JP2019113375A - Method of evaluating intestinal environment, method of evaluating risk of developing colitis, and method of screening for intestinal environment improving substances - Google Patents
Method of evaluating intestinal environment, method of evaluating risk of developing colitis, and method of screening for intestinal environment improving substances Download PDFInfo
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Abstract
Description
本発明は、腸内環境評価方法、大腸炎罹患リスク評価方法、及び腸内環境改善物質のスクリーニング方法に関する。 The present invention relates to an intestinal environment evaluation method, a colitis risk evaluation method, and a screening method of an intestinal environment improving substance.
腸内には非常に多くの種類の常在菌が存在している。腸内常在菌のバランスは、年齢によっても変化する。腸内常在菌のバランスは、肥満等の健康の様々な面に関与するとされている。腸内細菌であるＥｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓの投与により、大腸内容物中にフリーラジカルが検出されたことが報告されている（非特許文献１）。腸内に存在するフリーラジカルは、腸壁等の細胞を破壊し、ＤＮＡを損傷させ、ガン化等のリスクを高める恐れがある。 There are numerous types of indigenous bacteria in the intestine. The balance of intestinal enterobacteria also changes with age. The balance of intestinal indigenous bacteria is believed to be involved in various aspects of health such as obesity. It has been reported that free radicals were detected in the contents of the large intestine by administration of the enterobacteria Enterococcus faecalis (Non-patent Document 1). Free radicals present in the intestine may destroy cells such as the intestinal wall, damage DNA, and increase the risk of canceration and the like.
ＥＳＲ装置は、生体試料中のフリーラジカル量の測定に用いられている（例えば非特許文献１、２）。特許文献１には、抗酸化物質の生体内での実際の抗酸化能を評価するために、ＥＳＲ装置を用いて生体内でのフリーラジカル分解能を判定する方法が開示されている。 The ESR apparatus is used to measure the amount of free radicals in a biological sample (for example, Non-Patent Documents 1 and 2). Patent Document 1 discloses a method of determining free radical resolution in vivo using an ESR apparatus in order to evaluate the actual antioxidant capacity of an antioxidant in vivo.
腸内に存在するフリーラジカル量を把握し、コントロールすることは、宿主の健康改善等の効果につながる可能性がある。しかしながら生体において腸内のフリーラジカル量を直接評価するために腸管内容物を試料として用いることは現実的な手法ではない。 Understanding and controlling the amount of free radicals present in the intestine may lead to effects such as improvement of host health. However, it is not a practical method to use intestinal contents as a sample to directly evaluate the amount of free radicals in the intestine in a living body.
本発明は、非侵襲で腸内環境を評価可能な方法を提供することを目的とする。 An object of the present invention is to provide a method capable of noninvasively evaluating the intestinal environment.
本発明者らは意外にも、ＥＳＲ（Ｅｌｅｃｔｒｏｎ Ｓｐｉｎ Ｒｅｓｏｎａｎｃｅ）装置を用いて測定される糞便中のフリーラジカル量が、腸内のフリーラジカル量と強く相関していることを新たに見出した。 The inventors surprisingly found that the amount of free radicals in feces measured using an ESR (Electron Spin Resonance) apparatus is strongly correlated with the amount of free radicals in the intestine.
本発明の腸内環境評価方法は、ＥＳＲ装置を用いて糞便中のフリーラジカル量を測定することを含む。当該方法により、非侵襲で簡便に腸内のフリーラジカル量を評価することができる。 The intestinal environment evaluation method of the present invention includes measuring the amount of free radicals in feces using an ESR apparatus. According to the method, the amount of free radicals in the intestine can be evaluated noninvasively and easily.
本発明の大腸炎罹患リスク評価方法は、ＥＳＲ装置を用いて糞便中のフリーラジカル量を測定することを含む。当該方法により、糞便中のフリーラジカル量を指標として、非侵襲で簡便に大腸炎への罹患リスクを評価することができる。 The colitis risk assessment method of the present invention includes measuring the amount of free radicals in feces using an ESR apparatus. According to the method, the risk of colitis can be evaluated noninvasively and easily using the amount of free radicals in feces as an index.
本発明の腸内環境改善物質のスクリーニング方法は、被験物質を摂取した後の被験動物の糞便中のフリーラジカル量を、ＥＳＲ装置を用いて測定することを含む。当該方法により、被験物質の摂取が腸内環境に与える影響を評価することができ、腸内環境を改善する効果のある物質を簡便にスクリーニングすることができる。 The screening method of the substance for improving intestinal environment according to the present invention comprises measuring the amount of free radicals in feces of a test animal after intake of the test substance using an ESR apparatus. According to this method, the influence of intake of a test substance on the intestinal environment can be evaluated, and a substance having an effect of improving the intestinal environment can be conveniently screened.
上記方法は、測定対象である糞便とスピントラップ剤とを混合することを含んでいてよい。 The method may include mixing the feces to be measured and the spin trap agent.
本発明の腸内環境評価方法により、非侵襲で腸内環境を評価することができる。 The intestinal environment evaluation method of the present invention enables non-invasive evaluation of the intestinal environment.
以下、本発明を実施するための形態について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではない。 Hereinafter, modes for carrying out the present invention will be described in detail. The present invention is not limited to the following embodiments.
本実施形態に係る腸内環境評価方法は、ＥＳＲ装置を用いて糞便中のフリーラジカル量を測定することを含む。 The intestinal environment evaluation method according to the present embodiment includes measuring the amount of free radicals in feces using an ESR apparatus.
本明細書において糞便とは、体外に排出されたものをいい、腸管内に残存している状態のものは含まない。フリーラジカルは反応性が非常に高いため、従来、腸内に存在するフリーラジカルは、糞便とともに体外に排出されると、酸化反応等が生じた結果、腸内のフリーラジカル量を反映したものではなくなると考えられていた。しかしながら本発明者らは、糞便中のフリーラジカル量は意外にも腸内のフリーラジカル量を反映していることを見出した。 In the present specification, feces refer to those excreted outside the body, and do not include those which remain in the intestinal tract. Since free radicals have very high reactivity, conventionally, free radicals existing in the intestine, when excreted outside the body together with feces, cause an oxidation reaction etc., and as a result reflects the amount of free radicals in the intestine. It was believed to be gone. However, the present inventors have found that the amount of free radicals in feces unexpectedly reflects the amount of free radicals in the intestine.
本実施形態に係る腸内環境評価方法は、腸を試料として直接測定する必要がなく、採取しやすい糞便を試料として用いることができるため、非侵襲で簡便に腸内環境を評価することができる。また、糞便を試料として用いることにより、後述するスピントラップ剤を体内に投与する必要なく腸内環境を評価することができる。 The method for evaluating the intestinal environment according to the present embodiment does not have to directly measure the intestine as a sample, and can easily use the non-invasive and easy evaluation of the intestinal environment because feces easy to collect can be used as a sample. . In addition, by using feces as a sample, it is possible to evaluate the intestinal environment without the need to administer a spin trap agent described later into the body.
ＥＳＲ装置は、電子スピン共鳴を用いた装置であり、ラジカルの定性及び定量分析に用いられるものである。ＥＳＲ装置では生体試料から直接フリーラジカル量を測定することが感度的に困難であるため、寿命が短いフリーラジカルを捕捉するために、スピントラップ剤が用いられる。スピントラップ剤は、フリーラジカルと素早く反応し、スピンアダクトを生成する。生成したスピンアダクトをＥＳＲ装置で測定することにより、得られたスペクトルのピーク長から、フリーラジカルの相対量を調べることができる。ピークが長いほど、フリーラジカル量が多いことを示す。また、得られたスペクトルのピーク幅等を解析することによりフリーラジカル種を同定することも可能である。ＥＳＲはいずれの周波数のものであってもよく、例えばＸバンドＥＳＲであってよい。ＥＳＲ装置としては市販の装置を用いることができ、例えばＸ１０ＳＡ（キーコム株式会社製）、ＥＭＸ−ｐｌｕｓ（ブルカーバイオスピン株式会社製）が挙げられる。 The ESR apparatus is an apparatus using electron spin resonance and is used for qualitative and quantitative analysis of radicals. Since it is sensitively difficult to measure the amount of free radicals directly from a biological sample in an ESR apparatus, a spin trapping agent is used to trap free radicals having a short lifetime. Spin trap agents react rapidly with free radicals to produce spin adducts. By measuring the generated spin adduct with an ESR apparatus, the relative amount of free radicals can be determined from the peak length of the obtained spectrum. The longer the peak, the higher the amount of free radicals. It is also possible to identify free radical species by analyzing the peak width etc. of the obtained spectrum. The ESR may be of any frequency, for example the X band ESR. A commercially available apparatus can be used as the ESR apparatus, and examples thereof include X10SA (manufactured by Keycom Co., Ltd.) and EMX-plus (manufactured by Bruker Biospin Co., Ltd.).
測定対象である糞便と混合されるスピントラップ剤としては、例えば、ＰＯＢＮ（α−（４−ｐｙｒｉｄｙｌ−１−ｏｘｉｄｅ）−Ｎ−ｔ−ｂｕｔｙｌｎｉｔｒｏｎｅ）、ＣＹＰＭＰＯ（２−（５，５−Ｄｉｍｅｔｈｙｌ−２−ｏｘｏ−２λ５−［１，３，２］ｄｉｏｘａｐｈｏｓｐｈｉｎａｎ−２−ｙｌ）−２−ｍｅｔｈｙｌ−３，４−ｄｉｈｙｄｒｏ−２Ｈ−ｐｙｒｒｏｌｅ １−ｏｘｉｄｅ）、ＤＭＰＯ（５，５−ｄｉｍｅｔｈｙｌ−１−ｐｙｒｒｏｌｉｎｅ−Ｎ−ｏｘｉｄｅ）、Ｍ４ＰＯ（３，３，５，５−Ｔｅｔｒａｍｅｔｈｙｌ−１−ｐｙｒｒｏｌｉｎｅ−Ｎ−ｏｘｉｄｅ）、ＰＢＮ（Ｎ−ｔｅｒｔ−Ｂｕｔｈｙｌ−α−ｐｈｅｎｙｌｎｉｔｒｏｎｅ）、ＤＢＮＢＳ（３，５−Ｄｉｂｒｏｍｏ−４−ｎｉｔｒｏｓｏｂｅｎｚｅｎｅｓｕｌｆｏｎｉｃ ａｃｉｄ ｓｏｄｉｕｍ ｓａｌｔ）等が挙げられる。スピントラップ剤としては、ＰＯＢＮ又はＣＹＰＭＰＯを用いることが好ましい。ＰＯＢＮは、フリーラジカル検出感度がよいため好ましい。ＣＹＰＭＰＯは、ラジカル種の同定がしやすいため好ましい。 As a spin-trapping agent mixed with feces to be measured, for example, POBN (α- (4-pyridyl-1-oxide) -N-t-butylnitrone), CYPMPO (2- (5,5-Dimethyl-2) -Oxo-2λ5- [1,3,2] dioxaphosphinan-2-yl) -2-methyl-3,4-dihydro-2H-pyrrole 1-oxide), DMPO (5,5-dimethyl-1-pyrroline-N) -Oxide), M4PO (3,3,5,5-Tetramethyl-1-pyrroline-N-oxide), PBN (N-tert-butyl-α-phenylnitrone), DBNBS (3,5-dibromo-4-nitrosobenzene) ulfonic acid sodium salt), and the like. It is preferable to use POBN or CYPMPO as a spin trap agent. POBN is preferred because of its high free radical detection sensitivity. CYPMPO is preferable because it is easy to identify radical species.
測定されるフリーラジカルは、生体内に存在するいずれの種類であってもよく、例えば、スーパーオキシド、ヒドロキシラジカル等の活性酸素種、アルコキシルラジカル、ヒドロペルオキシラジカル、ペルオキシルラジカルなどであってよい。本実施形態に係る方法において測定されるフリーラジカル量は、種類を特に区別せずにフリーラジカルの総量であってもよく、特定又は一部の種類のフリーラジカルの量であってもよい。フリーラジカル量の算出に用いるピークとしては、得られたスペクトルから任意のピークを選択することができる。なお、サンプル間で比較する場合は、ｇ値に基づいて選択するピークを揃える。 The free radical to be measured may be any type existing in the living body, and may be, for example, active oxygen species such as superoxide, hydroxy radical, alkoxyl radical, hydroperoxy radical, peroxyl radical and the like. The amount of free radicals measured in the method according to the present embodiment may be the total amount of free radicals without particular distinction of types, or may be the amount of specific or partial types of free radicals. As a peak used for calculation of the amount of free radicals, any peak can be selected from the obtained spectrum. When comparing samples, select peaks are aligned based on the g value.
ＥＳＲ測定用試料は、例えば、糞便試料にスピントラップ剤を添加することにより調製することができる。測定に用いる糞便試料は、採取した糞便そのものであってよく、糞便を水、緩衝液等に懸濁させた懸濁液であってもよい。糞便は体外に排出された後速やかに採取されたものであることが好ましい。糞便は測定時まで冷凍又は冷蔵（例えば０〜１０℃）で保存されることが好ましい。 The sample for ESR measurement can be prepared, for example, by adding a spin trap agent to a stool sample. The stool sample used for the measurement may be the collected stool itself, or may be a suspension in which the stool is suspended in water, buffer solution or the like. It is preferable that feces are collected immediately after being excreted from the body. The stool is preferably stored frozen or refrigerated (eg 0 to 10 ° C.) until the time of measurement.
ＥＳＲ測定用試料としては、例えば、糞便試料と、スピントラップ剤を緩衝液に溶解した溶液とを混合し、所定時間経過後に混合液に遠心分離を行って得られる上清を用いることができる。スピントラップ剤は糞便試料に対して過剰の量を用いることが好ましい。例えば糞便試料１ｇに対して、１ｍＭ〜１Ｍのスピントラップ剤溶液を５〜５０ｇ混合することによりＥＳＲ測定用試料を調製してもよい。ＥＳＲ測定用試料の調製は、複数試料を比較評価する場合には、例えば試料濃度、スピントラップ剤の種類及び濃度等の条件を試料間で揃えることが好ましい。特に、糞便試料にスピントラップ剤を添加してから、ＥＳＲ装置で測定するまでの時間を試料間で揃えることが好ましい。糞便試料にスピントラップ剤を添加してからＥＳＲ装置により測定するまでの時間は、評価対象である動物が例えばマウス等である場合は、例えば５分〜２時間としてよく、１０分〜１時間としてもよく、２０〜４０分とすることが好ましい。上記時間内とすることにより、より精度よく腸内環境を反映した結果を得ることができる。 As a sample for ESR measurement, for example, a supernatant obtained by mixing a feces sample and a solution in which a spin trap agent is dissolved in a buffer solution and centrifuging the mixed solution after a predetermined time has elapsed can be used. Preferably, the spin-trapping agent is used in excess with respect to the stool sample. For example, the sample for ESR measurement may be prepared by mixing 5 to 50 g of 1 mM to 1 M spin trap agent solution with 1 g of stool sample. In the preparation of a sample for ESR measurement, when a plurality of samples are compared and evaluated, it is preferable that the conditions such as the sample concentration and the type and concentration of the spin trapping agent be the same among the samples. In particular, it is preferable to equalize the time from the addition of the spin trap agent to the stool sample to the time of measurement by the ESR apparatus among the samples. The time from addition of the spin trap agent to the stool sample to measurement by the ESR apparatus may be, for example, 5 minutes to 2 hours, and 10 minutes to 1 hour, for example, when the animal to be evaluated is a mouse etc. It is also preferable to set it to 20 to 40 minutes. By setting the time within the above-mentioned time, it is possible to obtain a result reflecting the intestinal environment more accurately.
調製したＥＳＲ測定用試料をＥＳＲ装置によって測定することにより、試料中のフリーラジカルを検出してスペクトルを得、スペクトルを解析することにより、フリーラジカルの量及び必要に応じてフリーラジカル種を把握することができる。 By measuring the prepared sample for ESR measurement using an ESR apparatus, free radicals in the sample are detected and a spectrum is obtained, and by analyzing the spectrum, the amount of free radicals and, if necessary, free radical species are grasped. be able to.
上記方法における評価対象は、糞便を採取できる動物であればいずれの動物であってもよい。評価対象である動物は例えば哺乳類であってよく、例えば、イヌ及びネコ等のコンパニオン動物、ウマ、ウシ、ブタ及びトリ等の家畜、ヒト、マウス、ラット、ハムスター、サル、ウサギ、モルモット等を挙げることができる。 The evaluation target in the above method may be any animal as long as it can collect feces. The animals to be evaluated may be, for example, mammals, for example, companion animals such as dogs and cats, domestic animals such as horses, cattle, pigs and birds, humans, mice, rats, hamsters, monkeys, rabbits, guinea pigs, etc. be able to.
腸内環境評価方法における評価対象としての腸は、小腸又は大腸であってよい。小腸は、十二指腸、空腸及び回腸からなる群から選ばれる少なくとも１種であってよい。大腸は、盲腸、結腸（上行結腸、横行結腸、下行結腸）、Ｓ状結腸及び直腸からなる群から選ばれる少なくとも１種であってよい。評価対象は大腸であることが好ましい。大腸におけるフリーラジカル量は、糞便中のフリーラジカル量との相関がより強いため、大腸が評価対象であると、より精度よく腸内環境を評価できる。 The intestine as an evaluation target in the intestinal environment evaluation method may be the small intestine or the large intestine. The small intestine may be at least one selected from the group consisting of duodenum, jejunum and ileum. The large intestine may be at least one selected from the group consisting of a cecum, a colon (an ascending colon, a transverse colon, a descending colon), a sigmoid colon and a rectum. It is preferable that an evaluation object is a large intestine. Since the amount of free radicals in the large intestine has a stronger correlation with the amount of free radicals in feces, the intestinal environment can be evaluated more accurately if the large intestine is to be evaluated.
腸内環境の評価とは、例えば、腸内環境がより良いか否かを評価することを含み、具体的には例えば、腸内に存在するフリーラジカルの量を予測することをいう。腸内環境がよりよいとは、具体的には例えば、腸内の特定種類のフリーラジカルの量又はフリーラジカルの総量がより少ないことをいう。腸内のフリーラジカル量がより少ないと、腸管壁等の細胞及び又はＤＮＡ損傷を防ぎ、大腸炎、大腸ガン等の疾患のリスクを軽減することにつながると考えられる。 The evaluation of the intestinal environment includes, for example, the evaluation of whether or not the intestinal environment is better, and specifically refers to, for example, predicting the amount of free radicals present in the intestine. A better intestinal environment specifically means, for example, that the amount of specific types of free radicals in the intestine or the total amount of free radicals is lower. A smaller amount of free radicals in the intestine is considered to prevent damage to cells and / or DNA such as intestinal wall and to reduce the risk of diseases such as colitis and colon cancer.
したがって、本実施形態に係る腸内環境評価方法は、測定したフリーラジカル量に基づき、腸内環境を評価することを含んでいてもよい。 Therefore, the intestinal environment evaluation method according to the present embodiment may include evaluating the intestinal environment based on the measured amount of free radicals.
（大腸炎罹患リスク評価方法）
本発明者らは、後述する実施例に示すように、糞便中のフリーラジカル量が、大腸炎への罹りやすさを反映していることを見出した。したがって上述の腸内環境評価方法は、大腸炎罹患リスク評価方法として応用することができる。
(Method to evaluate colitis risk)
The present inventors have found that the amount of free radicals in feces reflects the susceptibility to colitis, as shown in the examples described later. Therefore, the above-mentioned intestinal environment evaluation method can be applied as a colitis risk evaluation method.
具体的には、被験動物の糞便中のフリーラジカル量が多いほど、当該被験動物が大腸炎に罹患する可能性が高く、糞便中のフリーラジカル量が少ないほど、当該被験動物が大腸炎に罹患する可能性が低いと考えられる。このように、被験動物の糞便中のフリーラジカル量を、当該被験動物の大腸炎への罹患しやすさの指標とすることができる。すなわち、本実施形態に係る大腸炎罹患リスク評価方法は、ＥＳＲ装置を用いて糞便中のフリーラジカル量を測定することを含む。大腸炎は、例えば潰瘍性大腸炎であってよい。本実施形態に係る大腸炎罹患リスク評価方法は、測定したフリーラジカル量及び／又は種類に基づき、大腸炎罹患リスクを評価することを含んでいてよい。 Specifically, the higher the amount of free radicals in the feces of the test animal, the higher the possibility that the test animal suffers from colitis, and the lower the amount of free radicals in the feces, the more the test animal suffers from colitis. It is considered unlikely to be Thus, the amount of free radicals in the feces of the subject animal can be used as an indicator of the susceptibility of the subject animal to colitis. That is, the colitis risk evaluation method according to the present embodiment includes measuring the amount of free radicals in feces using an ESR apparatus. The colitis may be, for example, ulcerative colitis. The colitis risk evaluation method according to the present embodiment may include assessing colitis risk based on the measured amount and / or type of free radicals.
（腸内環境改善物質スクリーニング方法）
本発明はまた、腸内環境改善物質のスクリーニング方法を提供する。腸内環境改善物質スクリーニング方法は、被験物質を摂取した後の被験動物の糞便中のフリーラジカル量を、ＥＳＲ装置を用いて測定することを含む。上述のとおり、糞便中のフリーラジカル量は、腸内のフリーラジカル量を反映したものであるため、被験物質を摂取した後の糞便中のフリーラジカル量を指標として、摂取した物質が腸内の環境に与える影響を評価することができる。腸内環境改善物質とは、例えば、摂取により腸内のフリーラジカル量を低減することができる物質をいう。上記方法により、腸内環境を改善することができる物質を短期間に簡便にスクリーニングすることができる。
(Intestinal environmental improvement substance screening method)
The present invention also provides a method of screening for a substance for improving intestinal environment. The enteric environment improving substance screening method comprises measuring the amount of free radicals in the feces of the test animal after taking the test substance using an ESR apparatus. As described above, since the amount of free radicals in feces reflects the amount of free radicals in the intestine, the ingested substance is in the intestine using the amount of free radicals in feces after intake of the test substance as an index. Environmental impact can be assessed. The enteric environment improving substance refers to, for example, a substance that can reduce the amount of free radicals in the intestine by ingestion. By the above method, substances capable of improving the intestinal environment can be conveniently screened in a short period of time.
上記スクリーニング方法は、例えば、被験動物に被験食品を摂取させること、摂取後の当該被験動物の糞便を採取すること、ＥＳＲ装置を用いて当該糞便中のフリーラジカル量を測定すること、測定されたフリーラジカル量に基づいて当該被験動物の腸内環境を評価すること、及び、評価結果に基づいて、摂取させた被験物質の腸内環境改善効果を評価することを含んでいてもよい。上記スクリーニング方法は、腸内環境の改善に有効と評価された物質を選抜することを含んでいてもよい。上記スクリーニング方法により腸内環境改善に有効と判定された物質は、腸内環境改善用として用いることができる。腸内環境の評価は、フリーラジカル量に加えてフリーラジカルの種類を指標としてもよい。 The screening method includes, for example, feeding a test animal with a test food, collecting feces of the subject animal after ingestion, measuring an amount of free radicals in the feces using an ESR apparatus, and measuring The method may include evaluating the intestinal environment of the subject animal based on the amount of free radicals, and evaluating the intestinal environment improving effect of the test substance ingested based on the evaluation result. The screening method may include selecting a substance that has been evaluated to be effective for improvement of the intestinal environment. A substance determined to be effective for intestinal environment improvement by the above screening method can be used for intestinal environment improvement. The evaluation of the intestinal environment may be based on the type of free radicals in addition to the amount of free radicals.
被験物質は例えば、化合物、組成物、食品、食品成分、食品組成物、サプリメント等であってよい。食品は飲料であってもよい。被験物質としては１種を単独で用いてもよく、複数種を組み合わせて用いてもよい。 The test substance may be, for example, a compound, a composition, a food, a food component, a food composition, a supplement and the like. The food may be a beverage. As the test substance, one type may be used alone, or two or more types may be used in combination.
被験動物は例えば、マウス、ラット、ウサギ等の一般に用いられる実験動物であってよい。候補となる被験物質は、例えば被験動物に与える飲用水、餌等に混ぜることによって摂取させることができる。 The test animal may be, for example, a commonly used experimental animal such as a mouse, a rat, or a rabbit. The candidate test substance can be taken, for example, by mixing it with drinking water, food, etc. given to the subject animal.
以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be more specifically described based on examples. However, the present invention is not limited to the following examples.
［試験例１：糞便と消化管との比較］
８週齢のｄｄＹマウス（ｎ＝６）（日本ＳＬＣ）から糞便を回収した。回収した糞便はクラッシュアイス上で測定時まで保管した。スピントラップ剤にはＮ−ｔｅｒｔ−Ｂｕｔｙｌ−α−（４−ｐｙｒｉｄｙｌ）ｎｉｔｒｏｎｅ Ｎ’−Ｏｘｉｄｅ（ＰＯＢＮ）（東京化成）を用いた。糞便に、１００ｍＭ ＰＯＢＮ溶液（Ｄ−ＰＢＳ−ＥＤＴＡ、ｐＨ７．０）を糞便重量の９倍量添加し、１分間スパチュラにて懸濁した。１５秒間ボルテックスミキサーにより攪拌した後、３７℃で静置した。糞便にスピントラップ剤を添加してから３０分後に、１３，０００ｒｐｍで２分間遠心分離を行い、上清をＥＳＲ用扁平石英ホルダーに封入した。ＥＳＲ分光器はＸバンドのＸ１０ＳＡ（キーコム株式会社）を用いた。ＥＳＲの測定は次のパラメータ設定で行った。周波数９．５ＧＨｚ、変調周波数１００ｋＨｚ、マイクロウェーブパワー１６ｍＷ、掃引巾１６ｍＴ、時定数０．０３ｓｅｃ、スキャン数５回である。Ｍｎマーカーをスタンダードとして利用した。
[Test Example 1: Comparison of Feces and Gastrointestinal Tract]
Feces were collected from 8-week-old ddY mice (n = 6) (Japan SLC). The collected feces were stored on crushed ice until measurement. As a spin trap agent, N-tert-Butyl-α- (4-pyridyl) nitrone N′-Oxide (POBN) (Tokyo Kasei Co., Ltd.) was used. To the feces was added 100 mM POBN solution (D-PBS-EDTA, pH 7.0) at 9 times the weight of the feces, and suspended with a spatula for 1 minute. After stirring with a vortex mixer for 15 seconds, it was allowed to stand at 37 ° C. Thirty minutes after adding the spin trap agent to the feces, centrifugation was performed at 13,000 rpm for 2 minutes, and the supernatant was sealed in a flat quartz holder for ESR. The ESR spectrometer used X10SA (Keycom Co., Ltd.) of X band. The measurement of ESR was performed with the following parameter settings. The frequency is 9.5 GHz, the modulation frequency is 100 kHz, the microwave power is 16 mW, the sweep width is 16 mT, the time constant is 0.03 sec, and the number of scans is five. The Mn marker was used as a standard.
また、８週齢のｄｄＹマウスにペントバルビタール（東京化成）を投与することにより麻酔を行った。上記マウスを開腹した後、小腸（盲腸入口から５〜１５ｃｍの間）（ｎ＝７）又は大腸（盲腸出口から２〜７ｃｍ）（ｎ＝６）の両端を外科用糸で縛った。２２Ｇの留置針を用いて、１００ｍＭ ＰＯＢＮ溶液を、小腸には８００μｌ、大腸には７００μｌそれぞれ充填した。充填３０分後に小腸内容物及び大腸内容物を切り取って回収し、１３，０００ｒｐｍ、２分間遠心した。上清を採取し、上記と同じ条件にてＥＳＲ測定を実施した。なお、大腸内容物を採取した個体は、糞便を試験した個体と同一である。 In addition, anesthesia was performed by administering pentobarbital (Tokyo Kasei) to 8-week-old ddY mice. After the mice were opened, both ends of the small intestine (between 5 and 15 cm from the cecal entrance) (n = 7) or the large intestine (2 and 7 cm from the ceca exit) (n = 6) were tied with surgical threads. Using a 22G indwelling needle, the 100 mM POBN solution was filled with 800 μl in the small intestine and 700 μl in the large intestine. After filling for 30 minutes, the contents of the small intestine and the large intestine were cut and collected, and centrifuged at 13,000 rpm for 2 minutes. The supernatant was collected and ESR measurement was performed under the same conditions as described above. The individual from which the contents of the large intestine were collected is the same as the individual whose feces was tested.
試験例１における、小腸、大腸及び糞便におけるフリーラジカルスペクトルの一例を図１に示す。図１のスペクトルにおいて、ａＮは１．５２ｍＴ、ａＨは０．２２ｍＴと計測された。超微細結合定数から、ヒドロキシラジカルのアダクトが検出されたと推測される。 An example of the free radical spectrum in the small intestine, the large intestine and the feces in Test Example 1 is shown in FIG. In the spectrum of FIG. 1, aN was measured to be 1.52 mT and aH was measured to be 0.22 mT. From the hyperfine coupling constant, it is inferred that adducts of hydroxy radicals have been detected.
各サンプルのフリーラジカル量の比較は、図１中の＊印を付したピークを用い、Ｍｎ由来ピーク長、凍結乾燥重量、及びスピントラップ注入量（反応容量）の値を用いて次の計算式により補正して行った。
（ＰＯＢＮのピーク長／Ｍｎピーク長）÷（凍結乾燥重量／反応容量）
The comparison of the amount of free radicals of each sample uses the peak marked * in FIG. 1 and the following formula using the value of Mn-derived peak length, freeze-dried weight, and spin trap injection amount (reaction volume) Corrected by
(POBN peak length / Mn peak length) ÷ (lyophilized weight / reaction volume)
糞便におけるヒドロキシルラジカル量を１とした場合の相対比較の結果を図２に示す。糞便中のフリーラジカル量は、小腸及び大腸、特に大腸におけるフリーラジカル量を反映していることが示された。 The result of relative comparison when the amount of hydroxyl radicals in feces is 1 is shown in FIG. It has been shown that the amount of free radicals in feces reflects the amount of free radicals in the small intestine and the large intestine, particularly the large intestine.
［試験例２：マウスの系統間比較］
８週齢のｄｄＹマウス、ＩＣＲマウス及びＣ５７ＢＬ／６マウス（日本ＳＬＣ）から糞便を回収した。回収した糞便をクラッシュアイス上で測定時まで保管した。スピントラップ剤としては、２−（５，５−Ｄｉｍｅｔｈｙｌ−２−ｏｘｏ−２λ５−［１，３，２］ｄｉｏｘａｐｈｏｓｐｈｉｎａｎ−２−ｙｌ）−２−ｍｅｔｈｙｌ−３，４−ｄｉｈｙｄｒｏ−２Ｈ−ｐｙｒｒｏｌｅ １−ｏｘｉｄｅ（ＣＹＰＭＰＯ、株式会社司代システム）を用いた。
[Test Example 2: Inter-strain comparison of mice]
Feces were collected from 8-week-old ddY mice, ICR mice and C57BL / 6 mice (Japan SLC). The collected feces were stored on crushed ice until measurement. As a spin trapping agent, 2- (5,5-Dimethyl-2-oxo-2λ5- [1,3,2] dioxaphosphonan-2-yl) -2-methyl-3,4-dihydro-2H-pyrrole 1- oxide (CYPMPO, Inc., Shidai System, Inc.) was used.
糞便に、１００ｍＭのＣＹＰＭＰＯ溶液（Ｔｒｉｓ−ＥＤＴＡ、ｐＨ８．０）を糞便重量の９倍量添加し、１分間スパチュラにて懸濁した。懸濁液を１５秒間ボルテックスミキサーで攪拌した後、室温にて静置した。糞便にスピントラップ剤を添加してから３０分後に、３，０００ｒｐｍで１分間遠心分離を行い、上清をＥＳＲ用扁平ディスポーザブルセル（ラジカルリサーチ株式会社）に封入した。ＥＳＲ分光器はＸバンドのＥＭＸ−ｐｌｕｓ（ブルカーバイオスピン株式会社）を用いた。ＥＳＲの測定は次のパラメータ設定で行った。周波数９．８ＧＨｚ、変調周波数１００ｋＨｚ、マイクロ波パワー６ｍＷ、掃引巾２０ｍＴ、時定数０．１ｓ、スキャン数１０回である。 To the feces was added 100 mM of CYPMPO solution (Tris-EDTA, pH 8.0) at a volume of 9 times the fecal weight, and suspended with a spatula for 1 minute. The suspension was stirred with a vortex mixer for 15 seconds and then allowed to stand at room temperature. Thirty minutes after addition of the spin-trapping agent to feces, centrifugation was performed at 3,000 rpm for 1 minute, and the supernatant was sealed in a flat disposable cell for ESR (radical research corporation). The ESR spectrometer used X-band EMX-plus (Bruker Biospin Co., Ltd.). The measurement of ESR was performed with the following parameter settings. The frequency is 9.8 GHz, the modulation frequency is 100 kHz, the microwave power is 6 mW, the sweep width is 20 mT, the time constant is 0.1 s, and the number of scans is 10 times.
試験例２における、フリーラジカルスペクトルの一例を図３に示す。超微細結合定数を算出した結果、ヒドロキシルラジカル（aN: 1.37, 1.35 mT, aH: 1.36, 1.23 mT, aP: 4.87, 4.68 mT）及びスーパーオキシド（aN: 1.27, 1.27 mT, aH: 1.11, 1.06 mT, aP: 5.24, 5.09 mT）が同定された。各群の比較は、一元配置分散分析を実施した後、Ｔｕｋｅｙの多重比較検定により行った。ヒドロキシラジカルの相対比較結果を図４（ａ）に、スーパーオキシドの相対比較結果を図４（ｂ）に示す。３種のマウスではｄｄＹマウスの糞便においてヒドロキシルラジカル及びスーパーオキシドがともに多いことが確認された。ｄｄＹマウスの腸内においてフリーラジカル量がその他の系統マウスと比較して多いことが示唆された。 An example of the free radical spectrum in Test Example 2 is shown in FIG. As a result of calculating the hyperfine coupling constant, hydroxyl radical (aN: 1.37, 1.35 mT, aH: 1.36, 1.23 mT, aP: 4.87, 4.68 mT) and superoxide (aN: 1.27, 1.27 mT, aH: 1.11, 1.06 mT) , aP: 5.24, 5.09 mT) were identified. Comparison of each group was performed by Tukey's multiple comparison test after performing one-way analysis of variance. The relative comparison result of hydroxy radicals is shown in FIG. 4 (a), and the relative comparison result of superoxide is shown in FIG. 4 (b). It was confirmed that both hydroxyl radicals and superoxide were high in the feces of ddY mice in three mice. It was suggested that the amount of free radicals in the intestine of ddY mice was higher compared to other strain mice.
［試験例３：大腸炎の誘発］
６週齢のｄｄＹマウス、ＩＣＲマウス及びＣ５７ＢＬ／６（日本ＳＬＣ）を各１０匹ずつ導入し、１週間予備飼育を行った。その後、分子量２５，０００のＤＳＳ（デキストラン硫酸ナトリウム）を４％濃度となるように飲水に添加し、マウスに自由摂取させた。ＤＳＳ摂取開始後の生存個体数を記録した。結果を図５に示す。ＤＳＳは大腸炎誘発試薬として一般に用いられる薬剤である。評価したマウスの生存日数は通常の寿命より著しく短かったことから、マウスはＤＳＳによって誘発された大腸炎に起因して死亡したものと推測できる。ＤＳＳ摂取開始後の生存個体数の減少が最も早いのはｄｄＹマウスであった。試験例２の結果と併せると、通常時の糞便中フリーラジカル量が多い系統のマウスは、糞便中フリーラジカル量がより少ない他の系統のマウスと比較して、大腸炎誘発時に大腸炎を発症しやすい傾向にあることが示唆された。試験例２及び３の結果は、糞便中のフリーラジカル量に基づいて大腸炎への罹りやすさを予測可能であることを示している。
[Test Example 3: Induction of colitis]
Ten weeks old each of ddY mice, ICR mice and C57BL / 6 (Japan SLC) were introduced and pre-fed for one week. Thereafter, DSS (dextran sodium sulfate) having a molecular weight of 25,000 was added to the drinking water to a 4% concentration, and the mice were allowed to freely take it. The number of surviving individuals after initiation of DSS intake was recorded. The results are shown in FIG. DSS is a drug commonly used as a colitis inducing agent. Since the survival days of the mice evaluated were significantly shorter than the normal lifespan, it can be inferred that the mice died due to DSS-induced colitis. It was ddY mice that the fastest decline in the number of survivors after DSS intake began. In combination with the results of Test Example 2, mice with a strain with a large amount of free radicals in feces usually develop colitis at the time of colitis induction compared with mice with other strains with a smaller amount of free radicals in stools. It was suggested that it was easy to do. The results of Test Examples 2 and 3 indicate that the susceptibility to colitis can be predicted based on the amount of free radicals in feces.
Claims (4)
The method according to any one of claims 1 to 3, comprising mixing the feces to be measured and the spin trap agent.
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