JP2020203856A - Method for producing tomatidine-containing extract - Google Patents
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- 239000000284 extract Substances 0.000 title claims abstract description 79
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Abstract
Description
本発明は、トマチジンの含有量が多いトマチジン含有抽出物の製造方法に関する。 The present invention relates to a method for producing a tomatidine-containing extract having a high content of tomatidine.
トマチンは、トマトの葉茎部や青い果実に豊富に含まれるアルカロイド配糖体である。トマチンは、トマト萎凋病菌（Fusarium oxysporum f. lycopersici）に対する抗菌物質として1940年代に発見された。トマチンには毒性がある一方で、抗癌作用や抗腫瘍作用など様々な生理活性をもつことが報告されている。トマチンから糖が外れて生じるトマチジンは、毒性が無い一方で、トマチンと同様に、抗癌・抗炎症作用（非特許文献１）を有する。さらに近年では、トマチジンには、血中コレステロール低下作用（非特許文献２）、筋萎縮の予防作用（非特許文献３）、デング熱に対する抗ウイルス作用（非特許文献４）を持つことも報告されている。 Tomatine is an alkaloid glycoside that is abundant in the leaf stems of tomatoes and blue fruits. Tomatine was discovered in the 1940s as an antibacterial agent against Fusarium oxysporum f. Lycopersici. While tomatine is toxic, it has been reported to have various physiological activities such as anticancer and antitumor effects. Tomatine, which is produced by removing sugar from tomatine, is not toxic, but has anti-cancer and anti-inflammatory effects (Non-Patent Document 1) like tomatine. Furthermore, in recent years, it has been reported that tomatidine has a blood cholesterol lowering effect (Non-Patent Document 2), a muscular atrophy preventive effect (Non-Patent Document 3), and an antiviral effect on dengue fever (Non-Patent Document 4). There is.
トマチジンは、トマチンを加水分解することによって得ることができる。特許文献１には、トマチンから糖鎖を除去してトマチジンへ変換する効率的な方法として、トマトの成熟果実を除く葉、茎、脇芽、花奔、未成熟果実、および根を裁断したもの、または乾燥後粉砕したものの重量当たり３〜１５倍量の水を加え、さらに酸溶液を加えてｐＨ２〜５に調整した後、室温から８０℃の温度下で３０〜１２０分間撹拌混合してグリコアルカロイドのデヒドロトマチンを含むトマチンを抽出し、該抽出液にアルカリ溶液を添加して室温においてｐＨ８以上且つｐＨ１０未満に調整することによってトマチンを沈殿させ、該トマチン沈殿物を回収した後、０．５〜２Ｎの酸溶液に溶解した後、６０〜８０℃で１〜４時間加熱してトマチンをトマチジンへ変換させ、ふたたびアルカリ溶液を用いてｐＨ８以上且つｐＨ１０未満に調整してトマチジンを沈澱させ、該沈殿物を更に７０％濃度以上のエタノール溶液に溶解して混在する不純物を沈殿させて除去した後に、エタノールを蒸発させることを特徴とするトマチジンの製造法が記載されている。特許文献１では、具体的には、脇芽や葉を少なくとも含むトマト材料を用いて、トマチジンが製造さえたことが示されている。 Tomatidine can be obtained by hydrolyzing tomatine. In Patent Document 1, as an efficient method for removing a sugar chain from tomatin and converting it into tomatidine, leaves, stems, armpit buds, flowers, immature fruits, and roots excluding mature tomatoes are cut. Or, after drying and crushing, add 3 to 15 times the amount of water per weight, add an acid solution to adjust the pH to 2 to 5, and then stir and mix for 30 to 120 minutes at a temperature of 80 ° C. from room temperature to control glyco Tomatin containing the alkaloid dehydrotomatine is extracted, and an alkaline solution is added to the extract to adjust the pH to 8 or more and less than 10 at room temperature to precipitate the tomatin. After recovering the tomatine precipitate, 0.5 After dissolving in an acid solution of ~ 2N, the tomatin is converted to tomatidine by heating at 60 to 80 ° C. for 1 to 4 hours, and the pH is adjusted to 8 or more and less than 10 using an alkaline solution again to precipitate the tomatidine. A method for producing tomatidine is described, which comprises further dissolving a precipitate in an ethanol solution having a concentration of 70% or more to precipitate and remove mixed impurities, and then evaporating ethanol. Patent Document 1 specifically shows that tomatodin was even produced using a tomato material containing at least side buds and leaves.
トマチンは、トマトの青果実よりも、脇芽や葉にはるかに多く含まれている。特許文献１の方法で実際にトマチジン含有抽出物が製造されたのは、脇芽や葉といった、トマチンが豊富に含まれている部分のみである。 Tomatine is much more abundant in armpit buds and leaves than in tomato fruits and vegetables. The tomatine-containing extract was actually produced by the method of Patent Document 1 only in the tomatine-rich parts such as armpit buds and leaves.
本発明者は、あえて、トマチンの含有量が少ないトマトの青果実からのトマチジン含有抽出物の製造を試みた。すると、トマトの青果実から従来の方法を用いて得られるトマチジン含有抽出物では、トマチジン含有量が少なく、多くのトマチンが残存しているという課題に直面した。 The present inventor dared to try to produce a tomatine-containing extract from tomato fruits and vegetables having a low tomatine content. Then, the tomatine-containing extract obtained from tomato fruits and vegetables by a conventional method faced a problem that the tomatine content was low and a large amount of tomatine remained.
そこで本発明は、トマトの青果実からトマチジン含有抽出物を製造する方法であって、トマチジン含有量が向上した抽出物を得る方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for producing a tomatodin-containing extract from tomato fruits and vegetables, and for obtaining an extract having an improved tomatodin content.
本発明者は鋭意検討の結果、トマトの青果実からのトマチンの抽出において、エタノール沈殿法を用いて不要物を除去した後に酸処理を行うことで、得られるトマチジン抽出物中のトマチジン含量が向上することを見出した。本発明はこの知見に基づいてさらに検討を重ねることにより完成したものである。 As a result of diligent studies, the present inventor improved the tomatine content in the obtained tomatine extract by performing acid treatment after removing unnecessary substances by using an ethanol precipitation method in the extraction of tomatine from tomato fruits and vegetables. I found out to do. The present invention has been completed by further studies based on this finding.
即ち、本発明は、下記に掲げる態様の発明を提供する。
項１． 青トマト果実の搾汁及び／又は水抽出物をエタノールと混合し、沈殿物を除去し、トマチンを含む上清画分を得る工程１と、前記上清画分を酸で処理する工程２と、を含む、トマチジン含有抽出物の製造方法。
項２． 前記工程１において、前記エタノールが、前記搾汁及び水抽出物の総量の１倍体積以上の量で用いられる、項１に記載のトマチジン含有抽出物の製造方法。
項３． 前記工程２において、前記酸が０．７〜０．９Ｎの終濃度となるように用いられる、項１又は２に記載のトマチジン含有抽出物の製造方法。
項４． 前記工程２で得られた酸処理物を、中性条件下での精製に供する工程３を更に含む、項１〜３のいずれかに記載のトマチジン含有抽出物の製造方法。
項５． 前記工程３において、前記精製をクロマトグラフィーにより行う、項４に記載のトマチジン含有抽出物の製造方法。
That is, the present invention provides the inventions of the following aspects.
Item 1. Step 1 of mixing the juice and / or water extract of green tomato fruit with ethanol to remove the precipitate to obtain a supernatant fraction containing tomatine, and step 2 of treating the supernatant fraction with acid. A method for producing a tomatine-containing extract, which comprises.
Item 2. Item 2. The method for producing a tomatidine-containing extract according to Item 1, wherein in the step 1, the ethanol is used in an amount of 1 times or more the total volume of the juice and the water extract.
Item 3. Item 2. The method for producing a tomatidine-containing extract according to Item 1 or 2, wherein the acid is used so as to have a final concentration of 0.7 to 0.9N in the step 2.
Item 4. Item 3. The method for producing a tomatidine-containing extract according to any one of Items 1 to 3, further comprising a step 3 of subjecting the acid-treated product obtained in the above step 2 to purification under neutral conditions.
Item 5. Item 4. The method for producing a tomatidine-containing extract according to Item 4, wherein the purification is performed by chromatography in the step 3.
本発明によれば、トマトの青果実を原料としながらも、トマチジン含有量が向上した抽出物を得ることができる。 According to the present invention, it is possible to obtain an extract having an improved tomatidine content while using tomato fruits and vegetables as a raw material.
本発明のトマチジン含有抽出物の製造方法は、青トマト果実の搾汁及び／又は水抽出物をエタノールと混合し、沈殿物を除去し、トマチンを含む上清画分を得る工程１と、前記上清画分を酸で処理する工程２と、を含むことを特徴とする。以下、本発明のトマチジン含有抽出物の製造方法について詳述する。 The method for producing a tomatine-containing extract of the present invention comprises the step 1 of mixing the juice and / or water extract of green tomato fruits with ethanol, removing the precipitate, and obtaining a supernatant fraction containing tomatine. It is characterized by comprising a step 2 of treating the supernatant fraction with an acid. Hereinafter, the method for producing the tomatidine-containing extract of the present invention will be described in detail.
工程１
工程１では、青トマト果実の搾汁及び／又は水抽出物をエタノールと混合し、沈殿物を除去し、トマチンを含む上清画分を得る。
Process 1
In step 1, the juice and / or water extract of green tomato fruit is mixed with ethanol to remove the precipitate and obtain a supernatant fraction containing tomatine.
原料となる青トマトとしては、完熟及び未熟を問わず、果実が青いトマトであればいかなる品種ものも用いることができる。例えば、未熟果実である青トマト（完熟果実は赤トマト又は黄トマト）として、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ ｖａｒ． ｃｅｒａｓｉｆｏｒｍｅ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｐｉｍｐｉｎｅｌｌｉｆｏｌｉｕｍ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｃｈｅｅｓｍａｎｉｉ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｃｈｅｅｓｍａｎｉｉ ｆ．ｍｉｎｏｒ等が挙げられる。また、完熟果実である青トマトとしては、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｐａｒｖｉｆｌｏｒｕｍ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｃｈｍｉｅｌｅｗｓｋｉｉ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｐｅｒｕｖｉａｎｕｍ、Ｌｙｃｏｐｅｒｓｉｃｏｎ ｃｈｉｌｅｎｓｅ、Ｓｏｌａｎｕｍ ｌｙｃｏｐｅｒｓｉｃｏｉｄｅｓ、Ｓｏｌａｎｕｍ ｒｉｃｋｉｉ、及びＳｏｌａｎｕｍ ｏｃｈｒａｎｔｈｕｍ等が挙げられる。 As the raw material, any kind of green tomato can be used as long as the fruit is blue, regardless of whether it is ripe or immature. For example, as green tomatoes which are immature fruits (ripe fruits are red tomatoes or yellow tomatoes), Lycopersicon esculentum Mill, Lycopersicon esculentum var. cerasiforme, Lycopersicon pineappleform, Lycopersicon cheesemanii, Lycopersicon cheesemanii f. Minor and the like can be mentioned. Examples of the green tomatoes that are ripe fruits include Lycopersicon paraviflorum, Lycopersicon chmielewskii, Lycopersicon peruvianum, Lycopersicon chilense, Solanum lycopersicon, Solanum lycopersicon, etc.
青トマト中のトマチンの含有量は、品種によって様々であるが、１００ｇ湿重量（ｆｒｅｓｈ ｗｅｉｇｈｔ）中、２ｍｇ以上、好ましくは４ｍｇ以上、より好ましくは１５ｍｇ以上、さらに好ましくは２０ｍｈ以上、一層好ましくは２５ｍｇ以上が挙げられる。 The content of tomatine in green tomatoes varies depending on the variety, but in 100 g wet weight (fresh weight), 2 mg or more, preferably 4 mg or more, more preferably 15 mg or more, still more preferably 20 mh or more, still more preferably 25 mg. The above can be mentioned.
青トマト果実の搾汁を得る方法としては特に限定されないが、例えば、果実をミキサー等で粉砕してピューレを調製し、ピューレを圧搾することによって得ることができる。また、青トマト果実の水抽出物を得る方法としても特に限定されないが、例えば、果実を粉砕してピューレを調製し、ピューレを水に分散させて圧搾することによって得てもよいし、上記の搾汁の調製によって得られた圧搾残渣を０．８〜２倍体積、好ましくは０．８〜１．５倍体積の水に分散させ、２０分〜１時間撹拌した後、圧搾することによって得てもよい。ここで、抽出溶媒としての水は、水に酸を添加して調製した酸水溶液ではない。青トマト果実の搾汁及び水抽出物は、室温、好ましくは１５〜２５℃で得ることができる。また、青トマト果実の搾汁及び水抽出物は、流動性を向上させて調製を容易にする観点から加熱しても良く、具体的な加熱温度としては、上記の室温超８５℃以下、好ましくは５０〜８０℃が挙げられる。 The method for obtaining the juice of the green tomato fruit is not particularly limited, but for example, it can be obtained by crushing the fruit with a mixer or the like to prepare a puree and squeezing the puree. Further, the method for obtaining a water extract of green tomato fruit is not particularly limited, but for example, the fruit may be crushed to prepare a puree, and the puree may be dispersed in water and pressed. Obtained by dispersing the squeezed residue obtained by preparing the juice in 0.8 to 2 times the volume, preferably 0.8 to 1.5 times the volume of water, stirring for 20 minutes to 1 hour, and then squeezing. You may. Here, the water as the extraction solvent is not an acid aqueous solution prepared by adding an acid to water. The juice and water extract of green tomato fruits can be obtained at room temperature, preferably 15-25 ° C. Further, the juice and water extract of green tomato fruits may be heated from the viewpoint of improving fluidity and facilitating preparation, and the specific heating temperature is preferably above room temperature of 85 ° C. or lower. Is 50 to 80 ° C.
上記の搾汁及び／又は水抽出物はエタノールと混合する。エタノールによって、搾汁及び／又は水抽出物中の夾雑物の少なくとも一部を沈殿させる。沈殿させられる成分には、糖、タンパク質等が含まれる。なお、トマチジン含有量がより向上した抽出物を得る観点から、上記の搾汁及び／又は水抽出物に対しては、酸の添加を行わない。エタノールの使用量としては特に限定されないが、夾雑物をより効率的に沈殿させる観点から、上記の搾汁及び水抽出物の総量の好ましくは１倍体積以上、より好ましくは２倍体積以上、さらに好ましくは３倍体積以上、一層このましくは３．５体積以上、特に好ましくは４倍体積以上の量で用いられる。エタノールの使用量の上限としては特に限定されないが、例えば５倍体積以下、好ましくは４．５倍体積以下が挙げられる。 The above juice and / or water extract is mixed with ethanol. Ethanol precipitates at least a portion of the contaminants in the juice and / or water extract. The components to be precipitated include sugars, proteins and the like. From the viewpoint of obtaining an extract having a higher tomatidine content, no acid is added to the above-mentioned squeezed juice and / or water extract. The amount of ethanol used is not particularly limited, but from the viewpoint of more efficiently precipitating impurities, the total amount of the above-mentioned juice and water extract is preferably 1 times or more, more preferably 2 times or more, and further. It is preferably used in an amount of 3 times or more, more preferably 3.5 times or more, and particularly preferably 4 times or more. The upper limit of the amount of ethanol used is not particularly limited, and examples thereof include 5 times volume or less, preferably 4.5 times volume or less.
生じた沈殿物は除去される。沈殿物の除去方法としては固液分離法であれば特に限定されないが、沈殿物を効率的に捕捉し必要画分を効率的に回収する観点から、吸引濾過法が好ましい。また、濾材としては、沈殿物を効率的に捕捉し必要画分を効率的に回収する観点から、好ましくはガラス濾紙又はガラスフィルター、より好ましくはガラス濾紙が挙げられる。好ましい濾材の保持粒子径としては、例えば０．６５〜０．９５μｍ、好ましくは０．７〜０．９μｍ、より好ましくは０．７５〜０．８５μｍが挙げられる。ここで、保持粒子径とは、ＪＩＳ Ｚ ８９０１で規定された、７種粉体分散水を自然濾過した時、９０％以上を保持できる粒子径をいう。 The resulting precipitate is removed. The method for removing the precipitate is not particularly limited as long as it is a solid-liquid separation method, but a suction filtration method is preferable from the viewpoint of efficiently capturing the precipitate and efficiently recovering the necessary fraction. Further, as the filter medium, a glass filter paper or a glass filter is preferable, and a glass filter paper is more preferable, from the viewpoint of efficiently capturing the precipitate and efficiently recovering the necessary fraction. A preferable holding particle size of the filter medium is, for example, 0.65 to 0.95 μm, preferably 0.7 to 0.9 μm, and more preferably 0.75 to 0.85 μm. Here, the retained particle size refers to a particle size that can retain 90% or more when the 7-kind powder-dispersed water specified in JIS Z 8901 is naturally filtered.
沈殿物を除去することで、上清をトマチンを含む画分として得ることができる。得られた上清は、トマチンをさらに精製した後に後述の工程２に供することもできるが、トマチジン含量が一層向上した抽出物を得る観点から、好ましくは、得られた上清をそのままで、好ましくは溶媒留去により濃縮して、後述の工程２に供する。上清を濃縮する場合、濃縮率としては、｛搾汁及び水抽出物の総体積とエタノールの体積との総量／濃縮後の上清体積｝の比で、たとえば１０〜２０倍、好ましくは１３〜１７倍が挙げられる。本発明では、上記の上清からトマチンをさらに精製した画分、上記の上清そのもの、及び上記の上清を濃縮した画分をいずれも上清画分と称する。 By removing the precipitate, the supernatant can be obtained as a tomatine-containing fraction. The obtained supernatant can be subjected to step 2 described later after further purifying tomatine, but from the viewpoint of obtaining an extract having a further improved tomatine content, the obtained supernatant is preferably left as it is. Is concentrated by distilling off the solvent and subjected to step 2 described later. When the supernatant is concentrated, the concentration ratio is the ratio of {total volume of juice and water extract to total volume of ethanol / supernatant volume after concentration}, for example, 10 to 20 times, preferably 13. ~ 17 times can be mentioned. In the present invention, a fraction obtained by further purifying tomatine from the above supernatant, the above supernatant itself, and a fraction obtained by concentrating the above supernatant are all referred to as a supernatant fraction.
工程２
工程２では、工程１で得られた上清画分を酸処理する。酸処理によって、上清画分に含まれていたトマチンがトマチジンへ変換され、酸処理物が得られる。
Process 2
In step 2, the supernatant fraction obtained in step 1 is acid-treated. By the acid treatment, tomatine contained in the supernatant fraction is converted to tomatine, and an acid-treated product is obtained.
酸処理に用いる酸としては特に限定されず、例えば、酢酸、塩酸、硫酸等が挙げられ、好ましくは塩酸が挙げられる。酸処理系中における酸濃度（終濃度）としては、トマチジン含量が一層向上した抽出物を得る観点から、たとえば０．７〜０．９Ｎ、好ましくは０．７〜０．８５Ｎ、より好ましくは０．７８〜０．８２Ｎが挙げられる。酸処理温度としては、トマチジン含量が一層向上した抽出物を得る観点から、たとえば９０〜１００℃、好ましくは９５〜１００℃が挙げられる。酸処理時間としては、反応スケール等によっても異なりうるが、例えば２〜４時間が挙げられる。 The acid used for the acid treatment is not particularly limited, and examples thereof include acetic acid, hydrochloric acid, sulfuric acid, and the like, and hydrochloric acid is preferable. The acid concentration (final concentration) in the acid treatment system is, for example, 0.7 to 0.9 N, preferably 0.7 to 0.85 N, more preferably 0, from the viewpoint of obtaining an extract having a further improved tomatidine content. Examples include .78 to 0.82N. The acid treatment temperature is, for example, 90 to 100 ° C., preferably 95 to 100 ° C. from the viewpoint of obtaining an extract having a further improved tomatidine content. The acid treatment time may vary depending on the reaction scale and the like, but examples thereof include 2 to 4 hours.
工程３
工程３では、工程２で得られた酸処理物を、中性条件下での精製に供する。つまり、工程３では、まず、工程２で得られた酸処理物のｐＨを中性条件に調整する。この場合、具体的なｐＨとしては、６．５〜７．５、好ましくは６．８〜７．２、最も好ましくは７が挙げられる。中性条件に調整する方法としては特に限定されず、当業者によって適宜選択される。好ましくは、水酸化カリウム、水酸化ナトリウム等の塩基を用いて中和することによって調整することができる。
Process 3
In step 3, the acid-treated product obtained in step 2 is subjected to purification under neutral conditions. That is, in step 3, first, the pH of the acid-treated product obtained in step 2 is adjusted to a neutral condition. In this case, the specific pH is 6.5 to 7.5, preferably 6.8 to 7.2, and most preferably 7. The method for adjusting to the neutral condition is not particularly limited, and is appropriately selected by those skilled in the art. Preferably, it can be adjusted by neutralizing with a base such as potassium hydroxide and sodium hydroxide.
中和された酸処理物からは、トマチジンが精製される。トマチジンの精製方法としては特に限定されず、カラムクロマトグラフィー（逆相、疎水、イオン交換、ゲルろ過、金属キレートアフィニティー）、透析（好ましくは電気透析）等が挙げられる。これらの精製方法の中でも、トマチジン含有量がより向上した抽出物を得る観点から、好ましくはカラムクロマトグラフィーが挙げられ、より好ましくは逆相カラムクロマトグラフィーが挙げられる。 Tomatidine is purified from the neutralized acid-treated product. The method for purifying tomatidine is not particularly limited, and examples thereof include column chromatography (reverse phase, hydrophobicity, ion exchange, gel filtration, metal chelate affinity), dialysis (preferably electrodialysis) and the like. Among these purification methods, column chromatography is preferable, and reverse phase column chromatography is more preferable, from the viewpoint of obtaining an extract having a more improved tomatidine content.
カラムクロマトグラフィーを用いて精製を行う場合、使用するカラム担体の量としては、中和された酸処理物の体積の０．１〜２倍が挙げられる。また、酸処理物は、必要に応じ予め濃縮されていてもよい。カラムクロマトグラフィーを用いて精製を行う場合の具体的な手順としては特に限定されないが、例えば、中和された酸処理物をカラム担体に吸着させ、担体を水で洗浄した後、エタノール又はエタノール水溶液を用いて溶出することができる。溶出にエタノール水溶液を用いる場合、エタノール濃度としては、トマチジン含有量がより向上した抽出物を得る観点から、好ましくは７０ｖ／ｖ％以上、好ましくは８０ｖ／ｖ％以上が挙げられる。エタノール水溶液中のエタノール濃度の上限としては、例えば９５ｖ／ｖ％以下、好ましくは９０ｖ／ｖ％以下、より好ましくは８５ｖ／ｖ％以下が挙げられる。 When purification is performed using column chromatography, the amount of column carrier used may be 0.1 to 2 times the volume of the neutralized acid-treated product. Further, the acid-treated product may be pre-concentrated if necessary. The specific procedure for purification using column chromatography is not particularly limited, but for example, a neutralized acid-treated product is adsorbed on a column carrier, the carrier is washed with water, and then ethanol or an aqueous ethanol solution is used. Can be eluted using. When an aqueous ethanol solution is used for elution, the ethanol concentration is preferably 70 v / v% or more, preferably 80 v / v% or more, from the viewpoint of obtaining an extract having a more improved tomatidine content. Examples of the upper limit of the ethanol concentration in the aqueous ethanol solution include 95 v / v% or less, preferably 90 v / v% or less, and more preferably 85 v / v% or less.
洗浄に用いる水の量としては特に限定されず、非吸着物を溶出するために必要な量であればよいが、トマチジン含有量がより向上した抽出物を得る観点から、好ましくは２〜４ＣＶ（カラム体積）、より好ましくは２．５〜３．５ＣＶが挙げられる。溶出に用いるエタノール又はエタノール水溶液の量としては特に限定されず、吸着させたトマチジンを溶出するために必要な量であればよいが、トマチジン含有量がより向上した抽出物を得る観点から、好ましくは２〜４ＣＶ、より好ましくは２．５〜３．５ＣＶが挙げられる。 The amount of water used for washing is not particularly limited as long as it is an amount necessary for eluting the non-adsorbent, but from the viewpoint of obtaining an extract having a higher tomatidine content, it is preferably 2 to 4 CV ( Column volume), more preferably 2.5 to 3.5 CV. The amount of ethanol or an aqueous ethanol solution used for elution is not particularly limited as long as it is an amount necessary for eluting the adsorbed tomatidine, but it is preferable from the viewpoint of obtaining an extract having a more improved tomatidine content. 2 to 4 CV, more preferably 2.5 to 3.5 CV.
溶出画分は、必要に応じて脱塩を行い、濃縮することができる。これによって、トマチジン含有抽出物が得られる。トマチジン含有抽出物の形態としては、半固形（軟エキス）及び固形（乾燥エキス）が挙げられ、好ましくは固形が挙げられる。 The eluted fraction can be desalted and concentrated as needed. This gives a tomatidine-containing extract. Examples of the form of the tomatidine-containing extract include semi-solid (soft extract) and solid (dry extract), and preferably solid.
トマチジン含有抽出物中のトマチジンの含有比率としては、原料である青トマトが有していたトマチンの量によっても異なるが、乾燥重量換算量で、例えば０．１重量％以上、好ましくは１重量％以上、より好ましくは１０重量％以上、さらに好ましくは３０重量％以上、一層好ましくは３５重量％以上、特に好ましくは３７重量％以上が挙げられる。トマチジンの含有比率が多いほど好ましいため、当該範囲の上限としては特に限定されないが、例えば７０重量％以下、６０重量％以下、又は５０重量％以下が挙げられる。 The content ratio of tomatine in the tomatodin-containing extract varies depending on the amount of tomatine contained in the raw material green tomato, but in terms of dry weight equivalent, for example, 0.1% by weight or more, preferably 1% by weight. As described above, more preferably 10% by weight or more, further preferably 30% by weight or more, still more preferably 35% by weight or more, and particularly preferably 37% by weight or more. Since it is preferable that the content ratio of tomatidine is large, the upper limit of the range is not particularly limited, and examples thereof include 70% by weight or less, 60% by weight or less, or 50% by weight or less.
より具体的には、トマチジン含有抽出物中のトマチジンの含有比率としては、原料である青トマト中のトマチンの含有量１００重量部当たり、例えば２０重量部以上、好ましくは３０重量部以上、より好ましくは３５重量部以上、さらに好ましくは３７重量部以上が挙げられる。トマチジンの含有比率が多いほど好ましいため、当該範囲の上限としては特に限定されないが、例えば６０重量部以下、又は５０重量部以下が挙げられる。 More specifically, the content ratio of tomatine in the tomatine-containing extract is, for example, 20 parts by weight or more, preferably 30 parts by weight or more, more preferably, per 100 parts by weight of the content of tomatine in the raw material green tomato. Is 35 parts by weight or more, more preferably 37 parts by weight or more. Since it is preferable that the content ratio of tomatidine is large, the upper limit of the range is not particularly limited, and examples thereof include 60 parts by weight or less or 50 parts by weight or less.
また、トマチジン含有抽出物中にはトマチンが実質的に含まれていないことが好ましい。トマチンが実質的に含まれていないとは、超高速高分離液体クロマトグラフィー（ＵＰＬＣ）での検出限界以下であることをいい、具体的には、１×１０-3重量％以下、好ましくは１×１０-5重量％以下、より好ましくは１×１０-7重量％以下、さらに好ましくは１×１０-8重量％以下であることをいう。 Further, it is preferable that the tomatine-containing extract does not substantially contain tomatine. The fact that tomatine is substantially not contained means that it is below the detection limit by ultra high performance liquid chromatography (UPLC), specifically, 1 × 10 -3 % by weight or less, preferably 1. It means that it is × 10 -5 % by weight or less, more preferably 1 × 10 -7 % by weight or less, and further preferably 1 × 10 -8 % by weight or less.
トマチジンは、抗癌・抗炎症作用、血中コレステロール低下作用、筋萎縮の予防作用、デング熱に対する抗ウイルス作用等の様々な生理活性を有する。このため、上記のトマチジン含有抽出物は、機能性食品及び外用組成物といった様々なへの利用が有効である。上記のトマチジン含有抽出物は、例えば、清酒、発酵調味料、飲料、食品、健康補助食品、化粧料等の各種組成物に添加して使用することができる。このように、トマチジン含有抽出物が添加された組成物は、トマチジン基づく有用効果、即ち、抗癌・抗炎症作用、血中コレステロール低下作用、筋萎縮の予防作用、デング熱に対する抗ウイルス作用等の有用な効果が期待できる。 Tomatidine has various physiological activities such as anti-cancer / anti-inflammatory action, blood cholesterol lowering action, muscular atrophy preventive action, and antiviral action against dengue fever. Therefore, the above-mentioned tomatidine-containing extract is effective for various purposes such as functional foods and external compositions. The above-mentioned tomatidine-containing extract can be used by adding it to various compositions such as sake, fermented seasonings, beverages, foods, dietary supplements, and cosmetics. As described above, the composition to which the tomatidine-containing extract is added has useful effects based on tomatidine, that is, anticancer / anti-inflammatory effects, blood cholesterol lowering effects, muscular atrophy preventive effects, antiviral effects against dengue fever, and the like. Can be expected to have a positive effect.
上記のトマチジン含有抽出物の各種組成物への添加量については、抽出物中のトマチジンの含有量、該組成物の種類や形態、期待される効果等に応じて適宜設定すればよい。例えば、該組成物の総量当たりのトマチジン含量が０．００１〜１０重量％となるように上記トマチジン含有抽出物を目的の組成物に添加すればよい。 The amount of the above-mentioned tomatidine-containing extract added to various compositions may be appropriately set according to the content of tomatidine in the extract, the type and form of the composition, the expected effect, and the like. For example, the tomatidine-containing extract may be added to the target composition so that the tomatidine content per total amount of the composition is 0.001 to 10% by weight.
以下に実施例を示して本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.
実施例１
（工程１）
果汁用の熟した青いトマト５４６ｇを、ミキサー（１０００ｒｐｍ）で３分粉砕し、ピューレを得た。ピューレをろ布で圧搾し、搾汁を調製した。更に、搾汁残渣に１倍体積量の水を添加して分散させ、室温で、ミキサー（１０００ｒｐｍ）で３０分間攪拌し、ろ布で圧搾することで、水抽出液を得た。搾汁と水抽出液とを混合し、９３０ｍＬの搾汁液を得た。これにエタノール３７２０ｍＬを添加して、沈殿物を生じさせた。ガラス濾紙（ＡＤＶＡＮＴＥＣ ＧＡ−２００、保持粒子径０．８μｍ）で吸引ろ過することにより沈殿物を除去し、上清を得た。得られた上清を、エバポレーターで３００ｍＬまで濃縮した。
Example 1
(Step 1)
546 g of ripe blue tomatoes for fruit juice were pulverized with a mixer (1000 rpm) for 3 minutes to obtain puree. The puree was squeezed with a filter cloth to prepare a juice. Further, 1 times the volume of water was added to the juice residue to disperse it, and the mixture was stirred at room temperature with a mixer (1000 rpm) for 30 minutes and squeezed with a filter cloth to obtain a water extract. The squeezed juice and the water extract were mixed to obtain 930 mL of the squeezed liquid. 3720 mL of ethanol was added to this to form a precipitate. The precipitate was removed by suction filtration with a glass filter paper (ADVANTEC GA-200, holding particle diameter 0.8 μm) to obtain a supernatant. The obtained supernatant was concentrated to 300 mL with an evaporator.
（工程２）
濃縮された上清に、１．２Ｎ塩酸６００ｍＬ（終濃度は０．８Ｎ）を添加し、沸騰浴（１００℃）上で３時間反応させた。その後、反応混合物を冷却し、酸処理物を得た。
(Step 2)
To the concentrated supernatant, 600 mL of 1.2N hydrochloric acid (final concentration: 0.8N) was added, and the mixture was reacted in a boiling bath (100 ° C.) for 3 hours. Then, the reaction mixture was cooled to obtain an acid-treated product.
（工程３）
酸処理物に、１Ｎ水酸化ナトリウム水溶液を加えてｐＨ７となるように中和し、中和された酸処理物７００ｍｌを得た。カラムにＣｏｓｍｏｓｉｌ １４０Ｃ１８−ＯＰＮを１００ｍｌ充填し、中和された酸処理物７００ｍｌを通液した。その後、３ＣＶの水で担体を洗浄後、３ＣＶの８０ｖ／ｖ％エタノール水溶液で溶出した。溶出液を脱塩し、エバポレーターで濃縮することで溶媒をすべて留去した。その結果、１６４ｍｇの粉末をトマチジン含有出物として得た。
(Step 3)
A 1N aqueous sodium hydroxide solution was added to the acid-treated product to neutralize the pH to 7, and 700 ml of the neutralized acid-treated product was obtained. The column was filled with 100 ml of Cosmosil 140C18-OPN and 700 ml of the neutralized acid-treated product was passed through. Then, the carrier was washed with 3 CV of water and eluted with 3 CV of 80 v / v% ethanol aqueous solution. The eluate was desalted and concentrated on an evaporator to distill off all the solvent. As a result, 164 mg of powder was obtained as a tomatidine-containing product.
（青トマト中のトマチン及びトマチジン含有出物中のトマチン及びトマチジンの測定方法）
上述のようにして得られた青トマトの搾汁、及びトマチジン含有出物を、それぞれ、液体クロマトグラフィー／質量分析法（ＬＣ／ＭＳ法）による以下の条件に供し、トマチン含有量及びトマチジン含有量を測定した。
(Measuring method of tomatine and tomatine in tomatine and tomatine-containing products in green tomato)
The green tomato juice obtained as described above and the tomatine-containing product were subjected to the following conditions by liquid chromatography / mass spectrometry (LC / MS method), respectively, and the tomatine content and tomatine content were subjected to the following conditions. Was measured.
装置：ACQUITY UPLC及びXevo G2-S QTof（いずれもWaters社製）
カラム：ACQUIRY UPLC BEH C18（50×2.1 mm I.D.、1.6 μm）
移動相：Ａ；０．１ｖ／ｖ％ギ酸を含む水、Ｂ；０．１ｖ／ｖ％ギ酸を含むアセトニトリル
グラジエント：Ａ／Ｂの組成（体積比）を９５／５から５．５分で０／１００に変化させ、０／１００の組成でさらに１分溶出させた。
流速：０．８ｍＬ／分
カラム温度：５５℃
検出波長：２１０〜５００ｎｍ
イオン化：ＥＳＩ（ポジティブ）
インジェクション：２μＬ
内部標準：レセルピン
検出限界：０．１ｎｇ／ｇ
Equipment: ACQUITY UPLC and Xevo G2-S QTof (both manufactured by Waters)
Column: ACQUIRY UPLC BEH C18 (50 x 2.1 mm ID, 1.6 μm)
Mobile phase: A; water containing 0.1 v / v% formic acid, B; acetonitrile gradient containing 0.1 v / v% formic acid: A / B composition (volume ratio) 0 in 95/5 to 5.5 minutes It was changed to / 100 and eluted with a composition of 0/100 for an additional 1 minute.
Flow rate: 0.8 mL / min Column temperature: 55 ° C
Detection wavelength: 210-500 nm
Ionization: ESI (positive)
Injection: 2 μL
Internal standard: Reserpine detection limit: 0.1 ng / g
得られた測定結果は以下の通りである。
青トマト中のトマチン含有量：１００ｇ湿重量中２８ｍｇ
トマチジン抽出物中のトマチン含有量：検出限界以下
トマチジン抽出物中のトマチジン含有量：３７重量％（粉末トマチジン含有抽出物１００ｇ中、トマチジン含有量が３７ｇ）
The obtained measurement results are as follows.
Tomatine content in green tomatoes: 28 mg in 100 g wet weight
Tomatine content in tomatine extract: below the detection limit Tomatine content in tomatine extract: 37% by weight (tomatine content is 37 g in 100 g of powdered tomatine-containing extract)
比較例１
実施例１で用いたものと同じ品種の果汁用の熟した青いトマト５４６ｇに、３倍体積量（１，３６８ｍＬ）の水を添加して撹拌し、さらに４Ｎ塩酸を添加してｐＨを３．５に調整した後、６０℃で６０分間撹拌してトマチンを抽出した。抽出液を５０メッシュのろ過器でろ過し、得られたろ液を４Ｎ水酸化ナトリウム溶液でｐＨ９．０に調整することでトマチンを沈殿させた。その後、遠心分離（３，０００ｒｐｍ×１０分）によって沈殿物を回収した。沈殿物を３００ｍＬの水に溶解し、さらに１．２Ｎ塩酸を６００ｍＬ（終濃度０．８Ｎ）添加して、沸騰浴（１００℃）中で３時間保持することでトマチンをトマチジンへ変換した。得られた反応物を冷却し、１Ｎ水酸化ナトリウム水溶液でｐＨ７になるように中和した。中和液を遠心分離（３，０００ｒｐｍ×１０分）し、不溶物を除去した後、カラムにＣｏｓｍｏｓｉｌ １４０Ｃ１８−ＯＰＮを充填した。中和液を通液した後、水（３ＣＶ）で洗浄し、８０ｖ／ｖ％エタノール（３ＣＶ）で溶出した。得られた溶離液を脱塩した後、エバポレーターで濃縮して、溶媒をすべて留去した。その結果、５５２ｍｇの粉末をトマチジン含有抽出物として得た。
Comparative Example 1
To 546 g of ripe blue tomatoes for fruit juice of the same variety as that used in Example 1, a triple volume (1,368 mL) of water was added and stirred, and 4N hydrochloric acid was further added to adjust the pH. After adjusting to 5, tomatine was extracted by stirring at 60 ° C. for 60 minutes. The extract was filtered through a 50 mesh filter, and the obtained filtrate was adjusted to pH 9.0 with a 4N sodium hydroxide solution to precipitate tomatine. Then, the precipitate was collected by centrifugation (3,000 rpm × 10 minutes). The precipitate was dissolved in 300 mL of water, 600 mL of 1.2N hydrochloric acid (final concentration 0.8N) was added, and the mixture was kept in a boiling bath (100 ° C.) for 3 hours to convert tomatine to tomatine. The resulting reaction was cooled and neutralized with 1N aqueous sodium hydroxide solution to pH 7. The neutralized solution was centrifuged (3,000 rpm x 10 minutes) to remove insoluble matters, and then the column was filled with Cosmosil 140C18-OPN. After passing the neutralizing solution, the mixture was washed with water (3CV) and eluted with 80v / v% ethanol (3CV). After desalting the obtained eluent, it was concentrated with an evaporator to distill off all the solvent. As a result, 552 mg of powder was obtained as a tomatidine-containing extract.
実施例１と同様にしてトマチジン含有抽出物を測定した結果、トマチジン含有抽出物中のトマチン量は２４ｇ／１００ｇ、トマチジン量は０．５ｍｇ／１００ｇであった。 As a result of measuring the tomatine-containing extract in the same manner as in Example 1, the amount of tomatine in the tomatine-containing extract was 24 g / 100 g, and the amount of tomatine was 0.5 mg / 100 g.
比較例２
実施例１で用いたものと同じ品種の果汁用の熟した青いトマト５４６ｇを、ミキサーで粉砕しピューレを得た。ピューレをろ布で圧搾し、搾汁を調製した。さらに、搾汁残渣に1倍体積量の水を添加して分散させ、ミキサー（１０００ｒｐｍ）で３０分間攪拌し、ろ布で圧搾することで、水抽出液を得た。搾汁ろ布で圧搾し、元の搾汁液と混合して搾汁液を得た。これに、５０ｖ／ｖ％のメタノールと２ｖ／ｖ％の酢酸とを含む水溶液を添加して、５０℃で２０分間保持して抽出を行った。抽出液を、ガラス濾紙（ＡＤＶＡＮＴＥＣ ＧＡ−２００）で吸引ろ過した後、エバポレーターで３００ｍＬまで濃縮した。この濃縮液に１．２Ｎ塩酸を６００ｍＬ（終濃度０．８Ｎ）を添加して、沸騰浴中で３時間保持した。得られた反応物を冷却し、１Ｎ水酸化ナトリウム水溶液でｐＨ７になるように中和し、中和液を得た。カラムにＣｏｓｍｏｓｉｌ １４０Ｃ１８−ＯＰＮを充填し、中和液を通液した後、水（３ＣＶ）で洗浄し、８０ｖ／ｖ％エタノール（３ＣＶ）で溶出した。得られた溶離液を脱塩した後、エバポレーターで濃縮して、溶媒をすべて留去した。その結果、４０６ｍｇの粉末をトマチジン含有抽出物として得た。
Comparative Example 2
546 g of ripe blue tomatoes for fruit juice of the same variety as that used in Example 1 were pulverized with a mixer to obtain puree. The puree was squeezed with a filter cloth to prepare a juice. Further, 1 times the volume of water was added to the juice residue to disperse it, and the mixture was stirred with a mixer (1000 rpm) for 30 minutes and squeezed with a filter cloth to obtain a water extract. It was squeezed with a juice filter cloth and mixed with the original juice to obtain a juice. An aqueous solution containing 50 v / v% methanol and 2 v / v% acetic acid was added thereto, and the mixture was held at 50 ° C. for 20 minutes for extraction. The extract was suction-filtered with a glass filter paper (ADVANTEC GA-200) and then concentrated to 300 mL with an evaporator. 600 mL (final concentration 0.8N) of 1.2N hydrochloric acid was added to this concentrated solution, and the mixture was kept in a boiling bath for 3 hours. The obtained reaction product was cooled and neutralized with a 1N aqueous sodium hydroxide solution to a pH of 7, to obtain a neutralized solution. The column was filled with Cosmosil 140C18-OPN, passed through a neutralizing solution, washed with water (3CV), and eluted with 80v / v% ethanol (3CV). After desalting the obtained eluent, it was concentrated with an evaporator to distill off all the solvent. As a result, 406 mg of powder was obtained as a tomatidine-containing extract.
実施例１と同様にしてトマチジン含有抽出物を測定した結果、トマチジン含有抽出物中のトマチン量は１６ｇ／１００ｇ、トマチジン量は４ｇ／１００ｇであった。 As a result of measuring the tomatine-containing extract in the same manner as in Example 1, the amount of tomatine in the tomatine-containing extract was 16 g / 100 g, and the amount of tomatine was 4 g / 100 g.
結果
実施例１、比較例１及び比較例２で得られたトマチジン含有抽出物の対比を下記表に示す。下記表から明らかなとおり、比較例１及び２によるトマチジン含有抽出物では、原料由来のトマチンが多く残存し、トマチジンの割合が非常に少ないことに対し、実施例１によるトマチジン含有集出物では、原料由来のトマチンが実質的に含まれておらず、トマチジンの割合が顕著に向上した。
Results The table below shows the comparison of the tomatidine-containing extracts obtained in Example 1, Comparative Example 1 and Comparative Example 2. As is clear from the table below, in the tomatine-containing extracts according to Comparative Examples 1 and 2, a large amount of tomatine derived from the raw material remains and the proportion of tomatine is very small, whereas in the tomatine-containing extract according to Example 1, The raw material-derived tomatine was substantially not contained, and the proportion of tomatine was significantly improved.
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