200411057 玖、發明說明 、實施方式及圖式簡單說明）· ，以及利用此 (發明說明應敘明:發明所屬之技術領域、先前技術、內容 一、發明所屬之技術領域 本發明係關於一種高氏柴胡之細胞系 細胞系生產柴胡多醣體之方法。 二、先前技術200411057 玖, description of the invention, the embodiment, and a simple description of the drawing), and the use of this (the description of the invention should state: the technical field to which the invention belongs, prior art, content 1, the technical field to which the invention belongs. A method for producing Bupleurum polysaccharides from the cell line of Bupleurum.
傳統上中藥的活性成份都由野生或是田間栽培的藥 用植物以水蒸煮或疋以酒精萃取取得。許多且有特玫、、么 ㈣果的稀有藥用植物，由於生長緩慢或需要特殊㈣ 境才能生長，或是雖然容易栽培取得但是受到重金屬、 農藥等環境之污染，影響到藥材的品f。植物組織及細 胞培養技術被視為另-種生產植物生理活性成份的優良 :具。它具有可全年生產、不受地區、緯度、氣候、季 $ 襄/亏柒重金屬、農藥、病蟲害的威脅等因素之 優點。更由於細胞培養法的製程易於控制，並且產物之 分離、純化步驟簡便且低汙染等特點，而逐漸受到重視。Traditionally, the active ingredients of traditional Chinese medicine are obtained by boiling wild or field-grown medicinal plants with water or by extracting with alcohol. Many of the rare medicinal plants with special rosacea, mango fruits have slow growth or require special environment to grow, or although they are easily cultivated but are contaminated by the environment such as heavy metals and pesticides, they affect the quality of medicinal materials. Plant tissue and cell culture technology is regarded as another kind of excellent plant physiologically active ingredients. It has the advantages that it can be produced all year round, and is not affected by factors such as regions, latitudes, climates, seasons / defective heavy metals, pesticides, and the threat of pests and diseases. Moreover, due to the easy control of the cell culture process, and the simple steps of product isolation and purification, and low pollution, it has gradually attracted attention.
長久以來植物來源的多醣體被廣泛應用於食品、 粧品及醫藥品，中草藥水煮後溶入大量的多醋體是中 療，的主要成份之-。㈣是傳統中藥處方中最重要 生藥之一’具有良好的解熱、鎮痛、止痛、止咳、制 等及抗肝炎作^其根所含的果#多糖體，經由文獻: 告具有抗潰瘍、抗腫瘤及免疫力的效果。日本研究指』 三島㈣含有的果膠多Mbupleuran 2IIe，具抗潰癌 抗腫瘤及免疫力的效果。此外，柴胡尚含有多種細 甘（saikosaponin ) a，c，d，e，等成分。傳統上，柴胡藥木 Γ" *> 7 200411057 侍是由野生或是田間栽培“2年的柴胡植株取其 二因為其生長期受到氣候、土壤污染、重金屬、農藥、 病軸害等不良環境因素的影響，藥材品質不容易掌握。 有鑑於此’本發明之目的即在於透過植物組織及細 胞培養技術，生產具有特殊治療效果之柴胡多膽體。透 :此生產方法’同時產率可大幅提昇，更因為使用細胞 。養方法而易於控制所生產多醣體之品質。 三、發明内容 /根據上述目的，本發明係提供一種高氏柴胡細胞 系，係自高氏柴胡植株取得之培植體，在適當之培養基 上使用細胞培養技術誘導產生癒合組織所獲得。 本發明係又提供一種生產柴胡多醣體的方法，其包 括以下步驟：將上述癒合組織置於培養液内懸浮= 養，使該癒合組織形成懸浮細胞；經培養的細胞在種^ 胞培養液中長期馴化及進行進一步的培養；接著由培養 細胞後之培養液回收離柴胡多醣體。 本發明之咼氏柴胡⑺叩/⑼Liu)係為臺灣的 固有原生種。文獻上顯示，高氏柴胡含有高量的柴胡d， 在保肝及肝病治療上效果良好。因此，本發明選用高氏 柴胡細胞培養生產柴胡多醣體有其獨特的市場競爭S。 咼氏柴胡細胞在本發明所使用的培養條件下，培養的矣 胞能直接將多醣體釋放到培養液中，於常溫下即可取= 柴胡多醣體，製程易於掌控，所生產之多醣體品質高。仔 200411057 因此，藉由本發明之方法，其在人為控制的條件下， 即可快速生產吾人需要之多醣體，不似傳統的中藥材需 要由野生或是田間栽培的藥用植物利用高溫蒸煮後萃取 才能產出。再者，藉由本發明之方法，其每一批次的生 產時間只需2週左右，優於現有的在田間需要栽培1-2年 才能採收藥材的生產方法，同時產率大幅提昇，更因為 使用細胞培養方法而易於控制所生產多醣體之品質 由表1柴胡多醣體生產方法之比較，可清楚地了解本 發明之效益。 表1 :本發明與習知柴胡多St體生產方法之比較 項 目 習知生產方法 本發明細胞培養方法 原料來源 田間栽培或野生植物體 反應器細胞培養 產率 35克/公斤/年 186.8克/公斤/2週 產率提高 田間育種選拔，栽培1-2 年才能進行產率比較 藉製程改進及細胞系改 良可以快速提高 產物取得 南溫洛煮 常溫細胞釋放在培養液 内 品質管制 受外在生長環境影響，藥 材來源及品質不易控制 在反應器内培養製程易 控制，品質易掌控 四、實施方式 本發明所採用之方法中，高氏柴胡培植體之取得植 株部位並無限制，較佳為取自其葉片、枝條或花梗。 本發明所採用之方法中，至少一培養基之成分係選 自一由無機鹽類、硫胺素鹽酸鹽（Thiamine · HC1 )、菸 驗酸（Nicontinic acid)、^哆醇鹽酸鹽（Pyridoxine · HC1 )、酿蛋白水解產物（Casein hydrolysate )、肌醇 200411057 (Myo-inositol )、嚴糖及植物生長調節素（Plant growth regulator ; PGR)組成之群組。其中該無機鹽類係選自一 由 KN〇3、（NH4)2S04、MgS04 · 7H20、ZnS04 · 7H20、 MnS04-4H20、CuS04*5H20、CaCl2-2H20、KI、CoCl2· 6H2〇、NaH2P04、H3B03、Na2Mo04 · 2H20、Na-EDTA、 以及FeS04· 7H20所組成之群組。又其中該生長調節素係 選自一由 3 -口引 口朵基醋酸（3-indoleacetic acid; IAA )、 α 〜萘酷酸（a -naphthalene acetic acid ; NAA)以及2,4-一氣苯氧基醋酸（2,4-dichlorophenoxyacetic acid ·’ 2,4 D ) 所組成之群組之一者或其混合物，其中較佳為NAA以及 2,4七。 本發明將藉由以下的較佳具體實施例而作 更進一步地詳細說明，但這些具體實施例僅是作 為舉例說明，而非用以限定本發明之範疇。 例一、高氏柴胡細胞系之建立 取柴胡的枝條及花梗，以1-3%次氯酸鈉消毒3分 鐘’再用無菌水沖洗三次，而後將該培植體在無菌操作 台中，切成約1公分左右之小片塊，然後再移植至無菌的 固體培養基中，培養基為含有2,4D的Gamborg，O.L. B5改 良培養基，置於25°C及黑暗或弱光下，誘導癒合組織產 生。該培植體約2-3週會在其表面生長出白色的癒合組 織’將此癒合組織繼代培養，以此建立高氏柴胡細胞系。 200411057 多醣體之^^ 1、鬲氏柴胡細胞之懸浮培養系統建立 選取生長旺盛之癒合組織約15-2 〇g，置入含有 :amb〇rg，〇丄.比改良培養液之三角瓶内，進行懸浮培 。更進一步將該懸浮培養細胞長期繼代培養進行馴 。該馴化過程係以稀釋比例加權方式補充培養液，亦 即馴化過程中取出部分培養液，再補充新的培養液。 2、咼氏柴胡細胞之柴胡多醣體誘導 取上述之高氏柴胡細胞，懸浮培養於含有〇·5_2ρρηι 或ΝΑΑ的Bs培養液中進行培養，誘導多醣體產出。 问氏柴胡的細胞懸浮培養過程，培養液的黏度會逐漸增 阿，批次（batch culture)培養2週左右含量即可達到最高， 、圖1所示。若進行培養過程中適當的添加蔗糖則可繼續 增加多酶體的含量。 3、柴胡多醣體之提取及其分子量測定 取上述所培養過南氏柴胡細胞之培養液，經高速離 心（10,000rpm/5min)後，取其上層液，並以酒精粹取上層 液即可得柴胡多醣體。 將收集到之柴胡多醣體以GPC管柱進行分子量分 析’其結果顯示高氏柴胡細胞培養產出的多醣體分子量 分布在20,000-1，800,000之間，如圖2所示。 200411057 實施例二、柴胡多醣體對B犁肝炎病毒細胞表面抗原 (HBsAg)及e抗原（HBeAg)之抑制效杲 柴胡多醣體經過MTT試驗方法後，以三株不同不同B 型肝炎病毒之細胞系為試驗材料，分別添加柴胡多醣體 培養1〜5天後，進行B型肝炎表面抗原及e抗原抑制效果之 測定。試驗結果顯示，本發明產出的柴胡多醣體對B型肝 炎病毒表面抗原及e抗原具有相當程度的抑制效果，如圖 3所示。 雖然本發明已以較佳實施例揭露如上，然其並非用以限 定本發明，任何熟悉此技藝者，在不脫離本發明之精神和範 圍外，當可作各種之更動與潤飾。因此，本發明之保護範圍， 當視後附之申請專利範圍而所界定者為準。 200411057 五、圖式簡单說明 圖1係本發明高氏柴胡細胞培養過程產出柴胡多醣體與 時間之變化對應圖。 圖2係本發明所獲得之柴胡多醣體之GPC分析結果。 圖3係本發明所獲得之柴胡多醣體對B型肝炎病毒之表面 抗原（HBsAg)以及e抗原（HBeAg)之抑制效果。 圖號說明For a long time, plant-derived polysaccharides have been widely used in food, cosmetics, and pharmaceuticals. Chinese herbal medicine is a major component of traditional Chinese medicine after it is dissolved in a large amount of polyvinegar. ㈣ is one of the most important crude drugs in traditional Chinese medicine prescriptions. 'It has good antipyretic, analgesic, analgesic, cough, systemic, and anti-hepatic effects. ^ Fruits contained in its roots. And immune effects. Japanese research indicates that the pectin contained in Mishima maggot is Mbupleuran 2IIe, which has anti-cancer, anti-tumor and immunity effects. In addition, Bupleurum contains many saikosaponin a, c, d, e, and other components. Traditionally, Chaihu medicinal wood Γ " * > 7 200411057 The Chaihu plant was cultivated in the wild or in the field. "The 2 years of Chaihu plant is the second one because its growing season is affected by climate, soil pollution, heavy metals, pesticides, disease and other harmful environments." Due to the influence of factors, the quality of medicinal materials is not easy to grasp. In view of this, 'the purpose of the present invention is to produce Bupleurum bile with special therapeutic effects through plant tissue and cell culture technology. The production rate of this method can be It is greatly improved, and it is easy to control the quality of the produced polysaccharides due to the use of cells. Culture methods. III. Summary of the Invention / According to the above purpose, the present invention provides a Gao Bupleurum cell line obtained from a Gao Bupleurum plant. The cultured body is obtained by using cell culture technology to induce the production of healing tissues on an appropriate medium. The present invention also provides a method for producing Bupleurum polysaccharides, which includes the following steps: the above-mentioned healing tissues are suspended in a culture solution = culture , So that the healing tissue forms suspension cells; the cultured cells are acclimated in the cell culture medium for a long time and further Cultivate; then recover Bupleurum polysaccharides from the culture solution after culturing the cells. The Bupleurum Bupleurum / Bei Liu) of the present invention is an inherent native species of Taiwan. The literature shows that Gao Bupleurum contains high amounts of Bupleurum. Hu d has a good effect on liver protection and treatment of liver diseases. Therefore, the use of Gao's Bupleurum cell culture to produce Bupleurum polysaccharide has its unique market competition S. Cultivation condition of Bupleurum Bupleurum cells in the present invention The cultured spores can release the polysaccharides directly into the culture medium, and can be taken at room temperature = Bupleurum polysaccharides, the process is easy to control, and the quality of the polysaccharides produced is high. Tsai 200411057 Therefore, by the method of the present invention, Under artificially controlled conditions, it can quickly produce polysaccharides that we need, unlike traditional Chinese medicinal materials, which need to be extracted by wild or field-cultivated medicinal plants after high-temperature cooking. Furthermore, the present invention The production method of each batch only takes about 2 weeks, which is better than the existing production methods that require 1-2 years of cultivation in the field to harvest medicinal materials, and the yield is greatly improved. It is easy to control the quality of the produced polysaccharides by using the cell culture method. The benefits of the present invention can be clearly understood from the comparison of the polysaccharide production methods of Chaihu in Table 1. Table 1: Production of the present and conventional Chaihu poly-St Comparison of methods. Known production methods. Cell culture method of the present invention. Source of raw materials. Field cultivation or wild plant body reactor. Cell culture yield. 35 g / kg / year. 186.8 g / kg. Yield comparison can only be carried out in 2 years. Process improvement and cell line improvement can quickly increase the product. South Wenluo cooked room temperature cells are released. The quality control in the culture medium is affected by the external growth environment. The source and quality of medicinal materials are not easy to control. The manufacturing process is easy to control and the quality is easy to control. 4. Embodiments In the method adopted by the present invention, there are no restrictions on the plant parts that can be obtained from Gao's Bupleurum, and it is preferably taken from its leaves, branches or pedicels. In the method used in the present invention, at least one component of the culture medium is selected from the group consisting of inorganic salts, Thiamine · HC1, Nicontinic acid, and Pyridoxine. · HC1), Casein hydrolysate, Myo-inositol 200411057, strict sugar, and plant growth regulator (PGR). The inorganic salt is selected from the group consisting of KN〇3, (NH4) 2S04, MgS04 7H20, ZnS04 7H20, MnS04-4H20, CuS04 * 5H20, CaCl2-2H20, KI, CoCl2 6H20, NaH2P04, H3B03, Na2Mo04 · 2H20, Na-EDTA, and FeS04 · 7H20. The growth regulator is selected from the group consisting of 3-indoleacetic acid (IAA), α-naphthalene acetic acid (NAA), and 2,4-monophenyloxyl. One of the group consisting of 2,4-dichlorophenoxyacetic acid · '2,4 D or a mixture thereof. Among them, NAA and 2,4 are preferred. The present invention will be further described in detail through the following preferred specific embodiments, but these specific embodiments are only for illustration, rather than limiting the scope of the present invention. Example 1: Establishment of Gao's Bupleurum cell line Take the branches and pedicels of Bupleurum, and sterilize them with 1-3% sodium hypochlorite for 3 minutes', then rinse with sterile water three times, and then cut the implants into about 1 Small pieces of about centimeters are then transplanted into a sterile solid medium. The medium is a modified medium containing 2,4D Gamborg, OL B5, which is placed at 25 ° C and dark or weak light to induce the production of healing tissue. The cultured body will grow a white healing tissue on its surface in about 2-3 weeks, and this healing tissue is subcultured to establish a Gao Bupleurum cell line. 200411057 ^^ 1. Suspension culture system of Bupleurum Bupleurum cells was established. About 15-20 g of vigorously growing healing tissue was selected and placed in a triangular flask containing: amborg, 0. , Suspension culture. Furthermore, the suspension culture cells were subjected to long-term subculture. In the domestication process, the culture medium is supplemented in a weighted manner in a dilution ratio, that is, a part of the culture medium is taken out during the domestication process, and then a new culture medium is replenished. 2. Induction of polysaccharides from Bupleurum Bupleurum Cells Take the above Bupleurum Bupleurum cells and suspend and culture them in a Bs culture solution containing 0.5-2ρρηι or NAA to induce the production of polysaccharides. In the process of cell suspension culture of Bupleurum Bupleurum, the viscosity of the culture solution will gradually increase. The batch culture can reach the highest content in about 2 weeks, as shown in Figure 1. If the appropriate addition of sucrose during the cultivation process can continue to increase the content of polyenzymes. 3. Extraction of polysaccharide from Bupleurum chinense and determination of its molecular weight Take the culture solution of Bupleurum cultivar Cultivated above and take high-speed centrifugation (10,000 rpm / 5min), then take the upper liquid, and take the upper liquid with alcohol Bupleurum polysaccharides are available. The molecular weight analysis of the collected Bupleurum polysaccharides was performed on a GPC column. The results showed that the molecular weight distribution of the polysaccharides produced by G. bupleurum cell culture was between 20,000 and 1,800,000, as shown in FIG. 2. 200411057 Example 2. Inhibitory effect of Bupleurum polysaccharides on B surface hepatitis virus cell surface antigen (HBsAg) and e antigen (HBeAg). After the Bupleurum polysaccharides were tested by MTT, three different types of hepatitis B virus were tested. Cell lines were used as test materials. After being cultured with Bupleurum polysaccharides for 1 to 5 days, the inhibitory effects of hepatitis B surface antigen and e antigen were measured. The test results show that the Bupleurum polysaccharide produced by the present invention has a considerable degree of inhibitory effect on hepatitis B virus surface antigen and e antigen, as shown in FIG. 3. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and retouching without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the attached patent application. 200411057 V. Brief description of the diagram Fig. 1 is a graph corresponding to the change of Bupleurum polysaccharides produced by B. euphorbia cell culture according to the present invention and time. FIG. 2 is a GPC analysis result of the polysaccharide of Bupleurum chinensis obtained by the present invention. Fig. 3 shows the inhibitory effect of Bupleurum polysaccharides obtained by the present invention on the surface antigen (HBsAg) and e antigen (HBeAg) of hepatitis B virus. Drawing number description