TWI737639B - 藉由酶法從人蔘的皂苷選擇性地製備化合物k 及化合物y 的方法 - Google Patents
藉由酶法從人蔘的皂苷選擇性地製備化合物k 及化合物y 的方法 Download PDFInfo
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- TWI737639B TWI737639B TW105134031A TW105134031A TWI737639B TW I737639 B TWI737639 B TW I737639B TW 105134031 A TW105134031 A TW 105134031A TW 105134031 A TW105134031 A TW 105134031A TW I737639 B TWI737639 B TW I737639B
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- ginseng
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- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 48
- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 48
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- HYPFYJBWSTXDAS-UHFFFAOYSA-N Ginsenoside Rd Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C4CCC5C(C)(C)C(CCC5(C)C4CC(O)C23C)OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C HYPFYJBWSTXDAS-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
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- PFSIGTQOILYIIU-UHFFFAOYSA-N ginsenoside Rb3 Natural products CC(=CCCC(C)(O)C1CCC2(C)C3CCC4C(C)(C)C(CCC4(C)C3CC(OC5OC(COC6OCC(O)C(O)C6O)C(O)C(O)C5O)C12C)OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C PFSIGTQOILYIIU-UHFFFAOYSA-N 0.000 description 2
- JDCPEKQWFDWQLI-LUQKBWBOSA-N ginsenoside Rc Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@@H](CO)[C@H](O)[C@H]1O JDCPEKQWFDWQLI-LUQKBWBOSA-N 0.000 description 2
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Abstract
本發明涉及從人蔘的皂苷選擇性地製備原本以微量存在於人蔘中的化合物K及化合物Y的方法,更詳細地,涉及使用特定酶對獲自人蔘的皂苷進行處理來轉換所述皂苷的結構,從而能夠以高收率獲得所需的目標化合物,即能夠以高收率獲得化合物K及化合物Y的方法。
Description
本發明涉及從人蔘的皂苷選擇性地製備原本以微量存在於人蔘中的化合物K及化合物Y的方法,更詳細地,涉及使用特定酶對獲自人蔘的皂苷進行處理來轉換所述皂苷的結構,從而能夠以高收率獲得所需的目標化合物,即能夠以高收率獲得化合物K及化合物Y的方法。
人蔘的皂苷具有與其它植物中發現的皂苷不同的特殊的化學結構,因此其藥理功效也特殊,因此作為人蔘(ginseng)配糖體(glycoside)的意思也被稱為“人蔘皂苷(ginsenoside)”。人蔘的皂苷的具體種類有作為人蔘二醇類的人蔘皂苷Rb1、Rb2、Rc、Rd、化合物K、化合物Mc、化合物O等,及作為人蔘三醇類的人蔘皂苷Re、Rf、Rg1、Rg3、Rg5、Rh1、Rh2等,這些人蔘的皂苷分別顯示出不同的功效。
如上述反應式1中所示,尤其是作為人蔘二醇類皂苷的人蔘皂苷Rb1、Rb2、Rc等能夠藉由微生物代謝而轉換成其它人蔘的皂苷,因此,作為由人蔘的皂苷轉換成其它種類的特定的人蔘的皂苷的方法,一直使用的是利用酶的方法。
然而,就利用現有酶的轉換反應而言,由於酶對基質的非特異性強,導致與使用的皂苷基質相比,需要使用大量的酶,此外,生成的所需人蔘的皂苷的收率非常低。
現有的獲得人蔘的皂苷的方法所採用的解決技術問題的方案為,不是僅轉換成所需的特定人蔘的皂苷,而是藉由萃取等來獲得經過轉換的多種人蔘的皂苷後,對其進行精製來僅分離出所需的人蔘的皂苷。
然而,這種現有方法由於為了獲得純的特定人蔘的皂苷,而需要用於精製的附加費用及時間等,因此只會導致人蔘的皂苷的售價升高,由此使得在相關產品中大量使用人蔘的皂苷方面存在限制。
尤其,在化合物K的情況下,其在由人蔘的皂苷轉換的化合物
的路徑中相當於最後生成的化合物,因此需要能夠使中間代謝物組全部進行反應的多種酶,此外,反應中生成的代謝產物會使得代謝物組和酶之間的反應性降低。不僅如此,中間代謝物組之間的反應液內的凝聚現象會導致收率降低的問題。
現有技術文獻
專利文獻
1.韓國公開專利第10-2014-0107865號(2010年10月06日公開)
採用現有的方法來獲得特定的人蔘的皂苷不僅需要花費大量的費用,還難以大量地獲得所需的人蔘的皂苷。因此,需要提供能夠大量生產目標人蔘的皂苷的同時能夠降低費用的製備方法。
因此,本發明的目的在於,提供能夠以高收率獲得所需的特定人蔘的皂苷且容易實施的人蔘的皂苷轉換方法。
為了實現上述目的,本發明提供以高收率製備化合物K及化合物Y的方法,該方法是利用選自從麯黴屬中分離的果膠酶、柚皮苷酶、纖維素酶及半纖維素酶中的一種以上,及選自從木黴屬中分離的果膠酶及纖維素酶中的一種以上來轉換人蔘的皂苷,從而以高收率地製備化合物K及化合物Y。
利用本發明的轉換為人蔘的皂苷的方法能夠以高收率且容易地獲得所需的人蔘的皂苷。
第1圖為示出藉由矽膠柱色譜法來確認在進行轉換為人蔘的皂
苷的反應之後所生成的化合物K及化合物Y的結果。
較佳實施方式
本發明涉及藉由酶法從人蔘的皂苷選擇性地製備化合物K(化學式1)及化合物Y(化學式2)的方法。
在本發明的方法中,藉由使用源自微生物的酶,使得從人蔘的皂苷,具體為人蔘的人蔘二醇類皂苷,更具體為人蔘皂苷Rb1、Rb2、Rc、Rd或它們的混合物,能夠有效地進行所需人蔘的皂苷的轉換,從而能夠以高收率獲得化合物K及化合物Y。
具體地,本發明中使用的酶較佳獲自麯黴屬的微生物及木黴(Trichoderma)屬的微生物,更佳地,獲自選自黑麯黴(Aspergillus niger)、棘孢麯黴(Aspergillus aculeatus)、琉球麯黴(Aspergillusluchuensis)及米麯黴(Aspergillus oryzae)中的一種以上的麯黴屬微生物,及選自侵佔木黴(Trichoderma aggressivum)、哈茨木黴(Trichoderma harzianum)、里氏木黴(Tricho derma reesei)及綠色木黴(Trichoderma viride)中的一種以上的木黴屬微生物,最佳地,獲自棘孢麯黴及里氏木黴。
此外,本發明中使用的酶可以為由上述微生物分離的柚皮苷酶、半纖維素酶、β-葡聚糖酶、乳糖酶、纖維素酶、β-半乳糖苷酶或果膠酶,較佳為果膠酶、柚皮苷酶、半纖維素酶或纖維素酶。
即使大部分是起相同作用的同一種類的酶,但是根據作為酶的來源的微生物種類,酶在基質內起到作用的部位會不同,由此會產生基質特異性的差異。因此,本發明中最佳同時使用選自從棘孢麯黴中獲得的果膠酶、柚皮苷酶、纖維素酶及半纖維素酶中的一種以上,及選自從里氏木黴中獲得的果膠酶、纖維素酶或它們的混合物中的一種以上。
本發明中,將人蔘的皂苷以0.01~20重量%的量溶解於溶劑中後,使用上述酶並藉由酶法將皂苷轉換成所需的其它皂苷。此時,較佳使用不會降低酶的活性的溶劑,例如可以使用水或緩衝溶液等水性溶劑,或者可以使用水或緩衝溶液等水性溶劑與有機溶劑的混合液。具體地,此時使用的緩衝溶液可以是乙酸、檸檬酸(citrate)、磷酸或檸檬酸-磷酸等,有機溶劑可以是乙腈、二惡烷(dioxane)、二甲基亞碸、甲醇、乙醇、1-丙醇、2-丙醇等。較佳使用的溶劑的pH值範圍為2.5~7.5,較佳為3~6,更佳為3.5~5.5。
在本發明的方法中,相對於所使用的基質的量,所使用的酶的總量較佳為1~500重量%,更佳為10~400重量%,進一步較佳為10~200重量%。
反應溫度應為不會使酶失活的溫度條件,較佳維持30~60℃,更佳為維持35~60℃,進一步較佳為維持40~55℃範圍的溫度。
此外,就反應時間而言,只要是能夠維持酶活性的時間,則不受特別的限制,較佳地,可以攪拌1~120小時,較佳可以攪拌1~96小時,更佳可以攪拌24~96小時,進一步較佳可以攪拌24~72小時並進行反應。
酶反應可以以將2種酶同時添加到基質中的方式進行,或者可以以使一種酶先進行反應後再加入剩餘的酶的按次序方式來實行。
之後,可以使用如在沸騰的水浴槽中進行加熱等習知方法來使酶失活,從而能夠獲得大量含有化合物K及化合物Y的反應液。
具體實施方式
以下,將藉由實施例來更詳細地說明本發明。但是,本發明的範圍並不限定於下述實施例。
參考例1:人蔘精製皂苷的製備
在2kg的紅參、白參、水參、尾參或它們的人蔘葉、人蔘花、人蔘果實中加入20L的乙醇,進行3次回流萃取後,在15℃下浸漬6天。之後,藉由濾布過濾和離心分離對殘渣和濾液進行分離,並對分離的濾液進行減壓濃縮而獲得萃取物,將獲得的萃取物懸浮于水中,然後用1L的乙醚萃取5次,從而去除色素,用1L的1-丁醇對水層進行萃取3次。用5%的KOH對由此獲得的總的1-丁醇層進行處理後,用蒸餾水洗滌,然後進行減壓濃縮來獲得1-丁醇萃取物,將其溶解於少量的甲醇中,然後加入到大量的乙酸乙酯中,並對生成的沉澱物進行乾燥,從而獲得40~80g的人蔘的精製皂苷(包括人蔘皂苷Rb1、Rb2、Rc、Rd、Re、Rg1、Rf等)。
實施例1:藉由酶反應來製備化合物K及化合物Y
將10g的上述參考例1的人蔘精製皂苷(包括人蔘皂苷Rb1、Rb2、Rc、Rd、Re、Rg1、Rf等)溶解於1L的水中。
然後,在上述混合液中同時添加從棘孢麯黴分離的果膠酶和從里氏木黴分離的果膠酶,此時,相對於基質,分別添加100重量%的所述酶,並在30℃下反應72小時。藉由薄層色譜法週期性地進行確認,待基質完全消失後在沸騰水浴槽中加熱10分鐘使得酶失活,從而終止反應。最後,在反應液中以1:1的比例(相對於反應液的體積比)添加乙酸乙酯並萃取3次後,進行濃縮,然後藉由矽膠柱色譜法(氯仿:甲醇=9:1)分離出化合物K及化合物Y(圖1)。
在10g的人蔘皂苷中含有2.66g的人蔘皂苷Rb1、0.73g的人蔘皂苷Rb2、1.23g的人蔘皂苷Rc及0.38g的人蔘皂苷Rd。化合物K是從人蔘皂
苷Rb1和人蔘皂苷Rc及人蔘皂苷Rd中以95%以上的收率轉換,化合物Y是從人蔘皂苷Rb2中以95%以上的收率轉換。
Claims (6)
- 一種製備化合物K及化合物Y的方法,其中,該方法是利用從棘孢麯黴(Aspergillus aculeatus)中分離的果膠酶及從里氏木黴(Trichoderma reesei)中分離的果膠酶來轉換人蔘的皂苷,從而製備化學式1所示之化合物K及化學式2所示之化合物Y;
- 如申請專利範圍第1項所述的方法,其中,同時或依次添加該從棘孢麯黴中分離的果膠酶及從里氏木黴中分離的果膠酶。
- 如申請專利範圍第1項所述的方法,其中,相對於基質的量,該酶的量分別為100重量%。
- 如申請專利範圍第1項所述的方法,其中,該酶的反應是在30℃的溫度下進行。
- 如申請專利範圍第1項所述的方法,其中,該酶的反應進行72小時。
- 如申請專利範圍第1項所述的方法,其中,該溶劑的pH值範圍為3.5~5.5。
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