtered product was used.

[0036] Furthermore, PSE-I was prepared as follows: Pomegranate seeds were powdered using a milling machine, extracted by permeation stirring with 99.5% ethanol for one day, and the supernatant obtained by centrifugation was dried under reduced pressure to obtain pomegranate seed ethanol extract (PSE: Punica granatum Seed Etanol extract). In this way, the extract was obtained by drying the supernatant obtained by centrifugation under reduced pressure using an evaporator without using any filter material or filter paper.

[0037] The supernatant was collected after centrifugation at room temperature at 3000 rpm for 20 minutes. A rotary evaporator was then used to dry the resulting supernatant under reduced pressure in a water bath set to 37°C.

[0038] Furthermore, regarding the preparation of the sample concentrations, the concentration of PSE-S after dissolution was adjusted to 400,000 μg / mL. The concentration of PSE-I after dissolution was adjusted to 200,000 μg / mL.

[0039] Using this as the basic concentration, as shown in Table 1 of the metabolome analysis results described below, the solution was adjusted to 100 µL or 200 µL with Milli-Q water, and then, for those requiring dilution when analyzed using each mode, dilution was performed before analysis. The term "metabolome" refers to the entire collection of low-molecular-weight chemical substances produced by intracellular metabolism, and besides nucleic acids (DNA) and proteins, it covers thousands of species including sugars, organic acids, and amino acids. Analysis of this metabolome may enable the prediction of disease onset. Table 1 shows the metabolome analysis results.

[0040] Table 1 is described as follows. A01 is a sample prepared using PSE-S, and B01 is a sample prepared using PSE-I. The sample amount indicates the injection volume into CE-TOFMS and LC-TOFMS (total volume combining the sample and Milli-Q water).

[0041] <CE-TOFMS Analysis> This is an apparatus that separates components using electric force and measures mass. CE (capillary electrophoresis) separates substances by flowing a sample through a narrow tube; when an electric field is applied, the migration speed differs for each component, enabling separation. TOFMS (time-of-flight mass spectrometry) is an apparatus that measures the mass of separated components. Examples of characteristic components that can be analyzed include amino acids and organic acids.

[0042] <LC-TOFMS Analysis> LC (liquid chromatography) is an apparatus that flows a sample through a narrow tube (column) and separates components based on chemical interactions. It can analyze a wide range of characteristic components including lipids, sugars, and flavonoids, and steroid hormones including estradiol can also be analyzed by LC (liquid chromatography).

[0043] The dilution factor indicates the dilution ratio used for each measurement mode (dilution is only applied to samples that require dilution after preparation with the initial sample amount and Milli-Q water (ultrapure water); in the present case, 5-fold dilution was used in Anion mode).

[0044] <Cation Mode> This is a mode for detecting cations. It is mainly used for analyzing cationic metabolites such as amino acids and polyamines.

[0045] <Anion Mode> This is a mode for detecting anions. It is mainly used for analyzing ionic metabolites such as organic acids and phosphorylated metabolites.

[0046] <Positive Ion Mode> This is a detection mode in which molecules are positively charged for detection in a mass spectrometer. Most metabolites are detected in this mode. It is mainly applied to the analysis of lipids, amino acids, saccharides and the like.

[0047] <Negative Ion Mode> This is a detection mode in which molecules are negatively charged for detection. It is mainly applied to the analysis of organic acids, fatty acids, sugar phosphate compounds, flavonoids and the like.

[0048] In measurement by LC-MS, if the sample concentration is too high, signal saturation and ion suppression will occur, so it is necessary to prepare the sample within an appropriate concentration range. For this reason, measurement is carried out after examining the dilution ratio for each measurement mode.

[0049] In CE-TOFMS analysis, separation is performed using an electric field by a method called capillary electrophoresis, so dilution with a buffer having appropriate ionic strength and dielectric constant is required, and Milli-Q water was used in the present study. In addition, in CE-TOFMS analysis, the injection volume of a sample into the analytical instrument is 200 μL, which means a larger amount of solvent than in LC analysis is used to ensure the sample conductivity and separation efficiency.

[0050] On the other hand, LC-TOFMS analysis enables high-sensitivity measurement, so the analysis was performed with a total sample volume of 100 μL.

[0051] Regarding the analysis of estradiol in PSE-S and PES-I, the results of metabolome analysis are shown in Table 2.

[0052]

[0053] In Table 2, ID consists of the initial letter of the measurement mode and a sequential number, where C indicates cation mode, A indicates anion mode, P indicates positive mode, and N indicates negative mode. ND stands for Not Detected, indicating that the sample was included in the analysis but was below the detection limit. NA stands for Not Available, indicating that the sample was included in the calculation but could not be calculated due to insufficient data.

[0054] Furthermore, † indicates candidate compounds obtained by matching the m / z of the detection peak with the HMT database using MT or RT. FA indicates fatty acids, AC indicates acylcarnitines, and the numbers in parentheses indicate the number of carbon atoms and double bonds. ¶: The ratio of the average detection values ​​between the two groups is calculated using the latter as the denominator.

[0055] In the analysis results, PSE stands for "Punica granatum Seed Extract." 17α-Estradiol was detected in the PSE-I sample, while "ND" indicates it was undetectable in the PSE-S sample. This is likely because PSE-S undergoes two filtration steps during its manufacturing process to prevent turbidity when incorporated into cosmetics, and the 17α-Estradiol is removed in the residue. PSE-I, on the other hand, does not undergo a filtration process, which is likely why 17α-Estradiol was detected. Although its physiological activity is weaker compared to the active β-form, it is an important component contributing to bone and skin health and can therefore be incorporated into functional foods.

[0056] Furthermore, since methyl alcohol is not used in the solvent extraction process, it can be incorporated into various compositions.

[0057] According to the present invention, it is suitable for manufacturing products such as cosmetics, health foods, or pharmaceuticals that contain estrogen-acting substances, and has high industrial value.

Claims

1. A method for removing estradiol contained in pomegranate seeds, comprising the steps of: extracting a solution from a pulverized sample obtained by crushing pomegranate seeds using an extraction solvent; centrifuging the solution; and drying the supernatant obtained by centrifuging under reduced pressure.

2. The method for removing residual estradiol according to claim 1, characterized in that the extraction solvent is ethanol.

3. The method for removing estradiol according to claim 1, characterized in that the centrifugal separation conditions are 1500 to 3000 rpm.

4. The method for residual estradiol according to claim 1, characterized in that the estradiol is 17α-estradiol or 17β-estradiol.

5. The method for retaining estradiol according to claim 1, characterized in that the supernatant is dried under reduced pressure using a rotary evaporator or a vacuum dryer.

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