TWI627279B - Method for cultivating plant callus rich in stilbene - Google Patents
Method for cultivating plant callus rich in stilbene Download PDFInfo
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Abstract
一種富含類二苯乙烯的植物癒傷組織之培養方法,其係包括以下 步驟:(a)活化步驟:將松屬植物的種子銳角處製造一切口,並將該種子放入活化液中以使該種子中之種胚活化;(b)逆境處理步驟:將活化後之該種胚從該種子中取出,並在胚軸處製造出一創傷缺口;(c)培養步驟:將上述逆境處理步驟(b)中所得之該種胚放入培養基中進行組織培養,該創傷缺口處會因細胞增生而形成癒傷組織;且該癒傷組織中含有兒茶素、原花青素、類二苯乙烯化合物及彼等混合物中之至少一種。 A method for cultivating plant callus rich in stilbene, which comprises the following Step: (a) activating step: making a mouth at the acute angle of the seeds of the pine plant, and placing the seed in the activation solution to activate the seed embryo in the seed; (b) stress treatment step: after activation The embryo is taken out from the seed and a wound gap is created at the hypocotyl; (c) a culture step: the embryo obtained in the above-mentioned stress treatment step (b) is placed in a medium for tissue culture, the wound The callus is formed by cell proliferation in the notch; and the callus contains at least one of catechin, proanthocyanidins, stilbene-like compounds, and mixtures thereof.
Description
本發明關於一種生產類二苯乙烯的方法,尤其係指以植物之癒傷組織生產類二苯乙烯的方法。 The present invention relates to a process for producing stilbene-like, and more particularly to a process for producing stilbene-like plants from plant callus.
癒傷組織因其具有可藉組織培養之方式大量生產之優點而廣受重視,其中又以植物二次代謝物之生產最為受到食品及工業的重視,如由植物體所誘導而產生之液晶聚合物原料:鄰羥基苯甲酸(p-hydroxybenzoic acid)相較於工業上所使用之Kolbe-Schmitt process則更具優勢(McQualter et al.,2005)。除此之外,藉由適當之誘導方式更可進一步使癒傷組織產生一般稱為植物防禦素(phytoalexins)之化合物,其中之類二苯乙烯物質(stilbenoids)(Hammerschmidt and Dann,1999)尤其受到注目。此類化合物不僅在植物防禦系統中占有重要的角色,於人體中亦可調整多種病理機制,如抗發炎、抗氧化、抗病毒、預防老化及心血管疾病、減少肥胖和神經元保護等(Aggarwal and Shishodia,2006;Losa,2003;Ray et al.,1999;Rice-Evans et al.,1995),被視為具有醫療及保健雙重功效之天然抗氧化物質。 Callus is widely recognized for its advantages of mass production by means of tissue culture. Among them, the production of plant secondary metabolites is most valued by food and industry, such as liquid crystal polymerization induced by plant bodies. Material: p- hydroxybenzoic acid is superior to the Kolbe-Schmitt process used in industry (McQualter et al., 2005). In addition, callus can be further produced by a suitable induction method as a compound commonly referred to as phytoalexins, in which stilbenoids (Hammerschmidt and Dann, 1999) are particularly affected. Attention. Such compounds not only play an important role in the plant defense system, but also can adjust a variety of pathological mechanisms in the human body, such as anti-inflammatory, anti-oxidation, anti-viral, prevent aging and cardiovascular diseases, reduce obesity and neuroprotection (Aggarwal And Shishodia, 2006; Losa, 2003; Ray et al., 1999; Rice-Evans et al., 1995), considered a natural antioxidant with dual medical and health benefits.
再者,因類二苯乙烯物質(stilbenoids)具有豐富之生理活性但含量稀少,即使配合可大量生產之組培技術仍難以滿足其需求,故目前研究多以逆境處理之方式,提高癒傷組織所產生之類二苯乙烯化合物之種類及含量,例如Fritzemeier等人(1983)以UV-C(ultraviolet-C)刺激誘導落花生癒傷組織產生類二苯乙烯化合物;Ku氏等(2005)利用UV 刺激落花生癒傷組織以增加白藜蘆醇(resveratrol)之產量及發現新型類二苯乙烯化合物piceatannol。 Furthermore, because stilbenoids are rich in physiological activity but rare in content, even with the tissue culture technology that can be mass-produced, it is still difficult to meet their needs. Therefore, the current research mostly uses adversity to improve callus. The type and content of the stilbene compound produced, for example, Fritzemeier et al. (1983) induced the stilbene-like compound by the UV-C (ultraviolet-C) stimulation to induce the groundnut callus; Ku et al. (2005) utilized UV. Stimulate the groundnut callus to increase the yield of resveratrol and discover the novel stilbene compound piceatannol.
根據以往文獻中顯示,松屬植物具多種且高當量之抗氧化活性物質,可視為新興保健產品之潛力原料。但因其生長環境等諸多限制,故其樣本難以大量取得而限縮其應用空間。為此,在考量生態環境的維護及新興保健產品開發之概念下,發展由植株部位所培養之具分生特性,可大量生產之癒傷組織便顯其重要性。 According to the previous literature, Pinus genus has a variety of high-equivalent antioxidant active substances, which can be regarded as potential raw materials for emerging health care products. However, due to many limitations such as its growth environment, it is difficult to obtain a large amount of samples and limit its application space. To this end, considering the maintenance of the ecological environment and the concept of the development of emerging health products, it is important to develop the meristematic characteristics cultivated by the plant parts, and the mass-produced callus.
綜上所述,本發明人利用松屬植物之種胚作為基礎,希冀利用組織培養的方式,建立松屬植物癒傷組織之培養系統,藉以達到可大量生產之目的以作為後續保健產品之開發研究及量產之用。並配合適當之誘導方式,探討癒傷組織中類二苯乙烯之含量變化,做為後續保健產品之開發之依據。 In summary, the present inventors used the embryo of Pinus genus as a basis, and hoped to establish a culture system of pine plant callus by means of tissue culture, thereby achieving mass production for development of a follow-up health product. Research and mass production. And with appropriate induction methods, explore the changes in the content of stilbene in callus, as the basis for the development of follow-up health products.
亦即,根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其係包括以下步驟:(a)活化步驟:將松屬植物的種子銳角處製造切口,並將該種子放入活化液中以使該種子中之種胚活化;(b)逆境處理步驟:將活化後之種胚從種子中取出,並在胚軸處製造一創傷缺口;(c)培養步驟:將上述逆境處理步驟(b)中所得之種胚放入培養基中進行組織培養,該創傷缺口處會因細胞增生而形成癒傷組織。 That is, according to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, which comprises the steps of: (a) an activation step: making an incision at an acute angle of a seed of a Pinus genus plant, And placing the seed in an activation solution to activate the embryo in the seed; (b) stress processing step: removing the activated embryo from the seed and creating a wound gap at the hypocotyl; (c) Culture step: the embryos obtained in the above-mentioned stress treatment step (b) are placed in a medium for tissue culture, and the wounds are formed into callus due to cell proliferation.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,該活化液為過氧化氫水溶液。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus, which is an aqueous hydrogen peroxide solution, can be provided.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,上述之過氧化氫水溶液的濃度並未特別加以限定,一般為0.5至3%之範圍;較佳為0.5至2%之範圍;特佳為0.5至1.5%之範圍;最佳為1%。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided. The concentration of the above aqueous hydrogen peroxide solution is not particularly limited, and is generally in the range of 0.5 to 3%; preferably A range of 0.5 to 2%; particularly preferably in the range of 0.5 to 1.5%; most preferably 1%.
根據本發明之另一實施例可以提供富含類二苯乙烯的植物癒傷組織之培養方法,上述活化步驟(a)中的活化時間並未特別加以限定,一般為1至5天,最佳為3天。 According to another embodiment of the present invention, a stilbene-rich plant callus culture method can be provided, and the activation time in the activation step (a) is not particularly limited, and is generally 1 to 5 days, preferably. It is 3 days.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該培養基包括固態培養基或液態培養基。 According to one aspect of the present invention, a method for culturing a stilbene-rich plant callus can be provided, wherein the medium comprises a solid medium or a liquid medium.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該固態培養基為以LPG(leaf protoplasts glutamine)為固態培養基(Anderson,1990)。 According to one aspect of the present invention, a method for culturing a stilbene-rich plant callus can be provided, wherein the solid medium is a solid medium (LPG (leaf protoplasts glutamine) (Anderson, 1990).
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基係由蔗糖、6-芐氨基嘌呤(6-Benzylaminopurine)、2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)、麩醯胺酸(Glutamine)、洋菜(Sigma type agar)及彼等混合物中之至少一種所組成。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the liquid medium is composed of sucrose, 6-Benzylaminopurine, 2,4-dichlorobenzene. It consists of at least one of 2,4-dichlorophenoxyacetic acid, Glutamine, Sigma type agar, and mixtures thereof.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基中之蔗糖的濃度並未特別加以限定,一般為在1至10%之範圍;較佳為在1至8%之範圍;更佳為在1至6%之範圍;特佳為在2至5%之範圍;最佳為3%。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of sucrose in the liquid medium is not particularly limited, and is generally in the range of 1 to 10%; Preferably, it is in the range of 1 to 8%; more preferably in the range of 1 to 6%; particularly preferably in the range of 2 to 5%; most preferably 3%.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基中之6-芐氨基嘌呤(6-Benzylaminopurine)的濃度並未特別加以限定,一般為在2μM至10μM之 範圍;較佳為在2至8μM之範圍;更佳為在2至6μM之範圍;特佳為在2至5μM之範圍;最佳為3至5μM之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of 6-Benzylaminopurine in the liquid medium is not particularly limited, and is generally From 2μM to 10μM The range; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基中之2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)的濃度並未特別加以限定,一般為在2μM至10μM之範圍;較佳為在2至8μM之範圍;更佳為在2至6μM之範圍;特佳為在2至5μM之範圍;最佳為3至5μM之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein a concentration of 2,4-dichlorophenoxyacetic acid in the liquid medium is It is not particularly limited and is generally in the range of 2 μM to 10 μM; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably 3 to 5 μM. The scope.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基中之麩醯胺酸(Glutamine)的濃度並未特別加以限定,一般為在2μM至20μM之範圍;較佳為在4至18μM之範圍;更佳為在6至16μM之範圍;特佳為在8至14μM之範圍;最佳為8至12μM之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of the glutamic acid (Glutamine) in the liquid medium is not particularly limited, and is generally 2 μM to A range of 20 μM; preferably in the range of 4 to 18 μM; more preferably in the range of 6 to 16 μM; particularly preferably in the range of 8 to 14 μM; most preferably in the range of 8 to 12 μM.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該液態培養基的pH值並未特別加以限定,一般為在5.0至7.0之範圍;較佳為在5.0至6.5之範圍;最佳為在5.5至6.5之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the pH of the liquid medium is not particularly limited, and is generally in the range of 5.0 to 7.0; preferably Range of 5.0 to 6.5; optimally in the range of 5.5 to 6.5.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該松屬植物為五葉松、赤松、山松、刺果松、長葉松、華山松、五針松、大果松、紅松、二葉松、馬尾松、或白皮松;較佳為五葉松、山松、長葉松、華山松、五針松、大果松、二葉松、馬尾松、或白皮松;更佳為五葉松、長葉松、華山松、五針松、二葉松、馬尾松、或白皮松;特佳為五葉松、華山松、五針松、馬尾松、或白皮松;最佳為五葉松、華山松、或馬尾松。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the pine plant is five-leaf pine, red pine, mountain pine, thorn pine, long-leaf pine, huashan pine, and five needles. Pine, big fruit pine, Korean pine, two-leaf pine, masson pine, or white pine; preferably pine, pine, pine, pine, pine, pine, pine, pine, pine or pine; Jiawei is a five-leaf pine, long-leaf pine, Huashan pine, five-pin pine, two-leaf pine, masson pine, or white pine; special is five-leaf pine, huashan pine, five-pin pine, masson pine, or white pine; the best is five-leaf pine, Huashan pine, Or masson pine.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其進一步包含一誘導步驟,係將葡萄糖水加入培養 步驟(c)所得之癒傷組織,再以紫外光照射一適當時間後,置於暗室中培養。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, which further comprises an inducing step of adding glucose water to the culture. The callus obtained in the step (c) is irradiated with ultraviolet light for a suitable period of time, and then cultured in a dark room.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,其中該紫外光之波長範圍為在220nm至350nm之範圍;較佳為在220nm至330nm之範圍;更佳為在240nm至310nm之範圍;更佳為在240nm至290nm之範圍;最佳為在240nm至280nm之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the ultraviolet light has a wavelength in the range of 220 nm to 350 nm; preferably in the range of 220 nm to 330 nm; Preferably, it is in the range of 240 nm to 310 nm; more preferably in the range of 240 nm to 290 nm; most preferably in the range of 240 nm to 280 nm.
根據本發明之一觀點可以提供一種富含類二苯乙烯的植物癒傷組織之培養方法,上述之適當時間一般為10至60分鐘之範圍;較佳為10至50分鐘之範圍;更佳為10至40分鐘之範圍;最佳為20至40分鐘之範圍。 According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, and the above-mentioned appropriate time is generally in the range of 10 to 60 minutes; preferably in the range of 10 to 50 minutes; more preferably A range of 10 to 40 minutes; preferably a range of 20 to 40 minutes.
另外,本發明可以提供一種富含類二苯乙烯之植物癒傷組織,其係由上述所記載之培養方法而得,且該癒傷組織中含有兒茶素、原花青素、類二苯乙烯化合物、及彼等混合物中之至少一種。 In addition, the present invention can provide a stilbene-rich plant callus obtained by the above-described culture method, and the callus contains catechin, proanthocyanidins, stilbene-like compounds, And at least one of the mixtures thereof.
根據本發明之其他實施例可以提供一種富含類二苯乙烯之植物癒傷組織,其係由上述所記載之培養方法而得,且該植物癒傷組織中的兒茶素之含量為3.0至4.7mg/Kg植物癒傷組織乾重之範圍;較佳為3.06至4.3mg/Kg植物癒傷組織乾重之範圍;更佳為3.20至4.16之範圍。 According to another embodiment of the present invention, a stilbene-rich plant callus obtained by the culture method described above may be provided, and the catechin content of the plant callus is 3.0 to The range of 4.7 mg/Kg plant callus dry weight; preferably from 3.06 to 4.3 mg/Kg of plant callus dry weight; more preferably from 3.20 to 4.16.
根據本發明之其他實施例可以提供一種富含類二苯乙烯之植物癒傷組織,其係由上述所記載之培養方法而得,且該植物癒傷組織中的原花青素之含量為1.1至2.5mg/Kg植物癒傷組織乾重之範圍;較佳為1.17至2.19之範圍;更佳為1.43至1.93之範圍。 According to another embodiment of the present invention, a stilbene-rich plant callus can be provided, which is obtained by the culture method described above, and the proanthocyanidin content in the plant callus is 1.1 to 2.5 mg. /Kg plant callus dry weight range; preferably in the range of 1.17 to 2.19; more preferably in the range of 1.43 to 1.93.
根據本發明之其他實施例可以提供一種富含類二苯乙烯之植物癒傷組織,其係由上述所記載之培養方法而得,且該植物癒傷組織中的類二苯乙烯之含量0.2至0.8mg/Kg植物癒傷組織乾重之範圍;較佳為0.35至0.65mg/Kg植物癒傷組織乾重之範圍;更佳為0.44至0.62之範圍。 According to another embodiment of the present invention, a stilbene-rich plant callus can be provided, which is obtained by the above-described culture method, and the stilbene content in the plant callus is 0.2 to The range of 0.8 mg/Kg plant callus dry weight; preferably from 0.35 to 0.65 mg/Kg of plant callus dry weight; more preferably from 0.44 to 0.62.
又,本發明可以提供一種類二苯乙烯之製備方法,其係包括以下步驟: Moreover, the present invention can provide a method for preparing stilbene, which comprises the following steps:
(a)活化步驟:將松屬植物的種子銳角處製造一切口,並將該種子放入活化液中以使該種子中之種胚活化; (a) activating step: making a mouth at an acute angle of the seeds of the pine plant, and placing the seed in an activating solution to activate the seed embryo in the seed;
(b)逆境處理步驟:將活化後之種胚從種子中取出,並於該種胚的胚軸處製造一創傷缺口; (b) a stress treatment step: removing the activated embryo from the seed and creating a wound gap at the hypocotyl of the embryo;
(c)培養步驟:將上述逆境處理步驟(b)中所得之種胚放入培養基中進行培養,該創傷缺口處會因細胞增生而形成癒傷組織。 (c) culturing step: the embryo obtained in the above-mentioned stress treatment step (b) is cultured in a medium in which callus is formed due to cell proliferation.
(d)萃取步驟:將上述誘導步驟(c)所得之癒傷組織進行萃取以得到類二苯乙烯化合物。 (d) Extraction step: The callus obtained by the above induction step (c) is subjected to extraction to obtain a stilbene-like compound.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中上述之萃取步驟(d)係將誘導步驟(c)所得之癒傷組織切碎後加入甲醇中進行研磨,然後以孔徑為0.22μm之濾碟過濾收集澄清之濾液。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the extracting step (d) is carried out by chopping the callus obtained in the inducing step (c), adding it to methanol, and then grinding. The clear filtrate was collected by filtration through a 0.22 μm pore size filter.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該活化液為過氧化氫水溶液。 According to another embodiment of the present invention, a method for producing a stilbene-like styrene may be provided, wherein the activation liquid is an aqueous hydrogen peroxide solution.
根據本發明之另一實施例可以提供一種類二苯乙烯之製備方法,上述之過氧化氫水溶液的濃度並未特別加以限定,一般為0.5至3%之範圍;較佳為0.5至2%之範圍;特佳為0.5至1.5%之範圍;最佳為1%。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided. The concentration of the above aqueous hydrogen peroxide solution is not particularly limited, and is generally in the range of 0.5 to 3%; preferably 0.5 to 2%. Range; particularly preferably in the range of 0.5 to 1.5%; optimally 1%.
根據本發明之另一實施例可以提供一種類二苯乙烯之製備方法,上述活化步驟(a)中的活化時間並未特別加以限定,一般為1至5天,最佳為3天。 According to another embodiment of the present invention, a method for producing stilbene-like styrene may be provided, and the activation time in the above activation step (a) is not particularly limited, and is usually 1 to 5 days, preferably 3 days.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該培養基包括固態培養基或液態培養基。 According to another embodiment of the present invention, a method for producing stilbene-like can be provided, wherein the medium comprises a solid medium or a liquid medium.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該固態培養基為LPG固態培養基。 According to another embodiment of the present invention, a method for preparing a stilbene-like styrene may be provided, wherein the solid medium is an LPG solid medium.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基係由蔗糖、6-芐氨基嘌呤(6-Benzylaminopurine)、2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)、麩醯胺酸(Glutamine)、Sigma type agar及彼等混合物中之至少一種所組成。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the liquid medium is composed of sucrose, 6-Benzylaminopurine, 2,4-dichlorophenoxyacetic acid (2, 4-dichlorophenoxyacetic acid, at least one of Glutamine, Sigma type agar, and mixtures thereof.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基中之蔗糖的濃度並未特別加以限定,一般為在1至10%之範圍;較佳為在1至8%之範圍;更佳為在1至6%之範圍;特佳為在2至5%之範圍;最佳為3%。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of sucrose in the liquid medium is not particularly limited, and is generally in the range of 1 to 10%; preferably at 1 to A range of 8%; more preferably in the range of 1 to 6%; particularly preferably in the range of 2 to 5%; most preferably 3%.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基中之6-芐氨基嘌呤(6-Benzylaminopurine)的濃度並未特別加以限定,一般為在2μM至10μM之範圍;較佳為在2至8μM之範圍;更佳為在2至6μM之範圍;特佳為在2至5μM之範圍;最佳為3至5μM之範圍。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of 6-Benzylaminopurine in the liquid medium is not particularly limited, and is generally from 2 μM to 10 μM. The range; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基中之2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)的濃度並未特別加以限定,一般為在2μM至10μM之範圍;較佳為在2至8μM之範圍;更佳為在2至6μM之範圍;特佳為在2至5μM之範圍;最佳為3至5μM之範圍。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein a concentration of 2,4-dichlorophenoxyacetic acid in the liquid medium is not particularly limited. It is generally in the range of 2 μM to 10 μM; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基中之麩醯胺酸(Glutamine)的濃度並未特別加以限定,一般為在2μM至20μM之範圍;較佳為在4至18μM之範圍;更佳為在6至16μM之範圍;特佳為在8至14μM之範圍;最佳為8至12μM之範圍。 According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of glutamic acid in the liquid medium is not particularly limited, and is generally in the range of 2 μM to 20 μM; Preferably, it is in the range of 4 to 18 μM; more preferably in the range of 6 to 16 μM; particularly preferably in the range of 8 to 14 μM; most preferably in the range of 8 to 12 μM.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該液態培養基的pH值並未特別加以限定,一般為在5.0至7.0之範圍;較佳為在5.0至6.5之範圍;最佳為在5.5至6.5之範圍。 According to another embodiment of the present invention, a method for preparing a stilbene-like syrup may be provided, wherein the pH of the liquid medium is not particularly limited, and is generally in the range of 5.0 to 7.0; preferably in the range of 5.0 to 6.5. The best is in the range of 5.5 to 6.5.
根據本發明中另一實施例可以提供一種類二苯乙烯之製備方法,其中該松屬植物為五葉松、赤松、山松、刺果松、長葉松、華山松、五針松、大果松、紅松、二葉松、馬尾松、或白皮松;較佳為五葉松、山松、長葉松、華山松、五針松、大果松、二葉松、馬尾松、或白皮松;更佳為五葉松、長葉松、華山松、五針松、二葉松、馬尾松、或白皮松;特佳為五葉松、華山松、五針松、馬尾松、或白皮松;最佳為五葉松、華山松、或馬尾松。 According to another embodiment of the present invention, a method for preparing stilbene-like stilbene can be provided, wherein the pine plant is Pinus sylvestris, Pinus koraiensis, Pinus koraiensis, Pinus sylvestris, Pinus sylvestris, Pinus sylvestris var. , Korean pine, pine, pine, or white pine; preferably pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine Ye Song, Huashan Pine, Wuzheng Pine, Pinus sylvestris, Pinus massoniana, or Pinus bungeana; Tejia Pine, Pinus armandi, Pinus sylvestris, Pinus massoniana, or Pinus bungeana; the best is Pinus bungeana, Pinus armandi, or Pinus massoniana.
圖1為本發明有關之實施例中植物癒傷組織的培養過程圖。 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the cultivation process of plant callus in the relevant embodiment of the present invention.
圖2為本發明有關之實施例中線上電化學式抗氧化活性分析並聯電噴灑質譜裝置示意圖。 2 is a schematic diagram of an on-line electrochemical antioxidant activity analysis parallel electrospray mass spectrometer in accordance with an embodiment of the present invention.
圖3為本發明有關之實施例中利用線上電化學式抗氧化活性分析系統的UV吸收圖譜及電化學掃描圖譜。 Figure 3 is a diagram showing the UV absorption spectrum and electrochemical scanning spectrum of an on-line electrochemical antioxidant activity analysis system in accordance with an embodiment of the present invention.
圖4為本發明有關之實施例中之植物癒傷組織經紫外光照射後,靜置不同時間的螢光圖譜。 Fig. 4 is a view showing the fluorescence spectrum of the plant callus in the relevant embodiment of the present invention after being irradiated with ultraviolet light for a different period of time.
以下,針對本發明的實施態樣列舉不同的具體實施例而更加詳盡地敘述與說明,以便使本發明的精神與內容更為完備而易於瞭解;然 而,本項技藝中具有通常知識者應當明瞭本發明當然不受限於此等實例而已,亦可利用其他相同或均等的功能與步驟順序來達成本發明。 Hereinafter, the embodiments of the present invention will be described in more detail with reference to the specific embodiments, so that the spirit and content of the present invention are more complete and easy to understand; It is to be understood by those skilled in the art that the present invention is not limited to the examples, and other equivalent or equivalent functions and steps can be used to achieve the invention.
《實施例1》 "Embodiment 1"
取25粒之台灣華山松種子,先於種子之銳角處製造一可以肉眼觀察到種胚之切口,如圖1a所示;接著,再將有切口之種子轉移至150毫升之1%過氧化氫水溶液(須每日更換過氧化氫水溶液以活化種胚),藉以活化種胚之生長活性,如圖1b所示。經活化處理後之種胚部位可明顯觀察到其開始膨大,代表種胚之生長活性已成功被活化且細胞開始進行分裂及生長。待三日後,將種子去除種殼後先以20%漂白水消毒3分鐘,再以70%乙醇水溶液消毒一分鐘。接著,再以無菌水清洗3次後,先於胚軸處製造一缺口,並利用解剖刀取出種胚並置於培養基上,如圖1c所示,然後每4週進行一次繼代。 Take 25 capsules of Taiwanese Pinus armandii seeds, and make an incision that can be observed by the naked eye at the acute angle of the seed, as shown in Figure 1a. Next, transfer the incisioned seeds to 150 ml of 1% hydrogen peroxide. An aqueous solution (aqueous hydrogen peroxide solution is required to be activated daily to activate the embryo) to activate the growth activity of the embryo, as shown in Figure 1b. After the activation treatment, the embryo part was obviously observed to expand, and the growth activity of the embryo was successfully activated and the cells began to divide and grow. After three days, the seeds were removed from the seed coat and then sterilized with 20% bleach for 3 minutes and then sterilized with a 70% aqueous solution of ethanol for one minute. Next, after washing three times with sterile water, a notch was made at the hypocotyl, and the embryos were taken out using a scalpel and placed on a medium, as shown in Fig. 1c, and then subcultured every 4 weeks.
上述之培養基係以LPG為基礎固態培養基並添加3%之蔗糖、4.4μM之6-芐氨基嘌呤(6-Benzylaminopurine)、4.5μM之2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)及10μM之麩醯胺酸(Glutamine),將培養基的pH值調整至為5.8後加入0.6%之洋菜(sigma type agar),並置於高壓滅菌爐中以1.1Kg/cm2之壓力、121℃之溫度下進行滅菌15分鐘。 The above medium is a solid medium based on LPG and added with 3% sucrose, 4.4 μM of 6-Benzylaminopurine, and 4.5 μM of 2,4-dichlorophenoxyacetic acid. And 10 μM of glutamic acid (Glutamine), the pH of the medium was adjusted to 5.8, then 0.6% sigma type agar was added, and placed in an autoclave at a pressure of 1.1 Kg/cm 2 , 121 Sterilize at a temperature of °C for 15 minutes.
經3天之培養後,可於種胚之創傷缺口處觀察到新生之細胞組織,如圖1d所示;再經一週之培養後,明顯可觀察到細胞之增生且膨大之情形,如圖1e所示。 After 3 days of culture, the new cell tissue can be observed in the wound gap of the embryo, as shown in Figure 1d; after a week of culture, the proliferation and enlargement of the cells can be clearly observed, as shown in Figure 1e. Shown.
然後,將新生之細胞組織取下後移至其他培養盤中,經6個月的繼代後,便完成華山松種胚癒傷組織之培養,所得之癒傷組織如圖1f所示。 Then, the newborn cell tissue was removed and transferred to other culture dishes. After 6 months of subculture, the callus culture of Pinus armandii was performed, and the resulting callus was as shown in Fig. 1f.
接著,秤取1.0g之癒傷組織置於研缽之中,加入2毫升之60%甲醇水溶液並進行研磨,將萃取物以滴管取出後,再以1毫升之60%甲醇水溶液將殘留於研缽中之樣品洗出後,先將萃取原液置於20毫升之樣品瓶中沉澱10分鐘待其澄清後,再取其上清液以0.22μm濾碟過濾。濾液收集後以定量瓶定量至5mL。 Next, 1.0 g of the callus was weighed and placed in a mortar, 2 ml of a 60% aqueous methanol solution was added and ground, and the extract was taken out as a dropper, and then left in 1 ml of a 60% aqueous methanol solution. After the sample in the mortar was washed out, the extract was placed in a 20 ml sample vial for 10 minutes to be clarified, and then the supernatant was filtered through a 0.22 μm filter. The filtrate was collected and quantified to 5 mL in a quantitative bottle.
然後,將所得濾液進行結構分析及抗氧化物質活性分析;如圖2所示,實驗儀器配置分為兩部份,樣品進樣後,利用HPLC pump推動移動相來帶動樣品進入層析管柱(Agilent Poroshell 120 PFP 4.6*150mm,2.7μm)分離,樣品經管柱分離後進入200:1分流器(QuickSplitTM)進行分流。 Then, the obtained filtrate is subjected to structural analysis and activity analysis of antioxidant substances; as shown in FIG. 2, the experimental instrument configuration is divided into two parts, and after the sample is injected, the mobile phase is pushed by the HPLC pump to drive the sample into the chromatography column ( Agilent Poroshell 120 PFP 4.6*150mm, 2.7μm) was separated and the sample was separated by a column and passed through a 200:1 splitter (QuickSplitTM) for splitting.
第一部份,進入紫外光-可見光偵測器偵測吸收值後直接進入電化學偵測器(ultraviolet-visible detector,Agilent HPLC 1200 Series instrument(Agilent Technologies,Waldbronn,Germary),G1314B UV-vis detector)偵測280nm吸收值後再進入電化學偵測器,以安培法作為分析方式施加氧化電壓0.7V偵測成分氧化電量。 The first part enters the ultraviolet-visible light detector to detect the absorption value and directly enters the electrochemical detector (Agilent HPLC 1200 Series instrument (Agilent Technologies, Waldbronn, Germary), G1314B UV-vis detector After detecting the absorption value at 280 nm, it enters the electrochemical detector, and the oxidizing voltage is applied to the oxidizing voltage of 0.7 V by the amperometric method.
第二部份則將流洗液進樣至電噴灑游離離子阱質譜儀(electrospray ionization-ion trap mass spectrometer,Finnigan LCQ advantage Electrospray ion-trap mass spectrometer(Thermal,San Jose,CA,USA))偵測,而質譜條件為:於負電模式下進行偵測,屏蔽氣流(sheath gas)流速為30arb,輔助氣體(auxiliary gas)流速為0arb,噴灑電壓(spray voltage)為3.25kV,毛細管溫度(capillary temp)為220℃。並以碰撞誘導解離(collision induced dissociation,CID)進行二次或多次質譜,以產生可作為判斷之離子碎片來進行結構鑑定。 In the second part, the flow washing liquid is injected into an electrospray ionization-ion trap mass spectrometer (Finnigan LCQ advantage Electrospray ion-trap mass spectrometer (Thermal, San Jose, CA, USA)). The mass spectrometry conditions were: detection in the negative mode, the sheath gas flow rate was 30 arb, the auxiliary gas flow rate was 0 arb, the spray voltage was 3.25 kV, and the capillary temperature (capillary temp) It is 220 °C. Two or more mass spectra were performed by collision induced dissociation (CID) to generate structural identifiable ion fragments for structural identification.
所使用之移動相由含有2.5mM醋酸銨之水溶液之移動相A與同樣含有2.5mM醋酸銨甲醇溶液之移動相B組成;當梯度為0min時, 移動相B為2%,維持5min後,移動相B於85min內以每分鐘上升1%上升至87%,之後移動相B以87%維持5min。 The mobile phase used consisted of mobile phase A containing an aqueous solution of 2.5 mM ammonium acetate and mobile phase B also containing 2.5 mM ammonium acetate in methanol; when the gradient was 0 min, The mobile phase B was 2%. After 5 min, the mobile phase B rose to 1% with an increase of 1% per minute in 85 min, after which the mobile phase B was maintained at 87% for 5 min.
上述之分析方法可直接針對具有抗氧化活性的成分結構進行解析,再經由文獻及資料庫比對以確定其結構,所得之UV吸收圖譜及電化學掃描圖譜如圖3所示;結果顯示,於華山松種胚癒傷組織之萃取物中共發現到八種具抗氧化活性之物質,即為圖3中之編號1-8。 The above analysis method can directly analyze the structure of the component having antioxidant activity, and then compare the literature and the database to determine the structure thereof, and the obtained UV absorption spectrum and electrochemical scanning spectrum are shown in FIG. 3; Eight kinds of substances with antioxidant activity were found in the extract of callus of Pinus armandii, which is numbered 1-8 in Figure 3.
接著,將鑑定結果與其抗氧化活性能力依層析條件中滯留時間(retention time;R.t.))之先後進行排列,紀錄於表1中。 Next, the identification results and their antioxidant activity capacities were ranked according to the retention time (R.t.) in the chromatographic conditions, and are recorded in Table 1.
結果顯示,在華山松種胚癒傷組織之萃取物中共發現到八種具抗氧化活性之物質,其中包含有兩種兒茶素類化合物,分別為表沒食子兒茶素(epigallocatechin,編號1)與兒茶素(catechin,編號5),由於兒茶素類化合物於以往文獻以證實其具有良好的生理活性,對人體健康之促進有極大的助益,顯示此癒傷組織對於成為保健產品之原料具有其可行性。 The results showed that eight kinds of antioxidant active substances were found in the extracts of callus of Pinus armandii, including two catechins, epigallocatechin (epigallocatechin). 1) With catechin (No. 5), since catechins have been proven to have good physiological activity in the past literature, they have greatly contributed to the promotion of human health, indicating that the callus is becoming health care. The raw materials of the products have their feasibility.
同樣的,於萃取物之分析圖譜中同樣發現由兒茶素所聚合而成之原花青素化合物存在,分別為Proanthocyanidin trimer isomer-1及-2(編號3及6)及B-type proanthocyanidinisomer-1及-2(編號2及4),原花青素除其具有高水溶性且抗氧化活性強之優點外,更可用於輔助維生素C與E之用。 Similarly, in the analysis of the extract, the proanthocyanidin compounds synthesized by catechin were also found to be Proanthocyanidin trimer isomer-1 and -2 (Nos. 3 and 6) and B-type proanthocyanidinisomer-1 and - 2 (Nos. 2 and 4), proanthocyanidins can be used to supplement vitamin C and E in addition to its high water solubility and strong antioxidant activity.
再者,除了觀察到兒茶素及其聚合物原花青素之存在外,亦從圖譜中比對到兩種類二苯乙烯化合物,其為白藜蘆醇(resveratrol)之衍生物,經比對結構後可知此兩種類二苯乙烯化合物分別為Resveratrol-hexose(編號7)與Methoxyresveratrol-hexose(編號8)。 Furthermore, in addition to the observation of the presence of catechin and its polymer proanthocyanidins, two stilbene-like compounds were also compared from the map, which are derivatives of resveratrol, after alignment It is known that these two stilbene-like compounds are Resveratrol-hexose (No. 7) and Methoxyresveratrol-hexose (No. 8), respectively.
然後,分別以標準品溶劑換算上述八種活性物質的含量,並紀錄於表2中。 Then, the contents of the above eight active substances were converted into standard solvents, respectively, and are reported in Table 2.
由上述結果可知:華山松種胚癒傷組織不僅含有常見之抗氧化物質兒茶素,更含有對人體生理活性具有極大助益之原花青素及類二苯乙烯化合物,且因其具有可大量生產之優點,若配合適當且系統之管理生產,將有助於華山松相關保健產品之生產及品質之提升。 It can be seen from the above results that the callus of Pinus armandii has not only the common antioxidant substance catechin, but also the proanthocyanidin and stilbene-like compounds which are very beneficial to the physiological activity of human body, and because of its mass production. Advantages, if properly managed and systematically managed, will contribute to the improvement of the production and quality of Huashan Pine related health care products.
《實施例2-4》 <<Example 2-4》
與實施例1同樣地培養華山松種子之癒傷組織,其差異在於實施例2-4的繼代時間為1個月,並在無菌條件下將2毫升之20g/L葡萄糖水溶液加入癒傷組織的培養基中,再以無菌操作平台(laminar flow)之紫外燈以波長254nm照射30分鐘後取出,分別置於暗室中以室溫進行培養12、24及96小時,然後將癒傷組織取出進行萃取。 The callus of Pinus armandii seeds was cultured in the same manner as in Example 1 except that the subculture time of Example 2-4 was 1 month, and 2 ml of 20 g/L aqueous glucose solution was added to the callus under aseptic conditions. The medium was irradiated with a laminar flow ultraviolet lamp at a wavelength of 254 nm for 30 minutes, and then taken out in a dark room at room temperature for 12, 24, and 96 hours, and then the callus was taken out for extraction. .
秤取1g之癒傷組織置於研缽之中,加入2毫升之60%甲醇水溶液並進行研磨,將萃取物以滴管取出後,再以1毫升之60%甲醇水溶 液將殘留於研缽中之樣品洗出後,先將萃取原液置於20毫升之樣品瓶中沉澱10分鐘待其澄清後,再取其上清液以0.22μm濾碟過濾。 1 g of calli was placed in a mortar, 2 ml of 60% aqueous methanol solution was added and ground, and the extract was taken out as a dropper, and then dissolved in 1 ml of 60% methanol. After washing the sample remaining in the mortar, the extract was placed in a 20 ml sample vial for 10 minutes to be clarified, and then the supernatant was filtered through a 0.22 μm filter.
濾液收集後以定量瓶定量至5mL並採用螢光偵檢器配合快速篩檢之流洗梯度分析類二苯乙烯化合物之含量,所使用之移動相組合為含有0.01%甲酸之水溶液(移動相A)與含有0.01%甲酸之氰甲烷溶液(移動相B)。所使用之梯度為0min時,移動相B為20%,於20min內上升至32%,再於10min內以上升至90%之後,移動相B以90%維持10min。 After the filtrate was collected, the quantitative flask was quantified to 5 mL and the content of the stilbene compound was analyzed by a flow detector with a flow detector gradient analysis. The mobile phase combination used was an aqueous solution containing 0.01% formic acid (mobile phase A). ) with a solution of 0.01% formic acid in cyanide (mobile phase B). When the gradient used was 0 min, the mobile phase B was 20%, rose to 32% in 20 min, and then moved to 90% in 10 min, and the mobile phase B was maintained at 90% for 10 min.
由圖4之螢光圖譜中可觀察到隨著靜置時間的增加,resveratrol-hexose之波峰面積並無太明顯之變化,但另一化合物methoxyresveratrol-hexose之波鋒面積會隨靜置時間變長而增加。 It can be observed from the fluorescence spectrum of Fig. 4 that the peak area of resveratrol-hexose does not change significantly with the increase of the standing time, but the wave front area of another compound methoxyresveratrol-hexose will become longer with the standing time. And increase.
接著,將結果積分並進行統計分析,並將結果紀錄於表3
由表3結果可知:Resveratrol-hexose於靜置不同時間後所得之積分面積並無明顯差異,意即該化合物之含量並未變化,顯示出癒傷組織生成Resveratrol-hexose之誘導原因並非為紫外光,其因素或為病原菌或其他化學物質。然而,隨照光後靜置時間之增加,另一類二苯乙烯化合物Methoxyresveratrol-hexose之波峰積分面積亦隨之增加,且於96小時後達到最大值,由此結果可知:照射紫外光可有效誘導華山松種胚癒傷 組織生成Methoxyresveratrol-hexose,進而提升其含量以達到大量生產之目的。 It can be seen from the results in Table 3 that there is no significant difference in the integrated area of Resveratrol-hexose after standing for different time, which means that the content of the compound does not change, indicating that the cause of callus formation of Resveratrol-hexose is not ultraviolet light. The factors are either pathogens or other chemicals. However, with the increase of the rest time after illumination, the peak integration area of another type of stilbene compound Methoxyresveratrol-hexose also increased, and reached the maximum value after 96 hours. From this result, it is known that ultraviolet light can effectively induce Huashan. Pine embryo callus The tissue produces Methoxyresveratrol-hexose, which in turn increases its content for mass production purposes.
當可理解上述實施方式與實施例僅為例示,且熟習此技藝者可對其進行各種修飾。上文提出之說明書、實施例與資料的目的在於使本說明書的結構完備,並作為實作本發明之例示。雖然本揭示內容已以實施方式揭露如上,然其並非用以限定本揭示內容,任何熟習此技藝者,在不脫離本揭示內容之精神和範圍內,當可作各種之更動與潤飾,因此本揭示內容之保護範圍當視後附之申請專利範圍所界定者為準。 It is to be understood that the above-described embodiments and examples are merely illustrative, and various modifications may be made by those skilled in the art. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.
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TW201100538A (en) * | 2009-06-25 | 2011-01-01 | Nat Univ Chiayi | Method for producing piceatannol and resveratrol using chitin |
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TW201100538A (en) * | 2009-06-25 | 2011-01-01 | Nat Univ Chiayi | Method for producing piceatannol and resveratrol using chitin |
CN104099287A (en) * | 2014-07-07 | 2014-10-15 | 福建省农业科学院农业工程技术研究所 | Acquiring and subculture maintaining method for high yield oligomeric proanthocyanidins vitis davidii callus |
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