201100538 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種同時生產雲杉醇（piceatannol) 及白藜蘆醇（resveratrol)的方法，尤其是使用曱殼素 (chitin)誘導落花生癒傷組織生產雲杉醇及白藜蘆醇 的方法。 【先前技術】 〇 植物是許多藥材或化學藥品的一種來源，人類 使用其對抗疾病。某些植物受到病原菌感染或其他 環境刺激後，會生成植物防禦素（phytoalexin)，而雲 杉醇（piceatannol，3,4,3',5'-tetrahydroxy-trans-stilbe ne)及白藜蘆醇（resveratrol, trans-3,5,4'-trihydroxyst ilbene)皆屬於植物防禦素，而以苯乙浠（stilbene)為 基本結構之低分子量多紛化合物。前述兩種化合物 對人體健康有多種益處例如抗氧化、抑制企小板凝 〇 集、預防心肌硬塞、腦栓塞、對缺氧心臟有保護作 用、抑制癌細胞增殖、抗發炎並能夠擴張動脈血管 及改善微循環。不過，至目前為止，發現含有雲杉 醇的植物稀少且含量偏低，即使在槳果含量較高， 也僅有在gg範圍内。由於含量太低，很難由植物直 接萃取雲杉醇，另外，高純度的白藜蘆醇於商業上 也相當昂貴，因此，如何得到價格合理且來源容易 的雲杉醇及白藜蘆醇是一大課題。 •201100538 相關研究顯示植物或真菌細胞壁之衍生物（酵 素、細胞壁碎片）會誘導植物產生植物防禦素。而真 菌侵染植物時，細胞壁為與植物最先接觸之構造， 許多真菌之細胞壁主要成分為曱殼素，且已有文獻 證明在受到病原菌侵染時，植物會產生一些酵素如 甲殼素酶（chitinase)及其他致病相關性蛋白質 (Pathogenesisrelated protein，PR Protein)，這些酵素 同時活化並直接抵抗病原菌侵染，當植物辨識病源 〇 菌侵染時，植物產生的曱殼素酶（chitinase)會分解病 原細胞壁，甲殼素便會被釋放並藉由去乙醯作用 (deacetylation)轉換成脫乙酿曱殼素（chitosan)。脫乙 醯曱殼素誘導許多植物不同的防禦反應，如豌豆莢 產生植物防禦素，水稻幼苗之葉片產生致病相關性 蛋白質，受傷之小麥葉片的木質化皆可抑制真菌生 長及侵染。露葉松（Drosophyllum lusitanicum Link) 之培養基中添加甲殼素誘導白花丹素（plumbagin)的 ❹ 產生。 換言之，若是甲殼素為細胞壁主成分的真菌感 染植物時，會使植物產生如曱殼素酶（chitinase)的致 病相關性蛋白質，植物藉由曱殼素酶分解真菌細胞 壁，真菌細胞壁的甲殼素便會轉換成脫乙醯甲殼素 (chitosan)，而已有研究發現脫乙醯曱殼素誘導許多 植物不同的防禦反應，如產生植物防禦素。 201100538 剷述相關研究顯示脫乙醯曱殼素（chitosan)直接 和植物防禦素的生成有關，且目前以曱殼素為誘導 源的文獻中’也是需將甲殼素溶解於酸中，使曱殼 素經由去乙酿作用轉換成脫乙醯曱殼素（chit〇san)， 再添加於培養基中，誘導植物防禦素之生成或探討 抑菌之活性。然而’目前尚無文獻是直接使用曱殼 素誘導癒傷組織產生植物防禦素，尤其是誘導落花 生的癒傷組織產生雲杉醇及白藜蘆醇。 【發明内容】 因此，本發明即致力於解決上述先前技術的缺 點。為解決前述習知技術之問題，本發明之目的即 在於^供一種同時生產雲杉醇（piceatann〇l)及白藜 廬醇（resveratr〇i)的方法，尤其是使用曱殼素（chitin) 誘導落花生癒傷組織生產雲杉醇及白藜蘆醇的方 法。 本發明的另一目的在使用組織培養方式大量生 產提供雲杉醇及白藜蘆醇的方法。 為達前述之目的，本發明使用曱殼素生產雲杉 醇及白藜蘆醇的方法，本發明方法包含：取一落花 生的胚軸做為一培植體，於該培植體上形成一傷 口，於該傷口形成的一癒傷組織上添加一曱殼素懸 浮水溶液；以及細碎及過濾該癒傷組織，藉以取得雲 杉醇及白藜蘆醇；其中該落花生的胚軸係由一脫^ 201100538 後種子經去離子水清洗、及1%次氯酸鈉加土溫20 乳化劑（TWEEN 20)後以超音波震盪2分鐘洗淨並培 養而成；接著在該培植體上切割出一傷口，並把該 培植體置於含有 0.1〜0.3 mg/L 萘乙酸 (1-1^卩11化71&。64。3(^(1)及0.05〜0.08111呂/1^苄氨基°票吟 (6-benzylamino purine)之培養基中於室溫中進行暗 培養，以形成癒傷組織，並進一步細碎研磨及過濾 癒傷組織以獲得雲杉醇及白藜蘆醇。 〇 本發明係直接使用甲殼素誘導癒傷組織，而不 需先把曱殼素（chitin)溶解於酸中，使其去乙隨 (deacetylation)轉換成脫乙臨曱殼素（chitosan)，再誘 導癒傷組織生成類二苯乙烯化合物。 本發明方法操作容易，曱殼素及落花生作為誘 導物及細胞培養的植物材料取得方便，能降低環境 影響，而獲得高濃度的雲杉醇及白藜蘆醇，其中曱 ^ 殼素懸浮水溶液濃度與雲杉醇（piceatannol)的產量 u 呈現正向劑量依附關係；且本發明於添加甲殼素懸 浮水溶液第4小時便能開始產生雲杉醇及白藜蘆醇 遠高於其餘文獻產生雲杉醇的速度，因此本發明提 供一種有效產生雲杉醇及白藜蘆醇的方法。 茲配合所附圖示、實施例之詳細說明及申請專 利範圍，將上述及本發明之其他目的與優點詳述於 後。然而，當可了解所附圖示純係為解說本發明之 201100538 精神而設，不當視為本發明範疇之定義。有關本發 明範疇之定義，請參照所附之申請專利範圍。 【實施方式】 為證實本發明能生產雲杉醇（piceatannol)及白 藜蘆醇（resveratrol)，本發明係藉由高效液相色層分 析儀（HPLC)分析不同濃度0.(H、0.02、0.04 g/mL甲 殼素懸浮水溶液處理之癒傷組織，偵測不同濃度甲 殼素懸浮水溶液誘導產生雲杉醇及白藜蘆醇的效 果。另外，也評估以曱殼素懸浮水溶液誘導癒傷組 織的時間長短對於雲杉醇及白藜蘆醇的影響。由該 些測試結果證實本發明使用曱殼素懸浮水溶液確能 生產雲杉醇及白藜蘆醇之功效。茲對前述實施方式 詳盡說明如下：201100538 VI. Description of the Invention: [Technical Field] The present invention relates to a method for simultaneously producing piceatannol and resveratrol, in particular using chitin to induce groundnut callus Tissue production of spruceol and resveratrol. [Prior Art] 〇 Plants are a source of many medicinal materials or chemicals that humans use to fight disease. Some plants are infected with pathogens or other environmental stimuli to produce phytoalexin, while piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbe ne) and resveratrol (resveratrol, trans-3,5,4'-trihydroxyst ilbene) are all plant defensins, and low molecular weight compounds with the basic structure of stilbene. The two compounds mentioned above have many benefits for human health such as anti-oxidation, inhibition of platelet aggregation, prevention of myocardial tamponade, cerebral embolism, protection against hypoxic heart, inhibition of cancer cell proliferation, anti-inflammatory and expansion of arterial vessels And improve microcirculation. However, until now, it has been found that plants containing spruce are rare and low in content, and even in the case of high acacia content, they are only in the gg range. Since the content is too low, it is difficult to directly extract spruceol from plants. In addition, high-purity resveratrol is also commercially expensive, so how to obtain a reasonable price and easy source of spruceol and resveratrol is A big topic. • 201100538 Related studies have shown that plant or fungal cell wall derivatives (enzymes, cell wall fragments) induce plant-derived plant defensins. When the fungus infects the plant, the cell wall is the first structure to contact with the plant. The main component of many fungal cell walls is chitin, and it has been documented that when infected by pathogenic bacteria, the plant produces some enzymes such as chitinase ( Chitinase and other pathogenesis related proteins (PR Protein), these enzymes are simultaneously activated and directly resistant to pathogen infection. When the plant identifies the pathogen, the chitinase produced by the plant will decompose. In the pathogenic cell wall, chitin is released and converted to chitosan by deacetylation. Deacetylase induces different defense responses in many plants, such as pea pods producing plant defensins, leaves of rice seedlings producing pathogenicity-related proteins, and lignification of injured wheat leaves inhibits fungal growth and infection. The addition of chitin to the medium of Drosophyllum lusitanicum Link induces the production of plutonium in plumbagin. In other words, if chitin is a fungal infection of the main component of the cell wall, the plant will produce a pathogenic protein such as chitinase, and the plant will decompose the fungal cell wall by chitinase, the chitin of the fungal cell wall. It will be converted to chitosan, and studies have found that quercetin induces different defense responses in many plants, such as the production of plant defensins. 201100538 Related research shows that chitosan is directly related to the production of plant defensins, and the current literature on the induction of chitin is also required to dissolve chitin in acid to make clam shells. It is converted into chitosan by chitosan, and then added to the medium to induce the formation of plant defensins or to investigate the antibacterial activity. However, there is currently no literature on the direct use of chitin to induce callus to produce plant defensins, especially the callus that induces fall flower production to produce spruce and resveratrol. SUMMARY OF THE INVENTION Accordingly, the present invention has been made in an effort to address the disadvantages of the prior art described above. In order to solve the problems of the prior art described above, the object of the present invention is to provide a method for simultaneously producing picateann〇l and resveratr〇i, especially using chitin to induce groundnuts. A method of producing spruce and resveratrol from callus. Another object of the present invention is to produce a method for providing spruce alcohol and resveratrol in large quantities using tissue culture. For the purpose of the foregoing, the present invention uses chitin to produce spruceol and resveratrol. The method of the present invention comprises: taking a hypocotyl of a groundnut as an implant, forming a wound on the implant. Adding a suspension solution of chitin to a callus formed by the wound; and finely chopping and filtering the callus to obtain spruceol and resveratrol; wherein the hypocotyl of the groundnut is from a decapitation 201100538 The seed is washed with deionized water and 1% sodium hypochlorite plus soil temperature 20 emulsifier (TWEEN 20), then washed and incubated for 2 minutes with ultrasonic vibration; then a wound is cut on the implant and the The culture body is placed in a medium containing 0.1 to 0.3 mg/L of naphthaleneacetic acid (1-1^卩11, 71 & 64. 3 (^(1) and 0.05 to 0.08111 len / 1 benzylamino °) (6-benzylamino purine) The medium is darkly cultured at room temperature to form callus, and the callus is further finely ground and filtered to obtain spruceol and resveratrol. The present invention directly uses chitin to induce callus. Without first dissolving chitin in acid It is converted into a chitosan by deacetylation, and then the callus is induced to form a stilbene-like compound. The method of the invention is easy to operate, and chitin and groundnut are used as inducers and cell culture. The plant material is convenient to reduce the environmental impact, and high concentrations of spruceol and resveratrol are obtained, wherein the concentration of the suspension aqueous solution of the chitin is positively dose-dependent with the yield of the piceatannol; Moreover, the present invention can start to produce spruceol and resveratrol at a temperature of 4 hours after the addition of the aqueous suspension of chitin, which is much higher than the rate of production of spruceol in the rest of the literature, so the present invention provides an effective production of spruceol and white cucurbit The above-mentioned and other objects and advantages of the present invention will be described in detail below with reference to the accompanying drawings, the detailed description of the embodiments, and the appended claims. In the case of the invention, the invention is not limited to the definition of the scope of the invention. For the definition of the scope of the invention, please refer to the attached patent application. In order to confirm that the present invention can produce piceatannol and resveratrol, the present invention analyzes different concentrations by high performance liquid chromatography (HPLC). (H, 0.02, 0.04 g/ The callus treated with mL chitin suspension aqueous solution was used to detect the effect of different concentrations of chitin suspension aqueous solution to induce the production of spruceol and resveratrol. In addition, the length of time for inducing callus with aqueous suspension of chitin was also evaluated. The effects of spruceol and resveratrol. From these test results, it was confirmed that the present invention can effectively produce spruceol and resveratrol using an aqueous solution of quercetin. The foregoing embodiment is described in detail as follows:
實施例1 洛花生癒傷組織之誘導生成 落花生種子由雲林農家提供之台南9號。脫莢 後之種子以清水洗淨，於無菌操作台中以1 %次氯酸 鈉加兩滴土溫20乳化劑（TWEEN 20)後，以超音波 震盪2分鐘，再以無菌方式播種於以MS (Murashige and Skoog，1962)為基礎鹽類之基礎培養基；基礎培 養基添加30g/L庶糖、2.5g/L水晶續_脂’培養液加 201100538 入瓊脂前，先以氳氧化鈉及鹽酸將pH值調整為5.8 ±0.3，再以 120°C，1.05 Kg/cm2 殺菌 15 分鐘，再 倒入培養皿中，冷卻備用。 於落花生播種一週後取胚軸做為培植體，裁切 成複數個培養片段，接於含有0.25 mg/L萘乙酸 (1-naphthylacetic acid) ' 0.075 mg/L 苄氨基 σ票吟 (6-benzylamino purine)之基礎培養基中，由切口誘 導出癒傷組織，每隔四週繼代一次。因癒傷組織需 在無光線、室溫下生長，故培養皿連同培植體放置 於培養環境為28±1°C之暗處理之生長箱。 實施例2 曱殼素誘導落花生癒傷組織中類二苯乙烯化合物生成及萃 取方法 甲殼素（chitin，講於sigma)先以0.22 //m篩子 過篩，再將其配製成〇.〇1、〇.〇2、0.04 g/mL之懸浮 水溶液，滅菌後冷卻備用。 取繼代培養三週之落花生癒傷組織，於落花生 癒傷組織上滴1 mL曱殼素懸浮水溶液或是無菌水 (H20)，再將做誘導處理及未做誘導處理（blank)之培 養皿置於28±1°C之暗處理之生長箱，培養12小時 後，秤取癒傷組織1 g加入100%甲醇（MeOH)研磨， 然後抽氣過濾共三次，再定量至5mL，最後以0.22 201100538 A m過濾碟過濾。 實施例3 高效液相色層分析儀(HP L C )測定癒傷組織誘導產生的雲 杉醇（piceatannol)及白藜蘆醇(resveratrol)的含量 本發明癒傷組織中雲杉醇及白藜蘆醇的測定， 係利用高效液相色層分析儀（HPLC)分析，其分析條 件係使用 L-7100 HPLC pump 、L7420 UV、L7485 〇 螢光偵測儀（Hitachi Co. Ltd·，Tokyo, Japan)、以及 Mightysil C18 分離管枉（250x4.6 mm，5pL)。螢光 偵測儀的光源激發與散發波長分別設計為343和 395 nm。以螢光層析圖譜的訊號計算雲杉醇及白藜 蘆醇的濃度。移動相為乙晴/含0.1 %甲酸的去離子水 (acetonitrile/ 〇·ι〇/〇 f〇rmic acid deionized water)，移 動相梯度為20%乙腈，經過20分鐘後為32%，再經 過1〇分鐘後為90%，最後以90%乙腈維持5分鐘。 ❾ 進行定量癒傷組織產生雲杉醇及白藜蘆醇的含 量前’先將雲杉醇及白藜蘆醇之標準品分別以100 % 曱醇稀釋成 20 ppb、100 ppb、250 ppb、500 ppb、 lOOOppb ;各濃度經HPLC分析後利用尖峰面積與濃 度製作出標準曲線。雲杉醇之標準曲線： y=427.0x-189.〇，相關係數（R2)為1.0 ;白藜蘆醇之 標準曲線：y=1308.6x-8116.4，相關係數（R2)為 201100538 0.9997。再由此標準曲線計算出癒傷組織中雲杉醇 及白藜蘆醇的含量。 實施例4 不同濃度甲殼素誘導癒傷組織產生雲杉醇及白藜蘆醇的測 定 以不同濃度甲殼素懸浮水溶液：〇.(Π、0.02、0.04 g/mL誘導落花生癒傷組織12小時後，分析所產生 〇 雲杉醇及白藜蘆醇的含量，結果如表一所示。 表一 濃度（ng/g鮮重） 曱殼素（g/mL)- 雲杉醇 白藜蘆醇 空白 ND ND h2o ND ND 0.01 582.64 ± 169.19 6266.35 ±4165.63 0.02 1056.27 ±379.66 3587.54 ±2028.83 0.04 2362.15 ±771.33 10090.78 ±758.46 三重複實驗取平均值 ND :未偵測出Example 1 Induction of Callus from Luoyang Peanut Seeds were collected from No. 9 Tainan provided by Yunlin Farm. The seeds after de-pod were washed with water, and then sterilized by ultrasonic wave with 2% sodium hypochlorite and two drops of soil temperature 20 emulsifier (TWEEN 20) in an aseptic table, and then aseptically sown in MS (Murashige and Skoog, 1962) is the basic medium for the basic salt; the base medium is added with 30g/L sucrose, 2.5g/L crystal continuation _ lipid 'culture solution plus 201100538 before agar, the pH is adjusted to 5.8 with sodium bismuth oxide and hydrochloric acid. ±0.3, and then sterilized at 120 ° C, 1.05 Kg / cm2 for 15 minutes, then poured into a Petri dish, cooled and set aside. One week after planting the groundnuts, the hypocotyls were taken as the culture body, and cut into a plurality of culture fragments, which were 0.25 mg/L naphthaleneacetic acid (1-naphthylacetic acid) '0.075 mg/L benzylamino σ 吟 (6-benzylamino In the basal medium of purine), callus was induced by the incision and subcultured every four weeks. Since the callus needs to grow in the absence of light and at room temperature, the culture dish is placed in a growth chamber with a dark environment of 28 ± 1 ° C in the culture environment. Example 2 Chitin-induced formation and extraction of stilbene-like compounds in groundnut callus Chitin (chitin, sigma) was first sieved through a 0.22 //m sieve and then formulated into 〇.〇1 〇.〇2, 0.04 g/mL suspension aqueous solution, sterilized and cooled for use. The peanut callus was subcultured for three weeks, and 1 mL of a suspension solution of quercetin or sterile water (H20) was placed on the callus of the groundnut, and then the culture vessel was subjected to induction treatment and no induction treatment (blank). Placed in a darkened growth chamber at 28 ± 1 ° C. After 12 hours of incubation, 1 g of callus was weighed and added to 100% methanol (MeOH) for grinding, then suction filtered for three times, then quantified to 5 mL, and finally to 0.22. 201100538 A m filter disc filter. Example 3 Determination of the content of picatinannol and resveratrol induced by callus by high performance liquid chromatography (HP LC) The spruce and white gourd in the callus of the present invention The alcohol was measured by a high performance liquid chromatography (HPLC) analysis using L-7100 HPLC pump, L7420 UV, L7485 〇 fluorescence detector (Hitachi Co. Ltd., Tokyo, Japan). And the Mightysil C18 separation tube (250x4.6 mm, 5pL). The source and excitation wavelengths of the fluorometer are designed to be 343 and 395 nm, respectively. The concentration of spruceol and resveratrol was calculated from the signal of the fluorescence chromatogram. The mobile phase is acetonitrile/acetic acid deionized water with 0.1% formic acid. The mobile phase gradient is 20% acetonitrile. After 20 minutes, it is 32%. After 90 minutes, it was 90% and finally maintained at 90% acetonitrile for 5 minutes.前 Before quantifying the content of spruce and resveratrol in callus, first dilute the standard of spruceol and resveratrol with 100% sterol to 20 ppb, 100 ppb, 250 ppb, 500 Ppb, lOOOOppb; each concentration was subjected to HPLC analysis to make a standard curve using peak area and concentration. Standard curve of spruceol: y=427.0x-189.〇, correlation coefficient (R2) is 1.0; standard curve of resveratrol: y=1308.6x-8116.4, correlation coefficient (R2) is 201100538 0.9997. The content of spruceol and resveratrol in the callus was calculated from this standard curve. Example 4 Determination of spruceol and resveratrol by callus induced by different concentrations of chitin. The suspension of aqueous solution of chitin at different concentrations: 〇. (Π, 0.02, 0.04 g/mL induced groundnut callus 12 hours later, The content of cedarol and resveratrol produced was analyzed, and the results are shown in Table 1. Table 1 concentration (ng/g fresh weight) Chitin (g/mL) - Spruceol Resveratrol blank ND ND h2o ND ND 0.01 582.64 ± 169.19 6266.35 ±4165.63 0.02 1056.27 ±379.66 3587.54 ±2028.83 0.04 2362.15 ±771.33 10090.78 ±758.46 Three replicates take the average ND: not detected
由表一可知，雲杉醇含量隨曱殼素懸浮水溶液 濃度增加而增加，雲杉醇含量分別為582.64 土 169.19、1056.27±379.66、2362.15±771.33 ng/g (鮮 201100538 重），白藜蘆醇含量以甲殼素懸浮水溶液濃度為〇 〇4 g/mL之誘導含量達最高，其次為〇 〇i g/mL，最低 為〇.〇2g/mL，其中白藜蘆醇含量分別為1〇〇9〇 78± 758.46 ng/g、6266.35 ± 4165.63 ng/g、3587.54 土 2028.83ng/g (鮮重）；對照組中未滴入任何液體（空 白）及滴入無菌纟（H20 ) ’則皆未偵測到雲杉醇及 白藜產醇。以高劑量曱殼素（〇 〇4 g/mL)誘導癒傷組 織，產生雲杉醇及白藜蘆醇效果最好，此結果顯示， Ο 甲殼素懸浮水溶液的確能誘導落花生絲組織生產 雲杉醇及白藜蘆醇，且曱殼素濃度高，誘導效果較 好。 實施例5 不同時間測定甲殼素誘導癒傷組織產生雲杉醇及白藜蘆醇 含量 〇 以固疋》辰度（〇.〇 1 g/mL)甲殼素懸浮水溶液誘導 落花生癒傷組織，於不同時間分析癒傷組織產生雲 杉醇及白藜蘆醇的含量變化，結果如表二所示。 表二、 濃度（ng/g鮮重） 時間（小時）_______ 雲杉醇 白藜蘆醇It can be seen from Table 1 that the content of spruceol increases with the concentration of aqueous suspension of chitin, and the content of spruceol is 582.64, 169.19, 1056.27±379.66, 2362.15±771.33 ng/g (fresh 201100538), resveratrol. The content of chitin suspension aqueous solution was 〇〇4 g/mL, which was the highest, followed by 〇〇ig/mL, the lowest was 〇.〇2g/mL, and the resveratrol content was 1〇〇9〇. 78± 758.46 ng/g, 6626.35 ± 4165.63 ng/g, 3587.54 soil 2028.83 ng/g (fresh weight); no liquid (blank) and drip into sterile sputum (H20) were not detected in the control group. Toceol and white peony produce alcohol. Callus was induced by high-dose chitin (〇〇4 g/mL), which produced the best effect of spruceol and resveratrol. The results showed that the aqueous suspension of chitin was able to induce the production of spruce in the ground. Alcohol and resveratrol, and the concentration of chitin is high, and the induction effect is good. Example 5 Determination of the content of spruceol and resveratrol in chitin-induced callus at different times 诱导Induction of groundnut callus by solid solution of 甲 疋 (〇.〇1 g/mL) chitin suspension aqueous solution, different Time analysis of callus produced changes in the content of spruce and resveratrol, the results are shown in Table 2. Table 2, Concentration (ng/g fresh weight) Time (hours) _______ Spruceol Resveratrol
0 KD ND 4 1480.18 ± 194.27 640.72 + 268.94 201100538 8 1069.27 ± 356.58 2724.34 ± 690.96 12 1023.08 ± 343.08 4382.72 ± 1981.86 24 2361.76 ± 269.75 4593.06 ± 669.80 72 948.16 ± 94.13 1104.39 ± 286.55 120 ND 507.26 ± 63.94 三重複實驗取平均值 ND :未偵測出 〇 由表二可知，落花生癒傷組織在甲殼素懸浮水 溶液誘導後4小時即產生雲杉醇及白藜蘆醇，並配 合參考第一圖，雲杉醇含量於第8小時有下降情 形’於第24小時含量為2361 76±269 75 ng/g (鮮 重）、第72小時含量降低至948 16±94 13 ng/g (鮮 重）’至12 0小時已無偵測到雲杉醇。 由第二圖可知，白藜蘆醇含量變化係由4小時 〇 直至第24小時逐漸增加’達最高含量4593.06土 669.80 ng/g (鮮重），隨後降低至第12〇小時含量為 507.26 ± 63.94 ng/g (鮮重）。此結果顯示，甲殼素 懸浮液誘導落花生癒傷組織後，於第4小時便能偵 測到雲杉醇及白藜蘆醇，且雲杉醇含量高於白藜蘆 醇而雲杉醇及白藜蘆醇兩者於誘導後第24小時便 達最高含量。 综上所述，本發明之方法直接使用曱殼素能有 12 201100538 效誘導落花生癒傷組織產生雲杉 .^ θ %及白藜盧醇，且 兩者的產1會隨著^殼㈣度增加而提升。 其#!一方面’本發明方法在室溫下使用-般培養 土更此培養瘡傷組織’使可提供雲杉醇及白黎彦醇 大量生成的需求。 π 惟，以上所述|，僅為本發明之較佳實施例， 並非用以限定本發明之範圍，任何熟習此技藝者， 〇 +脫離本發明之精神與範圍所作之等效修飾或變 更，皆應涵蓋於本發明之專利範圍中。 【圖式簡單說明】 ^圖係曱设素水懸浮液（〇.〇lg/mL)誘導落花生癒 ^ 傷組織’於不同時間内雲杉醇之含量變化。 第二圖係曱殼素水懸浮液（0.01g/mL)誘導落花生癒 傷組織，於不同時間内白藜蘆醇之含量變化。 【主要元件符號說明】 無0 KD ND 4 1480.18 ± 194.27 640.72 + 268.94 201100538 8 1069.27 ± 356.58 2724.34 ± 690.96 12 1023.08 ± 343.08 4382.72 ± 1981.86 24 2361.76 ± 269.75 4593.06 ± 669.80 72 948.16 ± 94.13 1104.39 ± 286.55 120 ND 507.26 ± 63.94 ND: Undetected 〇 According to Table 2, the groundnut callus produced spruceol and resveratrol 4 hours after the induction of aqueous suspension of chitin, and the reference to the first figure, the content of spruce in the 8th The hour has a decrease of '2361 76±269 75 ng/g (fresh weight) at the 24th hour and 948 16±94 13 ng/g (fresh weight) at the 72nd hour' to 12 hours. Spanning alcohol was detected. As can be seen from the second figure, the change in resveratrol content gradually increased from 4 hours to 24 hours 'up to the highest content of 4593.06 soil 669.80 ng / g (fresh weight), and then decreased to the 12th hour hour content was 507.26 ± 63.94 Ng/g (fresh weight). This result showed that after the chitin suspension induced the callus of the groundnut, spruceol and resveratrol were detected, and the content of spruceol was higher than that of resveratrol and spruce and white. Resveratrol reached the highest level at 24 hours after induction. In summary, the method of the present invention directly uses the chitin to have 12 201100538 effect-induced peanut ground callus to produce spruce. ^ θ % and chalk alfalfa, and the yield of both will follow the shell (four) degree Increase and increase. On the one hand, the method of the present invention uses the culture-like soil at room temperature to cultivate the sore tissue, which provides a demand for the mass production of spruceol and baclitaxel. The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and any equivalent modifications or alterations from the spirit and scope of the present invention will be apparent to those skilled in the art. All should be covered by the scope of the invention. [Simple description of the figure] ^ The system is designed to induce the change of the content of spruce in the groundnuts of the groundnuts. In the second figure, the aqueous suspension of chitin (0.01 g/mL) induced the callus of the groundnut, and the content of resveratrol changed at different times. [Main component symbol description] None