JP2002053488A5 - - Google Patents

Description

【００７３】
以上の結果から、冬虫夏草より単離された純粋な天然物Ｈ１−Ａは、インビトロではＰＨＡに活性化された末梢血単核球細胞よりもＩＬ−２の産生を抑制することができた。MRL lpr/lpr動物モデルでは、リンパ節の増殖を阻止し、抗ds-DNA自己抗体の産生を抑制することもできた。またMRL lpr/lprマウスの生存期間を延長することもできた。同時に、腎糸球体のメサンギウム増殖を抑制し、蛋白尿の進行を遅らせることができたが、驚くことに、本研究においては、免疫複合体の沈着に有意な変化が見られなかった。本研究の結果は、この化学構造が明確な単離された天然物が、MRL lpr/lprマウスの自己免疫病を治療する潜在力を持っていることを明らかにした。その他、血清中の抗ds-DNA自己抗体濃度と、腎臓組織病理変化と腎臓免疫複合体沈着との３者間は関連していないが、将来的に、全身性エリテマトーデス性腎炎の原因に対し、より多い検討空間を提供することが可能となる。
【００７４】
本発明のインビトロ試験結果は、Ｈ１−Ａが、ＰＨＡによって活性化された末梢血単球細胞によるＩＬ−２の産生を有効に抑制することが可能であることを示した。この薬物の効果は、周知の免疫抑制剤であるシクロスポリンＡと似ている。前述したように、冬虫夏草の原始複合物であるＨ１−Ａは、細胞免疫に対し、免疫抑制の作用を持っている。この結果は、シクロスポリンＡの皮膚移植における結果と類似している。換言すれば、Ｈ１−Ａは免疫調節剤と呼ばれたほうがよりふさわしい。シクロスポリンＡは、ヒトの全身性エリテマトーデスの治療に有効であると言われている。シクロスポリンＡは、Ｔリンパ球に対し、選択的に使用されている。したがって、Ｔ細胞の反応を主とする病変への治療に用いられる。シクロスポリンＡは、いくつかの抗体と関係している反応にも抑制作用があるが、その主な作用は、Ｔヘルパー細胞より生じた反応に対するものである。比較すると、シクロスポリンＡは、抗原が直接Ｂ細胞を刺激して生じた抗体反応に対する作用が少ない。Ｈ１−Ａは、抗ds-DNA抗体に対する抑制作用が顕著に見られるため、シクロスポリンＡと類似の作用メカニズムを持っているかもしれない。それは、抗ds-DNAの産生が、抗原の起動によるＴ細胞調節の過程であるためである。Ｈ１−Ａは、Ｔ細胞増殖を主な症状とするリンパ節増殖にも抑制作用を有する。全身性エリテマトーデスのメカニズムに関する新観点は、ＣＤ４^＋Ｔ細胞が、MRL lpr/lprマウス自己免疫疾患発生の主因であることである。したがって、現在、このような病気になる原因は、Ｔ細胞の機能のバランスが崩れたか、またはＴ細胞およびＢ細胞間の協力に欠如があるためである。抗ＣＤ３、抗ＣＤ４、および抗ＣＤ８モノクローナル抗体は、マウスのエリテマトーデスの治療で有効であると報告されている。したがって、Ｈ１−Ａ治療効果は、Ｔ細胞の産生を抑制すること、またはＴｈ１／Ｔｈ２の産生を抑制することによって発揮すると推測されている。
【００７５】
Ｔｈ１およびＴｈ２への抑制作用により、マウス体内での抗ds-DNA抗体の産生が顕著に下がった。腎臓局部Ｔｈ１の産生が抑制されたことによるものかもしれない。腎糸球体内のメサンギウム細胞の増殖はこれによって停止した。これらの仮説を、将来の研究でさらに証明することが必要である。本発明は、腎臓切片に免疫複合体沈着の変化が見られないことについて、３つの可能な解釈を提供した。
【００７６】
第１に、腎臓組織病理変化と免疫複合体沈着との間の不関連性は、臨床上で良く見られる。したがって、免疫複合体で全身性エリテマトーデス性腎炎の全ての現象を解釈するのが不可能である。その他の介入可能な因子は、細胞因子または化学因子を含んでいる。したがって、Ｈ１−Ａのメサンギウム増殖への抑制作用は、免疫複合体沈着を下げる以外のメカニズムを通じて発揮しているかもしれない。
【００７７】
第２に、既に沈着した免疫複合体の除去は、多くの時間を要する。ＩＶ類全身性エリテマトーデス性腎炎の臨床経験によると、患者がシクロホスファミドの治療を受けた後、疾病の症状の軽減に伴い、血液中の抗ds-DNA抗体値が先に低下する。腎臓組織中の免疫複合体の排除は、臨床の症状改善よりも遅い。通常、最低６ヶ月の治療を経過した後、腎臓組織に顕著な変化が初めて見られる。
【００７８】
第３に、Ｈ１−Ａは、マクロファージの、免疫複合体を排除する能力を改善しない。以上、Ｈ１−Ａは、ヒト全身性エリテマトーデスを治療するための潜在的な単離された天然物である。Ｈ１−Ａは、シクロスポリンＡよりも安全性が高い。冬虫夏草が中国伝統的な治療に用いられるのは、千年以上の歴史を持っているにもかかわらず、今までに、腎臓毒性に関する報告が全くなかったからである。反対に、冬虫夏草は、シクロスポリンＡおよびアミノグリコシドによってもたらされた腎臓毒性を有効に避けることができると報告されている。本研究も、このような観点を証明した。腎臓切片には、いかなる腎臓毒性を全く見ることがなく、逆にＨ１−ＡがMRL lpr/lprマウスの腎臓機能に対し、保護作用さえ有している。
【００７９】
免疫のメカニズムから見ると、最近、全身性エリテマトーデス患者のＴ細胞機能に欠陥があると多くの文献に述べられている。その中に、ＩＬ−２の産生が減少し、かつＩＬ−２によって誘発されたＴ細胞増殖の反応も顕著に下がることが含まれている。Gutierrez-Ramosらは、ＩＬ−２／ワクシニアの組換えワクチンで、MRL lpr/lprマウスの治療に成功した。いくつかのいわゆる免疫調節剤は、ＩＬ−２による欠陥を修正し、胸腺内の細胞分裂および末梢Ｔ細胞の活性を促進するといわれてきた。しかし、ある人は、完全に反対な意見を提出した。Cuadradoらは、全身性エリテマトーデス患者の血液中にＩＬ−４、ＩＬ−２、およびｓＩＬ−２Ｒの比較的正常かつ顕著な上昇が見られると報告した。これに対し、Razらは、ＩＬ−２ ｃＤＮＡをMRL lpr/lprマウスの体内に導入することで、その病状を悪化させ、さらに、自己抗体の産生も促進すると報告した。発明者らの結果は、後者の意見とほぼ一致している。インビトロで、Ｈ１−Ａは、末梢単核球細胞でのＩＬ−２の産生を抑制する；MRL lpr/lprマウス動物モデルでは、Ｈ１−Ａは、臨床の症状および腎臓の組織病変を改善することができる。
【００８０】
以上のように、本発明は、中国の伝統的な漢方薬である冬虫夏草から分離された、明確な構造を有する単離された天然物Ｈ１−Ａを用いて、自己免疫疾患モデルのMRL lpr/lpr体内の治療効果を上げることに成功した。
【００８１】
実施例３：誘発ＩｇＡ腎炎を持つマウスに対する冬虫夏草抽出物（１％および0.5％のＦ２）の効果
【００８２】
１．ＩｇＡ腎炎を誘発する方法：使用される抗原はＲ３６Ａであり、抗体はａ−Ｒ３６Ａ−ＩｇＡ−ｍＡｂである。まず、Balb/cマウスの腹部に２mgの抗体を注射し、２時間後、尾静脈に100μgの抗原を注射した。次いで、24時間ごとに100μgの抗原を注射し（１週間連続）、これにより、免疫複合体が腎間質に沈着し、最初の注射後約５日間、血尿および蛋白尿が現れた。
【００８３】
２．ＩｇＡ腎炎を誘発するマウスを、投薬群（ｎ＝６）および対照群（ｎ＝６）に分けた。投薬群は、１％および0.5％のＦ２を摂取させた。毎日、各マウスに約3ｇの正常飼料を摂取させた。試験群と対照群とのマウスを、毎日、血尿および蛋白尿を測定し、７日後、マウスを屠殺した。その腎臓を取り、組織病理学検査および免疫蛍光検査を行い、そして血清中のＡＬＴ、ＡＳＴ、および肝臓中のコレステロール、チトクロムＰ４５０の変化を観察した。
【００８４】
Ｉ．血尿および蛋白尿の測定：
尿液試験紙（Urine Strip）で検査した。
【００８５】
ＩＩ．腎臓組織病理学検査：
腎臓組織を10％中性ホルマリンで固定し、漸増濃度のアルコールで脱水した後、パラフィン包埋し、厚さ４μmの切片を切り、ヘマトキシリン-エオシンで染色し、光学顕微鏡にて観察した。各マウスについて50個の糸球体を観察し、糸球体の破壊程度を５等級に分け、点数が高ければ、糸球体の破壊程度が高くなる。最後に点数を累計し、総数（Total）で表示する。
【００８６】
ＩＩＩ．免疫蛍光検査：
腎臓組織をO.C.T.compoundで包埋し、−70℃の冷凍庫に保存した。免疫染色を行う際に、冷凍切片機で４μmの組織切片を切り出し、そしてアセトンで固定し、次いで、蛍光標識されたヤギ抗マウスＩｇＡ、ヤギ抗マウスＣ３、およびヤギ抗フィブリノーゲンで直接免疫蛍光の染色を実施し、蛍光顕微鏡で観察した。蛍光染色強度を４等級に分け、点数が高ければ、蛍光の強度が強くなり、ＩｇＡ、ＩＣ沈着およびＣ３補体の活性化が高くなることを表す。
【００８７】
３．統計方法：
血尿、蛋白尿、組織病理学検査および免疫蛍光検査は、χ２乗検定で分析を行った。血清中のＡＬＴ、ＡＳＴおよび肝臓中のコレステロール、チトクロムＰ４５０の統計は、Studentのｔ検定で分析を行った。P＜0.05は、統計学的に有意であるとみなす。
【００８８】
結果：
１．血尿および蛋白尿を表２に示す。
【００８９】
【表２】

【００９０】
抗原を注射した５日後、血尿および蛋白尿が現れた。７日目に、１％Ｆ２群および0.5％Ｆ２群では、血尿および蛋白尿の顕著な軽減が見られた（P＜0.05）。
【００９１】
２．腎臓組織病理学検査の結果を表３に示す。
【００９２】
【表３】

【００９３】
総数（Total）から見ると、１％Ｆ２群は、腎糸球体の破壊程度が有意に軽減したが（P＜0.05）、0.5％Ｆ２群では、統計学的有意差が認められなかった。
【００９４】
３．免疫蛍光検査の結果を表４および５に示す。
【００９５】
【表４】

【００９６】
【表５】

【００９７】
総数（Total）評価より、投薬の１％Ｆ２群および0.5％Ｆ２群は、対照群と比較すると、ＩｇＡ ＩＣ沈着またはＣ３補体の活性化の両方とも、薬剤量に比例して軽減されていた。１%Ｆ２群は、対照群と比較すると、統計学的有意差が認められた（P＜0.05）。
【００９８】
４．肝臓機能検査の結果を表６に示す。
【００９９】
【表６】

【０１００】
肝重量および体重について、投薬群と対照群とは、すべて差がなかった：100倍の肝重量対体重の比率から見ると、１％Ｆ２群は6.75±0.09であり、対照群は7.12±0.12であった。１％Ｆ２は肝重量は明らかに減少した（P＜0.05）。血清中のＡＬＴおよびＡＳＴは、投薬群と対照群との間に、顕著な変化がなかった。肝臓中のコレステロールの含有量は、投薬群と対照群との間に、統計学的有意差がなかった。肝臓中のチトクロムＰ４５０の含有量は、１％Ｆ２群が0.31±0.03nmol/mgタンパク質であり、対照群は0.23±0.01nmol/mgタンパク質であった。１％Ｆ２は、チトクロムＰ４５０の含有量を顕著に増加させた（P＜0.05）。肝重量対体重の比率から見ると、急性毒性試験では、２％Ｆ２を投与すると、肝臓腫脹が見られた。ＩｇＡ腎炎が誘発された動物モデルでは、１％Ｆ２を投与すると、肝臓の萎縮が見られた。これらは反対の結果を示している。急性毒性試験は、正常ICRマウスを対照群とし、その100倍肝重量対体重の比率は5.44±0.17であった。ＩｇＡ腎炎が誘発されたBalb/cマウス試験では、正常の飼料の飼育を対照群とした。予めＩｇＡ腎炎が誘発された動物自体は、その100倍肝重量対体重の比率が7.12±0.12であり、ＩｇＡ腎炎のマウスには、肝臓腫脹の現象が見られる。２つの試験に使用された動物は異なる品種であった。さらに、正常のBalb/cマウスに、「冬虫夏草抽出物で飼育する」方式で、正常飼料を飼育すると、最後には、100倍肝重量対体重の比率は5.50±0.24となり（図７を参照のこと）、これは、ICRマウスの正常な100倍肝重量対体重の比率（5.44±0.17）と一致していた。これは、ＩｇＡ ＩＣ自体が、顕著な肝臓腫脹現象を引き起こすことを示した（P＜0.05）。そのメカニズムは不明である。ただし、１％Ｆ２を投与すると、ＩｇＡ ＩＣ誘発の肝臓腫脹が有意に軽減されたが（P＜0.05）、正常値までに回復することができなかった。
【０１０１】
実施例４：体外でＨ１−Ａの細胞に対する安全性
Ｈ１−Ａの細胞に対する活性および毒性について検討し、その結果を図８に示した。最上方の図８（ａ）は、トリパンブルー染色で細胞の活性を測定した結果であり、72時間培養した３群（ヒトメサンギウム細胞のみ、ヒトメサンギウム細胞＋ＩＬ−１とＩＬ−６、およびヒトメサンギウム細胞＋ＩＬ−１とＩＬ−６および40μM/mlのＨ１−Ａ）の細胞活性に差異がないことを示した。死滅した細胞がLDHを放出するため、同じ(ａ)試験で、72時間後、培養液中のLDHの濃度を測定し、その結果を図８（ｂ）に示した。３群は、共に統計上の差異がなかった。細胞が生存すれば、メサンギウム細胞も増殖する。そこで、ＭＴＴ方法で、細胞増殖の状況を分析した。その結果を図８（ｃ）に示した。３群は共に統計上の差がなかったことが分かる。
【０１０２】
実施例５：ＩｇＡ腎炎が誘発されたマウスに対するＨ１−Ａの影響
実施例３で誘発したＩｇＡ腎炎マウスに、10mg/mlのＨ１−Ａを、腹部に１回注射し、７日間連続して注射した。
【０１０３】
治療群のＩｇＡ腎炎マウスとコントロール群の血尿／蛋白尿に対する影響を比較した。表７に示した結果から、治療群の血尿／蛋白尿が顕著に改善され、かつ統計学的に有意であると分かる。
【０１０４】
腎臓組織の病理変化について、コントロール群では、重篤なメサンギウム増生およびメサンギウム細胞増殖が見られたが、治療群はＨ１−Ａの治療を受けた後、組織病理変化の顕著な改善が見られた。治療群の腎臓切片は、ヘマトキシリン-エオシン染色で、僅かなメサンギウム増殖があっただけであり、蛍光染色の改善は表７に示されている。治療群には顕著な改善があり、かつ統計学的に有意である。
【０１０５】
【表７】

【０１０６】
【発明の効果】
冬虫夏草より分離されたＦ２は、体外において、腎間質細胞の活性化を抑制することが可能で、体内において、ＩｇＡ腎炎の発生または悪化を阻止することができる。Ｆ２は急性毒性がほとんどなく、かつＩｇＡ ＩＣより誘発された肝臓腫脹を軽減することもできる。Ｆ２より単離された活性天然物（Ｈ１−Ａ）は、「体内ＩｇＡ腎炎の発生」を阻止する動物モデルで、良好な治療効果を持っている。
【０１０７】
本発明は、好ましい実施例について説明しているものの、それらの種々の変更は、この明細書を読めば、当業者に明かなことが理解されるべきである。従って、ここで開示の発明は、添付の請求の範囲に入るようなこれらの変更を含むべく意図されていることが理解されるべきである。
【図面の簡単な説明】
【図１】
冬虫夏草から活性画分と活性化合物とを分離する工程を示すフローチャートである。
【図２】
本発明のＨ１−Ａが、末梢血単核球細胞によるＩＬ−２の産生に対する影響を示すグラフである。●：Ｈ１−Ａのみ ；▽：単核球細胞をＰＨＡ（10μg/ml）＋Ｈ１−Ａで刺激。
【図３】
Ｈ１−ＡのMRL lpr/lprマウスの生存期間に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図４】
本発明のＨ１−ＡのMRL lpr/lprマウスのリンパ病変に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図５】
本発明のＨ１−ＡのMRL lpr/lprマウスの蛋白尿に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図６】
本発明のＨ１−ＡのMRL lpr/lprマウスの腎臓機能に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図７】
本発明のＨ１−Ａの、MRL lpr/lprマウスにおける抗ds-DNA自己抗体の産生に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図８】
本発明のＨ１−Ａの細胞活性に対する影響を示すグラフである。■：対照群で、ヒトのメサンギウム細胞のみ；□：ヒトのメサンギウム細胞＋ＩＬ−１、ＩＬ−６；□：ヒトのメサンギウム細胞＋ＩＬ−１、ＩＬ−６およびＨ１−Ａ 40μM/mlの72時間の培養。（ａ）トリパンブルー染色による細胞の活性の観察；（ｂ）放出されたLDHの測定による細胞の死亡の観察；（ｃ）MTT試験による細胞の増殖の観察。 [Document name] statement
Patent application title: PHARMACEUTICAL COMPOSITIONS COMPRISING ACTIVE INGREDIENT COMPOUNDS AND ACTIVE COMPOUNDS OF HYDROPOD AND METHOD OF Improving Renal Function Using It
[Claim of claim]
    1. A pharmaceutical composition comprising an active fraction F2 isolated from Cordyceps sinensis, an active compound H1-A, and optionally a pharmaceutically acceptable base.
    2. The active fraction F2 was prepared by heating and drying the angiosperm portion of Cordyceps sinensis at 45 to 50 ° C., grinding it, immersing it in 20 times amount (w / v) of methanol, and concentrating the extract under reduced pressure Thereafter, the crude extract is subjected to silica gel column chromatography, eluted with a 1: 1 mixed solvent of n-hexane / ethyl acetate, and the extract is obtained by concentrating the eluate. object.
    3. The active compoundBut,The pharmaceutical composition according to claim 1, which is (24R) -ergosta-7,22-diene-3b, 5a, 6b-triol isolated from summer grass.
    4. The pharmaceutical composition is for improving renal function, and in vitro, production of IL-2 by monocyte cells in peripheral blood activated by PHA and proliferation of activated mesangial cells Inhibit the growth of lymph nodes, suppress the production of anti-ds-DNA autoantibodies, slow the progression of proteinuria, and prevent the aggravation of IgA nephritis in vivo in MRL lpr / lpr animal models The pharmaceutical composition according to claim 1, which is capable of
    5. The pharmaceutical composition according to claim 1, comprising a therapeutically effective amount of said F2 or H1-A for improving renal function.
    6. The pharmaceutical composition according to claim 2, which is for improving renal function for treating lupus erythematosus nephritis and / or IgA nephritis.
    7. The pharmaceutical composition according to claim 1, comprising an effective amount of said F2 or H1-A for preventing and / or treating lupus erythematosus nephritis.
    8. The pharmaceutical composition according to claim 1, comprising an effective amount of the F2 or H1-A for preventing and / or treating IgA nephritis.
Detailed Description of the Invention
  [0001]
    Field of the Invention
  The present invention relates to an active fraction of Cordyceps sinensis (Berk) Sacc and a pharmaceutical composition containing the active compound and a method for improving kidney function using this. The invention also relates to methods for preventing and / or treating lupus erythematosus nephritis and IgA nephritis.
  [0002]
    [Prior Art]
  Lupus erythematosus nephritis is a chronic disease that is multisystemic and systemic inflammatory. The disease is associated with immune system disorders and a widespread distribution of immune complexes. In patients with systemic lupus erythematosus autoantibodies against many autoantigens are found. These autoantigens are cell membranes, cell nuclei, or tissue components of cytoplasm, and autoantibodies produced include antinuclear antibodies, anti ds-DNA antibodies, anticardiolipin antibodies, anti Ro antibodies, anti La antibodies, etc. . The clinical feature of systemic lupus erythematosus is that the patient repeats the process of onset and palliation. However, its etiology has not been found yet. Therefore, the clinical diagnosis of systemic lupus erythematosus is currently performed based on "Revised Rheumatism Association Criteria for SLE" (Table I-I) in 1982. The pathogenesis of systemic lupus erythematosus is currently presumed to be related to genetic inheritance, environmental factors, foreign antigens (eg, viruses), hormones and stress. The symptoms are divided into systemic lesions or local lesions. Systemic lesions refer to the following conditions. That is, there is an imbalance in immune regulation, and B cells automatically proliferate and activate polyclonal without T cell regulation to generate a large amount of autoantibodies. Alternatively, there is an imbalance between T1 / Th2 T cells and a defect in T cell tolerance, which leads to Ag-driven T cell-induced B cell expansion, somatic mutation, and transformation. Will be brought. In both cases, many autoantibodies and immune complexes are produced. Deposition of these immune complexes induces local lesions. Thus, in the body of systemic lupus erythematosus patients, deposition of immune complexes is seen in each organ, and at the same time an inflammatory response co-evoked by cytoplasm, adhesion molecules and inflammatory cells is seen.
  [0003]
  The difference between systemic lupus erythematosus in children and adults is that the ratio of males and females in adults is 1:10, while that in children is 1: 5. Other than that, pre-adolescent children are less likely to see typical skin symptoms such as erythema and photosensitization, but blood (eg hemolytic anemia, thrombocytopenia) or kidney (proteinuria, hematuria) And symptoms of nephritic syndrome are relatively common. When nephritis is severe, especially with severe lupus erythematosus nephritis, the prognosis is poor, resulting in relatively high morbidity and mortality.
  [0004]
  According to the World Health Organization (WHO) classification of lupus erythematosus nephritis, lupus erythematosus nephritis is classified into five. Groups I to IV are classified based on the severity of mesangial cell proliferation and the degree of glomerulosclerosis. Class V, unlike other classes in pathological changes and clinical symptoms, may be simple membranous glomerular changes, but may also be pathologic changes in which the symptoms of classes II, III and IV coexist. Of these five types of nephritis, the class IV is the so-called membranoproliferative glomerulonephritis (MPGN), which is the poorest nephritis. Pathological sections of MPGN include endothelial cell proliferation, mesangial cell proliferation, capillary wall thickening, vascular embolism, diffuse cell necrosis, inflammatory cell infiltration, crescentic glomerulosclerosis, atrophy and fibrosis, etc. Symptoms are seen. Among these lesions, for example, mesangial cell proliferation, leukocyte infiltration, fibrotic necrosis, hyaline material thrombus and interstitial inflammation are related to the activity of the disease and are called activity index (activity index). There is. In addition, glomerulosclerosis, fibrotic crescents, renal tubular atrophy, interstitial fibrosis and the like are chronic and non-recoverable lesions, and are called chronicity index (chronicity index). The activity of the kidney sections and the height of the chronic index indicate the extent of nephritis and predict the possibility that the patient's condition may progress to end-stage nephritis.
  [0005]
  In the onset mechanism of systemic lupus erythematosus nephritis, glomerular mesangial cells, glomerular epithelial cells and renal matrix together play an important role for acute glomerular lesions and glomerulosclerosis. Among them, the action of mesangial cells is dominant. Mesangial cells are very interesting cells, 95% of which are derived from endogenous kidney cells. Its function is similar to vascular wall myocytes, macrophages. The other 5% are derived from bone marrow cells and transmigrate into the glomeruli during embryonic development. Activation of mesangial cells is largely associated with the pathogenesis mechanism of membranoproliferative glomerulonephritis and glomerulosclerosis. According to the literature, mesangial cells are activated by effects such as infection, stimulation of immune complexes, changes in blood flow dynamics, accumulation of metabolites and fats, or proliferation of residual kidney tissue due to loss of kidney tissue. In the mechanism of the onset of systemic lupus erythematosus nephritis, these external stimuli produce immune complex deposits formed by antigen and autoantibodies. After being activated by the immune complex, mesangial cells release cytokines, growth factors, and adhesion molecules. These substances have autocrine, paracrine and endocrine functions and not only lead to repopulation and reactivation of mesangial cells in the glomerulus, but also other cells in the kidney (eg, renal epithelial cells) , Tubular cells, vascular endothelial cells). With the activation of mesangial cells, renal epithelial cells and tubular cells, the kidney secretes more and more cytokines, growth factors, adhesion molecules, and more fibrocytes and inflammatory cells (eg, macrophages, B cells, etc.) T cells) and activated complement. The interaction between these endogenous kidney cells, exogenous inflammatory cells, fibrocytes and vascular endothelial cells leads to the progressive accumulation of mesangial matrix, and finally to renal glomerulosclerosis, tubular atrophy, and fibrosis of renal interstitial tissue To develop.
  [0006]
  In fact, systemic lupus erythematosus nephritis and stiffening of the glomerulus are fairly complex processes, but mesangial cells and renal epithelial cells also play a considerable role. The entire process is divided into three stages by cause, time, and order. The first step is the deposition of immune complexes. The second step is the activation of mesangial cells, renal epithelial cells and changes in a series of cytokine networks induced thereby. At this stage, the mesangial cells show the following changes: Accelerate cell turnover; 2. Change in appearance of cells; Change in secretory form of cells; Releasing the medium causing some inflammation; and Accelerate the synthesis of the renal matrix. In the third stage, mesangial cells produce more fibrotic cells than secretory cell factors and adhesion molecules, and at the same time stimulate angiogenesis. In addition to the proliferation of mesangial cells, the synthesis of the renal matrix, the fibrosis of tissues, and the formation of blood vessels, renal glomerulosclerosis results.
  [0007]
   According to clinical experience and the systemic lupus erythematosus animal model of MRL lpr / lpr, it is difficult to prevent or eliminate the immune buildup of systemic lupus erythematosus nephritis (ie, the first stage renal lesions) within a short period of time. As such, a relatively viable therapeutic strategy blocks the second stage of mesangial cell proliferation, activation, secretion and synthesis of the renal matrix and avoids the kidneys from entering third stage of glomerulosclerosis. It is.
  [0008]
  So far, methods of treatment of systemic lupus erythematosus nephritis are systemic immunosuppressive or immunomodulatory treatments with drugs, among which corticosteroids (corticosteroids), high doses of methylprednisolone, cyclopentanone. It includes hormonal treatments such as phosphamide, azathioprine, MTX, the antimalarial drug plaquenyl, non-steroidal anti-inflammatory drugs, high dose immunoglobulin or dehydroepiandrosterone (DHEA), tamoxifen and the like. In recent years, cyclosporin, FK506, is increasingly and widely used for the treatment of systemic lupus erythematosus nephritis.
  [0009]
  However, the above treatment methods can not effectively suppress the progression of systemic lupus erythematosus nephritis, and furthermore, nonselective immunosuppression can always cause clinically fatal complications. In many documents, different treatment plans were proposed one after another. One therapeutic model is to turn systemic immunotherapy for systemic lupus erythematosus nephritis into local therapy. There, heparin treatment improves blood flow in the renal region; the ACE inhibitor captopril suppresses the production of angiotensin II; the ornithine carboxylase inhibitor suppresses nitric oxide production in the kidney region. These therapeutic effects have been demonstrated in animal models of mice.
  [0010]
  Another therapeutic model is to selectively perform immunotherapy according to the pathogenesis of systemic lupus erythematosus nephritis. As mentioned above, systemic lupus erythematosus may cause the imbalance of the mechanism of T cells and cause the lack of cooperation between T cells and B cells or the imbalance between Th1 / Th2, so In the literature, we see the effect of neonatal thymectomy on animal models of systemic lupus erythematosus mice, such as for homing receptor monoclonal antibodies, IL-2 / vaccinia recombinant vaccines, introduction of transgenic T cell genes, etc. It has been reported that it can. However, the human use of these treatments must be carefully considered in terms of technology and complications. Therefore, the treatment of systemic lupus erythematosus nephritis in the future is to clearly understand the cause of its progressive deterioration, which allows appropriate treatment at an appropriate time, avoiding unnecessary systemic side effects. .
  [0011]
  On the other hand, in the clinic of IgA nephritis, paroxysmal hematuria and / or proteinuria always appeared in a chronic disease fashion. The cause of becoming IgA nephritis is that the immune complex of IgA is deposited on mesangial cells of the kidney glomerulus and activates mesangial cells. Activated mesangial cells release interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF alpha), and cause mesangial cells to proliferate. The expanded mesangial cells release IL-1, IL-6, platelet derived growth factor (PDGF), transforming growth factor β (TGF-β) and the like. The role of these cellular hormones and growth factors is the same as that of autocrine and viciously circulates the growth of mesangial cells.₂ ^-, H₂O₂, Platelet activating factor (PAF), prostaglandin E₂(PGE₂), Thromboxane B₂(TXB₂And neutral protease etc. can be released. Thus, mesangial proliferation hardens the glomeruli and damages the renal basement membrane. In humans, about 20% gradually develop uremia after getting sick. To date, it has not been possible to stop the disease with appropriate therapeutic agents. Therefore, research on therapeutic agents is the most important task.
  [0012]
  Immunomodulators are potent therapeutic agents that can suppress immune boosting and also treat immunodeficiency. There are many Chinese medicines with immunoregulatory function in China. One of them is the famous Cordyceps.
  [0013]
  Cordyceps sinensis (Cordyceps Sinensis (Berk) Sacc) is a complex of cilia and larval carcasses in which the worm fungus of the genus Orthopedic caterpillum caterpillars has parasitized on the bat larvae (Hepialus armoricanus Oberthur) as classified below is there. Since the host is an insect larva such as L. or Coleoptera, in winter, mycelium invades the larva body in the soil, causes the insect body to be filled with mycelium, and causes death; Come out. Therefore, it is called "Torcus summer grass". Its production area is Sichuan, Yunnan, Qinghai, Tibet etc., but it is very expensive because it is difficult to obtain it.
  [0014]
Classification (Taxonomy)
  Fungi (Fungi)
    Ascomycotina
      Fungus net (Pyrenomycetes)
        Tamakabi eyes (Sphaeriales)
          Er horn fungus (Clavicipitaceae)
            Cordyceps (Cordyceps)
  [0015]
  The following is a report on the therapeutic effect of Cordyceps sinensis.
  [0016]
(1) Effects on the immune system
  Cordyceps is said to have a bidirectional regulatory function on cellular immunity. In terms of promoting cell immunity, in 1990, Zhang et al. Reported that an aqueous extract of Cordyceps sinensis can promote mitosis of thymocytes and further promote the proliferation of T lymphocytes. In 1985, Zhang et al. Also reported that the extract can activate murine peritoneal macrophages. The combination of Cordyceps sinensis and peach fruit can cure hepatitis and cirrhosis, and can further increase T lymphocyte conversion, natural killer cell function, and CD4 / CD8 cell ratio (Zhu 1992). Cordyceps has a role of stimulated division in spleen lymphocytes, stimulates spleen cells of chronic kidney failure rats, increases IL-2 and lowers the level of urea nitrogen and creatinine in blood, blood cell count Can be raised. The results show that Cordyceps sinensis has a regulatory function on cellular immunity in chronic kidney failure rats (Cheng Q, 1992). In addition, Cordyceps can protect T helper cells, increase the value of Th / Ts and counteract the immunosuppressive action of steroids and cyclophosphamide. Therefore, Cordyceps sinensis is presumed to be a kind of immunomodulator or biological response modifier to cell immunity. Thus, in 1991, Chen et al. Reported that Cordyceps can treat patients who were immunocompromised or acquired immunosuppressed.
  [0017]
  On the other hand, Cordyceps sinensis has an action to suppress cellular immunity. Zhang et al. Reported in 1990 that mycelial powder of Cordyceps sinensis can suppress the uptake function of mouse peripheral white blood cells and proliferation of splenic lymphocytes. The extract suppresses the displacement of the xenograft heart or prolongs the survival time of the organ.
  [0018]
  Cordyceps sinensis also has a bi-directional regulatory effect on humoral immunity, and can promote humoral immunity, directly induce proliferation of Thy-1 cells, or increase the antibody content of IgG and IgM. . However, in 1992, Zhu et al. Pointed out that Cordyceps sinensis also has an inhibitory effect on humoral immunity. While the treatment with liver hardening after hepatitis has been observed to reduce IgG and IgA, Zhu et al. Have an immune action similar to that of cyclosporin A (5 mg / kg) in 1990. I also pointed out that.
  [0019]
(2) Effects on kidney function
  Cordyceps can avoid damage to the proximal tubule of gentamicin. One of its effects is to promote cell repair. In addition, in rats with kanamycin-induced nephrotoxic acute kidney failure model, Cordyceps sinensis significantly reduces the degree of acute tubular damage in rats, or early restores damage to kidney function in renal failure rats Discovered that. The cause is to reduce the acute damage of tubular cell⁺, K⁺-May be related to ATPase. This effect is associated with the reduction of cellular lipid peroxidation (Zhen, 1992). Cordyceps can reduce mortality in chronic renal failure rats and lower blood urea nitrogen and serum creatinine levels; its clinical application clearly appears to improve the renal status of renal failure. In addition, Cordyceps can improve nephrotoxicity caused by cyclosporin and improve kidney function.
  [0020]
(3) Antitumor activity of Cordyceps
  The solution of Cordyceps sinensis and the solution of artificially grown Cordyceps hyphae have a suppressive effect on mouse sarcoma 180, and can also enhance the anticancer activity of cyclophosphamide. Cordyceps and artificially grown Cordyceps hyphae have all significant suppressive effects on primary growth and spontaneous lung metastasis of mouse subcutaneous lung transplant Lewis lung.
  [0021]
  The possible mechanism is that cordycepin contained therein suppresses the synthesis of DNA and RNA, suppresses the methylation of nucleic acids, suppresses the activity of protein kinases, and promotes the differentiation of cells, thus the bladder It has anti-cancer effects against cancer, colon cancer and lung cancer.
  [0022]
(4) Effects on the cardiovascular system
  Wild Cordyceps is effective against arrhythmias in experimental animal models. Artificially grown Cordyceps hyphae have a role to significantly increase coronary blood flow, to increase coronary artery / brain and peripheral blood resistance, and to lower blood pressure in anesthetized dogs, and also to use muscarinic receptors. be excited. Its active ingredient is adenosine, which has a direct relaxation effect on vascular smooth muscle, which is the main mechanism for dilating blood vessels.
  [0023]
(5) Influence on blood
  Artificially grown Cordyceps sinensis alcohol extract has an inhibitory effect on the formation of blood clots in the abdominal aorta of rabbits. Wild Cordyceps and artificially grown Cordyceps hyphae promote platelet formation, and the formed platelets have the same ultrastructure as normal.
  [0024]
(6) Other clinical effects
  Cordyceps itself has a liver function improvement effect. The seroconversion of HbsAg has a certain effect, and it can also significantly improve the patient's plasma albumin. Cordyceps has a remarkable suppressive effect on the activity of monoamine oxidase (MAO-B) in the brain, and is useful for alleviating aging and maintaining old health. In addition, Cordyceps sinensis has actions such as sedation, prevention of oxygen deficiency, anti-asthma, anti-inflammation and the like, and also has an action similar to androgen.
  [0025]
(7) Toxicity
  When Cordyceps sinensis is injected into the abdominal cavity of a mouse, its LD₅₀The value is 21.7 ± 2.6 g / kg, and for intravenous injection, its LD₅₀The value is 24.5 ± 2.2 g / kg. Toxicity has been shown to be low for either intravenous, intraperitoneal or oral administration.
  [0026]
(8) Known constituents of Cordyceps and its activity
  At present, the following components can be isolated from Cordyceps: nucleosides and nucleotides: adenine, adenosine, uracil, uridine, thymidine, ribosylhypoxanthine, hypoxanthine, cordycepin (3'-deoxyadenosine), 3 '-L-lysylamino-3'-deoxyadenosine, N⁶It is-(2-hydroxy) -adenosine. Alkaloid: opiocordin (ophiocordin). Sugars: polysaccharide, D-mannitol. Sterol: Ergosterol, ergosteryl-β-D-glucopyranoside, 22-dihydroergosteryl-β-D-glucopyranoside, 5α, 8α-epidioxy-5α-ergosta-6,22-diene-3β-ol. Fatty acids: palmitic acid, stearic acid, oleic acid, linoleic acid, and cholesterol palmitate. In Cordyceps sinensis, natural products already isolated include adenine, adenosine, uracil, uridine, cholesterol palmitate, palmitic acid, and 5α, 8α-epidioxy-5α-ergosta-6, 22-dien-3β-ol.
  [0027]
    [Problems to be solved by the invention]
  Cordyceps sinensis is a drug with a very broad pharmacological action and a very low toxicity, and furthermore, its main action is immunomodulation and renal function improvement, so systemic immune lupus erythematosus nephritis caused by immunity, in particular Furthermore, it has a considerable effect on chronic mesangial proliferation and at the same time IV systemic lupus erythematosus nephritis in which renal function is degraded. Previous studies have only used either the compound of Cordyceps or its crude product.
  [0028]
    [Means for Solving the Problems]
  In the present invention, studies were conducted on the treatment and treatment effect of systemic lupus erythematosus nephritis using the isolated natural product.
  [0029]
  As a result, the present invention has found an active moiety isolated from Cordycops sinensis (Berk) Sacc, a pharmaceutical composition of the active compound, and methods for using them to improve kidney function. Furthermore, the present invention has also found a method for preventing and / or treating lupus erythematosus nephritis and / or IgA nephritis.
  [0030]
  Thus, the present invention provides a pharmaceutical composition comprising an active fraction F2 isolated from Cordyceps sinensis, the active compound H1-A, and optionally a pharmaceutically acceptable base.
  [0031]
  In a preferred embodiment, the active fraction F2 is prepared by heating and drying the angiosperm portion of Cordyceps sinensis at 45 to 50 ° C., grinding it, and soaking it in 20 times the amount (w / v) of methanol and concentrating the extract under reduced pressure The crude extract was subjected to silica gel column chromatography, eluted with 1: 1 n-hexane / ethyl acetate mixed solvent, and the eluate was concentrated to give an extract.
  [0032]
  In a more preferred embodiment, said active compoundIs (24R) -ergosta-7,22-diene-3b, 5a, 6b-triol isolated from Cordyceps sinensis.
  [0033]
  In a preferred embodiment, the pharmaceutical composition is for improving renal function, and in vitro, production of IL-2 by monocyte cells in peripheral blood activated by PHA and activated mesangial cells Inhibits proliferation, in vivo inhibits lymph node proliferation, suppresses the production of anti-ds-DNA autoantibodies, slows the progression of proteinuria, and blocks the aggravation of IgA nephritis in the MRL lpr / lpr animal model be able to.
  [0034]
  In a more preferred embodiment, it contains a therapeutically effective amount of said F2 or H1-A for improving renal function.
  [0035]
  In another preferred embodiment, the pharmaceutical composition is for improving renal function to treat lupus erythematosus nephritis and / or IgA nephritis.
  [0036]
  In a further preferred embodiment, the pharmaceutical composition comprises an effective amount of the aforementioned F2 or H1-A for preventing and / or treating lupus erythematosus nephritis.
  [0037]
  In a more preferred embodiment, the pharmaceutical composition comprises an effective amount of said F2 or H1-A for preventing and / or treating IgA nephritis.
  [0038]
    BEST MODE FOR CARRYING OUT THE INVENTION
  The invention will be further described with reference to the attached figures.
  [0039]
  The present invention provides a pharmaceutical composition for treating systemic lupus erythematosus nephritis and a method for treating systemic lupus erythematosus nephritis using the pharmaceutical composition, the pharmaceutical composition comprising It contains an active fraction and an active compound separated from Sinensis (Berk) Sacc). The active fraction and the active compound isolated from Cordyceps sinensis (Berk) Sacc have already been described in detail in Taiwan Patent No. 78584 by the present inventor.
  [0040]
  Extraction and separation of the active ingredient of Cordyceps sinensis is carried out by taking the fruiting body part of Cordyceps sinensis, drying (45 to 50 ° C.), grinding, soaking in 20 volumes of methanol (w / v) and extracting. Further, the solvent is distilled off with a vacuum evaporator to obtain an extract. Then, the extract is subjected to silica gel column chromatography, and three solvents of hexane, ethyl acetate and methanol are mixed at various ratios, and roughly divided into six fractions (F1 to F6) according to the magnitude of polarity. For example, the separation procedure is shown in FIG. As a result of testing the biological activity, most of the activity is observed in the eluate of F2 (hexane: ethyl acetate = 1: 1 (v / v)). Thus, F2 is subsequently separated, silica gel column chromatography is again carried out on F2, eluted with different polar solvents and subdivided by the polarity into 18 fractions (C1 to C18). As a result of testing the biological activity, most of the activity is found in the eluate of C11 (hexane: ethyl acetate = 1: 2 (v / v)). Therefore, it is separated using preparative silica gel thin layer chromatography for C11. Developer: Hexane / ethyl acetate = 1: 1 (v / v), and when developed twice, it is further subdivided into six fractions (T1 to T6).
  [0041]
  Since the activity of T4 (Rf: 0.53 to 0.72) is relatively strong as a result of testing the biological activity, T4 is further subjected to reverse phase high performance liquid chromatography (RP-HPLC) separation. The conditions used are as follows.
Column: Reversed-phase, 5C18 (half-ready, 250 x 8 mm)
Mobile phase: Methanol
Detection: UV, wavelength 254 nm
Flow rate: 2 ml / min
  [0042]
  Five major fractions (H1-H5) were collected using this HPLC method. As a result of measuring their activity, H1 (t_RThe peak appeared at 10.0 to 10.9 minutes). After that, it is completely purified by further high performance liquid chromatography. The conditions used are as follows.
Column: reversed phase type, cosmosil 5C18 (250 × 8 mm)
Protective column 10C 18 (50 × 4.6 mm)
Mobile phase: Methanol: H₂O = 90: 10 (v / v)
Detection: UV, wavelength 254 nm
Flow rate: 2 ml / min
  [0043]
  Finally, the A and B compounds are obtained. According to its activity assay, its main activity is in component A (hereinafter referred to as H1-A). The purity of H1-A is approximately 100% by HPLC purity analysis. Analysis and identification of chemical structures of purified H1-A compounds by mass spectrometry (MS) and spectral methods such as nuclear magnetic resonance (NMR)Do. moleculeThe formula is C₂₈H₄₂O₂, Molecular weight is 410.
  [0044]
  In the procedure for separating natural products of Cordyceps sinensis, the activity of each fraction is measured using the inhibitory effect on the growth of mesangial cells activated by IL-1β and PDGFBB (platelet-derived growth factor) as an index. The method is as follows.
  [0045]
  First, the cultured mesangial cells are trypsinized and washed three times with RPMI-1640 medium of fetal bovine serum, and finally, the cell suspension is treated with RPMI-1640 medium containing 2% fetal bovine serum. Incubate and adjust the concentration to 1 x 10 cells / ml. The mesangial cells adjusted to this concentration are placed in 96-well culture dishes (100 μl / well), and after 6 hours, 25 μl each of IL-1β (30 units / ml) and PDGF (500 units / ml) are added, Add 50 μl of fractions of various extracts of Cordyceps sinensis (eg, F1 to F6, C1 to C18, T1 to T6, H1 to H5). After 3 days of culture,³Add 20 μl of “H” thymidine (1 μCi / well) and culture for another 18 hours. Thereafter, the supernatant fluid of the cells is removed, the cells are washed with PBS, trypsin is added thereon, and the cells are suspended. In an automated cell harvester, collect the cells on glass fiber filter paper, dry the filter paper and place the filter paper in a counting vial. By a liquid scintillation counter, [2-³The amount of incorporation of [H] thymidine into mesangial cell DNA is measured. In theory, if there is more cell division, it is absorbed by the cell.³H "thymidine also increases. Therefore, the uptake rate is taken as an indicator of cellular DNA synthesis. After treatment with various components, the radioactivity value (expressed as cpm) contained in cellular DNA can be compared to determine whether this component has an inhibitory effect. Display the magnitude of suppression as a percentage.
Suppression percentage (%) =
  (Group without drug (cpm)-group with drug (cpm)) / group without drug (cpm) x 100
Percentage of inhibition:> 50% is considered to have a significant inhibitory effect.
  [0046]
  The separated active ingredient contains an active fraction and an active compound, and is used in a pharmaceutical composition. These active fractions or active compounds are used alone or in the form of pharmaceutical compositions. One or more conventional excipients may be added to the compound to make a pharmaceutical composition suitable for different uses. The active ingredients or active compounds according to the invention are, for example, made into tablets, capsules, granules, powders, solutions and other forms. Conventional excipients, binders, lubricants, colorants, disintegrants, and other materials can be used when making solid dosage forms for internal use.
  [0047]
  Excipients include lactose, starch, talc, magnesium stearate, microcrystalline cellulose, methyl cellulose, hydroxymethyl cellulose, glycerin, sodium alginate, art (gel) and the like. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, art, shellac, sucrose and the like. Examples of lubricants include talc, magnesium stearate and the like. Commonly used colorants and disintegrants may be suitably employed. The tablets can be coated by known methods.
  [0048]
  Liquid formulations can be prepared in the usual manner for preparing these formulations in the form of aqueous or oily suspensions, solutions, syrups or other forms. In preparing the injections, pH adjusters, buffers, stabilizers, tonicity agents, local anesthetics and the like can be added to the compounds of the present invention. In addition, subcutaneous, intramuscular or intravenous formulations for injection can be made in a conventional manner. Materials used as a substrate in making suppositories include, for example, oils, fats, cocoa butter, polyglycols, and the like.
  [0049]
  The amount used of the pharmaceutical composition prepared above varies depending on the indication, the weight and age of the patient, etc., and the same amount is not always used. In general, when used for adults, the amount of the compound of the present invention may be about 0.01 to 2000 mg per day. This usage may be once a day, but can also be taken 2 to 4 times a day.
  [0050]
  Systemic lupus erythematosus is a chronic autoimmune disease as described above. The main cause of the pathology is that various autoantibodies produced in the body cause the loss of function of each system and organ in the body. The phenomena seen in the clinic are lymph node hypertrophy, vasculitis and fatal systemic lupus erythematosus nephritis. MRL lpr / lpr mice are animal models of spontaneous autoimmune diseases, and during the development of mice, symptoms of human systemic lupus erythematosus develop, among which production of autoantibodies (anti-DNA antibodies), evening It includes spontaneous arthritis, prominent lymphatic lesions and glomerulonephritis. Most mice lose progressive kidney function at 3 to 6 months. At 20 weeks, 50% of mice die. Renal failure is the leading cause of mouse death.
  [0051]
  Pathological causes of MRL lpr / lpr lesions have not been elucidated at present. Numerous literatures have reported studies of the treatment of mice with various treatment methods, among which cyclophosphamide, cyclosporin, FK506, heparin, captopril, ornithine carboxylase inhibitors, neonatal thymectomy surgery, homing Included are monoclonal antibodies against the receptor, IL-2 / vaccinia recombinant vaccine, introduction of transgenic T cell genes, and treatment of traditional Chinese medicine called immunomodulation. As mentioned above, Cordyceps sinensis has been reported as an immunomodulatory herbal medicine, but it is also possible to increase the survival rate of systemic lupus erythematosus mice and also to report that it suppresses the production of anti-ds-DNA autoantibodies. is there. As described in the Examples below, according to the results of an in vitro test of MRL lpr / lpr mice using the isolated natural product H1-A, H1-A is PHA (phytohemagglutinin) -activated peripheral It effectively suppresses the production of IL-2 by blood monocytic cells.
  [0052]
  In addition, in the following examples, F2 isolated from Cordyceps sinensis can suppress the activation of renal stromal cells outside the body, and can also prevent the onset or deterioration of IgA nephritis in the body To indicate that F2 does not cause acute toxicity and reduces the phenomenon of liver swelling induced by IgA IC. Active natural products (H1-A) purified from F2 show a good effect in animal models to prevent "the occurrence of IgA nephritis in the body".
  [0053]
  Thus, the invention further provides a method of treating lupus erythematosus nephritis and IgA nephritis. Also included is the administration of a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient who desires these methods of treatment.
  [0054]
  The following further illustrates the invention with reference to examples, but the invention is not limited to these examples. In the examples, when necessary, the results are expressed as mean value ± standard error, and statistical analysis was performed using Student's t-test to compare differences between the two groups.
  [0055]
    【Example】
Example 1: Effect of H1-A on the production of IL-2 by peripheral blood mononuclear cells (PBMNC)
  PBMNC are peripheral blood mononuclear cells isolated from normal and healthy blood donor blood. Cell concentration 2 × 10⁶Adjust to cells / ml. Also, 2 × 10^FiveCells were plated in each well of 96 well culture dishes. Cells were cultured in RPMI-1640 medium of 2% fetal bovine serum. Half of the cells were simultaneously treated with PHA (10 μg / ml) and different concentrations of H 1 -A (0 μmol / L, 3.125 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 9.375 μmol / L). The other half of the cells were treated with different concentrations of H 1 -A (0 μmol / L, 3.125 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 9.375 μmol / L). After adding H1-A to the cells, 5% CO 2 at 37 ° C.₂The cells were cultured for 72 hours in the After 72 hours, the cell supernatant was taken and analyzed. The concentration of IL-2 was measured using the IL-2 ELISA Kit (T Cell Diagnosis Inc., Cambridge, Mass.), The lowest concentration being 3.0 × 10.^-11It was g / ml.
  [0056]
  The effect of H1-A on activated peripheral blood mononuclear cells or resting peripheral blood mononuclear cells,Figure 2Shown in. Resting peripheral blood mononuclear cells have low IL-2 production (262 ± 26.6 / ml), and H1-A has a marked effect on resting peripheral blood mononuclear cell production of IL-2. I did not have it. PHA (10 μg / mL) stimulated IL-2 production of peripheral blood mononuclear cells, whereas H1-A inhibited IL-2 production. The inhibitory effect is related to the amount of drug. Statistical results indicate that there is a statistically significant difference in the inhibitory effect at concentrations of 6.25 μmol / L and 12.5 μmol / L.
  [0057]
Example 2: Treatment of MLR lpr / lpr mice with simple natural product H1-A of Cordyceps
  Twelve MLR lpr / lpr mice were purchased from the Animal Center of Jackson Laboratories. All mice were born at the same age. All of these mice were 12 weeks old when included in this study and all had symptoms of lymphadenopathy, hair loss, vasculitis and proteinuria. Before starting the experiment, another identically born MLR lpr / lpr mouse was sacrificed and the change in renal pathology before the experiment was observed; the result already has high proliferative glomerulonephritis The pathology was similar to systemic lupus erythematosus nephritis of class IV.
  [0058]
  The mice were then randomly divided into two groups. In order to avoid the influence of internal hormones by gender, the sex ratio of the treatment group and the control group was 3: 3. Prior to the experiment, Cordyceps sinensis (primitive composite containing H1-A) was first fed to another MLR lpr / lpr mouse. The result is that the compound of Cordyceps can indeed improve systemic lupus erythematosus nephritis and was dose related. The lowest effective concentration was 1% (wt / wt). Furthermore, the concentration of H1-A was calculated based on the extraction ratio of H1-A extracted from Cordyceps sinensis. The lowest effective dose was equivalent to 40 μg / kg / day of H1-A. Thus, Hl-A was placed in a minimal amount of alcohol and fed to the treatment group of MLR lpr / lpr mice for 8 weeks. All mice were bred in a specific pathogen free (SPF) environment.
  [0059]
(1) Effects of H1-A on the survival time of MLR lpr / lpr mice
  The results of comparing the survival times of the treated and control mice are:Figure 3As it was. All mice in the treatment group survived to the end of the study. That is, although surviving up to 20 weeks, 2 mice in the control group died of disease at 17 and 18 weeks. At the time of death, mice exhibited symptoms of generalized edema, severe proteinuria, and manure. The survival rate up to 20 weeks was 66.6% in the control group mice as compared to 100% in the treatment group mice.
  [0060]
(2) Effects of H1-A on lymphatic lesions and proteinuria in mice
  Lymph node size was measured, scored and evaluated. The size of lymph nodes is recorded every two weeks, and the method for calculating the score is as follows. <5 mm is 0 point, 6 to 10 mm is 1 point, 11 to 15 mm is 2 points, 16 to 20 mm is 3 points, and 20 mm or more is 4 points. The score for each time was obtained by adding up the scores of the axillary and radial lymph nodes and used as an indicator of the size of the lymph node at the time of recording.
  [0061]
  The differences in the lymphatic lesion process between the treatment group mice and the control group mice were compared. Six weeks after treatment with H1-A, retrograde changes may occur in the lymphatic lesions, but a statistically significant suppressive effect is eight weeks after treatment of mice with H1-A (Figure 4checking).
  [0062]
  Urine fluid was obtained by a method of massaging the bladder because urine from MLR lpr / lpr mice is low and difficult to obtain. Urine fluid was collected every two weeks. The collected urine was stored in a -70 ° C freezer until analysis. For urine protein, the ratio of albumin (μg / ml) and creatinine (mg / kg) in urine was recorded. For quantification of albumin in urine, an ELISA kit of a diagnostic agent for albumin (Albumin Diagnostic Reagent) (Sigma 631-2; Sigma Chemical Co., ST. Louis, MO) was used. The standard is purified mouse albumin (Cappel, Organon Teknika, Durham, NC) diluted at different concentrations, the concentration range is 5 to 50 μg / ml.
  [0063]
  With regard to the effect of H1-A on mouse proteinuria, mice in the control group have progressive proteinuria, but in the treatment group, the albumin / creatinine levels in urine decrease sequentially after 4 weeks of H1-A treatment It was a trend. The difference in numerical value between the two groups was statistically significant when the mice reached 20 weeks. In the control and treatment urines, the ratio of albumin "μg / ml" to creatinine "mg / kg" was 0.88 ± 2.94 and 0.293 ± 0.19, respectively (P <0.05)Figure 5checking).
  [0064]
(3) Effects of H1-A on kidney function of MLR lpr / lpr mice
  In order to examine whether H1-A can improve the kidney function of mice and conversely, it becomes renal toxicity, it is a system to take blood from retro-orbital every two weeks, in MLR lpr / lpr mice. I got blood. Furthermore, serum was obtained by centrifugation at 1000 rpm for 5 minutes. The obtained sera were all stored in a -70 ° C freezer until testing. Different stages of serum from mice were obtained and blood urea nitrogen and creatinine measurements were performed. Measurement of serum creatinine was performed colorimetrically with picric acid (Sigma 555A). Measurement of urea nitrogen in blood was performed with Urease Assay Kit (Sigma 640-A). All are the results of double testing. Mean value ± standard error and displayed in mg / dl.
  [0065]
  In the control group MRL lpr / lpr mice, their renal function gradually worsened during the course of the test, and the value of urea nitrogen in their blood increased from 25 ± 18 mg / kg to 35.94 ± 3.75 mg / kg, and serum The creatinine value increased from 1.33 ± 0.09 mg / kg to 3.04 ± 0.47 mg / kg. Relatively speaking, the kidney function of the treatment group is better than the control group, and the values of urea nitrogen and creatinine in the mouse blood are 24 ± 1.8 mg / kg and 1.23 ±, respectively, when the mouse reaches 20 weeks. It was 0.19 mg / kg. At week 20, the 2 groups had statistically significant differences in blood urea nitrogen and creatinine levels (P <0.05) (P <0.05)Figure 6checking).
  [0066]
(4) Effects of H1-A on the production of anti-ds-DNA autoantibodies by MLR lpr / lpr mice
  Serum of 12, 16 and 20 weeks old mice was collected for analysis of anti-ds-DNA autoantibodies. Analysis of anti-ds-DNA IgG group autoantibodies in serum was performed by the method of ELISA. First, a 96 well plate (Corning 25801 ELISA Plate) was covered with 100 μl of methylated bovine serum albumin (10 μg / ml). After standing overnight at 4 ° C., the cells were washed 3 times with PBS containing 0.05% Tween 20. Then add 100 μl of fetal calf thymus DNA (concentration 2.5 μg / ml) containing 0.1 mmol / l phosphate per well, place overnight at 4 ° C. and additionally 3 times with PBS containing 0.05% Tween 20 After washing, it was dried. Then, each well was blocked with 200 μl of 1 mg / ml gelatin, and after 2 hours at 37 ° C., washed three times with PBS containing 0.05% Tween 20. Separately, pre-prepared mouse serum samples were diluted 1: 500, 100 μl added per well and washed after 1 hour. Then, 100 μl of goat anti-mouse IgG (H + L) -horseradish peroxidase (1: 2000 in PBS) is added and washed after 1 hour at 37 ° C., finally, 2,2′-azino-di- [ 100 μl of a base solution containing 3-ethylbenzthiazoline sulfonate (6)] and hydrogen peroxide was added. After 30 minutes at room temperature, 50 μl of 10% sodium dodecyl sulfate was added to stop the reaction. The absorbance was measured at 405 nm and the results were expressed as mean ± standard error.
  [0067]
  The results of comparing the differences of IgG and anti-ds-DNA autoantibodies in mouse serum of the treatment group and control group at 12 weeks, 16 weeks and 20 weeks are as follows:Figure 7As shown in. That is, the anti-ds-DNA value in mouse blood of the control group increased from 0.141 ± 0.036 to 0.198 ± 0.047 in absorbance at 405 nm after 8 weeks of tracking, but the absorbance at 405 nm in the treatment group was 0.172 ± 0.009 It dropped to 0.112 ± 0.015. After 8 weeks with H1-A treatment, ie 20 weeks in mice, anti-ds-DNA autoantibody levels in the blood of mice in the treatment and control groups are statistically significant (P <0.005) (Figure 7checking).
  [0068]
(5) The effect of H1-A on the changes in the kidney histopathology of MRL lpr / lpr mice
  All mice were sacrificed 20 weeks later by cutting the cervical spine except for 2 mice which died at 17 and 18 weeks. Mouse kidney tissue (including 17 and 18 week-old spontaneously dead mice) was removed. One half of the kidney tissue was flash frozen, and the other half was fixed with Carson-Milloning's solution and prepared for sectioning and electron microscopy. The snap frozen tissue was cut out of a 4 μm thick slice by a cryosection machine and prepared for examination of a fluorescence microscope.
  [0069]
  The fixed kidney tissue is first dehydrated with different concentrations of alcohol, and then paraffin-embedded 3 μm sections are cut out and then stained with periodic acid-Schiff's reagent and prepared for light microscopy examination did. Semi-quantitative analysis was performed by the method of Gesualdo et al. Briefly, a minimum of 40 kidney glomeruli are examined for pieces of glass in which the kidney cortex is placed, and 0 to 3 depending on the degree of proliferation of mesangial cells.⁺Counting of various grades of 0 indicates normal; trace amount indicates that 2-3 nuclei are found in the mesangial portion; 1⁺Indicates that mesangial cells are lightly proliferated and that each mesangial portion has four to six nuclei; 2⁺Indicates moderate proliferation of mesangial cells, with 4 to 6 nuclei in most of the mesangial parts and 7 or more nuclei in the other parts; 3⁺Indicates that remarkable growth is observed in mesangial cells, and that seven or more nuclei are present in each mesangial portion. Two judges (L.F.S. and W.H.L.) made their own pathology section judgments, and the entire study was designed in the form of a double blind study. The total number of cell growth counts for each sample was calculated in the following manner. Total proliferation index = (percentage when renal glomerular proliferation index is 0 × 0) + (percentage when renal glomerular proliferation index is small × 0.5) + (renal glomerular proliferation index is 1⁺Percentage ratio when it is × 1) + (renal glomerular proliferation index is 2⁺Percentage ratio when it is × 2) + (renal glomerular proliferation index is 3⁺Percentage when it is × 3). The total proliferation index of each sample was 0-300.
  [0070]
  For fluorescence microscopy, frozen sections were first air dried and fixed with acetone for 10 minutes at room temperature and then washed with PBS. Each section of each sample was stained with fluorescein isothiocyanate conjugated goat anti-mouse IgA, IgG, IgM and C3 (Cappel) and observed again with a fluorescence microscope (Olympus). The method of determination was based on the counting method described above. Briefly, an observer examines more than 40 kidney glomeruli, and 0-3 per kidney glomerulus⁺A grade count was performed (0 indicates no fluorescence observed; trace indicates fluorescence but not apparent; 1⁺Exhibits mild fluorescence; 2⁺Exhibits moderate fluorescence staining; 3⁺(Indicates strong fluorescent staining). The total fluorescence intensity index was calculated in the following manner. Total fluorescence intensity index = (renal glomerular percentage ratio when fluorescence intensity is 0 × 0) + (renal glomerular percentage ratio when fluorescence intensity is very small × 0.5) + (fluorescence intensity is 1⁺Kidney glomerular percentage ratio × 1) + (fluorescence intensity is 2⁺Kidney glomerular percentage ratio x 2) + (fluorescence intensity is 3⁺Kidney glomerular percentage ratio × 3). The total fluorescence intensity index of each sample was 0-300.
  [0071]
  In the kidney tissue sections of the control group mice, severe renal proliferation was observed in all of two spontaneously dead mice and mice sacrificed at 20 weeks. In the treatment group, marked histopathological changes were seen after treatment with H1-A (Figure 8See A and B)). There was a clear suppression of mesangial growth and also retrograde changes. The kidney sections of the treatment group were stained with hematoxylin-eosin and all showed mild mesangial proliferation. There were no significant differences between the treatment and control groups for renal canalicular, renal interstitial or vascular changes. Also with the fluorescent staining no statistical difference was found between the two groups (see Table 1).
  [0072]
[Table 1]

  [0073]
  From the above results, pure natural product H1-A isolated from Cordyceps sinensis was able to suppress the production of IL-2 more than PHA-activated peripheral blood mononuclear cells in vitro. In the MRL lpr / lpr animal model, it was also possible to block lymph node growth and suppress the production of anti-ds-DNA autoantibodies. It was also possible to prolong the survival of MRL lpr / lpr mice. At the same time, it was able to suppress the renal glomerular mesangial growth and slow the progression of proteinuria, but surprisingly, in this study, no significant change was observed in the deposition of immune complexes. The results of this study revealed that the isolated natural product with this clear chemical structure has the potential to treat autoimmune disease in MRL lpr / lpr mice. In addition, although there is no relationship between serum anti-ds-DNA autoantibody levels and renal histopathology and renal immune complex deposition, there is no relation between them in the future, but for the cause of systemic lupus erythematosus nephritis, It is possible to provide more study space.
  [0074]
  The in vitro test results of the present invention showed that H1-A can effectively suppress the production of IL-2 by PHA-activated peripheral blood monocytic cells. The effect of this drug is similar to that of the well-known immunosuppressant cyclosporin A. As described above, H1-A, which is a primitive complex of Cordyceps sinensis, has an immunosuppressive effect on cell immunity. This result is similar to that of cyclosporin A skin transplantation. In other words, H1-A is more likely to be called an immunomodulator. Cyclosporin A is said to be effective in the treatment of systemic lupus erythematosus in humans. Cyclosporin A is used selectively for T lymphocytes. Therefore, it is used for treatment of lesions mainly composed of T cell responses. Cyclosporin A also has a suppressive effect on the reactions associated with some antibodies, but its main action is on the response generated from T helper cells. In comparison, cyclosporin A has little effect on the antibody response generated by the antigen directly stimulating B cells. H1-A may have an action mechanism similar to that of cyclosporin A, since a remarkable suppressive action on anti-ds-DNA antibody is observed. That is because the production of anti-ds-DNA is a process of T cell regulation by antigen activation. H1-A also has a suppressive effect on lymph node proliferation mainly caused by T cell proliferation. A new perspective on the mechanisms of systemic lupus erythematosus is CD4⁺T cells are the main cause of MRL lpr / lpr mouse autoimmune disease development. Therefore, at present, the cause of becoming such a disease is because the function balance of T cells is lost or there is a lack of cooperation between T cells and B cells. Anti-CD3, anti-CD4 and anti-CD8 monoclonal antibodies have been reported to be effective in the treatment of lupus erythematosus in mice. Therefore, it is speculated that the H1-A therapeutic effect is exerted by suppressing the production of T cells or suppressing the production of Th1 / Th2.
  [0075]
  The suppressive action on Th1 and Th2 significantly reduced the production of anti-ds-DNA antibody in the mouse body. It may be due to suppression of the production of regional kidney Th1. The growth of mesangial cells in the glomeruli was thereby arrested. These hypotheses need to be further demonstrated in future studies. The present invention provided three possible interpretations of no change in immune complex deposition in kidney sections.
  [0076]
  First, the disassociation between renal histopathology changes and immune complex deposition is common in the clinic. Therefore, it is impossible to interpret all the phenomena of systemic lupus erythematosus nephritis with immune complexes. Other interventional factors include cellular factors or chemical factors. Thus, the suppressive action of H1-A on mesangial proliferation may be exerted through mechanisms other than lowering immune complex deposition.
  [0077]
  Second, removal of the already deposited immune complex takes a lot of time. According to the clinical experience of class IV systemic lupus erythematosus nephritis, after the patient has been treated with cyclophosphamide, anti-ds-DNA antibody levels in the blood are reduced first as the symptoms of the disease are alleviated. Elimination of immune complexes in kidney tissue is slower than clinical improvement. Usually, significant changes are seen in kidney tissue for the first time after at least six months of treatment.
  [0078]
  Third, H1-A does not improve the ability of macrophages to clear immune complexes. Thus, H1-A is a potential isolated natural product for treating human systemic lupus erythematosus. H1-A is more secure than cyclosporin A. Cordyceps sinensis is used for traditional Chinese treatment because, despite its history of over a thousand years, there have been no reports of nephrotoxicity to date. In contrast, Cordyceps has been reported to be able to effectively avoid the nephrotoxicity caused by cyclosporin A and aminoglycosides. This study also proved this point of view. On kidney sections, we do not see any nephrotoxicity at all, conversely H1-A has even a protective effect on the kidney function of MRL lpr / lpr mice.
  [0079]
  From the viewpoint of the mechanism of immunity, it has recently been described in the literature that T cell function in patients with systemic lupus erythematosus is defective. Among them is the fact that the production of IL-2 is reduced, and the response of IL-2 induced T cell proliferation is also significantly reduced. Gutierrez-Ramos et al successfully treated MRL lpr / lpr mice with a recombinant vaccine of IL-2 / vaccinia. Several so-called immunomodulators have been suggested to correct IL-2 defects and promote cell division in the thymus and activation of peripheral T cells. However, some submitted completely opposed opinions. Reported that relatively normal and significant elevations of IL-4, IL-2, and sIL-2R were found in the blood of systemic lupus erythematosus patients. In contrast, Raz et al. Reported that introduction of IL-2 cDNA into the body of MRL lpr / lpr mice aggravated the condition and further promoted the production of autoantibodies. The inventors' results are almost in agreement with the latter opinion. In vitro, H1-A suppresses the production of IL-2 in peripheral mononuclear cells; in MRL lpr / lpr mouse animal models, H1-A improves clinical symptoms and renal tissue pathology Can.
  [0080]
  As described above, according to the present invention, MRL lpr / lpr of an autoimmune disease model is isolated using isolated natural product H1-A having a clear structure, which is isolated from Chinese traditional herbal medicine, Cordyceps It succeeded in raising the therapeutic effect in the body.
  [0081]
Example 3: Effects of Cordyceps sinensis extract (1% and 0.5% F2) on mice with induced IgA nephritis
  [0082]
1. Method of inducing IgA nephritis: The antigen used is R36A and the antibody is a-R36A-IgA-mAb. First, 2 mg of antibody was injected into the abdomen of Balb / c mice, and 2 hours later, 100 μg of antigen was injected into the tail vein. Then, 100 μg of antigen was injected every 24 hours (one week continuously), whereby the immune complex was deposited in the renal interstitium, and hematuria and proteinuria appeared for about 5 days after the first injection.
  [0083]
2. Mice inducing IgA nephritis were divided into dosed groups (n = 6) and control groups (n = 6). Dosing groups received 1% and 0.5% F2. Each mouse was fed approximately 3 g of normal diet daily. The mice in the test group and the control group were measured daily for hematuria and proteinuria, and after 7 days, the mice were sacrificed. The kidney was taken, histopathological examination and immunofluorescence examination were performed, and changes in serum ALT, AST, and cholesterol in the liver, cytochrome P450 were observed.
  [0084]
I. Hematuria and proteinuria measurement:
It was examined with a urine test strip (Urine Strip).
  [0085]
II. Renal histopathology examination:
  Kidney tissue was fixed with 10% neutral formalin, dehydrated with increasing concentrations of alcohol, paraffin-embedded, sectioned 4 μm thick, stained with hematoxylin-eosin, and observed with a light microscope. For each mouse, 50 glomeruli are observed, and the degree of destruction of the glomerulus is divided into five grades. The higher the score, the higher the degree of destruction of the glomeruli. Finally, the points are accumulated and displayed in total.
  [0086]
III. Immunofluorescence test:
  Kidney tissue was embedded in O.C.T. compound and stored in a -70 <0> C freezer. When performing immunostaining, cut out 4 μm tissue sections on a cryosection machine and fix with acetone, then directly immunofluorescent staining with fluorescently labeled goat anti-mouse IgA, goat anti-mouse C3 and goat anti-fibrinogen Was performed and observed with a fluorescence microscope. The fluorescence staining intensity is divided into four grades, and the higher the score, the stronger the fluorescence intensity, which means that the activation of IgA, IC deposition and C3 complement is high.
  [0087]
3. Statistical method:
  Hematuria, proteinuria, histopathology and immunofluorescence were analyzed by Chi-square test. The statistics of ALT in serum, cholesterol in liver, and cytochrome P450 were analyzed by Student's t-test. P <0.05 is considered statistically significant.
  [0088]
Result:
1. Hematuria and proteinuria are shown in Table 2.
  [0089]
【Table 2】

  [0090]
  Five days after antigen injection, hematuria and proteinuria appeared. On day 7, significant reductions in hematuria and proteinuria were seen in the 1% F2 and 0.5% F2 groups (P <0.05).
  [0091]
2. The results of kidney histopathology examination are shown in Table 3.
  [0092]
[Table 3]

  [0093]
  In terms of the total number (Total), in the 1% F2 group, the degree of destruction of kidney glomeruli was significantly reduced (P <0.05), but in the 0.5% F2 group, no statistically significant difference was observed.
  [0094]
3. The results of the immunofluorescence test are shown in Tables 4 and 5.
  [0095]
[Table 4]

  [0096]
[Table 5]

  [0097]
  According to the Total evaluation, both the IgA IC deposition and C3 complement activation were reduced in proportion to the amount of drug compared to the control group in the 1% F2 and 0.5% F2 groups of dosing . The 1% F2 group showed a statistically significant difference as compared to the control group (P <0.05).
  [0098]
4. The results of liver function tests are shown in Table 6.
  [0099]
[Table 6]

  [0100]
  There was no difference between the administration group and the control group in terms of liver weight and body weight: when viewed from the ratio of 100 times liver weight to body weight, the 1% F2 group is 6.75 ± 0.09 and the control group is 7.12 ± 0.12 Met. Liver weight decreased obviously in 1% F2 (P <0.05). There were no significant changes in serum ALT and AST between the dosing and control groups. There was no statistically significant difference in the content of cholesterol in the liver between the dosed group and the control group. The content of cytochrome P450 in the liver was 0.31 ± 0.03 nmol / mg protein in the 1% F2 group, and 0.23 ± 0.01 nmol / mg protein in the control group. 1% F2 significantly increased the content of cytochrome P450 (P <0.05). In terms of the ratio of liver weight to body weight, in the acute toxicity test, liver swelling was observed when 2% F2 was administered. In an animal model in which IgA nephritis was induced, liver atrophy was observed when 1% F2 was administered. These show the opposite results. In the acute toxicity test, normal ICR mice were used as a control group, and the ratio of 100 times liver weight to body weight was 5.44 ± 0.17. In the Balb / c mouse test in which IgA nephritis was induced, feeding a normal diet was used as a control group. The animal itself, in which IgA nephritis is induced in advance, has a ratio of 100 times liver weight to body weight of 7.12 ± 0.12, and in mice with IgA nephritis, a phenomenon of liver swelling is observed. The animals used in the two trials were of different breeds. Furthermore, when rearing normal diet to a normal Balb / c mouse in the "rearing with Cordyceps sinensis extract" method, the ratio of 100 times liver weight to body weight is finally 5.50 ± 0.24 (Figure 7), Which is consistent with the normal 100-fold liver weight to body weight ratio (5.44 ± 0.17) of ICR mice. This indicated that the IgA IC itself causes a significant liver swelling phenomenon (P <0.05). The mechanism is unknown. However, administration of 1% F2 significantly reduced IgA IC-induced liver swelling (P <0.05), but failed to recover to normal values.
  [0101]
Example 4: Safety of H1-A to cells in vitro
  The activity and toxicity of H1-A to cells were examined, and the results wereFigure 8It was shown to. At the topFigure 8(A) shows the results of measurement of cell activity by trypan blue staining, and three groups (human mesangial cells only, human mesangial cells + IL-1 and IL-6, and human mesangial cells + IL-1 and 72 hours of culture) It showed that there was no difference in the cell activity of IL-6 and 40 μM / ml H1-A). Since dead cells release LDH, the concentration of LDH in the culture solution is measured after 72 hours in the same (a) test, and the results areFigure 8It showed to (b). All three groups had no statistical difference. If cells survive, mesangial cells also proliferate. Therefore, the state of cell proliferation was analyzed by the MTT method. The resultFigure 8It is shown in (c). It can be seen that all three groups had no statistical difference.
  [0102]
Example 5: Effect of H1-A on IgA nephritis-induced mice
  IgA nephritis mice induced with Example 3 were injected once with 10 mg / ml H1-A in the abdomen and continuously for 7 days.
  [0103]
  The effects of IgA nephritis mice in the treatment group and hematuria / proteinuria in the control group were compared. From the results shown in Table 7, it can be seen that hematuria / proteinuria in the treatment group is significantly improved and statistically significant.
  [0104]
  With regard to pathological changes in kidney tissue, severe mesangial hyperplasia and mesangial cell proliferation were observed in the control group, but marked improvement in histopathological changes was observed after treatment with H1-A in the treatment group. . The kidney sections of the treatment group were hematoxylin-eosin stained with only slight mesangial proliferation, the improvement of the fluorescent staining is shown in Table 7. The treatment group has significant improvement and is statistically significant.
  [0105]
[Table 7]

  [0106]
    【Effect of the invention】
  F2 isolated from Cordyceps sinensis can suppress the activation of renal stromal cells in vitro, and can prevent the onset or deterioration of IgA nephritis in the body. F2 has little acute toxicity and can also reduce liver swelling induced by IgA IC. The active natural product (H1-A) isolated from F2 has a good therapeutic effect in an animal model that prevents "the occurrence of in vivo IgA nephritis".
  [0107]
  Although the invention has been described with reference to preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover these modifications as fall within the scope of the appended claims.
Brief Description of the Drawings
    [Fig. 1]
  It is a flowchart which shows the process of isolate | separating an active fraction and an active compound from Cordyceps sinensis.
    [Figure 2]
  Fig. 7 is a graph showing the effect of H1-A of the present invention on the production of IL-2 by peripheral blood mononuclear cells. ●: H1-A only; ▽: Stimulation of mononuclear cells with PHA (10 μg / ml) + H1-A.
    [Figure 3]
  It is a graph which shows the influence of the H1-A on the survival time of the MRL lpr / lpr mouse. ■: treatment group to which H1-A was administered; ○: untreated control group.
    [Figure 4]
  It is a graph which shows the influence with respect to the lymphoid lesion of the MRL lpr / lpr mouse | mouth of H1-A of this invention. ■: treatment group to which H1-A was administered; ○: untreated control group.
    [Figure 5]
  It is a graph which shows the influence of the H1-A of this invention on the proteinuria of the MRL lpr / lpr mouse. ■: treatment group to which H1-A was administered; ○: untreated control group.
    [Figure 6]
  It is a graph which shows the influence on the renal function of the MRL lpr / lpr mouse | mouth of H1-A of this invention. ■: treatment group to which H1-A was administered; ○: untreated control group.
    [Figure 7]
  It is a graph which shows the influence of the H1-A of the present invention on the production of anti-ds-DNA autoantibodies in MRL lpr / lpr mice. ■: treatment group to which H1-A was administered; ○: untreated control group.
    [Figure 8]
  It is a graph which shows the influence with respect to the cell activity of H1-A of this invention. ■: control group, human mesangial cells only; □: human mesangial cells + IL-1, IL-6; □: human mesangial cells + IL-1, IL-6 and H1-A for 72 hours at 40 μM / ml culture. (A) observation of cell activity by trypan blue staining; (b) observation of cell death by measurement of released LDH; (c) observation of cell proliferation by MTT test.

Images