JP4024128B2 - Extracts of flower parts of Araceae and their uses - Google Patents

Description

【０００６】
【化６】

で表される新規化合物が提供される。
【０００７】
【発明の実施の形態】
本発明の肝疾患の予防又は治療剤は、ウコギ科（Araliaceae）ニンジン属（Panax)の植物の花部を用いて抽出した抽出液又は抽出エキスを有効成分として含有する。ウコギ科ニンジン属の植物は、例えば、日本、韓国、中国等の山地、例えば、特に雲南省、広西省等の標高８００〜１０００ｍ程度の地域に広く分布している植物であれば特に限定されるものではなく、サンシチニンジン（P. notoginseng (BURK.) F. H. CHEN.）、チクセツニンジン、オタネニンジン(P. ginseng）等が挙げられ、なかでも、サンシチニンジンが代表的に用いられる。
これらの植物の花部は、通常、乾燥して用いられるが、その形態等は特に限定されない。
【０００８】
ウコギ科植物の花部から抽出液を得るために、例えば、花部をそのまま、粉砕して及び／又は乾燥し、１〜５０倍（重量）程度、好ましくは２０〜３０倍程度の低級脂肪族アルコール又はその含水物を用いて抽出することが適当である。低級脂肪族アルコールとしては、炭素数１〜４の脂肪族アルコールが挙げられ、例えば、メタノール、エタノール、プロパノール、ｎ−ブタノール、ｔ−ブタノール等が挙げられる。なかでも、メタノール又はエタノールが好ましい。また、１〜３０重量％程度の水を含有する低級脂肪族アルコール含水物であってもよい。
【０００９】
抽出は、冷浸、温浸又は熱浸のいずれで行ってもよい。例えば、５０〜８５℃程度の温度で、振盪下又は非振盪下に、上述した花部を上述した溶媒に浸漬することによって行うことが適当である。振盪下に浸漬する場合には、３０分間〜１０時間程度行うことが適当であり、非振盪下に浸漬する場合は、１時間〜２０日間程度行うことが適当である。なお、抽出処理は、同一原料について１回のみ行ってもよいが、複数回、例えば、２〜５回程度行うことが好ましい。
【００１０】
得られた抽出液は、濃縮して抽出エキスとしてもよい。濃縮は、低温低圧下で行うことが好ましい。この濃縮は乾固するまで行ってもよい。なお、濃縮する前にろ過し、ろ液を濃縮してもよい。また、抽出エキスは、濃縮したままの状態であってもよいし、粉末状又は凍結乾燥品等としてもよい。濃縮する方法、粉末状及び凍結乾燥品とする方法は、当該分野で公知の方法を用いることができる。
【００１１】
得られた抽出液は、濃縮する前後に、精製処理に付してもよい。精製処理は、クロマトグラフ法、イオン交換樹脂を使用する溶離法、溶媒による分配抽出等を単独又は組み合わせて採用することができる。例えば、クロマトグラフ法としては、順相クロマトグラフィー、逆相クロマトグラフィー、高速液体クロマトグラフィー、遠心液体クロマトグラフィー、カラムクロマトグラフィー、薄層クロマトグラフィー等のいずれか又はそれらを組み合わせて行う方法が挙げられる。この際の担体、溶出溶媒等の精製条件は、各種クロマトグラフィーに対応して適宜選択することができる。
【００１２】
特に、抽出液を濃縮して抽出エキスとしたものを、酢酸エチル及び水を用いて分配抽出して、水可溶画分とすることが好ましい。また、抽出液が、メタノール、エタノール又はプロパノールで抽出して得られた場合には、抽出液を濃縮して抽出エキスとし、酢酸エチル／水を用いて分配抽出し、さらにその水相をブタノールで分配抽出して、ブタノール可溶画分とすることが好ましい。分配抽出は、当該分野で通常行われる方法にしたがって行うことができる。例えば、室温下、振盪下又は非振盪下に、抽出エキス等に対して、酢酸エチル又はブタノールと水とを１〜３０（重量）倍程度（１：１０〜１０：１）加えて行うことが適当である。
【００１３】
得られた各画分は、さらに濃縮してもよいし、精製処理に付してもよい。ここでの濃縮及び精製処理は、上述した方法と同様に行うことができる。
また、本発明においては、上記に示す式（ａ）〜式（ｅ）のいずれかで表される化合物が、新規化合物として見出された。なお、これらの化合物は可能な場合には、それらの金属塩、有機酸又は無機酸塩、有機塩基又は無機塩基の塩、溶媒和物の形態であってもよい。
これらの化合物は、上述したような低級脂肪族アルコール又はその含水物による抽出液を濃縮して得られた抽出エキスを、酢酸エチル／水で分配抽出した場合の水相を、さらにブタノールで分配抽出した際に、ブタノール可溶画分に主に含有される。これらの化合物は、肝障害抑制作用を有するため、肝疾患の予防又は治療剤として用いることができる。なお、予防又は治療剤とは、医薬品のほか、健康食品等としての適用をも含む。
【００１４】
上記のような抽出液又は抽出エキス、式（ａ）〜式（ｅ）の化合物は、それぞれそのままの状態又は適当な媒体で希釈して、医薬品等の製造分野において公知の方法によって、散剤、顆粒剤、錠剤、カプセル剤又は液剤等の種々の医薬品の形態で使用することができる。これらの形態においては、適当な媒体を添加してもよい。適当な媒体としては、医薬的に受容な賦形剤、例えば結合剤（例えばシロップ、アラビアゴム、ゼラチン、ソルビトール、トラガント又はポリビニルピロリドン）；充填剤（例えば乳糖、砂糖、トウモロコシ澱粉、リン酸カルシウム、ソルビトール又はグリシン）；錠剤用滑剤（例えばステアリン酸マグネシウム、タルク、ポリエチレングリコール又はシリカ）；崩壊剤（例えば馬鈴薯澱粉）又は受容な湿潤剤（例えばラウリル硫酸ナトリウム）等が挙げられる。錠剤は、通常の製薬の実際に周知の方法でコートしてもよい。液体製剤は、例えば水性又は油性の懸濁液、溶液、エマルジョン、シロップ又はエリキシルの形態であってもよく、使用前に水又は他の適切な賦形剤と混合する乾燥製品として提供してもよい。こうした液体製剤は、通常の添加剤、例えば懸濁剤（例えばソルビトール、シロップ、メチルセルロース、グルコースシロップ、ゼラチン水添加食用脂）；乳化剤（例えばレシチン、ソルビタンモノオレエート又はアラビアゴム）；（食用脂を含んでいてもよい）非水性賦形剤（例えばアーモンド油、分画ココヤシ油又はグリセリン、プロピレングリコール又はエチルアルコールのような油性エステル）；保存剤（例えばメチル又はプロピルｐ−ヒドロキシ安息香酸塩又はソルビン酸）及び所望により着色剤等を含んでいてもよい。
【００１５】
また、上記抽出液又は抽出エキス、式（ａ）〜式（ｅ）の化合物は、健康食品に利用することができる。健康食品とは、通常の食品よりも積極的な意味で、保健、健康維持・増進等の目的とした食品を意味し、例えば、液体又は半固形、固形の製品、具体的には、散剤、顆粒剤、錠剤、カプセル剤又は液剤等のほか、クッキー、せんべい、ゼリー、ようかん、ヨーグルト、まんじゅう等の菓子類、清涼飲料、お茶類、栄養飲料、スープ等の形態が挙げられる。これらの食品の製造工程において、あるいは最終製品に、上記抽出液等を混合又は塗布、噴霧などして添加して、健康食品とすることができる。
【００１６】
上記抽出液又は抽出エキス、式（ａ）〜式（ｅ）の化合物の使用量は、濃縮、精製の程度、活性の強さ等、使用目的、治療又は予防等の対象疾患、その疾患の程度、体重、年齢、症状等によって適宜調整することができ、例えば、成人１回につき抽出液又は抽出エキスでは精製度や水分含量等に応じて、１〜２００００ｍｇ程度が挙げられ、化合物では、１〜１００００ｍｇ程度が挙げられる。服用は、食前３０分位に１日３回行うのが望ましい。また、健康食品としての使用時には、食品の味や外観に悪影響を及ぼさない量、例えば、対象となる食品１ｋｇに対し、上記抽出液又は抽出エキス、式（ａ）〜式（ｅ）の化合物を、１０〜１０００００ｍｇ程度の添加することが適当である。
以下、本発明の抽出物、新規化合物及びそれらの作用についての実施例を具体的に説明する。
【００１７】
ウコギ科植物三七花の抽出
（１）三七花メタノール抽出エキスの調製
三七花４９３ｇを粉砕し、約１０倍量のメタノール（ナカライテスク社製、特級）（５Ｌ）を加え、加熱還流下、３時間抽出した。抽出後、ひだ折りろ紙（アドバンテック社製、No. ２のろ紙）にてろ過し、抽出残査にさらにメタノール（５Ｌ)を加え、３時間加熱還流し、同様にろ過作業をおこなった。合計３回の抽出をおこない、その抽出液をあわせ、ロータリーエバポレーターを用いて、減圧下溶媒留去し、三七花の抽出エキス２１１ｇ（生薬からの収率４２．８％）を得た。
【００１８】
（２）三七花メタノール抽出エキスの溶媒分画の調製
三七花メタノール抽出エキス（１２５ｇ）を水（２Ｌ)に懸濁させ、分液ロートを用いて２Ｌの酢酸エチル（ナカライテスク社製、特級）で分配抽出する。水層にさらに酢酸エチル（２Ｌ）を加えて、溶媒抽出し、同様の操作を計３回行い、酢酸エチル層を得た。酢酸エチル抽出液を合して、減圧下溶媒留去し、酢酸エチル移行部エキス（２２．６ｇ、４．６％）を得た。
水層について、さらに２Ｌの１−ブタノール（ナカライテスク社製、特級）を加えて溶媒抽出し、同様の操作を計３回行い、１−ブタノール層を得た。それぞれを同様に減圧下溶媒留去し、１−ブタノール移行部エキス（１４６．３ｇ、２９．２％）及び水移行部エキス（４４．０ｇ、８．９％）を得た。
【００１９】
（３）１−ブタノール移行部エキスの分離・精製
１−ブタノール移行部エキス（７３．８ｇ）を順相シリカゲルカラムクロマトグラフィー（富士シリシア社製、ＢＷ−２００、１５０〜３５０メッシュ、２．５ｋｇ、移動相：クロロホルム（ナカライテスク社製、特級）：メタノール：水（１０：３：１、下層）→メタノール）にて順次溶出し、フラクション１（０．８１ｇ）、２（３．７０ｇ）、３（１０．２０ｇ）、４（３０．３０ｇ）、５（２５．８０ｇ）、６（２．８４ｇ）を得た。
【００２０】
このうちフラクション４（３０．００ｇ）について、逆相オクタデシルシリル（以下ＯＤＳ）カラムクロマトグラフィー（富士シリシア社製、Chromatrex ODS DM1020T、１００〜２００メッシュ、９００ｇ、移動相：メタノール：水（７０：３０）→メタノール）にて分離、精製し、ジンセンノサイド−Ｒｂ₃（ginsenoside-Rb₃、２、２．８７％）を得た。さらに、高速液体クロマトグラフィー（以下ＨＰＬＣ）［検出器：島津示唆屈折型検出器RID-6A、ポンプ：島津LC-10A、HPLCカラム：YMC社製YMC Pack-ODS-A、２０ｍｍi.d.×２５０ｍｍ、移動相：メタノール：水（８０：２０）］にて分離、精製し、ジンセンノサイド−Ｒｂ₁（ginsenoside-Rb₁、１、０．２４％)、ジンセンノサイド−Ｒｂ₃（２、３．１５％）、ジンセンノサイド−Ｒｄ（３、０．０１０％）、ジンセンノサイド−Ｒｃ（４、１．７９％）、ギペノサイド−ＩＸ（gypenoside-IX、 ５、０．１１％)、ギペノサイド−XVII (６、０．１８％）及び新規化合物ノトジンセンノサイド−Ｔ（notoginsenoside-T、７、０．０１１％)とノトジンセンノサイド−Ｏ（８、０．０１０％）とを得た。
【００２１】
フラクション５（２５．００ｇ）について、ＯＤＳカラムクロマトグラフィー（５０ｇ、移動相：メタノール：水（６０：４０→７０：３０）→メタノール）及びＨＰＬＣ（移動相：メタノール：１％酢酸水溶液（６０：３５））にて分離、精製し、ノトジンセンノサイド−Ｆａ（notoginsenoside-Fa、９、０．４１％)、ノトジンセンノサイド−Ｄ（１０、０．００９％）及び新規化合物ノトジンセンノサイド−Ｑ（notoginsenoside-Q、１１、０．０１４％）と、ノトジンセンノサイド−Ｐ （１２、０．００８％）と、ノトジンセンノサイド−Ｓ（１３、０．０２９％）とを得た。
（４）新規化合物の構造及び物性
上記で得られた新規化合物の構造式を以下に示す。
【００２２】
【化７】

【００２３】
【化８】

【００２４】
▲１▼notoginsenoside-T （７）の物性値
性状：無色結晶（融点：196〜198℃）
旋光度：[α]_D ²⁸ ＋０．３° (ｃ＝1.30, MeOH)
高分解能質量分析(High-resolution positive-ion FAB-MS)：
理論値 C₅₂H₈₈O₂₁Na (M+Na)⁺ : 1071.5716
実測値 : 1071.5708
赤外吸収スペクトル (KBr, ｃｍ^-1): 3410, 1655, 1078
質量分析
positive-ion FAB-MS: m/z 1071 (M＋Na)⁺
negative-ion FAB-MS: m/z 1047 (M−H)^-
核磁気共鳴スペクトル：
¹H-NMR (500 MHz, ピリジン-ｄ₅):δ0.84, 0.98, 0.98, 0.99, 1.29, 1.63, 1.63, 1.68 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H₃), 4.89 (1H, d, J =7.7 Hz, 1'-H), 4.97 (1H, d, J=7.6 Hz, 1'’’-H), 5.09 (1H, d, J=7.6 Hz, 1''-H), 5.19 (1H, d, J=7.4 Hz, 1''''-H), 5.34 (1H, d like, 24-H).
¹³C-NMR (125 MHz, ピリジン-ｄ₅):δc （表１に示す）
【００２５】
▲２▼notoginsenoside-O （８）の物性値
性状：無色結晶（融点：194〜196 ℃）
旋光度：[α]_D ²⁸ ＋2.1° (c=1.00, MeOH)
高分解能質量分析(High-resolution positive-ion FAB-MS)：
理論値 C₅₂H₈₈O₂₁Na (M+Na)⁺ : 1071.5716
実測値 : 1071.5721
赤外吸収スペクトル (KBr, ｃｍ^-1): 3431, 1684, 1078
質量分析
positive-ion FAB-MS: m/z 1071 (M＋Na)⁺
negative-ion FAB-MS: m/z 1047 (M−H)^-
核磁気共鳴スペクトル：
¹H-NMR (500 MHz, ピリジン-ｄ₅):δ0.83, 0.98, 0.99, 1.00, 1.29, 1.63, 1.63, 1.67 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H₃), 4.89 (1H, d like, 1 ''''-H), 4.90 (1H, d like, 1'''-H), 4.91 (1H, d like, 1'-H), 5.09 (1H, d, J=7.7 Hz, 1''-H), 5.31 (1H, d like, 24-H).¹³C-NMR (125 MHz, ピリジン-ｄ₅):δc （表１に示す）
【００２６】
▲３▼notoginsenoside-Q （１１）の物性値
性状：無色結晶（融点：196〜198 ℃）
旋光度：[α]_D ²⁸ ＋6.8° (ｃ＝1.20, MeOH)
高分解能質量分析(High-resolution positive-ion FAB-MS)：
理論値 C₆₄H₁₀₈O₃₁Na (M+Na)⁺ : 1395.6772
実測値 : 1395.6759
赤外吸収スペクトル (KBr, ｃｍ^-1): 3410, 1655, 1076
質量分析
positive-ion FAB-MS: m/z 1395 (M＋Na)⁺
negative-ion FAB-MS: m/z 1371 (M−H)^-
核磁気共鳴スペクトル：
¹H-NMR (500 MHz, ピリジン-ｄ₅):δ0.83, 0.96, 0.98, 1.11, 1.28, 1.64, 1.64, 1.69 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H₃), 4.90 (1H, d, J =7.3 Hz, 1'-H), 5.08 (1H, d, J=7.0 Hz, 1'''''-H), 5.09 (1H, d, J=7.3 Hz, 1''''-H), 5.22 (1H, d, J=7.3 Hz, 1''''''-H), 5.34 (1H, d like, 24-H), 5.36 (1H, d, J=6.7 Hz, 1'''-H), 5.48 (1H, d, J=8.9 Hz, 1''-H).
¹³C-NMR (125 MHz, ピリジン-ｄ₅):δc （表１に示す）
【００２７】
▲４▼notoginsenoside-P (１２)の物性値
性状：無色結晶（融点：194〜196 ℃）
旋光度：[α]_D ²⁸ −0.6° (ｃ＝0.70, MeOH)
高分解能質量分析(High-resolution positive-ion FAB-MS)：
理論値 C₆₃H₁₀₆O₃₀Na (M+Na)⁺ : 1365.6667
実測値 : 1365.6681
赤外吸収スペクトル (KBr, ｃｍ^-1): 3410, 1655, 1076
質量分析
positive-ion FAB-MS: m/z 1365 (M＋Na)⁺
negative-ion FAB-MS: m/z 1341 (M−H)^-
核磁気共鳴スペクトル：
¹H-NMR (500 MHz, ピリジン-ｄ₅):δ0.81, 0.96, 0.96, 1.10, 1.27, 1.62, 1.62, 1.66 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H₃), 4.89 (1H, d, J =7.0 Hz, 1'-H), 4.89 (1H, d, J=7.0 Hz, 1'''''-H), 4.89 (1H, d, J=7.0 Hz, 1''''''-H), 5.08 (1H, d, J=7.3 Hz, 1''''-H), 5.31 (1H, d like, 24-H), 5.35 (1H, d, J=6.7 Hz, 1'''-H), 5.47 (1H, d, J=9.1 Hz, 1''-H).
¹³C-NMR (125 MHz, ピリジン-ｄ₅): δc （表１に示す）
【００２８】
▲５▼notoginsenoside-S (１３)の物性値
性状：無色結晶（融点：186〜188 ℃）
旋光度：[α]_D ²⁸ −8.7° (ｃ＝1.40, MeOH)
高分解能質量分析(High-resolution positive-ion FAB-MS)：
理論値 C₆₃H₁₀₆O₃₀Na (M+Na)⁺ : 1365.6667
実測値 : 1365.6653
赤外吸収スペクトル (KBr, ｃｍ^-1): 3410, 1655, 1076
質量分析
positive-ion FAB-MS: m/z 1365 (M＋Na)⁺
negative-ion FAB-MS: m/z 1341 (M−H)^-
核磁気共鳴スペクトル：
¹H-NMR (500 MHz, ピリジン-ｄ₅):δ0.81, 0.94, 0.96, 1.11, 1.27, 1.61, 1.63, 1.67 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H₃), 4.85 (1H, d, J =7.6 Hz, 1''''''-H), 4.89 (1H, d, J=7.3 Hz, 1'-H), 5.09 (1H, d, J=6.7 Hz, 1''''-H), 5.32 (1H, d like, 24-H), 5.35 (1H, d, J =6.4 Hz, 1'''-H), 5.46 (1H, d, J=7.6 Hz, 1''-H), 5.59 (1H, d like, 1 '''''-H).¹³C-NMR (125 MHz, ピリジン-ｄ₅):δc （表１に示す）
【００２９】
【表１】

【００３０】
Ｄ−ガラクトサミンとリポ多糖に誘起された肝障害に対する作用
（１）実験方法
Tiegsらの方法（Tiegs G., Wolter M., Wendel A., Biochem. Pharmacol., 38, 627−631 (1989)）に準じて実験を行った。
つまり、約２０時間絶食したｄｄＹ系雄性マウス（６週齢、体重約２５ｇ、紀和実験動物研究所社製）に対し、５％ (w/v) アラビアゴム末（ナカライテスク社製）により、水性懸濁液とした被験物質を腹腔内投与した。１時間後、生理食塩水に溶解したD-galactosamine hydrochloride (３５０ｍｇ／ｋｇ、和光純薬工業社製) 及びlipopolysaccharide (１０μｇ／ｋｇ、Sigma) 混液を１０ｍＬ／ｋｇ(ＢＷ) の液量で腹腔内投与した。１０時間、絶食、絶水下飼育した後、無麻酔下、眼窩静脈叢より採血し、血液サンプルを得た。
【００３１】
得られた血液サンプルを遠心分離（３，０００ｒｐｍ、１０分間、４℃）して得られた血清はトランスアミナーゼ活性を測定するまで凍結（−２０℃）して保存した。
血清中トランスアミナーゼ（ｓ−ＧＰＴ、ｓ−ＧＯＴ）活性の測定には市販キットであるエスティーエーテストワコー（和光純薬工業社製）を使用した。
得られた結果を表２〜表４に示す。
【００３２】
【表２】

【００３３】
【表３】

【００３４】
【表４】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an extract of a flower part of Araliaceae carrot (Panax) plant and use thereof, and more specifically, has an action of inhibiting liver damage contained in a flower part of a carrot family of Araceae. The present invention relates to a prophylactic or therapeutic agent for liver diseases and a novel compound comprising an extract or extract as an active ingredient.
[0002]
[Prior art]
A plant belonging to the family Aceraceae (Panax), Sancytin carrot (scientific name: notoginseng (BURK.) FH CHEN.) Is cultivated in the mountainous area of China, and its roots are dried ginseng, It is called Carrot 37, Sanzo 7, Tanashi 7, Tabana and so on. This root has been extensively studied, and many components and medicinal effects have been reported (for example, Non-Patent Documents 1 to 3).
In addition, the flower part of the sanchinin carrot is cut out during cultivation in order to keep nutrition at the root part, and is used as a high-grade tea agent to reduce blood pressure drop and throat inflammation.
However, in spite of being abundant in terms of resources and being readily available, such as hanabe, which is used for food as a tea and thinned out during cultivation, little research has been conducted.
[0003]
[Non-Patent Document 1]
Yoshikawa M., et. Al., Chem. Pharm. Bull., 45, 1039-1045 (1997)
[Non-Patent Document 2]
Yoshikawa M., et. Al., Chem. Pharm. Bull., 45, 1056-1062 (1997)
[Non-Patent Document 3]
Yoshikawa M., et. Al., Chem. Pharm. Bull., 49, 11452-1456 (2001)
[0004]
[Means for Solving the Problems]
The inventors of the present invention, as a result of intensive studies on the components contained in the flower parts of the genus Carrot family, found a new compound that has never been reported so far, and determined its chemical structure. Thus, the present inventors have newly found that an extract of the flower part of the Argiaceae plant has a conventionally known liver injury-suppressing effect, and the present invention has been completed.
That is, according to the present invention, there is provided a prophylactic or therapeutic agent for hepatic diseases comprising, as an active ingredient, an extract or extract obtained by extraction with a lower aliphatic alcohol or a hydrated product thereof from the flower part of the genus Carrot family. Provided.
According to the present invention, the following formulas (a) to (e)
[0005]
[Chemical formula 5]

[0006]
[Chemical 6]

A novel compound represented by the formula:
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The preventive or therapeutic agent for liver diseases of the present invention contains, as an active ingredient, an extract or extract extracted from the flower part of a plant of the family Araliaceae carrot (Panax). A plant belonging to the genus Carrotaceae is particularly limited as long as it is widely distributed in mountainous areas such as Japan, Korea, China, etc., for example, particularly in the area of about 800 to 1000 m above sea level such as Yunnan and Guangxi. It is not a thing, but sanchinin ginseng (P. notoginseng (BURK.) FH CHEN.), Chixetsuninjin, ginseng (P. ginseng), etc. are mentioned, and among them, sanchinin ginseng is typically used.
The flower parts of these plants are usually used after being dried, but the form and the like are not particularly limited.
[0008]
In order to obtain an extract from the flower part of the family Argiaceae, for example, the flower part is crushed and / or dried as it is, and is about 1 to 50 times (weight), preferably about 20 to 30 times lower aliphatic. It is appropriate to extract using alcohol or its hydrate. Examples of lower aliphatic alcohols include aliphatic alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, propanol, n-butanol, and t-butanol. Of these, methanol or ethanol is preferable. Further, it may be a lower aliphatic alcohol hydrate containing about 1 to 30% by weight of water.
[0009]
Extraction may be carried out by either cold immersion, digestion or thermal immersion. For example, it is appropriate to carry out by immersing the flower part described above in the solvent described above at a temperature of about 50 to 85 ° C. with or without shaking. When soaking under shaking, it is appropriate to carry out for about 30 minutes to 10 hours, and when soaking under non-shaking, it is appropriate to carry out for about 1 hour to 20 days. In addition, although an extraction process may be performed only once about the same raw material, it is preferable to perform several times, for example, about 2 to 5 times.
[0010]
The obtained extract may be concentrated to obtain an extract. Concentration is preferably performed under low temperature and low pressure. This concentration may be carried out until dry. In addition, you may filter before concentrating and may concentrate a filtrate. In addition, the extract may be in a concentrated state, or may be a powder or a freeze-dried product. Methods known in the art can be used for the method of concentrating, powdered and freeze-dried.
[0011]
The obtained extract may be subjected to purification treatment before and after concentration. As the purification treatment, a chromatographic method, an elution method using an ion exchange resin, partition extraction with a solvent, or the like can be employed alone or in combination. For example, examples of the chromatographic method include normal phase chromatography, reverse phase chromatography, high performance liquid chromatography, centrifugal liquid chromatography, column chromatography, thin layer chromatography and the like, or a method of performing a combination thereof. . In this case, purification conditions such as a carrier and an elution solvent can be appropriately selected according to various chromatographies.
[0012]
In particular, the extract obtained by concentrating the extract is preferably subjected to partition extraction using ethyl acetate and water to obtain a water-soluble fraction. When the extract is obtained by extraction with methanol, ethanol or propanol, the extract is concentrated to obtain an extract, which is partitioned and extracted with ethyl acetate / water, and the aqueous phase is further extracted with butanol. It is preferable to perform partition extraction to obtain a butanol-soluble fraction. The partition extraction can be performed according to a method usually performed in this field. For example, it may be performed by adding about 1 to 30 (weight) times (1:10 to 10: 1) of ethyl acetate or butanol and water to the extract or the like at room temperature, with shaking or without shaking. Is appropriate.
[0013]
Each obtained fraction may be further concentrated or subjected to purification treatment. The concentration and purification treatment here can be performed in the same manner as described above.
Moreover, in this invention, the compound represented by either of the formula (a)-formula (e) shown above was discovered as a novel compound. If possible, these compounds may be in the form of their metal salts, organic acids or inorganic acid salts, salts of organic bases or inorganic bases, and solvates.
These compounds are obtained by concentrating the extract obtained by concentrating the extract of the lower aliphatic alcohol or its hydrate as described above, and partitioning and extracting the aqueous phase with ethyl acetate / water, and then partitioning and extracting with butanol. When contained, it is mainly contained in the butanol-soluble fraction. Since these compounds have an inhibitory effect on liver damage, they can be used as preventive or therapeutic agents for liver diseases. In addition, the preventive or therapeutic agent includes application as a health food in addition to pharmaceuticals.
[0014]
The extract or extract as described above and the compounds of formulas (a) to (e) are diluted as they are or with an appropriate medium, respectively, and powders, granules are obtained by methods known in the field of manufacturing pharmaceuticals and the like. It can be used in the form of various pharmaceuticals such as an agent, a tablet, a capsule or a liquid. In these forms, an appropriate medium may be added. Suitable vehicles include pharmaceutically acceptable excipients such as binders (eg syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone); fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or Glycine); tablet lubricants (eg magnesium stearate, talc, polyethylene glycol or silica); disintegrants (eg potato starch) or acceptable wetting agents (eg sodium lauryl sulfate). The tablets may be coated in a manner well known in normal pharmaceutical practice. Liquid formulations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs and may be provided as a dry product that is mixed with water or other suitable excipients prior to use. Good. Such liquid preparations contain conventional additives such as suspending agents (eg sorbitol, syrup, methylcellulose, glucose syrup, gelatin edible fat); emulsifiers (eg lecithin, sorbitan monooleate or gum arabic); Non-aqueous excipients (such as almond oil, fractionated coconut oil or oily esters such as glycerin, propylene glycol or ethyl alcohol); preservatives (such as methyl or propyl p-hydroxybenzoate or sorbine) Acid) and optionally a colorant and the like.
[0015]
Moreover, the said extract or extract and the compound of Formula (a)-Formula (e) can be utilized for health food. Health food means a food that is more active than normal food, and is intended for health, health maintenance and promotion, for example, liquid or semi-solid, solid products, specifically powders, In addition to granules, tablets, capsules, liquids, and the like, confectionery such as cookies, rice crackers, jelly, yokan, yogurt, and manju, soft drinks, teas, nutritional drinks, soups and the like can be mentioned. In the production process of these foods or to the final product, the above extract or the like can be added by mixing, coating, spraying or the like to obtain a health food.
[0016]
The amount of the extract or extract and the compound of formula (a) to formula (e) used is the degree of concentration, the degree of purification, the strength of activity, the purpose of use, the target disease for treatment or prevention, the degree of the disease, etc. , Body weight, age, symptom and the like can be adjusted as appropriate. For example, in the case of an adult, an extract or extract may contain about 1 to 20000 mg depending on the degree of purification, water content, etc. About 10,000 mg is mentioned. It is desirable to take three times a day about 30 minutes before meals. In addition, when used as a health food, the extract or extract, the compound of formula (a) to formula (e) is added to an amount that does not adversely affect the taste and appearance of the food, for example, 1 kg of the target food. It is appropriate to add about 10 to 100000 mg.
Examples of the extract of the present invention, novel compounds and their actions will be specifically described below.
[0017]
Extraction of Arachnaceae plant plant (1) Preparation of Miso Flower Extract Extract 493 g of Miso Flower is pulverized, and about 10 times the amount of methanol (manufactured by Nacalai Tesque, special grade) (5 L) is added. Extracted for 3 hours. After extraction, the mixture was filtered with a fold-fold filter paper (No. 2 filter paper manufactured by Advantech Co., Ltd.), methanol (5 L) was further added to the extraction residue, and the mixture was heated to reflux for 3 hours, and similarly filtered. Extraction was carried out three times in total, and the extracts were combined, and the solvent was distilled off under reduced pressure using a rotary evaporator to obtain 211 g of an extract of Shichihana (yield from crude drug 42.8%).
[0018]
(2) Preparation of Solvent Fraction of Miso Flower Methanol Extract Extract Miso Flower Methanol Extract Extract (125 g) was suspended in water (2 L), and 2 L of ethyl acetate (manufactured by Nacalai Tesque, (Special grade). Ethyl acetate (2 L) was further added to the aqueous layer, followed by solvent extraction, and the same operation was performed 3 times in total to obtain an ethyl acetate layer. The ethyl acetate extracts were combined and the solvent was distilled off under reduced pressure to obtain an ethyl acetate migration extract (22.6 g, 4.6%).
Further, 2 L of 1-butanol (manufactured by Nacalai Tesque, special grade) was added to the aqueous layer to perform solvent extraction, and the same operation was performed three times in total to obtain a 1-butanol layer. Similarly, the solvent was distilled off under reduced pressure to obtain 1-butanol transfer part extract (146.3 g, 29.2%) and water transfer part extract (44.0 g, 8.9%).
[0019]
(3) Separation and Purification of 1-Butanol Migration Part Extract 1-Butanol Migration Part Extract (73.8 g) was subjected to normal phase silica gel column chromatography (manufactured by Fuji Silysia, BW-200, 150 to 350 mesh, 2.5 kg, Mobile phase: Chloroform (manufactured by Nacalai Tesque, special grade): methanol: water (10: 3: 1, lower layer) → methanol) sequentially eluted, fractions 1 (0.81 g), 2 (3.70 g), 3 (10.20 g), 4 (30.30 g), 5 (25.80 g), 6 (2.84 g) were obtained.
[0020]
Among these, about the fraction 4 (30.00g), reverse phase octadecyl silyl (henceforth ODS) column chromatography (Fuji Silysia Co., Ltd. Chromatrex ODS DM1020T, 100-200 mesh, 900g, mobile phase: methanol: water (70:30) → separation with methanol) and purified, ginseng Roh side -Rb ₃ (ginsenoside-Rb _3, to obtain a 2,2.87%). Furthermore, high performance liquid chromatography (hereinafter referred to as HPLC) [detector: Shimadzu suggested refraction type detector RID-6A, pump: Shimadzu LC-10A, HPLC column: YMC Pack-ODS-A manufactured by YMC, 20 mmi.d. × 250 mm , mobile phase: methanol: separation with water (80:20) and purified, ginseng Roh side _{-Rb 1 (ginsenoside-Rb 1,} 1,0.24%), ginseng Bruno side -Rb ₃ (2,3.15 %), Ginsenoside-Rd (3, 0.010%), ginsenoside-Rc (4, 1.79%), gypenoside-IX (gypenoside-IX, 5, 0.11%), gypenoside-XVII (6) 0.18%) and the novel compounds notoginsenoside-T (7, 0.011%) and notoginsenoside-O (8, 0.010%).
[0021]
For fraction 5 (25.00 g), ODS column chromatography (50 g, mobile phase: methanol: water (60: 40 → 70: 30) → methanol) and HPLC (mobile phase: methanol: 1% aqueous acetic acid solution (60:35) )) Separated and purified, notoginsenoside-Fa (notoginsenoside-Fa, 9, 0.41%), notoginsenoside-D (10, 0.009%) and the new compound notoginsenoside-Q (notoginsenoside -Q, 11, 0.014%), Notoginsennoside-P (12, 0.008%), and Notoginsenonoside-S (13, 0.029%).
(4) Structure and physical properties of the novel compound The structural formula of the novel compound obtained above is shown below.
[0022]
[Chemical 7]

[0023]
[Chemical 8]

[0024]
(1) physical properties of notoginsenoside-T (7) Property: colorless crystals (melting point: 196-198 ° C)
Optical rotation: [α] _D ²⁸ + 0.3 ° (c = 1.30, MeOH)
High-resolution positive-ion FAB-MS:
Theoretical value C ₅₂ H ₈₈ O ₂₁ Na (M + Na) ⁺ : 1071.5716
Actual value: 1071.5708
Infrared absorption spectrum (KBr, cm ^-1 ): 3410, 1655, 1078
Mass spectrometry
positive-ion FAB-MS: m / z 1071 (M + Na) ⁺
negative-ion FAB-MS: m / z 1047 (M−H) ^-
Nuclear magnetic resonance spectrum:
¹ H-NMR (500 MHz, pyridine-d ₅ ): δ0.84, 0.98, 0.98, 0.99, 1.29, 1.63, 1.63, 1.68 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H ₃ ), 4.89 (1H, d, J = 7.7 Hz, 1'-H), 4.97 (1H, d, J = 7.6 Hz, 1 '''-H), 5.09 (1H, d, J = 7.6 Hz, 1``-H), 5.19 (1H, d, J = 7.4 Hz, 1 ''''-H), 5.34 (1H, d like, 24-H).
<sup>13</sup> C-NMR (125 MHz, pyridine-d <sub>5</sub> ): δc (shown in Table 1)
[0025]
(2) physical properties of notoginsenoside-O (8) Property: colorless crystals (melting point: 194-196 ° C)
Optical rotation: [α] <sub>D</sub> <sup>28</sup> + 2.1 ° (c = 1.00, MeOH)
High-resolution positive-ion FAB-MS:
Theoretical value C <sub>52</sub> H <sub>88</sub> O <sub>21</sub> Na (M + Na) <sup>+</sup> : 1071.5716
Actual value: 1071.5721
Infrared absorption spectrum (KBr, cm <sup>-1</sup> ): 3431, 1684, 1078
Mass spectrometry
positive-ion FAB-MS: m / z 1071 (M + Na) <sup>+</sup>
negative-ion FAB-MS: m / z 1047 (M−H) <sup>-</sup>
Nuclear magnetic resonance spectrum:
<sup>1</sup> H-NMR (500 MHz, pyridine-d <sub>5</sub> ): δ0.83, 0.98, 0.99, 1.00, 1.29, 1.63, 1.63, 1.67 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H <sub>3</sub> ), 4.89 (1H, d like, 1 '''' -H), 4.90 (1H, d like, 1 '''-H), 4.91 (1H, d like, 1'-H ), 5.09 (1H, d, J = 7.7 Hz, 1 ''-H), 5.31 (1H, d like, 24-H). <sup>13</sup> C-NMR (125 MHz, pyridine-d <sub>5</sub> ): δc (Table 1) To show)
[0026]
(3) physical properties of notoginsenoside-Q (11) Property: colorless crystals (melting point: 196-198 ° C)
Optical rotation: [α] <sub>D</sub> <sup>28</sup> + 6.8 ° (c = 1.20, MeOH)
High-resolution positive-ion FAB-MS:
Theoretical value C <sub>64</sub> H <sub>108</sub> O <sub>31</sub> Na (M + Na) <sup>+</sup> : 1395.6772
Actual value: 1395.6759
Infrared absorption spectrum (KBr, cm <sup>-1</sup> ): 3410, 1655, 1076
Mass spectrometry
positive-ion FAB-MS: m / z 1395 (M + Na) <sup>+</sup>
negative-ion FAB-MS: m / z 1371 (M−H) <sup>-</sup>
Nuclear magnetic resonance spectrum:
<sup>1</sup> H-NMR (500 MHz, pyridine-d <sub>5</sub> ): δ0.83, 0.96, 0.98, 1.11, 1.28, 1.64, 1.64, 1.69 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H <sub>3</sub> ), 4.90 (1H, d, J = 7.3 Hz, 1'-H), 5.08 (1H, d, J = 7.0 Hz, 1 '''''-H), 5.09 (1H, d, J = 7.3 Hz, 1`` ''-H), 5.22 (1H, d, J = 7.3 Hz, 1`` ''''-H), 5.34 (1H, d like, 24-H), 5.36 (1H, d, J = 6.7 Hz, 1 '''-H), 5.48 (1H, d, J = 8.9 Hz, 1''-H).
<sup>13</sup> C-NMR (125 MHz, pyridine-d <sub>5</sub> ): δc (shown in Table 1)
[0027]
(4) Physical properties of notoginsenoside-P (12) Properties: colorless crystals (melting point: 194-196 ° C)
Optical rotation: [α] <sub>D</sub> <sup>28</sup> −0.6 ° (c = 0.70, MeOH)
High-resolution positive-ion FAB-MS:
Theoretical value C <sub>63</sub> H <sub>106</sub> O <sub>30</sub> Na (M + Na) <sup>+</sup> : 1365.6667
Actual value: 1365.6681
Infrared absorption spectrum (KBr, cm <sup>-1</sup> ): 3410, 1655, 1076
Mass spectrometry
positive-ion FAB-MS: m / z 1365 (M + Na) <sup>+</sup>
negative-ion FAB-MS: m / z 1341 (M−H) <sup>-</sup>
Nuclear magnetic resonance spectrum:
<sup>1</sup> H-NMR (500 MHz, pyridine-d <sub>5</sub> ): δ0.81, 0.96, 0.96, 1.10, 1.27, 1.62, 1.62, 1.66 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H <sub>3</sub> ), 4.89 (1H, d, J = 7.0 Hz, 1'-H), 4.89 (1H, d, J = 7.0 Hz, 1 '''''-H), 4.89 (1H, d, J = 7.0 Hz, 1`` ''''-H), 5.08 (1H, d, J = 7.3 Hz, 1 ''''-H), 5.31 (1H, d like, 24-H), 5.35 (1H, d, J = 6.7 Hz, 1 '''-H), 5.47 (1H, d, J = 9.1 Hz, 1''-H).
¹³ C-NMR (125 MHz, pyridine-d ₅ ): δc (shown in Table 1)
[0028]
(5) Physical properties of notoginsenoside-S (13) Properties: colorless crystals (melting point: 186-188 ° C)
Optical rotation: [α] _D ²⁸ −8.7 ° (c = 1.40, MeOH)
High-resolution positive-ion FAB-MS:
Theoretical value C ₆₃ H ₁₀₆ O ₃₀ Na (M + Na) ⁺ : 1365.6667
Actual value: 1365.6653
Infrared absorption spectrum (KBr, cm ^-1 ): 3410, 1655, 1076
Mass spectrometry
positive-ion FAB-MS: m / z 1365 (M + Na) ⁺
negative-ion FAB-MS: m / z 1341 (M−H) ^-
Nuclear magnetic resonance spectrum:
¹ H-NMR (500 MHz, pyridine-d ₅ ): δ0.81, 0.94, 0.96, 1.11, 1.27, 1.61, 1.63, 1.67 (3H each, all s, 19, 18, 30, 29, 28, 26, 21, 27-H ₃ ), 4.85 (1H, d, J = 7.6 Hz, 1`` ''''-H), 4.89 (1H, d, J = 7.3 Hz, 1'-H), 5.09 (1H , d, J = 6.7 Hz, 1`` ''-H), 5.32 (1H, d like, 24-H), 5.35 (1H, d, J = 6.4 Hz, 1 '''-H), 5.46 ( 1H, d, J = 7.6 Hz, 1 ''-H), 5.59 (1H, d like, 1 '''''-H). ¹³ C-NMR (125 MHz, pyridine-d ₅ ): δc (Table 1)
[0029]
[Table 1]

[0030]
Action on liver injury induced by D-galactosamine and lipopolysaccharide (1) Experimental method
Experiments were performed according to the method of Tiegs et al. (Tiegs G., Wolter M., Wendel A., Biochem. Pharmacol., 38, 627-631 (1989)).
In other words, 5% (w / v) gum arabic powder (manufactured by Nacalai Tesque Co., Ltd.) was added to the aqueous solution of ddY male mice (6 weeks old, body weight approximately 25 g, manufactured by Kiwa Experimental Animal Research Co., Ltd.) fasted for about 20 hours. The test substance in suspension was administered intraperitoneally. One hour later, a mixture of D-galactosamine hydrochloride (350 mg / kg, manufactured by Wako Pure Chemical Industries, Ltd.) and lipopolysaccharide (10 μg / kg, Sigma) dissolved in physiological saline was intraperitoneally administered at a volume of 10 mL / kg (BW). did. After 10 hours of fasting and rearing under water, blood was collected from the orbital venous plexus without anesthesia to obtain a blood sample.
[0031]
Serum obtained by centrifuging the obtained blood sample (3,000 rpm, 10 minutes, 4 ° C.) was frozen (−20 ° C.) and stored until the transaminase activity was measured.
For measurement of serum transaminase (s-GPT, s-GOT) activity, a commercially available kit, STEA Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
The obtained results are shown in Tables 2 to 4.
[0032]
[Table 2]

[0033]
[Table 3]

[0034]
[Table 4]

Claims (5)

  A preventive or therapeutic agent for liver diseases, comprising an extract or extract obtained by extraction from a flower part of a plant belonging to the family Carrotaceae with a lower aliphatic alcohol or a hydrate thereof as an active ingredient.   The preventive or therapeutic agent for liver disease according to claim 1, wherein the extract is further subjected to partition extraction with ethyl acetate and water, and the water-soluble fraction is an active ingredient. The extract or extract is at least the following formula (a) to formula (e):

The preventive or therapeutic agent of the liver disease of Claim 1 containing any one compound of these. The preventive or therapeutic agent for liver disease according to any one of claims 1 to 3, wherein a flower part of a plant belonging to the genus Carrotaceae is a flower part of Sancytin carrot. Formula (a)-Formula (e)

In the represented Rui Zureka one compound.