201026370 六、發明說明： 【發明所屬之技術領域】 本發明係與中藥成分之萃取有關，更詳而言之是指一 種土肉桂之類黃酮糖苷成分的高效萃取方法。 【先前技術】 按’肉桂係一種中國傳統常用之食材’近代醫學研究 發現’肉桂根皮％取液中的類黃酮糖皆成分具有抑制幽門201026370 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the extraction of a traditional Chinese medicine component, and more particularly to an efficient extraction method of a flavonoid glycoside component of a soil cinnamon. [Prior Art] According to the 'Cinnamon, a traditional Chinese food ingredient', modern medical research found that the flavonoids in the root extract of cinnamon roots have inhibitory pyloric
桿菌的作用，亦有文獻指出類黃酮糖苷成分不論在體内或 是體外模型皆具有類胰島素的作用及降血糖的功效，故肉 桂於傳統醫樂及近代醫學的研究中，佔有相當重要之地 位。傳統中醫藥材之萃取方法，一般係以水煎煮法、水醇 煎煮法、熱迴流提取、水提醇沉法等方式進行萃取，藉以 提取藥材當中之有效成分並製得相關產品，供使用者^ 保健之用。 萃取過程中須不斷地大量使用有 惟須說明的是’上述水煎煮法等加熱方式進行中藥成 分萃取’往往需耗費相當多之時間及能源，且在需反覆進 行加熱高溫萃取過訂1造成_ +之錄性有效 成分大量裂解，使熱敏性有效物f之活性受到破壞 =率下降和萃取液中含有許以㈣狀況；上述缺失對 ^貝源缺乏財草㈣而言，在製作成本上影響甚矩 = 遂有以有機溶劑為溶媒提取中藥成分之方法，ς 係可保持有效物質成分之活性，且萃取率相對提高，惟 藥 機溶劑，造成提取之中 3 201026370 ，對環 ===劑，對人體造成嚴重傷害 【發明内容】 有鑒於此 ^ ，本案發明人乃經詳思細索，並累積多年從 事相關行業之研究開發及處理經驗，終而有本發明之產 生八本之主要目的在於提供—種土肉桂葉類黃_糖普 分的活性。The role of bacilli has also been pointed out in the literature that flavonoid glycosides have insulin-like effects and hypoglycemic effects in both in vivo and in vitro models. Therefore, cinnamon plays an important role in the study of traditional medical music and modern medicine. . The extraction method of the traditional Chinese medicine material is generally extracted by means of water boiling method, hydrocoal boiling method, hot reflux extraction, water extraction and alcohol precipitation method, etc., thereby extracting the active ingredients in the medicine and preparing related products for the user. ^ Health care. The extraction process must be continuously used in large quantities. It is only necessary to say that the above-mentioned "water decoction and other heating methods for the extraction of traditional Chinese medicine ingredients" often take a considerable amount of time and energy, and it is necessary to repeatedly heat the high temperature extraction to make a reservation 1 + The large amount of the active component of the recording is cleavage, so that the activity of the heat sensitive active substance f is destroyed = the rate is decreased and the extract contains the (4) condition; the above deficiency affects the production cost of the rice source (four) Moment = 遂 There is a method for extracting traditional Chinese medicine ingredients by using an organic solvent as a solvent. The lanthanum can maintain the activity of the active substance component, and the extraction rate is relatively increased, but the solvent of the pharmaceutical machine causes the extraction to be 3 201026370, the ring === agent, Serious damage to the human body [Summary of the Invention] In view of this, the inventor of the present invention has been carefully researched and accumulated years of experience in research and development and processing in related industries, and the main purpose of the invention is to produce eight books. Providing the activity of the clay-like yellow _ sugar fraction of the soil.
、^ 去係可保持土肉桂葉類黃酮糖芽之有效成 士 、^發，之另一目的在於提供一種土肉桂葉類黃酮糖 苦成刀之萃取^法，係可降低有機溶繼留，且具有高萃 取率'製程簡單、快速之優點。 緣以達成上述之目的，本發明所提供之土肉桂葉類黃 明糖苦成》之萃取方法，包含以下步驟··步驟—：備置土 肉桂葉碎片’步驟二：以超臨界二氧化碳流體萃取去除土 肉桂葉中的脂溶性成分；步驟三：以鹽水萃取土肉桂葉中 的類黃酮糖苷成分，形賴細㈣萃取液；以及步驟四： 去除賴黃__萃取財_成分，並經賴，以得土 肉桂葉類相㈣混合物；㈣五:再㈣黃瞻皆混合 物’以半製備型减液相層析法收集純化出土肉桂葉中二 種類育酮料；步驟六:類黃嘯苦之彡列類料素活性測 性’來證實土肉桂葉巾類黃嶋料有促進類胰島素活 性；藉此，本發明係以水為溶媒，其製備過財不需反覆 加熱及大量使时機溶劑，故可保制錢材之有效成分 201026370 的活性及穩定性，且鹽水的加入係可提高萃取溶液的極 性’藉以降低其他雜質被提取的可能性’再者，藉由脫去 鹽成分以得到酚酸類物質之方式，係可降低有機溶劑殘留 之可能性且可使製備簡單化’如此係可降低純化之困難性。 ' 【實施方式】 以下茲舉本發明之較佳實施例，並配合下列圖式詳細 說明於后： ' 第一圖係本發明土肉桂葉類黃酮糖苷成分之萃取方 法的流程圖。 第二圖為酸鹼值（pH)對類黃酮糖苷遷移時間之影響。 第三圖為緩衝溶液濃度對類黃酮糖苷遷移時間之影響。 第四圖為電壓對類黃酮糖苷遷移時間之影響。 第五圖為kaempferitrin之氫圖譜。 第六圖為kaempferitrin之液相分析質譜儀（lc—mass) _ 圖譜。 第七圖為 kaempferol 3-〇 _p-D-glucopyran〇Syl-(l—4)-α -Lrhamnopyranosyl-7-o-a-L-rhaninopyraiioside 之氮圖譜。 第八圖為 kaempferol 3-o-P-D-glucopyran〇Syl-(l—4)-α -Lrhamnopy ranosyl-7-o-a-L-rhamnopyranoside 之液相分析質譜 儀圖譜。 第九圖為各不同濃度鹽水萃取類黃_糖普之影響。 第十圖為不同溶質與溶液比例下萃取類黃酮糖苷之影 5 201026370 第十一圖為以毛細管電泳測定（CE)分析鹽水萃取土肉 桂葉之類黃酮糖苷層析圓其類黃酮糖苷遷移時間順序為 C2>a。 第十二圖為脂肪細胞内三酸甘油脂分析流程圖。 第十一圖為九細胞分級分離（Subcellular fractionation ) 實驗流程圖。 請參照第-圖所示，係本發明一較佳實施例之土肉桂 葉類黃酮糖苷成分的萃取方法，其包含以下步驟： 步驟.取乾燥土肉桂葉片，經均質機破碎處理後， 製得土肉桂葉碎片。 步驟二：將土肉桂葉碎片入不鏽鋼萃取槽内，並使用 在壓力15GGpsi和溫度贼條件下之超臨界二氧化碳流體 萃取去除土肉桂葉碎片中的脂溶性精油成分；在此步驟 中，由於超臨界二氧化碳龍萃取具妓好的溶解性能， 故不需增加修飾細卩可取得揮發性成分，且超臨界二氧化 碳流體萃取所需溫度條件較之萃取方法低，因此可避 免本發明之萃取標的物，土肉桂葉類黃贿普成分，受熱 解而可刀離得到的有效成分係具有純度高、雜質少以 及無有機溶劑殘留之土肉桂葉碎片。 5步驟三：進—步運用鹽水萃取土肉桂葉碎片中的類黃 ，，苷成分，以製得土肉桂葉類黃酮糖苷鹽水萃取液；詳 細操作參數如下：配置G綱刚的鹽水，並將鹽水與土 肉桂葉以1 : 20至1 : 6〇g/mL之比例配置成溶液的條件下 201026370 進行約60分鐘料取；於本實補巾，較佳參數為鹽水濃 度為〇_1M ’溶質與溶劑比例為1 : 60 g/mL，萃取時間為 60刀鐘’且本步驟可經至少—次以上之重複實施提高萃取 效果。 步驟四·以電透析儀(Electrodialysis)將每次步驟三所 製得之土肉桂_黃_㈣水萃取液巾的鹽成分脫去， 並經減壓魏至制無⑽土肉桂葉類伽㈣萃取液的 程度。 藉此，本發明之步驟三及步驟四利用鹽水萃取方式提 取土肉桂細黃嶋絲質成分，係可大幅改善傳統以有 機溶劑提取土肉桂葉類黃贿苷物f成分所衍生的各項缺 失，例如.殘留於物質成分上的有機溶劑等；再者，鹽類 的加入土肉桂葉類黃嘯料取祕可提高前述萃取溶液 的極性，如此一來，係可降低其他雜質被提取的可能性。 步驟五：再經由半製備型高效液相層析儀分離純化， 固定移動相比例收集純化並濃縮乾燥土肉桂葉二種類黃酮 糖苦。 以上為土肉桂葉類黃酮糖苷成分的萃取方法，而在分 析方面，採用如下之毛細管電泳方法，以緩衝溶液之酸鹼 值、濃度及電壓來進行探討，以求快速、精確的分析方法。 步驟六：以毛細管電泳分析類黃酮糖苷以酸鹼值、緩衝 溶液濃度、電壓等因素，進行分離探討，如圖二至四所示，其 最佳萃取參數為pH 9.8、緩衝溶液濃度35 mM、電壓25 kv， 毛細管長度57cm (有效長度50 cm；)，偵測波長28〇 nm，而類 7 201026370 黃_終遷移時間為:C1 : 6 7 min、C2 : 6 15 min。 步驟七：以核磁共振儀（NMR)及電灑游離法正離子 核式質錢（ESI-Mass)作進-步之分析鑑定’分析結果 請參閱第五U ;其巾：可發現本實_之土肉桂葉類 s 嗣糖皆具有 kaempferitrin 、 kaempferol 3-〇 β D glucopyranosy 1-(1 ~^4)-a-Lrhamnopyranosy 1-7·ο -a-L-rhamn opyranoside ;接著再以電灑游離法正離子模式質譜儀做進 一步之鑑定，如第六、八圖所示，分別可觀察到 m/z=577[M-H]-、m/z=739[M-H]-的離子訊號，此為類黃酮 糖苷的質譜特性；由此可知，本發明之萃取方法係可成功 萃取出土肉桂葉類黃酮糖芽。 請再參照第十一圖，係以毛細管電泳測定經鹽水萃取 後之土肉桂葉類黃酮糖苷之層析圖，可知其内包含有 kaempferitrin ' kaempferol 3-o -P~D-glucopyranosyl-(l—>4)-α-Lrhamnopyranosyl-7-o-a-L-rhamnopyranoside 之成分，其半製 備沒尚效液相層析法收集純化kaempferitrin、kaempferol 3-〇 -p-D-glucopyranosyl-(l—^4)-a-Lrhamnopyranosyl-7-o-a-L-rhamn opyranoside 之產率分別為 0 06 %、0 〇4 〇/0。 由上述可知’本發明藉由簡單之設備、步驟’並以鹽 水於短時間内萃取土肉桂葉類黃酮糖苷成分，如此不僅可 獲得較高含量之有效成分，且製程更為簡便及迅速；再者， 本發明係以鹽水為溶媒，其製備過程中不需反覆加熱及大 量使用有機溶劑，故可保持原有藥材之有效成分的活性及 穩定性’且鹽水的加入係可提高萃取溶液的極性’藉以降 8 201026370 低其他雜質被提取的可能性’而且，藉由脫去趟成八…曰 到盼酸類物質之方式，係可降低有機溶劑殘留之 可使製備簡單化，據以降低純化之困難性。 此 • 【圖式簡單說明】 • 第一圖係本發明土肉桂葉類黃酿I糖苷成分之萃取方 法的流程圖。 第二圖為酸驗值（pH)對類黃鲷糖苷遷移時間之影塑。 φ 第三圖為緩衝溶液濃度對類黃酿I糖苷遷移時間之影響。 第四圖為電壓對類黃酮糖苷遷移時間之影響。 第五圖為kaempferitrin之氫圖譜。 第六圖為kaempferitrin之液相分析質譜儀（lc-MASS) 圖譜。 第七圖為 kaempferol 3-〇 -p-D-glucopyranosyl-G-^^-a-And ^ can be used to maintain the effective flavor of the flavonoids of the cinnamon leaves, and the other purpose is to provide a method for extracting the cinnabarin flavonoids of the soil, which can reduce the organic solvent retention. And with high extraction rate, the process is simple and fast. In order to achieve the above object, the extraction method of the soil cinnamon leaf yellowing sugar bitterness provided by the present invention comprises the following steps: steps: preparing the clay leaf fragments of the soil step 2: extracting by supercritical carbon dioxide fluid extraction The fat-soluble component in the cinnamon leaves; Step 3: Extracting the flavonoid glycoside from the cinnamon leaves with salt water, and taking the fine (IV) extract; and Step 4: Removing the Lai _ _ extracting _ ingredients, and relying on Mixture of Cinnamon Cinnamon Leaf Phase (4); (4) V: Re-(4) Huangzhan Mixture's collection and purification of two types of ketones from cinnamon leaves by semi-preparative liquid chromatography; Step 6:彡 类 活性 活性 活性 活性 活性 证实 证实 证实 证实 证实 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土 土Therefore, the activity and stability of the active ingredient 201026370 can be preserved, and the addition of brine can increase the polarity of the extraction solution 'to reduce the possibility of extraction of other impurities'. Further, by removing the salt component Phenolic acids obtained manner, the system can reduce the possibility of residual organic solvent and can be simplified preparation 'of such a system can reduce the difficulty of purification. [Embodiment] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the following drawings: 'The first figure is a flow chart of the extraction method of the flavonoid glycoside component of the cinnamon leaf of the present invention. The second graph shows the effect of pH on the migration time of flavonoid glycosides. The third panel shows the effect of buffer solution concentration on the migration time of flavonoid glycosides. The fourth panel shows the effect of voltage on the migration time of flavonoid glycosides. The fifth picture shows the hydrogen spectrum of kaempferitrin. The sixth picture is the liquid analysis mass spectrometer (lc-mass) _ map of kaempferitrin. The seventh picture is the nitrogen map of kaempferol 3-〇 _p-D-glucopyran〇Syl-(l-4)-α-Lrhamnopyranosyl-7-o-a-L-rhaninopyraiioside. The eighth figure is a liquid phase analysis mass spectrometer map of kaempferol 3-o-P-D-glucopyran〇Syl-(l-4)-α-Lrhamnopy ranosyl-7-o-a-L-rhamnopyranoside. The ninth figure shows the effect of extracting yellow-like sugar from different concentrations of brine. The tenth picture shows the extraction of flavonoid glycoside under different solute and solution ratios. 201026370 The eleventh figure shows the flavonoid glycoside migration time sequence of the flavonoid glycoside of brine extracted cinnamon leaves by capillary electrophoresis (CE). For C2>a. Figure 12 is a flow chart for analysis of triglycerides in fat cells. The eleventh figure is a flow chart of the subcellular fractionation experiment. Referring to FIG. 3, a method for extracting flavonoid glycoside components of cinnamon leaves according to a preferred embodiment of the present invention comprises the following steps: Step: taking dried cinnamon leaves and crushing by homogenizer to obtain Cinnamon leaf pieces. Step 2: The soil cinnamon pieces are placed into a stainless steel extraction tank, and the fat-soluble essential oil component in the cinnamon leaf fragments is removed by supercritical carbon dioxide fluid extraction under a pressure of 15 GGpsi and temperature thief; in this step, due to supercritical The carbon dioxide dragon extract has good solubility, so the volatile components can be obtained without adding modified fines, and the temperature conditions required for supercritical carbon dioxide fluid extraction are lower than the extraction method, so that the extraction target of the present invention can be avoided. Cinnamon leaves are yellow bristle components, and the active ingredients which can be obtained by pyrolysis can be obtained from the soil of cinnamon leaf fragments with high purity, less impurities and no organic solvent residue. 5 Step 3: Step-by-step extraction of the yellow-like and glucoside-like components in the cinnamon leaf fragments by using brine to prepare the cinnamic flavonoid glycoside extract of the soil cinnamon; the detailed operating parameters are as follows: The salt water and the soil cinnamon leaves are placed in a solution of 1:20 to 1:6〇g/mL in a ratio of about 20 minutes for the preparation of the solution; in the case of the real towel, the preferred parameter is the brine concentration of 〇_1M ' The ratio of solute to solvent is 1:60 g/mL, the extraction time is 60 knives' and this step can be repeated for at least one or more times to improve the extraction effect. Step 4: Take the electrodialysis instrument (Electrodialysis) to remove the salt component of the soil cinnamon-yellow_(tetra) water extract liquid towel obtained in each step three, and decompress the Wei to produce the (10) soil cinnamon leaf gamma (4) The extent of the extract. Therefore, in the third step and the fourth step of the present invention, the silky extracting method is used to extract the silky component of the cinnamon yellow scutellaria, which can greatly improve the defects derived from the traditional component of the organic solvent extracting clay leaf yellow saponin. An organic solvent or the like remaining on the substance component; further, the addition of the salt to the soil of the cinnamon leaf yellow whisker can increase the polarity of the extraction solution, thereby reducing the possibility of extraction of other impurities. Step 5: Separate and purify by semi-preparative high performance liquid chromatography, and collect and purify and concentrate the two kinds of flavonoids of dried cinnamon leaves. The above is a method for extracting flavonoid glycosides from cinnamon leaves, and in the analysis, the following capillary electrophoresis method is used to investigate the pH value, concentration and voltage of the buffer solution for rapid and accurate analysis. Step 6: Analysis of flavonoid glycosides by capillary electrophoresis with pH, buffer concentration, voltage and other factors, as shown in Figures 2 to 4, the optimal extraction parameters are pH 9.8, buffer concentration 35 mM, Voltage 25 kv, capillary length 57cm (effective length 50 cm;), detection wavelength 28〇nm, and class 7 201026370 yellow _ final migration time: C1: 6 7 min, C2: 6 15 min. Step 7: Analyze and identify the results of nuclear magnetic resonance (NMR) and electrospray free cation-type ESI-Mass. Please refer to the fifth U for the analysis results. The cinnamon leaf s 嗣 嗣 具有 has kaempferitrin, kaempferol 3-〇β D glucopyranosy 1-(1 ~^4)-a-Lrhamnopyranosy 1-7·ο -aL-rhamn opyranoside; Further identification by ion mode mass spectrometer, as shown in the sixth and eighth graphs, ion signals of m/z = 577 [MH]-, m/z = 739 [MH]-, respectively, which are flavonoid glycosides The mass spectrometry property; thus, it can be seen that the extraction method of the present invention can successfully extract the flavonoid glycoside of the cinnamon leaf. Please refer to the eleventh figure for the chromatogram of the flavonoid glycoside of cinnamon leaves extracted by brine after capillary electrophoresis. It is known that kaempferitrin ' kaempferol 3-o -P~D-glucopyranosyl-(l- >4)-α-Lrhamnopyranosyl-7-oaL-rhamnopyranoside, the semi-preparative liquid chromatography method for the purification and purification of kaempferitrin, kaempferol 3-〇-pD-glucopyranosyl-(l-^4)-a- The yields of Lrhamnopyranosyl-7-oaL-rhamn opyranoside were 0 06 % and 0 〇 4 〇/0, respectively. It can be seen from the above that the present invention extracts the cinnamic flavonoid glycoside component of the cinnamon leaf by a simple device and a step, and extracts the flavonoid glycoside component of the cinnamon leaf in a short time, so that not only a high content of the active ingredient can be obtained, but also the process is simpler and more rapid; The invention adopts brine as a solvent, and does not need to be heated repeatedly and uses a large amount of organic solvent in the preparation process, so that the activity and stability of the active ingredient of the original medicinal material can be maintained' and the addition of brine can increase the polarity of the extraction solution. 'By the possibility of lowering 8 201026370, the other impurities are extracted'. Moreover, by removing the arsenic into the acid, the organic solvent residue can be reduced to simplify the preparation, thereby reducing the purification. Difficulty. This is a simplified flow chart of the present invention. The first figure is a flow chart of the extraction method of the yellow-brown I glycoside component of the cinnamon leaf of the present invention. The second graph shows the effect of acid test value (pH) on the migration time of xanthine-like glycosides. φ The third figure shows the effect of buffer solution concentration on the migration time of the yellow-like I glycoside. The fourth panel shows the effect of voltage on the migration time of flavonoid glycosides. The fifth picture shows the hydrogen spectrum of kaempferitrin. The sixth picture shows the liquid phase analytical mass spectrometer (lc-MASS) map of kaempferitrin. The seventh picture is kaempferol 3-〇 -p-D-glucopyranosyl-G-^^-a-
Lrhamnopyranosyl-y-o-a-L-rhamnopyranoside 之氫圖譜。 第八圖為 kaempferol 3-o-p-D-glucopyranosyl-(l—>4)-α- ⑩ Lrhamnopy ranosyl-7-o-a-L-rhamnopyranoside 之液相分析質譜 儀圖譜。 ‘ 第九圖為各不同濃度鹽水萃取類黃酮糖苷之影響。 - 第十圖為不同溶質與溶液比例下萃取類黃酮糖苷之影 響。 第十一圖為以毛細管電泳測定（CE)分析鹽水萃取土肉 桂葉之類黃_糖苷層析圖其類黃酮糖苷遷移時間順序為 C2>cn。 9 201026370 第十二圖為脂肪細胞内三酸甘油脂分析流程圖。 第十三圖為次細胞分級分離（Subcellular fractionation ) 實驗流程圖。 【主要元件符號說明】Hydrogen map of Lrhamnopyranosyl-y-o-a-L-rhamnopyranoside. The eighth figure is a liquid phase analysis mass spectrometer map of kaempferol 3-o-p-D-glucopyranosyl-(l->4)-α- 10 Lrhamnopy ranosyl-7-o-a-L-rhamnopyranoside. ‘The ninth figure shows the effect of extracting flavonoid glycosides from different concentrations of salt water. - The tenth figure shows the effect of extracting flavonoid glycosides at different ratios of solute to solution. The eleventh figure shows the yellow-glycoside chromatogram of the extract of the brine by capillary electrophoresis (CE). The flavonoid glycoside migration time sequence is C2>cn. 9 201026370 The twelfth chart is a flow chart for the analysis of triglycerides in fat cells. The thirteenth picture is a flow chart of the subcellular fractionation experiment. [Main component symbol description]