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Ying-Chen Lin Chi-Ting Lin Yu-Chun Lin Ling Ling Yang

The protective effects of Chinese medicinal prescriptions against oxidative stresses on rat brain and liver tissue. Ying-Chen Lin Chi-Ting Lin Yu-Chun Lin Ling Ling Yang. Taipei Medical University. 自由基可能參與之疾患.

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Ying-Chen Lin Chi-Ting Lin Yu-Chun Lin Ling Ling Yang

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  1. The protective effects of Chinese medicinal prescriptions against oxidative stresses on rat brain and liver tissue Ying-Chen Lin Chi-Ting Lin Yu-Chun Lin Ling Ling Yang Taipei Medical University

  2. 自由基可能參與之疾患 There is evidence that free radical damage contributes to the etiology of many chronic health problems such as Parkinson's syndrome disease, emphysema, cardiovascular, inflammatory diseases and cancer.

  3. ,HBV Interferon herbs Corticosteroids + ++ Antioxidants Radiation, Drugs, Toxins Supplements 肝臟致病因子

  4. Potential Sources Antioxidants Exogenous Endogenous Compounds Enzymes Xenobiotics Mitochondria Vitamin E SOD Pollution Phagocytes Vitamin C GSH Radiation Oxidases Carotene Peroxidase MDA(TBA)2 TBA + Malondialdehyde Radicals Pesticides P-450 Catalases Oxidants -OH Lipid LH Lipid peroxidant LOOH Preventive antioxidants Oxidatively Protein Lipid radicals L Lipid Damaged Cell RNA, DNA O2 Constituents Cytotoxicity Carbohydrate Direct Repair Lipid/DNA Peroxidases Lipid peroxide radicals LOO SH reduction Others Chain breaking antioxidants Excretion of Biosynthesis Damage Removal irrversibly Protein Sythesis Proteasome Stable products DNA/RNA Polymerase Phospholipases A2 , C damaged Products Lipid acylation Redoxyendonucleases Free radicals and lipid peroxidation Conservation of Undamaged Bullding Blocks Amino Acids Fatty Acids Nucleic Acids Cellular defense mechanisms against free radical/oxidant damage

  5. Chinese Medicinal Prescription Tonifying prescriptions Recipes to reinforce vital function or replenlish viral essence , energy and blood in their deficiencies. 四物湯、四君子湯、人參養榮湯、參苓白朮散、十全大補湯 Mediation prescriptions Recipes which regulate the correlation of viscera , channels, vital energy and blood , so as to remove pathogenic factors and restore normal function. 黃芩湯、小柴胡湯、大柴胡湯、加味逍遙、葛根黃連黃芩湯 Febrifugal prescriptions Recipes which are composed of drugs cold or cool in nature and used to relieve fever or other heat symptoms by clearing up heat in the interor of the body. 龍膽瀉肝湯、黃連解毒湯

  6. Experimental 160000 I C R mice were killed and organ homogenates 1ml (10%,W/V) were prepared in (PBS) under 4℃. Contain protein 100mg/ml(liver),1mg/ml(brain) 140000 100 120000 100000 80 peak area 80000 y = 3584.7x + 1640.2 60 60000 2 R = 0.9917 Inhibition (%) 40000 40 20000 20 Incubation were iniated by the addition of 1mM t-BOOH and CTMPs incubated at 37℃ for 1hr 0 0 10 20 30 40 50 0 micro mole 0 50 100 150 200 250 300 TEP-(MDA)2 Standard Curve m conc. ( g/ml) 1.centrifugation,25ml aliquots of the super fluids were added to thiobarbituric acid Analysis of TBARS 2.Incubated in a boiling water bath for 1 hr. 3.Then add 5%NaOH/ MeOH under ice bath Trolox IC50=48.48 μg/ml Inhibitory rate of lipid peroxidation (IR%) HPLC assay Column: Lichrospher 100 RP-18e I - D I R % =------------- I - B Mobile phase: MeOH / KH2PO4=4 / 6 Rate=1ml/min, Inject: 20 ul, I = average of inducer MDA(TBA)2 value D = average of sample MDA(TBA)2 value B = average of Blank MDA(TBA)2 value) UV detector: 532 nm

  7. Experimental (in vivo) O.P. I C R mice were killed and organ homogenates 1ml (10%,W/V) were prepared in (PBS) under 4℃. 7 days Male ICR mice ( 25-30g , 55 weeks ) 1. N 2. C 3. F 4. F+C Incubation under 37℃ for 1hr N: normal saline C: Hwang-Lian-Jiee-Du- Tang F: Fe2+(100mM) centrifugation, Analysis of TBARS: as described Braughler et al.1986. 25ml aliquots of the super fluids were added to thiobarbituric acid incubated in a boiling water bath for 1 hr. Then add 5%NaOH/ MeOH under ice bath HPLC assay Lichrospher 100RP-18 column ,532 nm Mobile phase:MeOH/KH2PO4=4:6 rate=1ml/min Inject:20ml

  8. 不同鼠齡ICR mice腦均質液之脂質過氧化 • M *P<0.05 * MDA(TBA)2 0.5 * 0.4 0.3 0.2 0.1 0 6 17 55 67 (week) -0.1 n=3

  9. *P<0.05 • M * MDA(TBA)2 * 0.3 * 0.2 0.1 0 Blank Fe(NH4)2(SO4)2 FeSO4 FeCl3 n=3 100mM 17週齡之ICR鼷鼠腦部脂質過氧化誘導

  10. Fe(NH4)2(SO4)2誘導腦部脂質過氧化 mM MDA(TBA)2 0.3 * * 0.2 0.1 *P<0.05 n=3 0 mM 0 25 50 100

  11. ANTI-LIPID PEROXIDATION EFFECT of CHINESE MEDICINAL PRESCRIPTIONS ON LIVER 100 ** 90 ** 80 ** ** 70 60 * 50 I R％ * * * * 40 * 30 20 10 0 大 黃 黃 龍 人 小 十 葛 加 參 四 四 膽 柴 根 芩 連 味 參 柴 苓 君 物 全 Trolox （中藥處方） 黃 瀉 子 大 胡 湯 解 逍 養 胡 白 湯 毒 遙 肝 榮 湯 朮 湯 湯 連 補 黃 湯 散 湯 湯 散 湯 芩 *P<0.05,**P<0.01

  12. ANTI-LIPID PEROXIDATION EFFECT OF CHINESE MEDICINAL PRESCRIPTIONS ON BRAIN in vitro n=3 *P<0.05 Tian-Ma-Gou-Terng-Saan Tranqilizer Gou-Terng-Saan Tian-Wang-Bun-Shin-Dan Ren-Shen-Yeang-Rong-Tang Tonifying Hwan-Shao-Dan Chi-Bae-Mei-Raan-Dan Tzy-Shenn-Wan * Sheng-May-Saan Febrifugal Hwang-Lian-Jai-Du- Tang * Dah-Chair-Hwu- Tang Mediation * Sheau-Chair-Hwu- Tang 0 50 100 %

  13. 黃連解毒湯 對FeCl2誘導ICR鼷鼠腦均質液脂質過氧化之抑制作用 100 80 60 抑制率 IR% 黃連解毒湯 40 n=3 20 0 0 0.2 0.4 0.6 0.8 1 mg/ml

  14. Fe2+口服投與誘導17週齡ICR鼷鼠腦脂質過氧化值(in vivo) • M MDA(TBA)2 0.5 0.4 0.3 0.2 0.1 0 0.2 0.1 0 ml(100mM) n=3

  15. Anti-lipid peroxidantion effect of Hwang-Lian-Jai-Du- Tang on brain of ICR mice.(in vivo) Anti-lipid peroxidantion effect of Hwang-Lian-Jai-Du- Tang on brain of ICR mice induced by Fe2+ . (in vivo) MDA(TBA)2 MDA(TBA)2 • M • M *p< 0.05 *P< 0.05 0.5 0.3 0.4 * 0.2 * 0.3 * 0.2 0.1 0.1 0 0 Hwang-Lian-Jai-Du- Tang 200mg/ kg; Fe++ (0.1ml) Saline 100mM Fe++(0.1ml) 300 200 100 0 mg/kg n=3 n=3

  16. ANTI-LIPID PEROXIDATION EFFECT OFINGREDIENTS OF HWANG-LIAN-JAI-DU-TANG TANG ON BRAIN (1) 120 100 80 I R% 60 Radix Scutellariae : S Rhizoma Coptidist : C Cortex Phellodendri : P Fructus Gardeniae : G 40 20 0 1 2 3 4 5 1. S+C+P+G 2. S+P+G 3. S+C+P 4. S+C+G 5. C+P+G

  17. ANTI-LIPID PEROXIDATION EFFECT OFINGREDIENTS OF HWANG-LIAN-JAI-DU-TANG TANG ON BRAIN (2) 120 100 80 Radix Scutellariae : S Rhizoma Coptidist : C Cortex Phellodendri : P Fructus Gardeniae : G 60 I R% 40 20 0 1 2 3 4 5 6 7 2. S+C 3.S+G 4.S+P 5.C+G 6. C+P 7. P+G 1. S+C+P+G

  18. ANTI-LIPID PEROXIDATION EFFECT OFINGREDIENTS OF HWANG-LIAN-JAI-DU-TANG TANG ON BRAIN (3) 120 100 80 Radix Scutellariae : S Rhizoma Coptidist : C Cortex Phellodendri : P Fructus Gardeniae : G 60 I R% 40 20 0 1 2 3 4 5 S C G P S+C+P+G

  19. OCH 3 R O R O O O HO OH O OH O baicalein R=H wogonin R=H wogonin glucuronide R=GlcA baicalin R=GlcA OCH HO 3 R O H CO O O 3 R H CO R 3 OH O OH O oroxylin-A R=H skullcapflavone R=H I oroxylin-A glucuronide R=GlcA skullcapflavone II R=OCH 3 R1 R2 R3 R R4 1 berberine -O-CH -O- OCH 2 OCH 3 3 N coptisine -O-CH -O- -O-CH -O- R 2 2 2 R palmatine OCH OCH OCH OCH 3 3 3 3 3 jateorrhizine OCH OCH OCH OH 3 3 3 R 4 H CO 3 HO CH CH COOH CH 3 + N HO CH 3 H H CO HO 3 Ferulic acid magnoflorine H CO 黃連黃芩 黃連Rhizoma Coptidis, Huang Lian, 黃芩Radix Scutellariae, Huang Qin 3

  20. 黃芩對ICR鼷鼠腦均質液脂質過氧化抑制作用 IR% 100 80 60 40 黃芩 20 0 0 0.2 0.4 0.6 0.8 1 mg/ml

  21. 抗鼠脂質過氧化實驗的優點 1.本實驗的穩定性及再現性高。 2.以鼠肝均質液進行研究具有以下優點： (1)老鼠的繁殖期短易於掌控其的生長期及來源不易短缺。 (2)老鼠的價格便宜能降低實驗經費。 (3)以老鼠均質液進行實驗可同時檢測數種藥品及對照組比對， 降低生物個體的差異性。 3.脂質過氧化為引發多種疾病的原因之一，因此對於抗脂質過氧化的開發可助於各類疾病藥物的開發以及用於疾病的預防。

  22. RESULTS AND DISCUSSION 1.本實驗發現鼷鼠高齡組腦部脂質過氧化情行明顯高於年青組 顯示脂質過氧化與老化有密切關係,可作為老化之參考指標. 2.以二價鐵離子可成功誘導腦部之脂質過氧化值,可以此建立老 化之模式. 3.黃連解毒湯對腦脂質過氧化抑制作用較強. Using the generation of TBA-reactive substances as an index of lipid peroxidation , a number of TCMPs were found to be active as antiperoxidative agents. In our experiments , the antioxidant Vitamin -E be used as a standard, I R %= 67% . Based on the results , we find that Febrifugal recipes more effective than tonfying, and Scutellatiae Radix is the essential drug of Coptis Detoxicant Decotion on anti-lipid- peroxidation.

  23. 方劑組成原則及規律 目的：*藥有個性之特長，方有合群之妙用 3.降低毒性及副作用 1.增強藥物作用 2.適應病情況擴大治療範圍 黃柏、知母合用 滋陰降火 之效 提高茵陳配梔子 利膽作用提昇 生薑半夏同用 可消半夏之毒性 四君子湯主脾胃氣虛若加陳皮、半夏為 四君子湯可兼行氣、燥溼及化痰 主藥治療藥物、用量較大： 如麻黃湯中麻黃為主藥 佐 藥 臣 藥 君 藥 主藥治療藥物、用量較大： 如麻黃湯中麻黃為主藥 輔佐主藥增強治療效果： 如麻黃湯中桂枝增強麻黃發汗功能 佐助：協助君藥， 麻黃湯中杏仁助麻黃宣肺平喘功能 佐制：制約毒性， 十棗湯中大棗能緩甘遂峻下 反佐：藥性相反， 藥性溫熱之吳茱萸佐以黃連之寒 使 藥 引經：引導至病變部位 調和：如麻黃湯中甘草調合諸藥

  24. RESULTSAND DISCUSSION 黃連解毒湯對腦脂質過氧化抑制作用較強,檢討中藥方劑藥物 配伍研究結果,黃芩為君藥,黃連可輔佐黃芩以降低用藥量並 減低毒性增加應用範圍, It s well known that CTMPs are compound medicines which are combined with crude drugs, and the exhibition of their pharmacological action is the result the pharmacodynamic interaction of the combined drugs and their components. It is an important factor to consider in TCMPs curative effect and toxicity studies as wells in the assessment of drug-drug interaction in patients under TCMPs. The structure activity relationship of these CTMPs are thus still unknown and requires further. Clarification of the various anti- lipid peroxidation mechanisms of CTMPs will necessitate much more study.The natural antioxidants extracted from CTMPs have certain advantages over synthetic ones,such as their easy and economical acquisition and the slight or negligible side effects,Currently there are at least three aspects to their application :(1)to cure the diseases induced by free radicals; (2) as the rotective agents of drugs, food and transplanted organs; (3)as additives of food in order to prevent cancer and retard aging. In summary the anti-lipidperoxidation effect of CTMPs is a significant field of pharmacological study full of hope for the future.

  25. Acknowledgement This work was supported by Dr. Chi-Chin Huang Foundation in the USA (4152Z23-B30-3) and we also thanks to Topnotch Stroke Research Center grant, Taiwan Ministry of Education.

  26. Thank you for your attention

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