JP7398516B2 - How to extract Atsubanori - Google Patents
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- JP7398516B2 JP7398516B2 JP2022099480A JP2022099480A JP7398516B2 JP 7398516 B2 JP7398516 B2 JP 7398516B2 JP 2022099480 A JP2022099480 A JP 2022099480A JP 2022099480 A JP2022099480 A JP 2022099480A JP 7398516 B2 JP7398516 B2 JP 7398516B2
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Description
本発明は、アツバノリの抽出方法、アツバノリの抽出物及びその用途、特に、超臨界流体による抽出により製造されるアツバノリの抽出物に関する創作である。 The present invention is a creation relating to a method for extracting Nori spp., an extract of Nori spp. and its uses, particularly an extract of Nori spp. produced by extraction with a supercritical fluid.
アツバノリ（Ｓａｒｃｏｄｉａ）は、暗赤色の扁平状の藻体であり、現在、アツバノリには、食物繊維、ミネラル、人体に必須のアミノ酸、ビタミンＥ、オメガ脂肪酸その他の栄養素が豊富に含まれていることが知られている。よって、アツバノリは、人体の健康に有益である健康食品とされている。 Sarcodia is a dark red, flat-shaped algae that is currently rich in dietary fiber, minerals, amino acids essential to the human body, vitamin E, omega fatty acids, and other nutrients. It has been known. Therefore, Atsubanori is considered to be a health food that is beneficial to human health.
しかしながら、現在、アツバノリは、栄養素を豊富に有し、健康食品としての利用に適していることが知られているが、未だにアツバノリから高い経済価値を有する成分は抽出されていない。よって、如何に新たな抽出方法を提供して、アツバノリから高い経済価値を有する成分を抽出するのかは、依然として未解決の課題である。 However, although Atsuba nori is currently known to be rich in nutrients and suitable for use as a health food, components with high economic value have not yet been extracted from Atsubanori. Therefore, it is still an unresolved problem how to provide a new extraction method to extract components with high economic value from Atsubanori.
本発明の目的は、前記問題に対処し、（ａ）乾燥したアツバノリの粉末を提供する、（ｂ）二酸化炭素流体で当該乾燥したアツバノリの粉末に対して抽出を行って抽出アツバノリの抽出液を取得し、当該アツバノリの抽出液にアツバノリの抽出物と当該二酸化炭素流体溶剤とが含有され、抽出圧力は３００－６５０ｂａｒであり、抽出温度は４５－５５℃であり、抽出時間は２－６時間である、及び（ｃ）当該アツバノリの抽出液から当該アツバノリの抽出物を分離し、分離圧力は３０－１００ｂａｒである、前記（ａ）から（ｃ）の工程を含むことを特徴とするアツバノリの抽出物の抽出方法を提供することである。 The purpose of the present invention is to address the above-mentioned problems and to (a) provide a dried Atsubanori powder; (b) extract the dried Atsubanori powder with a carbon dioxide fluid to obtain an extracted Atsubanori extract; obtained, the extract of Aspergillus orientalis contains the extract of Aspergillus orensis and the carbon dioxide fluid solvent, the extraction pressure is 300-650 bar, the extraction temperature is 45-55°C, and the extraction time is 2-6 hours. and (c) separating the extract of Atsubanori from the extract of Atsubanori, and the separation pressure is 30-100 bar. An object of the present invention is to provide a method for extracting an extract.
上述に記載の方法の通り、抽出圧力は３５０ｂａｒであり、抽出温度は５５℃であり、抽出時間は２時間である。 As per the method described above, the extraction pressure is 350 bar, the extraction temperature is 55°C and the extraction time is 2 hours.
上述に記載の方法の通り、抽出圧力は３６５ｂａｒであり、抽出温度は５５℃であり、抽出時間は４時間である。 As per the method described above, the extraction pressure is 365 bar, the extraction temperature is 55°C and the extraction time is 4 hours.
上述に記載の方法の通り、抽出圧力は４２０ｂａｒであり、抽出温度は５５℃であり、抽出時間は４時間である。 As per the method described above, the extraction pressure is 420 bar, the extraction temperature is 55°C and the extraction time is 4 hours.
前記目的及びその他目的を達成するために、本発明は、当該アツバノリ抽出物を提供し、当該アツバノリ抽出物は以上に記載の抽出方法によって製造される。 In order to achieve the above object and other objects, the present invention provides the same type of extract, which is produced by the extraction method described above.
前記目的及びその他目的を達成するために、本発明は、当該アツバノリ抽出物の濃度は５μｇ／ｍＬ以上であることを特徴とする毛包細胞の成長活性を促進する薬物を調製する用途のための、前記アツバノリ抽出物を提供する。 In order to achieve the above object and other objects, the present invention provides a method for preparing a drug that promotes the growth activity of hair follicle cells, characterized in that the concentration of the Atsuba Nori extract is 5 μg/mL or more. , provides the above-mentioned Atsubanori extract.
前記目的及びその他目的を達成するために、本発明は、当該アツバノリ抽出物の濃度は０．５ｍｇ／ｍＬ以上であることを特徴とするアクネ桿菌の成長を抑制する薬物又は化粧品を調製する用途のための前記アツバノリ抽出物を提供する。 In order to achieve the above object and other objects, the present invention provides a method for preparing a drug or cosmetic product for inhibiting the growth of acnes bacteria, characterized in that the concentration of the Acne laver extract is 0.5 mg/mL or more. The present invention provides the above-mentioned Atsubanori extract.
前記目的及びその他目的を達成するために、本発明は、当該アツバノリ抽出物の濃度は１μｇ／ｍＬ以上であり、角質細胞の移動活性を増進する薬物又は化粧品を調製する用途のための前記アツバノリ抽出物を提供する。 In order to achieve the above object and other objects, the present invention provides a method for preparing a drug or cosmetic product, wherein the concentration of the Atsuba nori extract is 1 μg/mL or more, and the above-mentioned Atsuba nori extract is for use in preparing a drug or cosmetic that enhances the migratory activity of corneocytes. provide something.
前記目的及びその他目的を達成するために、本発明は、当該アツバノリ抽出物の濃度は６μｇ／ｍＬ以上であり、抽出物角質細胞の成長活性を増進する薬物又は化粧品を調製する用途のためのアツバノリの抽出物を提供する。 In order to achieve the above object and other objects, the present invention provides that the concentration of the Atsuba nori extract is 6 μg/mL or more, and the Atsuba nori extract is for use in preparing a drug or cosmetic that enhances the growth activity of corneocytes. provides an extract of
本発明は、上述のようなアツバノリの抽出物、その抽出方法及びその用途によって、新たな抽出方法を提供することで、アツバノリから高い経済価値を有する成分を抽出することができる。 The present invention provides a new extraction method using the above-mentioned Atsubanori extract, its extraction method, and its use, thereby making it possible to extract components with high economic value from Atsubanori.
以下、本発明の目的、特徴および効果を十分に理解するために、以下の具体的な実施例によって、添付された図面とともに、本発明について詳細について以下にて説明する。 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to fully understand the objects, features and effects of the present invention, the present invention will be described in detail below by way of specific examples and in conjunction with the accompanying drawings.
実施例１のアツバノリの抽出物の抽出方法 Extraction method for extract of Atsubanori of Example 1
本実施例１のアツバノリの抽出物の抽出方法の略述は、以下の通りである。 A brief description of the method for extracting the Atsubanori extract of Example 1 is as follows.
まず、乾燥したアツバノリの粉末を提供する。本実施例１における乾燥したアツバノリの粉末は、アツバノリの粉末の一般的な調製方法で製造されるが、市販のものを取得することも可能である。アツバノリの粉末の一般的な調製方法は、以下の通り、新鮮なアツバノリを収穫した後、洗浄し、アツバノリに乾燥処理を行い、乾燥処理の方法はアツバノリをオーブンに入れ５０℃で２４時間持続的に乾燥し行う、乾燥処理後のアツバノリは粉砕機で粉砕して粉にすることで、アツバノリの粉末を調製し、アツバノリの粉末の粒径は、より好ましくは、１－４ｍｍであるが、その他の必要に応じてアツバノリの粉末の粒径を調整することができる。 First, provide dried Atsubanori powder. The dried Atsubanori powder in Example 1 is produced by a general method for preparing Atsubanori powder, but it is also possible to obtain a commercially available powder. The general method for preparing Atsubanori powder is as follows: After harvesting fresh Atsubanori, the Atsubanori is washed, and the Atsubanori is subjected to a drying process. After the drying process, the Atsubanori powder is prepared by pulverizing the Atsubanori powder with a grinder, and the particle size of the Atsubanori powder is preferably 1 to 4 mm, but other The particle size of Atsubanori powder can be adjusted according to needs.
そして、超臨界流体の抽出設備を用意し、前記超臨界流体の抽出設備は金属工業研究発展センター（ＭＩＲＤＣ）により提供されたものであり、モデル番号はＳＦＥ－３５０Ｓ－１０００Ｒである。超臨界流体の抽出設備を使用して前記アツバノリの粉末からアツバノリの抽出物を抽出する。抽出方法は下記の通りであり、前記アツバノリの粉末を当該超臨界流体の抽出設備の抽出槽に入れ、アツバノリの粉末の使用量は抽出槽体の約９０－９９％の体積である（アツバノリの粉末の供給体積は８５０ｇから９５０ｇであり、抽出槽体の体積は１．２Ｌである）。そして、超臨界流体作を溶剤として、超臨界流体の抽出設備の操作マニュアルが提供する操作方法に基づき、当該乾燥したアツバノリの粉末に対して抽出を行い、本実施例１において使用される超臨界流体は二酸化炭素流体であり、抽出槽における抽出圧力は３５０ｂａｒであり（製造工程の必要に応じて３００ｂａｒ－６５０ｂａｒの間で調整することができる）、抽出槽における抽出温度は５５℃であり（抽出温度は、製造工程の必要に応じて、４５℃－５５℃の間で調整することができる）、抽出時間は２時間であり、二酸化炭素流体の流量は１０Ｌ／ｈに設定したが、抽出の製造工程の必要に応じて、二酸化炭素流体の流量は５～１５Ｌ／ｈの範囲の間に設定することができる。抽出完了すると、アツバノリの抽出液を取得することができ、当該アツバノリの抽出液にはアツバノリの抽出物と当該二酸化炭素流体溶剤が含有され、当該アツバノリの抽出物は当該二酸化炭素流体溶剤に溶解している。 A supercritical fluid extraction equipment was provided, and the supercritical fluid extraction equipment was provided by the Metal Industry Research and Development Center (MIRDC), and the model number was SFE-350S-1000R. A supercritical fluid extraction equipment is used to extract an extract of Aspergillus laver from the powder of Aspergillus laver. The extraction method is as follows: the Atsubanori powder is placed in the extraction tank of the supercritical fluid extraction equipment, and the amount of Atsubanori powder used is about 90-99% of the volume of the extraction tank (Atsubanori powder is The feed volume of powder is 850 g to 950 g, the volume of the extraction tank body is 1.2 L). Then, using supercritical fluid as a solvent, extraction was performed on the dried Atsubanori powder based on the operating method provided in the operating manual of the supercritical fluid extraction equipment, and the supercritical fluid used in Example 1 was extracted. The fluid is carbon dioxide fluid, the extraction pressure in the extraction tank is 350 bar (can be adjusted between 300 bar and 650 bar according to the needs of the manufacturing process), and the extraction temperature in the extraction tank is 55 °C (extraction The temperature can be adjusted between 45 °C and 55 °C according to the needs of the manufacturing process), the extraction time was 2 h, and the flow rate of carbon dioxide fluid was set at 10 L/h, but the Depending on the needs of the manufacturing process, the flow rate of the carbon dioxide fluid can be set between 5 and 15 L/h. When the extraction is completed, an extract of Atsubanori can be obtained, and the Atsubanori extract contains an extract of Atsubanori and the carbon dioxide fluid solvent, and the extract of Atsubanori is dissolved in the carbon dioxide fluid solvent. ing.
最後に、当該アツバノリの抽出液を当該超臨界流体の抽出設備の分離槽に送入して減圧を行い、分離圧力は３５ｂａｒであり（分離圧力はアツバノリの抽出物の分離要件に基づいて３０－１００ｂａｒの間で調整することができる）、これにより、当該アツバノリの抽出液からアツバノリの抽出物を分離することができ、当該アツバノリの抽出物と二酸化炭素流体溶剤とを分離した後、エタノールで当該超臨界流体の抽出設備の抽出配管を洗浄し、当該抽出配管に付着した当該アツバノリの抽出物を洗浄する、即ち、当該アツバノリの抽出物をエタノールに溶解させ、当該アツバノリの抽出物を含むエタノールを当該抽出配管から流出させ、これにより、当該アツバノリの抽出物を収集し、エタノールに融解した当該アツバノリの抽出物を室温下に置き、エタノールが室温で揮発するまで待てば、当該アツバノリの抽出物を取得することができる。 Finally, the extract of Atsubanori is sent to the separation tank of the supercritical fluid extraction equipment to reduce the pressure, and the separation pressure is 35 bar (the separation pressure is 30-bar based on the separation requirements of the extract of Atsubanori). (can be adjusted between 100 bar), thereby making it possible to separate the Atsubanori extract from the Atsubanori extract, and after separating the Atsubanori extract from the carbon dioxide fluid solvent, the The extraction piping of the supercritical fluid extraction equipment is cleaned, and the extract of Atsuba nori adhering to the extraction pipe is cleaned. In other words, the extract of Atsuba nori is dissolved in ethanol, and the ethanol containing the extract of Atsuba nori is dissolved. Collect the Atsubanori extract by discharging it from the extraction pipe, place the Atsubanori extract dissolved in ethanol at room temperature, wait until the ethanol evaporates at room temperature, and collect the Atsubanori extract. can be obtained.
実施例１のアツバノリの抽出物のＨＰＬＣ分析 HPLC analysis of Atsubanori extract of Example 1
実施例１のアツバノリの抽出物中の成分を分析するために、ＨＰＬＣカラムで分析を行う。分析条件は以下の通りである。ＨＰＬＣカラムＰｏｒｏｓｈｅｌｌＨＰＨ－Ｃ１８８（４．６ｘ１００ｍｍ、２．７μｍ）を使用し、流速は１ｍｌ／ｍｉｎであり、紫外線／可視光線検出器Ａｇｉｌｅｎｔ１２６０ＤＡＤを使用して検出を行い（検出過程は、Ａｇｉｌｅｎｔ１２６０ＤＡＤの操作マニュアルを参照されたい）、検出光は波長２１０ｎｍ、２５４ｎｍ及び２８０ｎｍのＵＶ光に設定した。アツバノリの抽出物のＨＰＬＣ指紋プロファイルは、図１－３に示す通りである。 In order to analyze the components in the Atsubanori extract of Example 1, analysis is performed using an HPLC column. The analysis conditions are as follows. HPLC column Poroshell HPH-C188 (4.6 x 100 mm, 2.7 μm) was used, the flow rate was 1 ml/min, and detection was performed using an ultraviolet/visible light detector Agilent 1260DAD (the detection process was performed according to the Agilent 1260DAD operation manual). ), the detection light was set to UV light with wavelengths of 210 nm, 254 nm, and 280 nm. The HPLC fingerprint profile of the Atsubanori extract is as shown in Figures 1-3.
実施例１のアツバノリの抽出物のＧＣ／ＭＳ分析 GC/MS analysis of Atsubanori extract of Example 1
前記アツバノリの抽出物をガスクロマトグラフ（ＧＣ）（Ａｇｉｌｅｎｔ７８９０ＢＧＣ／５９７７ＢＥＩＭａｓｓＳｐｅｃ検出器）に置いて分析を行い、より明瞭なＧＣプロファイルを取得するため、前記アツバノリの抽出物のサンプルを別途採取してシリル化の前処理を行い、同一の方法でガスクロマトグラフィー分析を行い、前記ガスクロマトグラフィーが使用する分析カラムはＺＢ－１ＸＴＳＩＭＤＩＳＴｃｏｌｕｍｎ（５ｍｘ０．５３ｍｍＩＤ、０．０９μｍ膜厚）であり、ガスクロマトグラフィーの分析結果は、図４に示す通りであり、当該アツバノリの抽出物にはミリスチン酸、パルミチン酸、ステアリン酸等の脂肪酸及び植物ステロールが含有されている。 The Atsubanori extract was analyzed by placing it in a gas chromatograph (GC) (Agilent 7890BGC/5977BEIMassSpec detector). In order to obtain a clearer GC profile, a sample of the Atsubanori extract was separately collected and analyzed for silylation. Pretreatment was performed, and gas chromatography analysis was performed using the same method. The analytical column used in the gas chromatography was ZB-1XTSIMDIST column (5 m x 0.53 mm ID, 0.09 μm film thickness), and the gas chromatography analysis results were as follows. , as shown in FIG. 4, and the extract of Norifolia oleracea contains fatty acids such as myristic acid, palmitic acid, and stearic acid, and plant sterols.
実施例１のアツバノリの抽出物によるヒト毛包真皮乳頭細胞の細胞成長を促進する試験 Test for promoting cell growth of human hair follicle dermal papilla cells using the extract of Atsubanori of Example 1
まず、前記抽出方法でアツバノリの抽出物を調製すると同時に、ヒト毛包真皮乳頭細胞（ＦｏｌｌｉｃｌｅＤｅｒｍａｌＰａｐｉｌｌａＣｅｌｌ）細胞株ＨＦＤＰＣ（ＰｒｏｍｏＣｅｌｌ社から購入したものであり、製品モデルはＣ１２０７１である）を用意する。 First, an extract of Atsubanori is prepared using the above extraction method, and at the same time, a human follicle dermal papilla cell cell line HFDPC (purchased from PromoCell, product model C12071) is prepared.
そして、９６穴の細胞培養皿を用意し、そのうちの９穴にＨＦＤＰＣ細胞を含有する細胞液（毛包真皮乳頭細胞の成長培養基ＦｏｌｌｉｃｌｅＤｅｒｍａｌＰａｐｉｌｌａＣｅｌｌＧｒｏｗｔｈＭｅｄｉｕｍ（ＰｒｏｍｏＣｅｌｌ、Ｃ－２６５０１））を加え、各穴には５－８×１０３個の細胞が含有されており、三穴を一群とし、前記ＨＦＤＰＣ細胞液を加えた９穴を制御群、実験群１及び実験群２に分け、制御群にはアツバノリの抽出物を完全に加えず、実験群１に最終濃度が５μｇ／ｍＬであるアツバノリの抽出物を加え、実験群２に最終濃度が２５μｇ／ｍＬであるアツバノリの抽出物を加えた。実験群１及び実験群２にアツバノリの抽出物を加えた後、細胞培養皿を細胞培養箱に入れて３７℃の環境で４８時間培養し、それぞれ培養から２４時間目及び４８時間目に、各穴からサンプルを採取して、細胞の生存率に関する試験（ＭＴＴ試験）により実験群１及び実験群２の細胞増加率を検査した。計算方法は（実験群の吸光値（１又は２）／制御群の吸光値）ｘ１００である。 Then, a 96-well cell culture dish was prepared, and a cell solution containing HFDPC cells (Follicle Dermal Papilla Cell Growth Medium (PromoCell, C-26501)) containing HFDPC cells was added to 9 of the holes, and 5 cells were added to each hole. - Contains 8 x 10 3 cells, 3 holes are divided into a group, 9 holes added with the HFDPC cell solution are divided into a control group, experimental group 1 and experimental group 2, and the control group contains Atsubanori extract. Without completely adding the substance, to experimental group 1, the extract of Atsuba nori with a final concentration of 5 μg/mL was added, and to the experimental group 2, the extract of Atsuba nori with a final concentration of 25 μg/mL was added. After adding Atsubanori extract to Experimental Group 1 and Experimental Group 2, the cell culture dishes were placed in a cell culture box and cultured for 48 hours at 37°C. Samples were taken from the holes and the cell growth rates of Experimental Group 1 and Experimental Group 2 were examined by a test for cell viability (MTT test). The calculation method is (experimental group absorbance value (1 or 2)/control group absorbance value) x 100.
試験結果は以下の表一及び図５に示すとおりであり、培養から２４時間目における実験１の制御群に対する細胞成長率は１０６．５％であり、培養から４８時間目における実験１の制御群に対する細胞成長率は１０８．３％であり、培養から２４時間目における実験２の制御群に対する細胞成長率は１１３．１％であり、培養から４８時間目における実験２の制御群に対する細胞成長率は１０９．２％であった。 The test results are as shown in Table 1 and Figure 5 below, and the cell growth rate for the control group in Experiment 1 at 24 hours after culture was 106.5%, and the cell growth rate for the control group in Experiment 1 at 48 hours after culture. The cell growth rate for the control group in Experiment 2 at 24 hours after culture was 113.1%, and the cell growth rate for the control group in Experiment 2 at 48 hours after culture. was 109.2%.
実施例２のアツバノリの抽出物によるヒト毛包真皮乳頭細胞の細胞成長を促進する試験 Test for promoting cell growth of human hair follicle dermal papilla cells using the extract of Atsubanori of Example 2
まず、実施例１のアツバノリの抽出方法に基づくが、抽出圧力を３６５ｂａｒに調整することで、実施例２のアツバノリの抽出物を調製すると同時に、ヒト毛包真皮乳頭細胞の細胞株ＨＦＤＰＣ（ＰｒｏｍｏＣｅｌｌ社から購入したものであり、製品モデルはＣ１２０７１である）を用意する。 First, based on the extraction method of Atsuba nori in Example 1, the extract of Atsuba nori in Example 2 was prepared by adjusting the extraction pressure to 365 bar. The product model is C12071).
そして、細胞培養皿を用意し、そのうちの９穴にＨＦＤＰＣ細胞を含有する細胞液を加え、三穴を一群とし、前記ＨＦＤＰＣ細胞液を加えた９穴を制御群、実験群１及び実験群２に分け、制御群にはアツバノリの抽出物を完全に加えず、実験群１に最終濃度が５μｇ／ｍＬであるアツバノリの抽出物を加え、実験群２に最終濃度が２５μｇ／ｍＬであるアツバノリの抽出物を加えた。実験群１及び実験群２にアツバノリの抽出物を加えた後、細胞培養皿を細胞培養箱に入れて３７℃の環境で４８時間培養し、それぞれ培養から２４時間目及び４８時間目に、各穴からサンプルを採取して、ＭＴＴ試験により各群の細胞増加率を検査した。 Then, a cell culture dish was prepared, and a cell solution containing HFDPC cells was added to 9 of the wells to form a group of three holes, and the 9 holes to which the HFDPC cell solution was added were a control group, an experimental group 1, and an experimental group 2. To the control group, the extract of Atsuba nori was not added completely, to the experimental group 1, the extract of Atsuba nori with a final concentration of 5 μg/mL was added, and to the experimental group 2, the extract of Atsuba nori with a final concentration of 25 μg/mL was added. Added extract. After adding Atsubanori extract to Experimental Group 1 and Experimental Group 2, the cell culture dishes were placed in a cell culture box and cultured for 48 hours at 37°C. Samples were taken from the holes and the cell increase rate of each group was examined by MTT test.
試験結果は図６に示す通りであり、培養から２４時間目における制御群に対する実験群１の細胞成長率は１０７．２％であり、培養から４８時間目における制御群に対する実験群１の細胞成長率は１１１．６％であり、培養から２４時間目における制御群に対する実験２の細胞成長率は１２５．７％であり、培養から４８時間目における制御群に対する実験２の細胞成長率は１４５．９％であった。 The test results are as shown in Figure 6, the cell growth rate of experimental group 1 compared to the control group at 24 hours after culture was 107.2%, and the cell growth rate of experimental group 1 compared to the control group at 48 hours after culture The cell growth rate in experiment 2 relative to the control group at 24 hours after culture was 125.7%, and the cell growth rate in experiment 2 relative to the control group at 48 hours after culture was 145. It was 9%.
実施例３のアツバノリの抽出物によるヒト毛包真皮乳頭細胞の細胞成長を促進する試験 Test for promoting cell growth of human hair follicle dermal papilla cells using the extract of Noriflora cerevisiae in Example 3
まず、実施例１のアツバノリの抽出方法に基づくが、抽出圧力を４２０ｂａｒに調整することで実施例３のアツバノリの抽出物を調製すると同時に、ヒト毛包真皮乳頭細胞の細胞株ＨＦＤＰＣ（ＰｒｏｍｏＣｅｌｌ社から購入したものであり、製品モデルはＣ１２０７１である）を用意した。 First, based on the extraction method of Atsubanori of Example 1, the extract of Atsubanori of Example 3 was prepared by adjusting the extraction pressure to 420 bar. (The product model is C12071) was prepared.
そして、細胞培養皿を用意し、そのうちの９穴にＨＦＤＰＣ細胞を含有する細胞液を加え、三穴を一群とし、前記ＨＦＤＰＣ細胞液を加えた９穴を制御群、実験群１及び実験群２に分け、制御群にはアツバノリの抽出物を完全に加えず、実験群１に最終濃度が５μｇ／ｍＬであるアツバノリの抽出物を加え、実験群２に最終濃度が２５μｇ／ｍＬであるアツバノリの抽出物を加えた。実験群１及び実験群２にアツバノリの抽出物を加えた後、細胞培養皿を細胞培養箱に入れて３７℃の環境で４８時間培養し、それぞれ培養から２４時間目及び４８時間目に、各穴からサンプルを採取して、ＭＴＴ試験により各群の細胞増加率を検査した。 Then, a cell culture dish was prepared, and a cell solution containing HFDPC cells was added to 9 of the wells to form a group of three holes, and the 9 holes to which the HFDPC cell solution was added were a control group, an experimental group 1, and an experimental group 2. To the control group, the extract of Atsuba nori was not added completely, to the experimental group 1, the extract of Atsuba nori with a final concentration of 5 μg/mL was added, and to the experimental group 2, the extract of Atsuba nori with a final concentration of 25 μg/mL was added. Added extract. After adding Atsubanori extract to Experimental Group 1 and Experimental Group 2, the cell culture dishes were placed in a cell culture box and cultured for 48 hours at 37°C. Samples were taken from the holes and the cell increase rate of each group was examined by MTT test.
試験結果は図７に示す通りであり、培養から２４時間目における制御群に対する実験群１の細胞成長率は１０５．６％であり、培養から４８時間目における制御群に対する実験群１の細胞成長率は１１０．３％であり、培養から２４時間目における制御群に対する実験２の細胞成長率は１１６．２％であり、培養から４８時間目における制御群に対する実験２の細胞成長率は１２６．１％であった。 The test results are as shown in Figure 7, the cell growth rate of experimental group 1 relative to the control group at 24 hours after culture was 105.6%, and the cell growth rate of experimental group 1 relative to the control group at 48 hours after culture was 105.6%. The cell growth rate in Experiment 2 relative to the control group at 24 hours after culture was 116.2%, and the cell growth rate in Experiment 2 relative to the control group at 48 hours after culture was 126. It was 1%.
実施例４の異なるアツバノリの抽出物のＨＰＬＣ指紋プロファイルの比較 Comparison of HPLC fingerprint profiles of different Atsubanori extracts of Example 4
まず、比較のサンプルとしてのアツバノリの抽出物Ａ－Ｅを提供する。 First, extracts A to E of Aspen laver are provided as samples for comparison.
アツバノリの抽出物Ａは、前記実施例１におけるアツバノリの抽出物の抽出方法に基づき、抽出条件を以下の条件の通り設定して調製したものである。抽出溶剤は二酸化炭素流体であり、抽出槽における抽出圧力は３５０ｂａｒであり、抽出槽における抽出温度は５５℃であり、抽出時間は６時間である。 The extract A of Lamium laver was prepared based on the method for extracting the extract of Lamina laver in Example 1, and the extraction conditions were set as follows. The extraction solvent is carbon dioxide fluid, the extraction pressure in the extraction tank is 350 bar, the extraction temperature in the extraction tank is 55°C, and the extraction time is 6 hours.
アツバノリの抽出物Ｂは、前記実施例１におけるアツバノリの抽出物の抽出方法に基づき、抽出条件を以下の条件の通り設定して調製したものである。抽出溶剤は二酸化炭素流体であり、抽出槽における抽出圧力は６５０ｂａｒであり、抽出槽における抽出温度は５５℃であり、抽出時間は２時間である。 The extract B of Lamina laver was prepared based on the method for extracting the extract of Lamina laver in Example 1, with the extraction conditions set as follows. The extraction solvent is carbon dioxide fluid, the extraction pressure in the extraction tank is 650 bar, the extraction temperature in the extraction tank is 55°C, and the extraction time is 2 hours.
アツバノリの抽出物Ｃは、酢酸エチルで抽出したアツバノリの抽出物であり、抽出温度は５０℃であり、抽出圧力は１ｂａｒであり、抽出時間は６時間である。 The extract C of Nori spp. is an extract of Nori spp. extracted with ethyl acetate, the extraction temperature is 50° C., the extraction pressure is 1 bar, and the extraction time is 6 hours.
アツバノリの抽出物Ｄは、純水で抽出したアツバノリの抽出物であり、抽出温度は５０℃であり、抽出圧力は１ｂａｒであり、抽出時間は６時間である。 The extract D of Lamium laver is extracted with pure water, the extraction temperature is 50°C, the extraction pressure is 1 bar, and the extraction time is 6 hours.
アツバノリの抽出物Ｅは、９５％エタノールで抽出したアツバノリの抽出物であり、抽出温度は５０℃であり、抽出圧力は１ｂａｒであり、抽出時間は６時間である。 The extract E of Lamium laver is extracted with 95% ethanol, the extraction temperature is 50°C, the extraction pressure is 1 bar, and the extraction time is 6 hours.
そして、前記アツバノリの抽出物Ａ－ＥのＨＰＬＣ分析を行った。分析条件は以下の通りである。ＨＰＬＣカラムＴＳＫｇｅｌＯＤＳ－１００Ｓ（２５０ｘ４．６ｍｍ）を使用し、流速は１ｍｌ／ｍｉｎであり、紫外線／可視光線検出器を使用して検出を行い、検出光は波長２８０ｎｍのＵＶ光に設定した。 Then, HPLC analysis of the above-mentioned Atsubanori extracts A to E was performed. The analysis conditions are as follows. HPLC column TSKgelODS-100S (250 x 4.6 mm) was used, the flow rate was 1 ml/min, and detection was performed using an ultraviolet/visible light detector, and the detection light was set to UV light with a wavelength of 280 nm.
分析結果は図８に示す通りであり、図中の各種アツバノリの抽出物のＨＰＬＣ指紋プロファイル波頭から見ると、同様に、超臨界流体を用いるが、異なる抽出圧力で抽出したアツバノリの抽出物Ａ、Ｂはその内容物の成分が比較的近く、酢酸エチル、水及びエタノールを溶剤として抽出したアツバノリの抽出物Ｃ－Ｅの内容物の成分は、アツバノリの抽出物Ａ、Ｂの内容物の成分と異なる。 The analysis results are as shown in Figure 8, and looking from the HPLC fingerprint profile wave front of various Atsubanori extracts in the figure, the Atsubanori extract A, which was similarly extracted using supercritical fluid but with different extraction pressures; The components of the contents of B are relatively similar, and the components of the contents of the Atsubanori extract CE extracted using ethyl acetate, water, and ethanol as a solvent are the same as those of the Atsubanori extracts A and B. different.
実施例５のアツバノリの抽出物のアクネ桿菌の成長を抑制する試験 Test for inhibiting the growth of P. acnes using the extract of Example 5
まず、実施例１のアツバノリの抽出方法に基づき、アツバノリの抽出物を調製すると同時に、当該アツバノリの抽出物の抑菌効果の検査標的としてアクネ桿菌を用意し、選用されたアクネ桿菌の菌種はプロピオニバクテリウム・アクネス菌（Ｐｒｏｐｉｏｎｉｂａｃｔｅｒｉｕｍａｃｎｅｓ）（ＢＣＲＣ１０７２３）である。並びに、四皿の強化クロストリジウム培養基（ＲＣＭｍｅｄｉｕｍ）を用意し、一つ目の培養基には濃度が５０μｇ／ｍＬである抗生物質ゲンタマイシン（Ｇｅｎｔａｍｙｃｉｎ）が含有されているものを制御群１とした。二つ目の培養基には別途成分を添加しないものを制御群２とした。三つ目の培養基には濃度が１ｍｇ／ｍＬである前記アツバノリの抽出物を添加したものを実験群１とした。四つ目の培養基には濃度が０．５ｍｇ／ｍＬである前記アツバノリの抽出物を添加したものを実験群２とした。 First, based on the extraction method of P. acnes in Example 1, an extract of P. acnes was prepared, and at the same time, Bacillus acnes was prepared as a test target for the bacteriostatic effect of the extract of P. acnes. It is Propionibacterium acnes (BCRC10723). In addition, four dishes of reinforced Clostridium culture medium (RCM medium) were prepared, and the first culture medium contained the antibiotic gentamycin at a concentration of 50 μg/mL as control group 1. Control group 2 was a second culture medium in which no additional components were added. Experimental group 1 was prepared by adding the above-mentioned Atsubanori extract at a concentration of 1 mg/mL to the third culture medium. Experimental group 2 was prepared by adding the above-mentioned Atsubanori extract at a concentration of 0.5 mg/mL to the fourth culture medium.
そして、アクネ桿菌を強化クロストリジウム液体培地（ＲＣＭ）培養液に接種し、アクネ桿菌の初期接種菌量は１．１５×１０５ＣＦＵ／ｍＬであり、３７℃で４８時間嫌気培養すると、アクネ桿菌菌液が得られ、前記４群の培養基に１００μＬのアクネ桿菌菌液を均等に塗り、アクネ桿菌菌液を塗った前記４群の培養基を３７℃の培養箱に入れて嫌気培養を４８時間行い、培養が完了した後、培養箱から前記４群の培養基を取り出し、前記４群のコロニー形成単位（Ｃｏｌｏｎｙ－ｆｏｒｍｉｎｇｕｎｉｔ、ＣＦＵ）を計算する。 Then, K. acnes was inoculated into a reinforced Clostridium liquid medium (RCM) culture solution, and the initial inoculum amount of K. acnes was 1.15 × 10 5 CFU/mL. After the liquid was obtained, 100 μL of the acnes bacteria solution was evenly applied to the culture medium of the 4 groups, and the culture medium of the 4 groups coated with the acnes bacteria solution was placed in a culture box at 37°C and anaerobically cultured for 48 hours. After the culture is completed, the culture media of the four groups are taken out from the culture box, and the colony-forming units (CFU) of the four groups are calculated.
実施例５の試験結果は以下表二に示す通りであり、制御群１に抗生物質を添加したため、殺菌効果を奏することができた。実施例５のアツバノリの抽出物の濃度が０．５ｍｇ／ｍＬに達したとき、制御群２と比較して、菌量成長を抑制する現象があった。実施例５のアツバノリの抽出物の濃度が１ｍｇ／ｍＬに達したとき、殺菌効果があった。 The test results of Example 5 are as shown in Table 2 below, and since antibiotics were added to Control Group 1, a bactericidal effect could be achieved. When the concentration of the Atsubanori extract of Example 5 reached 0.5 mg/mL, there was a phenomenon in which the bacterial growth was suppressed compared to control group 2. When the concentration of the extract of Example 5 reached 1 mg/mL, it had a bactericidal effect.
実施例６のアツバノリの抽出物の角質細胞移動を促進する能力に関する試験 Test on the ability of the extract of Example 6 to promote keratinocyte migration
まず、比較サンプルとしてアツバノリの抽出物Ｆ－Ｈを提供する。 First, a comparison sample is provided as an extract FH of Atsubanori.
アツバノリの抽出物Ｆは、前記実施例１におけるアツバノリの抽出物の抽出方法に基づき、抽出条件を以下の条件の通り設定して調製したものである。抽出対象は番号１０８１２０４の乾燥したアツバノリ細粉であり、抽出溶剤は二酸化炭素流体であり、抽出槽における抽出圧力は３６５ｂａｒであり、抽出槽における抽出温度は５５℃であり、抽出時間は４時間である。 The extract F of Lamina laver was prepared based on the method for extracting the extract of Lamina laver in Example 1, and the extraction conditions were set as follows. The extraction target is dried Atsubanori fine powder with number 1081204, the extraction solvent is carbon dioxide fluid, the extraction pressure in the extraction tank is 365 bar, the extraction temperature in the extraction tank is 55 ° C, and the extraction time is 4 hours. be.
アツバノリの抽出物Ｇは、前記実施例１におけるアツバノリの抽出物の抽出方法に基づき、抽出条件を以下の条件の通り設定して調製したものである。抽出対象は番号１０８１１０５－１の乾燥したアツバノリ細粉であり、抽出溶剤は二酸化炭素流体であり、抽出槽における抽出圧力は３６５ｂａｒであり、抽出槽における抽出温度は５５℃であり、抽出時間は４時間である。 The extract G of Lamina laver was prepared based on the extraction method of the Lamina laver extract in Example 1, with the extraction conditions set as follows. The extraction target is dried Atsubanori fine powder with number 1081105-1, the extraction solvent is carbon dioxide fluid, the extraction pressure in the extraction tank is 365 bar, the extraction temperature in the extraction tank is 55 ° C, and the extraction time is 4 It's time.
アツバノリの抽出物Ｈは、前記実施例１におけるアツバノリの抽出物の抽出方法に基づき、抽出条件を以下の条件の通り設定して調製したものである。抽出対象は番号１０８１１０５－２の乾燥したアツバノリ細粉であり、抽出溶剤は二酸化炭素流体であり、抽出槽における抽出圧力は４２０ｂａｒであり、抽出槽における抽出温度は５５℃であり、抽出時間は４時間である。 The extract H of Lamium laver was prepared based on the method for extracting the extract of Lamina laver in Example 1, with the extraction conditions set as follows. The extraction target is dried Atsubanori fine powder with number 1081105-2, the extraction solvent is carbon dioxide fluid, the extraction pressure in the extraction tank is 420 bar, the extraction temperature in the extraction tank is 55 ° C, and the extraction time is 4 It's time.
そして、９６穴の細胞培養皿及びＯｒｉｓＴＭＣｅｌｌＭｉｇｒａｔｉｏｎキットを用意し、そのうちの１５個の穴にＯｒｉｓＴＭＣｅｌｌＭｉｇｒａｔｉｏｎキットの中のストッパを挿入し、ストッパに前記１５個の穴に配置した後、それぞれ前記当該穴に２×１０４のヒト角質細胞株（ＡＴＣＣＣＲＬ－２３０９）をそれぞれ接種し、三穴を一群とし、前記ヒト角質細胞株を接種した１５穴を制御群１、制御群２、実験群１、実験群２及び実験群３に分けた。制御群１に０．０５％のＤＭＳＯを加え、制御群２に０．０５％のＤＭＳＯ及び１００ｎｇ／ｍＬの表皮成長因子（ＥＧＦ）（ＥＧＦはヒト角質細胞の移動を促進することができる）を加え、実験群１に０．０５％のＤＭＳＯ及び１μｇ／ｍＬのアツバノリの抽出物Ｆを加え、実験群２に０．０５％のＤＭＳＯ及び１μｇ／ｍＬのアツバノリの抽出物Ｇを加え、実験群３に０．０５％のＤＭＳＯ及び１μｇ／ｍＬのアツバノリの抽出物Ｈを加えた。前記五群に検査したサンプルを加え終わった後、前記９６穴の細胞培養皿を細胞箱に入れて、３７℃の環境で２４時間培養し、前記９６穴の細胞培養皿で２４時間培養した後に、各群の穴に配置されたストッパ取り出して、各群の穴の上清液を取り除き、５０μＬの生体蛍光染料（ＣａｌｃｅｉｎＡＭ）（１μＭ）を加えて１時間静置した。 Then, a 96-well cell culture dish and an Oris TM Cell Migration kit were prepared, and the stoppers in the Oris TM Cell Migration kit were inserted into 15 of the holes, and the stoppers were placed in the 15 holes. Each hole was inoculated with 2 x 10 4 human corneum cell lines (ATCC CRL-2309) to form a group of three holes, and 15 holes inoculated with the human corneum cell lines were divided into control group 1, control group 2, and experimental group 1. , divided into experimental group 2 and experimental group 3. Control group 1 was supplemented with 0.05% DMSO, control group 2 was supplemented with 0.05% DMSO and 100 ng/mL epidermal growth factor (EGF) (EGF can promote the migration of human corneocytes). In addition, 0.05% DMSO and 1 μg/mL Atsubanori extract F were added to experimental group 1, 0.05% DMSO and 1 μg/mL Atsubanori extract G were added to experimental group 2, and experimental group 3 was added with 0.05% DMSO and 1 μg/mL of Noriflorum extract H. After adding the tested samples to the five groups, put the 96-well cell culture dish into a cell box and culture in a 37°C environment for 24 hours. Then, the stoppers placed in the holes of each group were taken out, the supernatant liquid in the holes of each group was removed, 50 μL of biofluorescent dye (Calcein AM) (1 μM) was added, and the cells were allowed to stand for 1 hour.
最後に、蛍光顕微鏡ＯｌｙｍｐｕｓＣＫＸ５３で前記五群の穴の影像を撮影し、撮影した影像数値（即ち、蛍光信号の数値を選定した）から前記五群の穴のヒト角質細胞株の細胞移動率を計算した。 Finally, images of the holes in the five groups were photographed using a fluorescence microscope Olympus CKX53, and the cell migration rate of the human corneum cell line in the holes in the five groups was calculated from the numerical values of the photographed images (i.e., the numerical values of the fluorescence signals were selected). did.
試験結果は図９及び図１０に示す通りである。図９からわかる通り、ヒト角質細胞の移動を促進することができる成分を添加しない制御群１と比較して、実験群１、実験群２及び実験群３は明らかな細胞の移動傾向を有しており、図１０からわかる通り、実験群１、実験群２及び実験群３の細胞移動率はいずれも制御群１より高く、制御群１の移動率は１００％であり、実験群１の移動率は１６５．７８％であり、実験群２の移動率は１９０．２５％であり、実験群３の移動率は１５６．０８％であった。 The test results are shown in FIGS. 9 and 10. As can be seen from Figure 9, compared to control group 1, in which no ingredients capable of promoting the migration of human corneocytes were added, experimental group 1, experimental group 2, and experimental group 3 had a clear tendency for cell migration. As can be seen from Figure 10, the cell migration rates of experimental group 1, experimental group 2, and experimental group 3 are all higher than control group 1, and the migration rate of control group 1 is 100%. The migration rate of experimental group 2 was 190.25%, and the migration rate of experimental group 3 was 156.08%.
実施例７のアツバノリの抽出物の角質細胞の成長を促進する試験 Test for promoting the growth of keratinocytes of the Atsubanori extract of Example 7
まず、実施例１のアツバノリの抽出方法に基づきアツバノリの抽出物を調製すると同時に本試験の検査標的として、ヒト角質細胞株（ＡＴＣＣＣＲＬ－２３０９）を用意した。 First, an extract of Aspergillus orientalis was prepared based on the extraction method of Aspergillus orensis in Example 1, and at the same time, a human corneocyte cell line (ATCC CRL-2309) was prepared as a test target for this test.
そして、９６穴の細胞培養皿を用意し、そのうちの１５個の穴に２×１０４ヒト角質細胞株（ＡＴＣＣＣＲＬ－２３０９）を接種し、三穴を一群とし、前記接種したヒト角質細胞株の１５穴を制御群１、制御群２、実験群１、実験群２及び実験群３に分けた。制御群１に１％のＤＭＳＯを加え、制御群２に１％のＤＭＳＯ及び１００ｎｇ／ｍＬの表皮成長因子（ＥＧＦ）を加え、実験群１に１％のＤＭＳＯ及び１．２μｇ／ｍＬのアツバノリの抽出物を加え、実験群２に１％のＤＭＳＯ及び６．０μｇ／ｍＬのアツバノリの抽出物を加え、実験群３に１％のＤＭＳＯ及び２４．１μｇ／ｍＬのアツバノリの抽出物を加えた。 Then, a 96-well cell culture dish was prepared, and 2×10 4 human corneocyte cell lines (ATCC CRL-2309) were inoculated into 15 of the holes, and three holes were made into a group. The 15 holes were divided into control group 1, control group 2, experimental group 1, experimental group 2, and experimental group 3. Control group 1 received 1% DMSO, control group 2 received 1% DMSO and 100 ng/mL epidermal growth factor (EGF), and experimental group 1 received 1% DMSO and 1.2 μg/mL Atsubanori. The extracts were added, to experimental group 2 1% DMSO and 6.0 μg/mL Acerium japonica extract, and to experimental group 3 1% DMSO and 24.1 μg/mL Acer japonica extract.
前記五群に検査サンプルを加え終わった後、前記９６穴の細胞培養皿を細胞培養箱に入れて３７℃の環境で２４時間培養し、前記９６穴の細胞培養皿を２４時間培養した後、ＭＴＳ／ＰＭＳ試験で細胞の生存率を検査する。本実施例７におけるＭＴＳ／ＰＭＳ試験の大まかな過程は以下の通りである。９６穴の細胞培養皿にＭＴＳ／ＰＭＳ試薬を加えて４時間反応させ、波長４９０ｎｍの箇所の吸光値を測定して細胞の生存率を計算する。 After adding the test samples to the five groups, the 96-well cell culture dish was placed in a cell culture box and cultured for 24 hours at 37°C, and after the 96-well cell culture dish was cultured for 24 hours, Test cell viability with MTS/PMS test. The general process of the MTS/PMS test in Example 7 is as follows. The MTS/PMS reagent is added to a 96-well cell culture dish, reacted for 4 hours, and the absorbance value at a wavelength of 490 nm is measured to calculate the cell survival rate.
試験結果は図１１に示す通りであり、濃度が６μｇ／ｍＬ以上であるアツバノリの抽出物は角質細胞の成長を促進する効果を有する。制御群１のＯＤ４９０吸光値は１．５７に達し、制御群２のＯＤ４９０吸光値は１．６２に達し、実験群１のＯＤ４９０吸光値は１．５９に達し、実験群２のＯＤ４９０吸光値は１．６８に達し、実験群３のＯＤ４９０吸光値は１．８９に達した。これらの数値からわかる通り、実験群２（６．０μｇ／ｍＬのアツバノリの抽出物を含有する）は制御群２（表皮成長因子を添加した）に相当する効果を有する。 The test results are as shown in FIG. 11, and the extract of Atsuba nori at a concentration of 6 μg/mL or more has the effect of promoting the growth of corneocytes. The OD 490 absorption value of control group 1 reached 1.57, the OD 490 absorption value of control group 2 reached 1.62, the OD 490 absorption value of experimental group 1 reached 1.59, the OD of experimental group 2 490 absorbance value reached 1.68, and the OD 490 absorbance value of experimental group 3 reached 1.89. As can be seen from these values, the experimental group 2 (containing 6.0 μg/mL of extract of Noriflora japonica) has an effect comparable to that of the control group 2 (adding epidermal growth factor).
前記アツバノリの抽出物方法により、新たな抽出方法を提供することで、アツバノリから高い経済価値を有する成分を抽出する、即ち、前記方法より抽出したアツバノリの抽出物は、毛包細胞の成長活性を促進する薬物、アクネ桿菌の成長を抑制する薬物若しくは化粧品又は角質細胞の活性を増進する薬物若しくは化粧品を調製するために用いることができる。 The Atsubanori extract method provides a new extraction method to extract components with high economic value from Atsubanori. In other words, the Atsubanori extract extracted by the above method stimulates the growth activity of hair follicle cells. It can be used to prepare drugs or cosmetics that promote, inhibit the growth of P. acnes or enhance the activity of corneocytes.
本発明は前記で好ましい実施例を開示しているものの、前記実施例は本発明を記述するためのものに過ぎず、本発明の範囲を限定するものと捉えてはならないと、当業者は理解するものとする。注意すべきは、前記実施例の等価変更や置換は何れも本発明の範疇に含まれるものとする。従って、本発明の保護範囲は下記の特許請求の範囲で定義するものを基準とする。 Although the present invention has been disclosed in the foregoing preferred embodiments, those skilled in the art will understand that the foregoing embodiments are merely for describing the invention and should not be construed as limiting the scope of the invention. It shall be. It should be noted that any equivalent changes or replacements of the above embodiments are included within the scope of the present invention. Therefore, the scope of protection of the present invention is defined in the following claims.
無し none
Claims (4)
（ｂ）二酸化炭素流体で当該乾燥したアツバノリの粉末に対して抽出を行って抽出アツバノリの抽出液を取得する工程であって、当該アツバノリの抽出液にアツバノリの抽出物と当該二酸化炭素流体溶剤とが含有され、抽出圧力は３００－６５０ｂａｒであり、抽出温度は４５－５５℃であり、抽出時間は２－６時間である、工程と、
（ｃ）当該アツバノリの抽出液から当該アツバノリの抽出物を分離する工程であって、
分離圧力は３０－１００ｂａｒである、工程とを含む、
ことを特徴とするアツバノリの抽出物の抽出方法。 (a) providing dried Atsubanori powder;
(b) A step of extracting the dried Atsubanori powder with a carbon dioxide fluid to obtain an extracted Atsubanori extract, the step of adding an Atsubanori extract and the carbon dioxide fluid solvent to the Atsubanori extract. is contained, the extraction pressure is 300-650 bar, the extraction temperature is 45-55°C, and the extraction time is 2-6 hours ;
(c) a step of separating the Atsubanori extract from the Atsubanori extract;
and the separation pressure is 30-100 bar .
A method for extracting an extract of Atsuba Nori.
ことを特徴とする請求項１に記載の方法。 Method according to claim 1 , characterized in that the extraction pressure is 350 bar, the extraction temperature is 55°C and the extraction time is 2 hours.
ことを特徴とする請求項１に記載の方法。 Process according to claim 1 , characterized in that the extraction pressure is 365 bar, the extraction temperature is 55°C and the extraction time is 4 hours.
ことを特徴とする請求項１に記載の方法。 Method according to claim 1 , characterized in that the extraction pressure is 420 bar, the extraction temperature is 55°C and the extraction time is 4 hours.
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