200918085 九、發明說明： 發明所屬之技術領域 品用於治療 胃炎、胃潰 本發明係關於一種從薑的根提取的有效產 與幽門桿菌有關的疾病的新用途，例如病人的 瘍或十二指腸潰瘍。 先前技術 中國的生草藥或香料例如薑⑺叹如—★)、丁香 ⑽挪⑽卿如㈣及㈣猶_ _•叫在藥用或食物調 方面都是很普遍的材料。生葚 …、 嘔、袪痰、抗咳嗽 及/肖化益g的促進劑。, Ll ti- b 劑+乾的老薑亦用作為胃痛、胸痛、 下月痛亥漱及-般感冒的治療以及浮腫（又稱為水腫 治療。在美國專利第5804603號中提到，墓所含墓綱 ㈣—是辛辣味的主要來源，且墓醇考生墓 醇（shogaol)也具有辛香呋。 社 卜，量醇有強心、抑制老鼠 離體靜脈收縮、調控老鼠盥免 '、鬼子g因eicosanoid引起的收 縮等作用。薑醇與生薑醇 呷马薅大變性，而薑及薑酮則被發 現具抗誘突變的活性。生蕃酿 王量私具有鹿角菜膠（carageenan)誘 發的腳掌浮腫的抑制效果 、 竹剌政果及血小板凝集的抑制效果。 美國專利第6,855,347费掘+ , 唬揭不了—種治療胃潰瘍的組成 物及其製備方法，其中該細士、札^ λ 物匕含從为warwWc»·?和 奶仏㈣以的植物部 f矛從八種植物中所挑選的至 少一種的植物部份所獲得的—茧 曼仔的卒取物，其中該八種植物當 中一種是薑。較佳的，咳έ劣私 ^ 忒組成物為一種水萃取物。 5 200918085 曰本專利公開第2004-1 1 5536號揭示一種預防和治療 胃炎和胃和十二指腸的潰瘍的抗幽門桿菌的有效產品，其 tl含一楂或多種的從 Sop horae radix, Anisi ste!lati Frutus, Myristica fragrans, Isodon japonicus Hara, Swertia japonic a,佛羅倫薩茴香（Florence fennel), Zingiber siccatum, Atractylodes rhizome,薑（ginger), rad/x及 r/n’zoma所選出的草本製劑（gaienicals)。 發表在 Anticancer Research 2λ(S A ) · 3699-3702, 2003 年的 文研九中結δ*出包含薑醇的董根萃取物可以抑制幽門桿 菌CagA+菌株的成長。 發明内容 本發明的一目的是提供一 種從薑的根提取的有效產品 用於治療與幽門桿菌有關的疾病。較佳的，本發明所製備 的有效產品比從薑的根萃取的粗水萃取物或粗有機溶劑萃 取物具有顯著的增進功效。200918085 IX. INSTRUCTIONS: Field of the Invention The present invention relates to the treatment of gastritis and gastric ulcer. The present invention relates to a new use of a disease derived from the roots of ginger which is effective for producing a disease associated with Helicobacter pylori, such as a patient's ulcer or duodenal ulcer. Prior Art Chinese herbal medicines or spices such as ginger (7) sigh - ★), cloves (10) (10) Qing (4) and (4) _ _ _ _ is called a medicinal or food adjustment is a very common material. An agent for oysters, vomiting, phlegm, anti-cough and//Xiao Huayi g. , Ll ti- b + dry ginger is also used as a stomachache, chest pain, next month pain and treatment of the common cold and edema (also known as edema treatment. mentioned in US Patent No. 5804063, the tomb Containing tombs (4) - is the main source of spicy taste, and the shogaol of the tomb alcohol also has scented fur. Sibu, the amount of alcohol has a strong heart, inhibits the in vitro vein contraction of mice, regulates mouse forgiveness, and devils g Due to the contraction caused by eicosanoid, gingerol and ginger alcohol have a large degeneration, while ginger and zingerone have been found to have anti-mutagenic activity. The raw material of the king has a carageenan-induced foot. The inhibitory effect of edema, the inhibition effect of radix sylvestris and platelet aggregation. US Patent No. 6,855,347 Fei Jin +, 唬 唬 — — — — — 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗From the plant parts of the plant parts f for the warwWc»·? and the milk thistle (four), obtained from the plant parts of at least one of the eight plants, the one of the eight plants is one of the eight plants. Ginger. Better, coughing ^ 忒 Composition is an aqueous extract. 5 200918085 曰 Patent Publication No. 2004-1 1 5536 discloses an effective product for preventing and treating gastritis and ulcers of the stomach and duodenum, which contains one or more From Sop horae radix, Anisi ste!lati Frutus, Myristica fragrans, Isodon japonicus Hara, Swertia japonic a, Florence fennel, Zingiber siccatum, Atractylodes rhizome, ginger (ginger), rad/x and r/n'zoma Selected gaienicals. Published in Anticancer Research 2λ(SA) · 3699-3702, 2003, Wenyan Nine, δ*, Donggen extract containing gingerol can inhibit the growth of the Helicobacter pylori CagA+ strain. SUMMARY OF THE INVENTION It is an object of the present invention to provide an effective product for extracting from the root of ginger for treating diseases associated with Helicobacter pylori. Preferably, the effective product prepared by the present invention is more crude than the crude water extract extracted from the root of ginger. Or crude organic solvent extracts have significant enhanced efficacy.
是有利的，因 且為相對 上不穩定的化學品。 的極性低到—足夠使所獲得的沖提液不含有 I醇時，該沖提液具有最佳的藥效。此發現 為6-薑醇及6_生薑醇都是芳香成分且為 200918085 本么月的較佳具體貫施例包含（但不限於）下列項目： 1 · 一種用於治療與幽門桿菌有關的一種疾病的醫藥組 成物 § /α療有效罝的從薑的根製 備的有效產品，該有效產品係經由包含下列步驟的一方法 所製備： 〇從薑的根製備一含有6-薑醇及6-生薑醇的粗製液； b) 將該粗製液導入一逆相層析管柱或正相層析管柱， " 序以第在洗液及第二流洗液流洗該管柱，以獲得第 机洗液的沖提液及第二流洗液的沖提液，其中a)當該逆 層析f柱被使用時，該第一流洗液的極性小於水，及該 第一流洗液的極性小於該第一流洗液的極性，或B)當該正 相層析官柱被使用時，該第二流洗液的極性小於乙酸乙酯 -、甲醇的重昼比i:丨的一混合液，及該第一流洗液的極性 小於該第二流洗液的極性；及 c) 揮發移除該第一流洗液的沖提液中的第一流洗液以 ‘旱第/農縮沖提液，該第一濃縮沖提液可作為該有效 產’或揮發移除該第二流洗液的沖提液中的第二流洗液 而得第二濃縮沖提液，該第二濃縮沖提液可作為該有效 產品； ~ 其中該步驟a)包含下列步驟i)至iv)，步驟1：)，步驟r) 或步驟p)，該步驟i)至iv)為： 〇將薑的新鮮根莖粉碎及過濾，而得到—濾液及一 部份； ' U)將該濾液以一第一有機溶劑萃取，取出所獲得的第 200918085 一有機洛劑萃取液，並揮發移除其中的第一有機溶劑而獲 得一濃縮的第一有機溶劑萃取液； ni)將該殘渣部份以一第二有機溶劑萃取，取出所獲得 的第二有機溶劑萃取液，並揮發移除其中的第二有機溶劑 而獲得一濃縮的第二有機溶劑萃取液；及 N)合併該濃縮的第一有機溶劑萃取液及該濃縮的第二 有機溶劑萃取液而獲得該粗製液； 該步驟I)為： & 〇以與該第二有機溶劑相同的一有機溶劑萃取被粉碎 的薑的乾燥根莖’ #出所獲得的該有機溶劑萃取液，並揮 發移除其中的該有機溶劑而獲得該粗製液；及 該步驟Γ)為： 以水蒸氣蒸餾被粉碎的薑的乾 称很呈，减壓濃縮所 獲得的餾出物而獲得該粗製液；及 該步驟I”）為： I ”）以超臨尺- 界一氧化碳萃取被粉碎的薑 揮發移除其中的-与y 量的乾秌根里，並 、一氧化碳而獲得該粗製液。 2·如前述第1項的醫藥組成物，其 3.如前诂馀， 〆庆闲马月潰瘍。 J之弟1項的醫藥組成物，其中 驟丨）至iv)。 /、〒的步驟a)包含步 其中該第一有機溶劑 甲醇、乙醇、曱醇與 、或其等之混合。 其中該第二有機溶劑 ⑴处弗3項的醫藥組 為乙醚，及該第二有機溶劑為 水的-混合液、乙醇與水的一 5 ·如剐述第4項的醫藥組 8 200918085 為丙酮。It is advantageous because it is a relatively unstable chemical. The polarity is so low that it is sufficient for the obtained extract to contain no alcohol, and the extract has the best efficacy. This finding is that both 6-gingerol and 6-gingerol are aromatic components and is a preferred embodiment of 200918085. This includes, but is not limited to, the following items: 1 · One treatment for Helicobacter pylori A pharmaceutical composition of a disease § /α therapeutically effective 制备 effective product prepared from the root of ginger, the effective product is prepared by a method comprising the following steps: 〇 Preparation of a ginger-containing root containing 6-gingerol and 6 - a crude liquid of ginger alcohol; b) introducing the crude liquid into a reverse phase chromatography column or a normal phase chromatography column, " sequentially washing the column with the washing liquid and the second flow washing liquid, Obtaining a rinse solution of the first machine wash liquid and a second flow wash liquid, wherein a) when the reverse chromatography f column is used, the polarity of the first flow wash liquid is less than water, and the first flow wash The polarity of the liquid is less than the polarity of the first stream washing liquid, or B) when the normal phase chromatography column is used, the polarity of the second stream washing liquid is less than the weight ratio of ethyl acetate-, methanol to i: 丨a mixture, and the polarity of the first stream washing liquid is less than the polarity of the second stream washing liquid; and c) volatilizing to remove the first stream washing The first flowing lotion in the liquid eluent is used as 'dry/agricultural extracting liquid, and the first concentrated extracting liquid can be used as the effective production' or volatilized to remove the second flowing washing liquid. The second stream of washing liquid obtains a second concentrated extract liquid, and the second concentrated extract liquid can be used as the effective product; wherein the step a) comprises the following steps i) to iv), step 1:), step r Or step p), the steps i) to iv) are: 粉碎 crushing and filtering the fresh rhizome of the ginger to obtain a filtrate and a part; 'U) extracting the filtrate as a first organic solvent, and removing the Obtaining the 200918085 organic extract extract, and volatilizing to remove the first organic solvent to obtain a concentrated first organic solvent extract; ni) extracting the residue portion with a second organic solvent, and removing the solution Obtaining a second organic solvent extract, and volatilizing the second organic solvent to obtain a concentrated second organic solvent extract; and N) combining the concentrated first organic solvent extract and the concentrated second The crude solvent is obtained by extracting the organic solvent; the step I) is: & Extracting the dried organic rhizome of the pulverized ginger with an organic solvent identical to the second organic solvent, and extracting the organic solvent extract obtained by volatilization, and removing the organic solvent to obtain the crude liquid; and the step Γ) For the steam dry distillation of the smashed ginger, the dry weight is obtained, and the obtained distillate is concentrated under reduced pressure to obtain the crude liquid; and the step I") is: I") is extracted by ultra-fine-bound carbon monoxide The pulverized ginger is volatilized to remove the - and y amount of the dried roots, and carbon monoxide is used to obtain the crude liquid. 2. The pharmaceutical composition according to the above item 1, 3. As in the previous stage, the Zhaoqing leisure horse month ulcer. The medical composition of one of J's brothers, among them, 丨)). Step (a) comprising the step wherein the first organic solvent is a mixture of methanol, ethanol, decyl alcohol, or the like. Wherein the second organic solvent (1) is in the form of diethyl ether, and the second organic solvent is a mixture of water, a mixture of ethanol and water, and the pharmaceutical group 8 of the fourth item is 2009. .
驟I)。Step I).
、乙醇與水的一混 酮、甲醇、乙醇、甲醇與水的一混合液、 〜 合液、或其等之混合。 8. 如前述第7項的醫藥組成物，其中该第-有機溶劑 為丙酮、乙醇、或乙醇與水的一混合液° 9. 如前述第1項的醫藥組成物，其中该逆相層析管柱 被使用，及該第一流洗液的極性小於具有40°/〇乙醇的乙醇 與水的'^混合液。 1 0 _如前述第9項的醫藥組成物，其中該第一流洗液為 甲醇、曱醇與水的一混合液、乙醇、乙酵與水的一混合液、 丙酮與水的一混合液、或其等之混合。 11. 如前述第1〇項的醫藥組成物，其中該第一流洗液 為乙醇與水的一混合液。 12. 如前述第11項的醫藥組成物，其中在以該第—流 洗液對該逆相層析管柱進行流洗之前先以水或具有2 〇 %乙 醇的乙㈣水的一混合液進行流洗。 液被作為該有效產品， 1 3 .如岫述第9項的醫藥組成物，其中該第一濃縮沖提 及§亥第一濃縮沖提液含有6 -薑醇及 6-生薑醇。 11或12項的醫藥組成物，其 該第二濃縮沖提液被作為該有 其中 14.如前述第1、10、 該逆相層析管柱被使用， 200918085 產品，及該第一流洗液的極性小於具有4〇%乙醇的乙醇與 水的一混合液。 15. 如丽述第14項的醫藥組成物，其中該第二流洗液 為丙酮、丙酮與水的一混合液、丙酮與曱醇的一混合液、 丙酮與乙醇的一混合液、丙酮與C4_C6烷的—混合液、 C4-C6烷、C4-C6烷與甲醇的一混合液、C4_C6烷與乙醇的 一混合液、或其等之混合。 16. 如前述第15項的醫藥組成物，其中該第二流洗液 為丙酮。 1 7.如前述第1 4項的醫藥組成物，其中該第二濃縮沖 提液不含有6-薑醇或6-生薑醇。 1 8.如蝻述第1 4項的醫藥組成物’其中該第二濃縮沖 提液不含有6-薑醇及6-生薑醇。 1 9.如前述第1項的醫藥組成物，其中該正相層析管柱 被使用’該第一濃縮沖提液被作為該有效產品，及該第一 流洗液的極性小於正己烷與乙酸乙酯的重量比6:4的一混 合液。 20.如前述第19項的醫藥組成物，其中該第一流洗液 的極性近似於正己烷與乙酸乙酯的重量比9:丨的一混合液。 2 1 ·如前述第2 〇項的醫藥組成物，其中該第一流洗液 為正己院與乙酸乙酯的重量比9:1的一混合液。 22_如前述第19項的醫藥組成物，其中該第一濃縮沖 提液不含有6-薑醇或6_生薑醇。 2 3 .如前述第1 9項的醫藥組成物’其中該第一濃縮沖 10 200918085 提液不含有6 -薑醇及6 -生薑醇。 24.如前述第1項的醫藥組成物，其中 "八切，兵干該正相層析 被使用，該第二濃縮沖提液被作 茌 彼W马该有效產品，該第二产 洗液的極性近似於正己烷盥 一 _ 疋^、^酸乙酯的重量比6:4的—$ 合液，及該第二濃縮沖提液含右 此 狀3有6_薑醇及6-生薑醇。 25.如前述第24項的醫 正己烷與乙酸乙酯的重量 本發明的醫藥組成物可 或載體。 為 劑 藥組成物’其中該第二流洗液 比6:4的—混合液。 以進一步含有一稀釋劑、賦形 實施方式 本案申請人於英國專利公開案GB 2306565揭示了 — 種從薑的根製備抗發炎、抗血小板凝集或抗菌活性的有效 f分的方法’包含下列步驟：從薑（z咖的根 莖以有機溶劑或超臨界i氧化碳萃取或以纟蒸氣蒸餘製備 《 一粗製液；將該粗製液導入一逆相層析管柱，並依序以水、 第一流洗液及第二流洗液流洗，收集其中的第一流洗液的 沖提液及第二流洗液的沖提液，其中該第一流洗液的極性 大於該第二流洗液，該第二流洗液的極性大於氯仿；揮發 移除所獲得沖提液中的第一流洗液及第二流洗液而得可作 為該有效成分第一濃縮沖提液及第二濃縮沖提液。此qb 2366565的内容藉由參考方式被併入本案。 本發明將藉以下實施例被進一步瞭解，該等實施例僅 為說明之用而非用於限制本發明範圍。 200918085 本說明書所提及的百分比及份量除非另外指明否則皆 以重量為基準。而各百分比範圍的總合為i 00%。 實施例1: 將切片的乾薑打碎並以網目1 〇的篩網過篩。過篩後的 粉末以1份乾薑重對8份9 5 %酒精的比例混合，加熱迴流 一小時後並過濾得一濾液A及殘渣。該殘渣以重量比1:8 的比例與9 5 %酒精的比例混合，加熱迴流一小時後並過濾、 得一濾液B。合併濾液a及B並於70°C水浴下減壓濃縮 (Rotavapor R_22〇, BOCHI，Switzerland)得一濃縮物。將該濃縮物導 入一填充有該濃縮物乾重20倍重的Diaion® HP-20樹脂 (Mitsubishi, Japan)的逆相色層層析管柱（7 1 cm χ go cm)，依序以$ 倍床體積（bed volume)的水、4倍床體積的40%酒精及4倍 床體積的丙酮流洗，收集40%酒精沖提液、及丙酮沖提液。 §玄 40¾酒精沖提液減麗漠縮（R〇tavap〇r R_22〇, b〇CHI, Switzerland) 至乾，再於80°C水浴下減壓（4〇 m bar)濃縮一小時得樣品1。 該丙酮沖提液減壓濃縮（Rotavapor R_22〇，bOCHI，Switzerland)至 乾’再於80°C水洛下減壓（4〇 mbar)濃縮一小時得樣品2。 實施例2 : 將40%酒精改成70%酒精，重複實施例1的步驟。由 該70%酒精沖提液得樣品3。由該丙酮沖提液濃縮物得樣品 4 〇 12 200918085 實施例3 : 將40〇/〇酒精改成95%酒精，重複實施例i的”。由 該95%酒精沖提液濃縮物得樣品5。 實施例4: 將切片的乾薑打碎並以網目1〇的篩網過篩。過篩後的 粉末以1份乾薑重對8份95%酒精的比例混合，加熱迴流 一小時後並過濾得一濾液A及殘渣。該殘渣以重量'比 的比例與9 5 /〇酒精的比例混合，加熱迴流一小時後並過濾 侍一濾液B。合併濾液a及B並於7〇c>c水浴下減壓濃縮 (Rotavapor R-220, B0CH丨，Switzer|and)至原合併濾液的 1/5 重而得 一濃縮物。對該濃縮物加入其4倍重量的水而恢復其濃縮 月'J原重，再將所獲得的液體導入一填充有該濃縮物乾重2〇 倍重的Diaion® HP-20樹脂（Mitsubishi, Japan)的逆相色層層析 官柱（7·1 cm X 90 cm)，依序以i倍床體積的2〇%酒精及4倍床 體積的70%酒精流洗，收集7〇%酒精沖提液。該7〇%酒精 冲 k 液減壓濃縮（Rotavapor R_22〇，bCICHI，Switzerland)至乾，再於 80°C水浴下減壓（4〇 mbar)濃縮一小時得樣品6。 實施例5: 將70%酒精改成95%酒精，重複實施例4的步驟。由 該9 5 %酒精沖提液濃縮物得樣品7。 貫施例6: 13 200918085 將切片的乾薑打碎並以網目丨0的篩網過篩。過篩後的 粉末以1份乾薑重對8份95%酒精的比例混合，所獲得的 混合物以均質機（Type X 5〇m，Ystral, Germany)於 2000 rpm 攪拌 1 小日寸並過濾得—濾液A及殘渣。該殘渣以重量比1:8的比 例與9 5 °/Q酒精的比例混合，所獲得的混合物以均質機於 2 000 rpm搜拌1小時並過濾得一濾液b。合併濾液a及B 並於 70°C 水浴下減壓濃縮（Rotavapor R-220, BClCHI, Switzerland)， 再將所獲得的濃縮物液體導入一填充有該濃縮物乾重2〇 倍重的Diaion® HP-20樹脂（Mitsubishi, Japan)的逆相色層層析 官柱（7.1 cm X 90 cm)，依序以1倍床體積的20%酒精及4倍床 體積的70%酒精流洗’收集7〇%酒精沖提液。該7〇%酒精 沖提液減壓濃縮（Rotavapor R-220, BClCHI，Switzerland)至乾，再於 8 0°C水浴下減壓（40 mbar)濃縮一小時得樣品8。 實施例7: 將70%酒精改成95%酒精，重複實施例6的步驟。由 該95%酒精沖提液濃縮物得樣品9。 實施例8: 將切片的乾薑打碎並以網目1 〇的篩網過篩。過篩後的 粉末以1份乾薑重對8份丙酮的比例混合，加熱迴流一小 時後並過濾得一濾液A及殘渣。該殘渣以重量比u的比 例與丙酮比例混合，加熱迴流一小時後並過濾得一濾液 B。合併濾液A及B並於70°C水浴下減壓濃縮（Rotavapor R-220, 14 200918085 BCICHI, Switzerland)得一濃縮物。將該濃縮物導入一填充有該濃 細物乾重20倍重的Diaion® HP-20樹脂（Mitsubishi, Japan)的逆 相色層層析管柱（7.1 cm X 90 cm) ’依序以5倍床體積的水、4 倍床體積的40%酒精及2倍床體積的丙酮流洗，收集丙酮 沖提液。該丙酮沖提液減壓濃縮（R〇tavap〇r R_22〇 B0CHI， Switzerland)至乾，再於80°C水浴下減壓(40 mbar)濃縮一小時 付樣品1 0。 實施例9: 將40%酒精改成7〇%酒精，重複實施例8的步驟。該 70〇/〇 酒精沖提液減壓濃縮（R〇tavap〇r R_22〇，b〇ch丨，Switze「丨and)至 乾，再於80°C水浴下減壓（40 mbar)濃縮一小時得樣品1 i。 實施例1 0: 將萃取薑粉末及殘渣的丙酮改成乙酸乙酯，重複實施 例8的步驟得樣品12。 實施例11: 將萃取薑粉末及殘渣的丙酮改成乙酸乙酯，重複實施 例9的步驟得樣品13。 實施例12: 將切片的乾薑打碎並以網目1 〇的篩網過篩。過篩後的 粉末以1份乾薑重對8份95%酒精的比例混合，加熱迴流 15 200918085 一小時後並過濾得一濾液A及殘渣。該殘渣以重量比1:8 的比例與95%酒精的比例混合’加熱迴流一小時後並過濾 得一滤液B。合併濾液a及B並於70〇C水洛下減壓濃縮 (Rotavapor R_22〇’ b〇CH丨，Switzerland)得一濃縮物。將該濃縮物導 入一填充有該濃縮物乾重20倍重的矽膠6〇 (Merck, Germany) 的正相色層層析管柱，依序以4倍床體積的正己烷與乙酸 乙酯的重量比9:1的一混合液、3倍床體積的正己烷與乙酸 乙醋的重量比6:4的一混合液及2倍床體積的乙酸乙酯與 曱醇的重量比1:1的一混合液流洗。收集正己烧與乙酸乙 醋的重量比9:1的混合液的沖提液，並減壓濃縮（R〇tavap〇r R-220, BClCHI，Switzerland)至乾，再於 80。(：水浴下減壓（40 mbar) 濃縮一小時得樣品1 4。收集正己烷與乙酸乙酯的重量比6:4 的混s液的沖提液’並減壓濃縮（Rotavapor R-220, BClCHI, Switzerland)至乾，再於80〇c水浴下減壓（4〇 mbar)濃縮一小時 得樣品15。收集乙酸乙酯與甲醇的重量比ι:1的的混合液 的沖提液，並減壓濃縮（Rotavap〇rR_22〇 B0CHI，Switzerland)至乾， 再於80°C水浴下減壓（40 mbar)濃縮一小時得樣品16。 實施例1 3 : 將卒取薑粉末及殘渣的9 5 %酒精改成丙酮外，重複實 施例12的步驟’分別從正己烷與乙酸乙酯的重量比9:丨的 混合液沖提液、正己烷與乙酸乙酯的重量比6_4的混合液 沖提液及乙酸乙酯與甲醇的重量比丨：丨的的混合液沖提液 得樣品1 7、1 8及1 9。 16 200918085 實施例1 4 : 將切片的乾薑打碎並以網目1 〇的篩網過篩。過篩後的 粉末以1份乾薑重對8份95%酒精的比例混合，加熱迴流 一小時後並過濾得一濾液A及殘渣。該殘渣以重量比1: 8 的比例與9 5 %酒精的比例混合，加熱迴流一小時後並過濾 得一濾液B。合併濾液a及B以一薄膜真空濃縮機（CEp_L, Okawam Mfg. Co., japan)濃縮至原合併濾液的丨/5重而得一濃縮 物。將該濃縮物導入一填充有該濃縮物乾重2〇倍重的 Dia1〇n® HP-20 樹脂（Mitsubishi, japan)的逆相色層層析管柱（34 5 cmx10〇cm)，依序以2倍床體積的95%酒精及2倍床體積的 丙酮流洗。收集丙酮沖提液，並減壓濃縮（R〇tavap〇r r_22〇, BUCHI’Switzer丨and)至乾，再於8〇〇c水浴下減壓（4〇mbar)濃縮 一小時得樣品2 0。 HPLC分析·· 檢品製備： 精秤樣品於樣品瓶中，加入甲醇溶解，使濃度成為1〇 mg/ml，即為檢品。檢品以0.45 μπι濾膜過濾，所得濾液進 行HPLC分析。 心1 17 200918085 HPLC分析條件： 分析管柱：Cosmosil 5C18-MS-II 4.6 X 250 mm (Nacalai Tesque Inc.) 前置管柱：Lichrospher® 100 RP-18e(5pm) (Merck) 流速：1 ml/min 樣品注射量：1 0 μ 1 時間（分） 0 10 80 乙腈（％) 40 40 100 0.1%磷酸溶液*(%) 60 60 0 管柱溫度 37°C 偵測波長 204 nm 0.1 %磷酸溶液配製：取磷酸（85%) 2.3 5 ml以純水溶解並定 容至 2000 ml。 以上實施例所製備的樣品1至20的HPLC分析結果示 於圖1至20。從HPLC分析所計算得到的樣品中6-薑醇及 6-生薑醇的含量（mg/g)被示於表1，其中同時列出製備該等 樣品的萃取及流洗的條件。 18 200918085 6-薑醇的 含量 (mg/g) 6-生薑醇 的含量 (mg/g)) 萃取條件 流洗條件 樣品1 … --- 95%酒精迴流 1)水流洗後以40%酒精流洗 樣品2 23.11 67.20 1"， 1)之後以丙酮流洗 樣品3 31.14 33.76 mt 2)水流洗後以70%酒精流洗 樣品4 … 68.36 nn 2)之後以丙酮流洗 樣品5 35.36 54.32 (III 水流洗後以95%酒精流洗 樣品6 23.85 85.18 MM 20%酒精流洗後以70%酒精流洗 樣品7 94.79 55.80 ΜΗ 20%酒精流洗後以95%酒精流洗 樣品8 84.53 於均質機以95% 酒精攪拌 20%酒精流洗後以70%酒精流洗 樣品9 8.98 87.15 1"1 20%酒精流洗後以95%酒精流洗 樣品10 39.28 67.53 丙_迴流 水、40%酒精流洗後以丙酮流洗 樣品11 66.94 103.48 m， 水流洗後以70%酒精流洗 樣品12 38.22 66.16 乙酸乙酯迴流 水、40%酒精流洗後以丙酮流洗 樣品13 112.97 34.37 Μ Μ 水流洗後以70%酒精流洗 樣品14 … --- 95%酒精迴流 3)正己烧:乙酸乙酯=9:1流洗 樣品15 69.48 96.95 ΜΗ 4)接著3)後正己烷:乙酸乙酯=6:4流洗 樣品16 … 接著4)後乙酸乙酯:曱醇=1:1流洗 樣品17 — --- 丙酮迴流 5)正己烷:乙酸乙酯=9:1流洗 樣品18 84.37 105.68 Μ Μ 6)接著5)後正己烷:乙酸乙酯=6:4流洗 19 200918085 6-薑醇的 含量 (mg/g) 6-生薑醇 的含量 (mg/g)) 萃取條件 ----------- 流洗條件 樣品19 HM 接著6)後乙酸乙酯：甲醇=1:卜流洗 樣品20 — ~~----- 95 /ί»酒精迴流 —--- ~—---- 95%酒精流洗後以丙_流洗 樣品1至13及20的製備係使用逆相色層層析管枝。 比較圖i i 4的肌C分析圖譜可以看出以鄉酒精流洗 所得到的沖提液（樣品丨）含有比6_薑醇極性更高的化合 物，以7〇%酒精流洗所得到的沖提液（樣口口口 3)含有比6_蔓: 及6-生薑醇極性更高的化合物’以丙酮流洗所得到的沖提 液（樣2及4)則含有殘留下來的極性相對較低的化合物。 從圖6及圖7可以看出當以95%酒精流洗相較於卿。酒精 流洗者在圖譜的右側顯現出較多的波峰。類似圖6及7 = 趨勢也可以在圖8及9觀察到，雖然其中的萃取條件不同。 圖及12所顯示的圖譜類似於圖2所示者，其中萃取條 件不同但流洗條件相同。圖丨丨及13所顯示的圖譜類似於 圖3所示者，其中萃取條件不同但流洗條件相同。樣品μ 所有的化合物大多為低極性者且纟Ηριχ分析令偵測不到 6-薑醇及6_生薑醇，如圖2〇所示，此結果示卩95%酒精流 洗已經把6-薑醇及6-生薑醇及極性比它們更高的化合物從 逆相色層層析嘗柱沖提出。樣品丨3至丨5的HpLc分析結 果類似於樣品16至19者（圖π至19)，其中萃取條件不同 但對正相色層層析管柱的流洗條件相同。使用正己烷與乙 20 200918085 酸乙醋的重…:1的混合液流洗所得到的樣品i4 (樣品 16)含有大部份非高極性化合物且其中_不到墓醇及 6-生蔓醇’如圖14 (圖16)所示。使用正己烧與乙酸乙酉旨的 重量比6:4 @混合液流洗所得到的樣口口口 15 (樣品⑺含有顯 著量的6-薑醇及6_生甚醇，如_ 15 (圖17)所示。 1.抗菌實驗 樣品對幽門桿菌（ATCC 435〇4)的抑制作用根據 Malanosk G· J.等人的石花菜方法加以評估（则議^ g j以 al. Effect of pH variation on the susceptibility of Helicobacter pylori to three marcolide antimicrobial agents and temafloxacin. Eur. J. Clin. Microbial Infect Dis. 12: pp. 131-133， 1993.) ’並以最小抑菌濃度表示 inhibitory concentration; MIC)。樣品以二甲亞砜（dimethy][ sulfoxide, (DMSO))溶解並進行系列稀釋。幽門桿菌（ATCC 43504)則以每毫升5 x 105菌落形成單位（c〇1〇ny f〇rmati〇n units/ml，CFU/ml)的濃度懸浮於腦心浸出營養液（brain heart infusion broth)中。實驗以48孔培養盤來進行。測試 時將0.01 ml的樣品加入〇. 99 m 1含7%去血纖維蛋白兔血 的Columbia石花菜基（agar base)。培養液中DMSO的最高 濃度為1 %。測試樣品的最高濃度則為300 pg/ml。培養盤 於35C微氧（N2 85%，C〇2 10%和〇2 5%)的情況下培養72 小時，隨後以肉眼檢查對幽門桿菌菌落的抑制作用，溶媒 及gentamicin則分別作為空白及陽性對照。每一個測試皆 21 200918085 以兩重覆的方式進行。結果示於表2，表2中同時示出6-薑醇及6-生薑醇的最小抑菌濃度。 表2 樣品 最小抑菌濃度（MIC, pg/ml) 樣品1 — 樣品2 30 樣品3 30 樣品4 30 樣品5 — 樣品6 30 樣品7 30 樣品8 30 樣品9 30 樣品1 0 30 樣品11 30 樣品1 2 100 樣品1 3 10 樣品1 4 30 樣品1 5 10 樣品1 6 30 樣品1 7 30 樣品1 8 10 22 200918085 樣品〜 — 樣品19 ---- 樣品2 0 .—— — 最小抑菌濃度（MIC, pg/ml) 300 10 6-薑醇 --—^ 6_生薑醇 ------ 10 10 — . 2.幽門桿菌引起的胃潰瘍 樣A對幽η桿菌所引起的胃潰瘍的治療效果根據 Marchett1，Μ•等人的方法加以評估（Marchetti, Μ·, Arico, Β.， Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P.A mixture of ethanol and water, methanol, ethanol, a mixture of methanol and water, a mixture of liquids, or the like. 8. The pharmaceutical composition according to the above item 7, wherein the first organic solvent is acetone, ethanol, or a mixture of ethanol and water. 9. The pharmaceutical composition according to the above item 1, wherein the reverse phase chromatography The column is used, and the polarity of the first stream wash is less than the mixture of ethanol and water with 40 ° / 〇 ethanol. The pharmaceutical composition according to the above item 9, wherein the first flowing liquid is methanol, a mixture of decyl alcohol and water, ethanol, a mixture of ethylene and water, a mixture of acetone and water, Or a mixture of them. 11. The pharmaceutical composition according to the above item 1, wherein the first aqueous washing solution is a mixture of ethanol and water. 12. The pharmaceutical composition according to the above item 11, wherein a mixture of water or B (tetra) water having 2% by weight of ethanol is used before the reverse phase chromatography column is subjected to flow washing with the first stream washing liquid. Flow wash. The liquid is used as the effective product, and the pharmaceutical composition according to item 9, wherein the first concentrated extracting solution and the first concentrated extracting liquid contain 6-gingerol and 6-gingerol. a pharmaceutical composition of item 11 or 12, wherein the second concentrated extract is used as the one of 14. The first, tenth, the reverse phase chromatography column is used, the 200918085 product, and the first flow lotion The polarity is less than a mixture of ethanol and water having 4% by weight of ethanol. 15. The pharmaceutical composition of Lithium No. 14, wherein the second washing liquid is acetone, a mixture of acetone and water, a mixture of acetone and decyl alcohol, a mixture of acetone and ethanol, acetone and a mixture of a C4_C6 alkane, a C4-C6 alkane, a mixture of C4-C6 alkane and methanol, a mixture of C4_C6 alkane and ethanol, or the like. 16. The pharmaceutical composition according to item 15 above, wherein the second stream washing liquid is acetone. The pharmaceutical composition according to the above item 14, wherein the second concentrated extract contains no 6-gingerol or 6-gingerol. 1 8. The pharmaceutical composition of item 14 wherein the second concentrated extract does not contain 6-gingerol and 6-gingerol. The pharmaceutical composition according to the above item 1, wherein the normal phase chromatography column is used as the first concentrated extract liquid as the effective product, and the first flow wash liquid has a polarity less than n-hexane and acetic acid. A mixture of ethyl esters having a weight ratio of 6:4. 20. The pharmaceutical composition according to the above item 19, wherein the first stream washing liquid has a polarity similar to a mixture of n-hexane and ethyl acetate in a weight ratio of 9: hydrazine. The pharmaceutical composition according to the above item 2, wherein the first aqueous washing liquid is a mixed liquid of 9:1 by weight of Zhenghexin and ethyl acetate. The pharmaceutical composition according to the above item 19, wherein the first concentrated extract contains no 6-gingerol or 6-glycol alcohol. 2 3. The pharmaceutical composition according to the above item 19, wherein the first concentrated rushing 10 200918085 extract does not contain 6-gingerol and 6-gingerol. 24. The pharmaceutical composition according to the above item 1, wherein the "eight cut, the dried stem is used for normal phase chromatography, and the second concentrated extract is used as the effective product, the second wash The polarity of the liquid is similar to that of n-hexane 盥 疋 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Ginger alcohol. 25. The weight of medical hexane and ethyl acetate according to item 24 above. The pharmaceutical composition of the present invention may be a carrier. It is a mixture of the drug composition 'where the second stream of washing liquid is 6:4. In order to further contain a diluent, the present invention is disclosed in the British Patent Publication No. GB 2306565, which discloses a method for preparing an effective f-score against anti-inflammatory, anti-platelet aggregation or antibacterial activity from the root of ginger'. Preparing a crude liquid from the roots of ginger (Z coffee roots extracted with organic solvent or supercritical i-oxide or steaming with hydrazine steam; introducing the crude liquid into a reverse phase chromatography column, followed by water, first flow The washing liquid and the second flowing washing liquid are washed, collecting the extract of the first flowing washing liquid and the extracting liquid of the second flowing washing liquid, wherein the polarity of the first flowing washing liquid is greater than the second flowing washing liquid, The polarity of the second stream washing liquid is greater than that of the chloroform; the first stream washing liquid and the second stream washing liquid in the obtained extract liquid are volatilized to obtain the first concentrated extract liquid and the second concentrated extract liquid which are the active ingredients. The contents of this reference are incorporated herein by reference to the following disclosure of the disclosure of the disclosure of the disclosure of Percentage And parts are by weight unless otherwise indicated. The sum of the percentage ranges is i 00%.Example 1: The sliced dried ginger is broken up and sieved through a sieve of mesh 1 。. The powder was mixed in a ratio of 1 part by weight of dried ginger to 8 parts of 95% by weight of alcohol, and heated under reflux for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed at a ratio of 1:8 by weight to 95% by weight of alcohol. After heating under reflux for one hour and filtering, a filtrate B was obtained, and the filtrates a and B were combined and concentrated under reduced pressure (Rotavapor R_22 〇, BOCHI, Switzerland) in a water bath at 70 ° C. The concentrate was introduced into a filling. A reverse phase chromatography column (7 1 cm χ go cm) of Diaion® HP-20 resin (Mitsubishi, Japan) having a dry weight of 20 times the concentrate, in order of bed volume The water, 4 times bed volume of 40% alcohol and 4 times bed volume of acetone are washed, collecting 40% alcohol extract and acetone extract. § Xuan 403⁄4 alcohol extract liquid reduction (R〇tavap 〇r R_22〇, b〇CHI, Switzerland) To the dry, concentrated in a water bath at 80 ° C under reduced pressure (4 〇 m bar) for one hour to obtain sample 1. The acetone extract was concentrated under reduced pressure (Rotavapor R_22®, bOCHI, Switzerland) to dryness and concentrated under reduced pressure (4 mbar) at 80 ° C for one hour to obtain sample 2. Example 2: 40% alcohol was changed The procedure of Example 1 was repeated in 70% alcohol. Sample 3 was obtained from the 70% alcohol extract. Samples were obtained from the acetone extract concentrate. 4 〇12 200918085 Example 3: 40 〇/〇 alcohol was changed to 95% alcohol, repeat the example i". Sample 5 was obtained from the 95% alcohol extract concentrate. Example 4: Sliced dried ginger was broken up and sieved through a mesh 1 mesh screen. The sieved powder was mixed in a ratio of 1 part by weight of dried ginger to 8 parts of 95% alcohol, and heated under reflux for one hour, and filtered to obtain a filtrate A and a residue. The residue was mixed in a weight ratio of 9 5 / 〇 alcohol, heated to reflux for one hour, and filtered to filter the filtrate B. The filtrates a and B were combined and concentrated under reduced pressure (Rotavapor R-220, B0CH 丨, Switzer|and) under a water bath of 7 〇c>c to a weight of 1/5 of the original combined filtrate to obtain a concentrate. The concentrate was added with 4 times by weight of water to recover its concentrated monthly 'J original weight, and the obtained liquid was introduced into a Diaion® HP-20 resin (Mitsubishi, which was filled twice the dry weight of the concentrate). Japan) Reverse Phase Chromatography Column (7·1 cm X 90 cm), sequentially washed with 2 times bed volume of 2% alcohol and 4 times bed volume of 70% alcohol, collecting 7〇% alcohol Effervescent solution. The 7 wt% alcohol solution was concentrated under reduced pressure (Rotavapor R_22®, bCICHI, Switzerland) to dryness, and concentrated under reduced pressure (4 Torr) in a water bath at 80 ° C for one hour to obtain sample 6. Example 5: The procedure of Example 4 was repeated by changing 70% alcohol to 95% alcohol. Sample 7 was obtained from the 95% alcohol extract concentrate. Example 6: 13 200918085 The sliced dried ginger was broken up and sieved through a sieve of mesh 丨0. The sieved powder was mixed in a ratio of 1 part by weight of ginger to 8 parts of 95% alcohol, and the obtained mixture was stirred by a homogenizer (Type X 5〇m, Ystral, Germany) at 2000 rpm for 1 hour and filtered. - filtrate A and residue. The residue was mixed at a weight ratio of 1:8 to a ratio of 95 ° / Q alcohol, and the obtained mixture was mixed with a homogenizer at 2 000 rpm for 1 hour and filtered to obtain a filtrate b. The filtrates a and B were combined and concentrated under reduced pressure in a water bath at 70 ° C (Rotavapor R-220, BClCHI, Switzerland), and the obtained concentrate liquid was introduced into a Diaion® filled with a dry weight of the concentrate 2 times the weight. Reverse phase chromatography chromatography column (7.1 cm X 90 cm) of HP-20 resin (Mitsubishi, Japan), sequentially washed with 1 bed volume of 20% alcohol and 4 times bed volume of 70% alcohol. 7〇% alcohol extract. The 7〇% alcohol extract was concentrated under reduced pressure (Rotavapor R-220, BClCHI, Switzerland) to dryness, and concentrated under reduced pressure (40 mbar) in a water bath at 80 ° C for one hour to obtain sample 8. Example 7: The procedure of Example 6 was repeated by changing 70% alcohol to 95% alcohol. Sample 9 was obtained from the 95% alcohol extract concentrate. Example 8: Sliced dried ginger was broken up and sieved through a mesh 1 mesh screen. The sieved powder was mixed in a ratio of 1 part by weight of dried ginger to 8 parts of acetone, and heated under reflux for one hour, and filtered to obtain a filtrate A and a residue. The residue was mixed with acetone in a ratio of weight ratio u, heated to reflux for one hour, and filtered to obtain a filtrate B. The filtrates A and B were combined and concentrated under reduced pressure (Rotavapor R-220, 14 200918085 BCICHI, Switzerland) in a water bath at 70 ° C to give a concentrate. The concentrate was introduced into a reverse phase chromatography column (7.1 cm X 90 cm) of Diaion® HP-20 resin (Mitsubishi, Japan) filled with a weight of 20 times the dry weight of the concentrate. The acetone extract was collected by double bed volume of water, 4 bed volumes of 40% alcohol, and 2 bed volumes of acetone. The acetone extract was concentrated under reduced pressure (R〇tavap〇r R_22〇 B0CHI, Switzerland) to dryness, and concentrated under reduced pressure (40 mbar) in a water bath at 80 ° C for one hour to give sample 10 . Example 9: The procedure of Example 8 was repeated by changing 40% alcohol to 7% alcohol. The 70 〇/〇 alcohol extract was concentrated under reduced pressure (R〇tavap〇r R_22〇, b〇ch丨, Switze “丨and) to dryness, and concentrated under reduced pressure (40 mbar) in a water bath at 80 ° C for one hour. Sample 1 i was obtained. Example 1 0: The acetone extracted from the ginger powder and the residue was changed to ethyl acetate, and the procedure of Example 8 was repeated to obtain Sample 12. Example 11: The extraction of ginger powder and the residue of acetone was changed to acetic acid B. Ester, the procedure of Example 9 was repeated to obtain Sample 13. Example 12: The sliced dried ginger was broken up and sieved through a mesh of 1 mesh. The sieved powder was 1 part dry ginger to 8 parts 95% Mixing the proportion of alcohol, heating and refluxing 15 200918085 After one hour, it is filtered to obtain a filtrate A and a residue. The residue is mixed with a ratio of 1:8 by weight to 95% alcohol, and heated to reflux for one hour and filtered to obtain a filtrate B. The filtrates a and B were combined and concentrated under reduced pressure (Rotavapor R_22〇' b〇CH丨, Switzerland) under a water pressure of 70 ° C to obtain a concentrate. The concentrate was introduced into a concentrate filled with the concentrate to a weight of 20 times. a normal phase chromatography column of 6 〇 (Merck, Germany), sequentially with 4 bed volumes of n-hexane and B The weight ratio of acid ethyl ester to a mixture of 9:1, 3 times bed volume of a mixture of n-hexane and ethyl acetate in a weight ratio of 6:4 and 2 times bed volume of ethyl acetate to furfuryl alcohol A 1:1 mixture was washed, and a mixture of 9:1 by weight of hexanone and ethyl acetate was collected and concentrated under reduced pressure (R〇tavap〇r R-220, BClCHI, Switzerland) to Dry, and then at 80. (: decompressed in a water bath (40 mbar) for one hour to obtain sample 14 4. Collect a mixture of n-hexane and ethyl acetate in a weight ratio of 6:4 mixture and concentrate under reduced pressure. (Rotavapor R-220, BClCHI, Switzerland) to dryness, and concentrated under reduced pressure (4 mbar) in a water bath of 80 ° C for one hour to obtain sample 15. Collect a mixture of ethyl acetate and methanol in a weight ratio of 1 :1 The extract was concentrated under reduced pressure (Rotavap 〇rR_22〇B0CHI, Switzerland) to dryness, and concentrated under reduced pressure (40 mbar) in a water bath at 80 ° C for one hour to obtain sample 16. Example 1 3: Stretching ginger The procedure of Example 12 was repeated except that the powder of the powder and the residue was changed to acetone. The mixture of the mixture of n-hexane and ethyl acetate was 9: 丨. The ratio of the weight ratio of n-hexane to ethyl acetate of 6_4 and the weight ratio of ethyl acetate to methanol 丨: the mixed solution of hydrazine to obtain samples 17 , 18 and 19 . 16 200918085 1 4 : The sliced dried ginger was broken and sieved through a mesh 1 mesh screen. The sieved powder was mixed in a ratio of 1 part by weight of dried ginger to 8 parts of 95% alcohol, and heated under reflux for one hour, and filtered to obtain a filtrate A and a residue. The residue was mixed at a ratio of 1:8 by weight to 95% alcohol, heated to reflux for one hour, and filtered to obtain a filtrate B. The combined filtrates a and B were concentrated in a film vacuum concentrator (CEp_L, Okawam Mfg. Co., japan) to the weight of the original combined filtrate to give a concentrate. The concentrate was introduced into a reverse phase chromatography column (34 5 cm x 10 〇 cm) of Dia1〇n® HP-20 resin (Mitsubishi, japan) filled with 2 times the dry weight of the concentrate, in order. Wash with 2 bed volumes of 95% alcohol and 2 bed volumes of acetone. The acetone extract was collected, concentrated under reduced pressure (R〇tavap〇r r_22〇, BUCHI'Switzer丨and) to dryness, and concentrated under reduced pressure (4 mbar) in an 8 〇〇c water bath for one hour to obtain a sample 2 0 . HPLC analysis··Preparation of the test sample: The sample of the precision scale is placed in the sample vial and dissolved in methanol to make the concentration 1 〇 mg/ml, which is the test sample. The test product was filtered through a 0.45 μm filter, and the obtained filtrate was subjected to HPLC analysis. Heart 1 17 200918085 HPLC Analytical Conditions: Analytical column: Cosmosil 5C18-MS-II 4.6 X 250 mm (Nacalai Tesque Inc.) Front column: Lichrospher® 100 RP-18e (5pm) (Merck) Flow rate: 1 ml/ Min Sample injection volume: 10 μl Time (minutes) 0 10 80 Acetonitrile (%) 40 40 100 0.1% phosphoric acid solution* (%) 60 60 0 Column temperature 37 °C Detection wavelength 204 nm 0.1% phosphoric acid solution preparation : Take phosphoric acid (85%) 2.3 5 ml Dissolve in pure water and dilute to 2000 ml. The results of HPLC analysis of the samples 1 to 20 prepared in the above examples are shown in Figs. The contents (mg/g) of 6-gingerol and 6-gingerol in the samples calculated from the HPLC analysis are shown in Table 1, in which the conditions for the extraction and washing of the samples were also listed. 18 200918085 6-Gingerol content (mg/g) 6-Ginger alcohol content (mg/g)) Extraction conditions Flow washing conditions Sample 1 --- 95% alcohol reflux 1) Water wash after 40% alcohol Rinse the sample 2 23.11 67.20 1", 1) After washing the sample with acetone 3 31.14 33.76 mt 2) Wash the sample with 70% alcohol after washing with water 7 ... 68.36 nn 2) After washing the sample with acetone 5 35.36 54.32 (III After washing with water, wash the sample with 95% alcohol. 6.23 85.18 MM 20% alcohol wash and wash the sample with 70% alcohol 7 94.79 55.80 ΜΗ 20% alcohol wash and then wash the sample with 95% alcohol 8 84.53 in the homogenizer 95% alcohol stirred 20% alcohol flow wash and 70% alcohol flow wash sample 9 8.98 87.15 1"1 20% alcohol wash and then wash the sample with 95% alcohol 10 39.28 67.53 C _ reflux water, 40% alcohol after washing The sample was washed with acetone at a rate of 11 66.94 103.48 m, washed with water and then washed with 70% alcohol. 12 38.22 66.16 Ethyl acetate reflux water, 40% alcohol flow wash, and acetone wash sample 13 112.97 34.37 Μ Μ After washing, 70 % alcohol wash sample 14 ... --- 95% alcohol reflux 3) is burned: ethyl acetate = 9:1 flow wash sample 15 69.48 96.95 ΜΗ 4) followed by 3) after n-hexane: ethyl acetate = 6:4 flow wash sample 16 ... followed by 4) ethyl acetate: decyl alcohol = 1:1 flow wash sample 17 -- --- acetone reflux 5 ) n-hexane: ethyl acetate = 9:1 flow wash sample 18 84.37 105.68 Μ Μ 6) followed by 5) n-hexane: ethyl acetate = 6:4 flow wash 19 200918085 6-glycol content (mg/g) 6-Ginger alcohol content (mg/g)) Extraction conditions ----------- Flow wash condition sample 19 HM Next 6) After ethyl acetate: methanol = 1: Bu rinse sample 20 - ~~----- 95 /ί»Alcohol reflux----~----- 95% alcohol wash after washing with C-flow sample 1 to 13 and 20 using reverse phase chromatography Pipe branch. Comparing the muscle C analysis chart of Figure ii 4, it can be seen that the extract (sample 丨) obtained by washing with the domestic alcohol contains a compound having a higher polarity than the 6-gingerol, and the rinsing obtained by washing with 7 〇% alcohol The extract (sample mouth 3) contains a compound which is more polar than 6-man: and 6-ginger alcohol. The extract obtained by washing with acetone (samples 2 and 4) contains residual polar relative Lower compound. It can be seen from Fig. 6 and Fig. 7 that when washed with 95% alcohol, it is compared with Qing. The alcohol rinser showed more peaks on the right side of the map. Similar to Figures 6 and 7 = trends can also be observed in Figures 8 and 9, although the extraction conditions are different. The maps shown in Figures 12 and 12 are similar to those shown in Figure 2, in which the extraction conditions are different but the flow wash conditions are the same. The maps shown in Figures 13 and 13 are similar to those shown in Figure 3, in which the extraction conditions are different but the flow wash conditions are the same. Sample μ Most of the compounds were low-polarity and the 纟Ηριχ analysis prevented the detection of 6-gingerol and 6-gingerol, as shown in Figure 2〇. This result shows that 95% alcohol flushing has taken 6- Gingerol and 6-gingerol and compounds with higher polarities are proposed from reverse phase chromatography. The HpLc analysis results of samples 丨3 to 丨5 were similar to those of samples 16 to 19 (Figs. π to 19), in which the extraction conditions were different but the flow washing conditions for the normal phase chromatography column were the same. The sample i4 (sample 16) obtained by washing the mixed solution of n-hexane and ethylene 20 200918085 acid ethyl vinegar with a weight of ...:1 contains most of the non-highly polar compounds and wherein _ less than tomb alcohol and 6-shengcanol 'As shown in Figure 14 (Figure 16). The mouth of the sample was obtained by using a 6:4 @mixture flow ratio of the ratio of hexanyl acetate to ethyl acetate (sample (7) contains a significant amount of 6-gingerol and 6_shengol, such as _ 15 (Fig. 17) ). 1. The inhibition of Helicobacter pylori (ATCC 435〇4) by antibacterial test samples was evaluated according to the method of the broccoli method of Malanosk G. J. et al. (eg, gj by al. Effect of pH variation on the susceptibility) Eur. J. Clin. Microbial Infect Dis. 12: pp. 131-133, 1993.) 'and indicates inhibitor concentration; MIC). The sample was dissolved in dimethy [sulfoxide, (DMSO)) and serially diluted. Helicobacter pylori (ATCC 43504) is suspended in brain heart infusion broth at a concentration of 5 x 105 colony forming units per ml (c〇1〇ny f〇rmati〇n units/ml, CFU/ml). in. The experiment was carried out in a 48-well culture plate. At the time of the test, 0.01 ml of the sample was added to Co. 99 m 1 of the Columbia agar base containing 7% defibrinated rabbit blood. The highest concentration of DMSO in the culture solution was 1%. The highest concentration of the test sample was 300 pg/ml. The culture plates were cultured for 72 hours under 35C micro-oxygen (N2 85%, C〇2 10% and 〇2 5%), followed by visual inspection of the inhibition of Helicobacter pylori colonies, and the vehicle and gentamicin were blank and positive, respectively. Control. Each test is 21 200918085 in a double-overlap. The results are shown in Table 2, which also shows the minimum inhibitory concentration of 6-gingerol and 6-gingerol. Table 2 Minimum inhibitory concentration of sample (MIC, pg/ml) Sample 1 - Sample 2 30 Sample 3 30 Sample 4 30 Sample 5 - Sample 6 30 Sample 7 30 Sample 8 30 Sample 9 30 Sample 1 0 30 Sample 11 30 Sample 1 2 100 Sample 1 3 10 Sample 1 4 30 Sample 1 5 10 Sample 1 6 30 Sample 1 7 30 Sample 1 8 10 22 200918085 Sample ~ — Sample 19 ---- Sample 2 0 .—— — Minimum inhibitory concentration (MIC) , pg/ml) 300 10 6-Gingerol---^ 6_Gingerol ------ 10 10 — . 2. Therapeutic effect of gastric ulcer-like A caused by Helicobacter pylori on gastric ulcer caused by η η According to the method of Marchett 1, Μ, et al. (Marchetti, Μ·, Arico, Β., Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P.
Development of a m〇use m〇dei 〇f p少/on- infection that mimics human disease. Science 267: 1655_1658, 1995·)。每組使用5隻體重24 ± 2克的公CD-I (CW.)小既，禁食〗8小時後，將臨床分離得到的幽門桿菌 (3.2 X 1〇9 CFU/0.4 ml/小鼠）懸浮液胃内投予，投予後一 小時開始將樣品或溶媒（2% CMC，1 0 ml/kg )以每天兩次 (上午1 〇點，下午4點）的方式口服給藥，連續給予7天； 陽性對照藥 〇meparz〇le (1 111§/]^)與 CUrithr〇mycin (ι〇 mg/kg)則1天1次合併口服給予7天。第&天時，所有動 物隔夜禁食，犧牲後將胃取出並沿著胃大彎剖開，胃潰瘍 則依出血程度及潰瘍損傷的嚴重度加以評分： 0分=外觀正常 1分=輕微紅斑 2分=中度紅斑及/或出血斑 23 200918085 3分=顯著出企斑 對幽門桿菌引起胃潰瘍的抑制百分比則依下列公式計算： 〔溶媒組分數一實驗組分數〕/〔溶媒組分數〕X 1 00% 結果示於表3。 表3 樣品 劑量(mg/kg) 對幽門桿菌引起胃潰瘍的抑制百分比 樣品1 300 17 樣品2 300 66 樣品3 300 83 樣品4 300 73 樣品5 300 50 樣品6 300 64 樣品7 300 71 100 57 樣品8 300 57 樣品9 300 57 樣品1 0 300 47 樣品11 300 73 樣品1 2 300 67 樣品1 3 300 73 樣品14 300 58 樣品1 5 300 67 樣品16 300 58 樣品1 7 300 58 樣品1 8 300 75 樣品1 9 300 65 24 200918085Development of a m〇use m〇dei 〇f p less / on- infection that mimics human disease. Science 267: 1655_1658, 1995·). Each group used 5 male CD-I (CW.) with a body weight of 24 ± 2 g, and the clinically isolated Helicobacter pylori (3.2 X 1〇9 CFU/0.4 ml/mouse) after 8 hours of fasting. The suspension was administered intragastrically. One hour after the administration, the sample or vehicle (2% CMC, 10 ml/kg) was orally administered twice a day (1 am, 4 pm), and continuously administered 7 times. Day; the positive control drug 〇meparz〇le (1 111§/]^) and CUrithr〇mycin (ι〇mg/kg) were administered orally once a day for 7 days. At the time of & day, all animals were fasted overnight. After sacrifice, the stomach was removed and dissected along the stomach. The gastric ulcer was scored according to the degree of bleeding and the severity of the ulcer injury: 0 points = normal appearance 1 point = slight erythema 2 points = moderate erythema and / or bleeding spots 23 200918085 3 points = significant percentage of inhibition of gastric ulcer caused by Helicobacter pylori is calculated according to the following formula: [number of components of the solvent - the number of experimental components] / [number of components of the solvent] The X 1 00% results are shown in Table 3. Table 3 Sample dose (mg/kg) Percent inhibition of gastric ulcer caused by Helicobacter pylori Sample 1 300 17 Sample 2 300 66 Sample 3 300 83 Sample 4 300 73 Sample 5 300 50 Sample 6 300 64 Sample 7 300 71 100 57 Sample 8 300 57 Sample 9 300 57 Sample 1 0 300 47 Sample 11 300 73 Sample 1 2 300 67 Sample 1 3 300 73 Sample 14 300 58 Sample 1 5 300 67 Sample 16 300 58 Sample 1 7 300 58 Sample 1 8 300 75 Sample 1 9 300 65 24 200918085
本發明已參照實施例的特定内容被描述如上 容不應視為下列中請專利範圍所界^的本發明範圍㈣内 制。可以理解的是可利心上所描述内容作出多種”和 變化。 圓式簡單說明 圖1至圖20為本發明的樣品1至樣品2〇的高效液相 層析（high performance liquid chromatography，簡稱 hplc) 的結果。 25The present invention has been described with reference to the specific content of the embodiments, and is not to be construed as limiting the scope of the invention as defined in the following claims. It is to be understood that a variety of "changes" can be made in the context of the description. Figure 1 to Figure 20 show high performance liquid chromatography (hplc) of samples 1 to 2 of the present invention. The result of .) 25