CN102977165A - Optimized purification preparation method for momordica grosvenori flavine - Google Patents
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Abstract
Description
技术领域 technical field
本发明涉及的是一种生物医药技术领域的方法,具体是一种罗汉果黄素的优化提纯制备方法。The invention relates to a method in the technical field of biomedicine, in particular to an optimized purification and preparation method of mogroside.
背景技术 Background technique
罗汉果黄素为黄酮苷类化合物,目前发现该成分仅局限分布在葫芦科植物罗汉果SiraitiagrosvenoriiSwingl(MomordicagrosvenoriiSwingle)的成熟果实中。其结构式如:Mogrosvenin is a flavonoid glycoside compound, and it is currently found that this component is only distributed in the mature fruit of the Cucurbitaceae plant SiraitiagrosvenoriiSwingl (MomordicagrosvenoriiSwingle). Its structural formula is as follows:
罗汉果为中国特有经济、药用植物。罗汉果黄素为罗汉果的特征性成分,具有抗氧化、抗菌、抗补体等活性,可作为理想的天然黄色素用于食品、饮料的着色剂,色度高,安全无毒副作用;高纯度的罗汉果黄素可作为化学对照品用于罗汉果药材、含罗汉果制剂的定性鉴别及定量分析。罗汉果黄素在罗汉果中含量较低。因此,如何从罗汉果中制备罗汉果黄素具有重要意义。Luo Han Guo is a unique economic and medicinal plant in China. Luo Han Guo flavin is a characteristic component of Luo Han Guo, which has anti-oxidation, antibacterial, anti-complement and other activities. It can be used as an ideal natural yellow pigment for food and beverage colorants. It has high chroma, is safe and has no toxic side effects; Flavin can be used as a chemical reference substance for the qualitative identification and quantitative analysis of Luo Han Guo medicinal materials and preparations containing Luo Han Guo. The content of Luo Han Guo flavins is low in Luo Han Guo. Therefore, how to prepare mogrosides from Luo Han Guo is of great significance.
《药学学报》于1994年2月(29卷第2期),158页-160页,名称为“鲜罗汉果中黄酮甙的分离及结构测定”公开了一种从鲜罗汉果分离罗汉果黄素的方法,即:新鲜罗汉果用水提取,水溶液经大孔树脂柱层析、硅胶柱层析后,重结晶得到罗汉果黄素。这种方法要求使用新鲜罗汉果,而市售商品罗汉果均为干果;同时,采用该方法制备罗汉果黄素收率低于十万分之二,操作繁琐,成本高,不利于大量制备罗汉果黄素。"Acta Pharmaceutica Sinica" in February 1994 (Volume 29, Issue 2), pages 158-160, titled "Isolation and Structure Determination of Flavonoid Glycosides in Fresh Monk Fruit" disclosed a method for isolating mogrosvenin from fresh Luo Han Guo , That is: fresh Luo Han Guo is extracted with water, and the aqueous solution is subjected to macroporous resin column chromatography and silica gel column chromatography, and then recrystallized to obtain Mo Han Guo flavin. This method requires the use of fresh Luo Han Guo, and the commercially available Luo Han Guo is all dried fruit; meanwhile, the yield of Luo Han Guo xanthin prepared by this method is less than 2/100,000.
发明内容 Contents of the invention
本发明针对现有技术存在的上述不足,提出一种罗汉果黄素的优化提纯制备方法,具有纯度高、得率高、工艺简单、成本低等优点。Aiming at the above-mentioned deficiencies in the prior art, the present invention proposes an optimized purification and preparation method of mogroside flavin, which has the advantages of high purity, high yield, simple process, and low cost.
本发明是通过以下技术方案实现的:本发明通过将罗汉果水煮液采用大孔树脂吸附并经洗脱得到罗汉果黄素浸膏,再将罗汉果黄素浸膏进行Sephadex LH-20柱层析并洗脱后收集其中流份并重复层析洗脱操作至少一次,得到纯度大于95%以上的罗汉果黄素精制品,具体步骤如下:The present invention is achieved through the following technical schemes: the present invention obtains Luo Han Guo xanthophyll extract by using macroporous resin to absorb and elute the Luo Han Guo boiled liquid, and then carries out Sephadex LH-20 column chromatography on the Luo Han Guo xanthophyll extract. After elution, the fractions were collected and the chromatographic elution operation was repeated at least once to obtain a refined product of monk fruit xanthophyll with a purity greater than 95%. The specific steps are as follows:
1)先将罗汉果粉碎,加入水煎煮,离心,取上清液;1) First crush the Luo Han Guo, add water to decoct, centrifuge, and take the supernatant;
2)将步骤1)中的上清液过大孔树脂,50%(v/v)酒精洗去杂质后采用60-80%(v/v)酒精洗脱,回收溶剂,收集其中流份,即罗汉果黄素浸膏;2) Pass the supernatant in step 1) through the macroporous resin, wash with 50% (v/v) alcohol to remove impurities, and then use 60-80% (v/v) alcohol to elute, recover the solvent, and collect the fractions. That is, Monk Fruit Extract;
3)将步骤2)所得的罗汉果黄素浸膏,进行Sephadex LH-20柱层析,60%-100%(v/v)的甲醇或其水溶液洗脱,回收甲醇并收集其中流份,得到罗汉果黄素粗品;3) Perform Sephadex LH-20 column chromatography on the mogroside extract obtained in step 2), elute with 60%-100% (v/v) methanol or its aqueous solution, recover methanol and collect the fractions to obtain Crude Monk Fruit Xanthin;
4)取罗汉果黄素粗品进行Sephadex LH-20柱层析,收集其中流份得到纯度大于95%以上的罗汉果黄素精制品。4) Take the crude monk fruit xanthophyll and carry out Sephadex LH-20 column chromatography, collect the fractions to obtain the refined monk fruit xanthin product with a purity greater than 95%.
本发明中:In the present invention:
所述的罗汉果为干燥、成熟果实。Described Momordica grosvenori is dry, ripe fruit.
所述的溶剂即水,提取1-2次。The solvent is water, which is extracted 1-2 times.
所述的大孔树脂为HZ-801或D101型。The macroporous resin is HZ-801 or D101 type.
所述的收集其中流份是指:采用TLC检测,其中:展开剂采用氯仿-甲醇-水为60:35:5的体积比配置;显色剂采用10%(v/v)硫酸-乙醇;通过加热显色进行检测,合并含罗汉果黄素的所有流份。The fractions in the collection refer to: detection by TLC, wherein: the developing agent is configured with a volume ratio of chloroform-methanol-water of 60:35:5; the developing agent is 10% (v/v) sulfuric acid-ethanol; Detection was carried out by heating chromogenicity, and all fractions containing mogroside were combined.
有益效果Beneficial effect
(1)通过罗汉果干果水煎煮、然后将水煎液通过大孔树脂、Sephadex LH-20柱即可得到高纯度罗汉果黄素。采用上述方法制备的罗汉果黄素,纯度高,在95%以上;工艺简便、得率高,可达0.1%以上。(1) High-purity monk fruit flavin can be obtained by decocting dried monk fruit with water, then passing the decoction through macroporous resin and Sephadex LH-20 column. The monk fruit flavin prepared by the above method has a high purity of more than 95%; the process is simple and the yield is high, which can reach more than 0.1%.
(2)采用水煎提取的方法,去除了罗汉果中大部分脂溶性成分(如油脂、蜡质);经济安全,同时也提高了提取的效率。(2) The decoction extraction method is used to remove most of the fat-soluble components (such as oil and wax) in Luo Han Guo; it is economical and safe, and also improves the extraction efficiency.
(3)采用大孔树脂纯化,先用低浓度的乙醇洗脱,除去了大部分皂苷、糖等水溶性杂质的干扰;同时,乙醇又可回收反复使用,节约了成本。(3) Purify with macroporous resin, elute with low concentration of ethanol first, remove most of the interference of saponin, sugar and other water-soluble impurities; at the same time, ethanol can be recycled and reused, saving cost.
(4)罗汉果黄素浸膏先进行1次Sephadex LH-20柱层析,去除了剩余的罗汉果皂苷,方便获得罗汉果黄素粗品;粗品再次进行Sephadex LH-20柱层析,即可获得高纯度的罗汉果黄素。整个过程中样品损失少,Sephadex LH-20填料又可再生后使用、洗脱溶剂甲醇可回收后反复使用,大大降低了成本。(4) Mogroside xanthin extract is subjected to Sephadex LH-20 column chromatography once to remove the remaining mogrosides, and it is convenient to obtain the crude product of Mogrosin xanthin; the crude product is subjected to Sephadex LH-20 column chromatography again to obtain high purity of Mogrosin. During the whole process, the sample loss is small, the Sephadex LH-20 filler can be regenerated and used, and the elution solvent methanol can be recycled and used repeatedly, which greatly reduces the cost.
附图说明 Description of drawings
图1为罗汉果黄素核磁共振图谱:1H-NMR(500MHZ,DMSO-d6)Figure 1 is the nuclear magnetic resonance spectrum of Mogroside flavin: 1 H-NMR (500MHZ, DMSO-d6)
图2为罗汉果黄素核磁共振图谱:13C-NMR(125MHZ,DMSO-d6)Figure 2 is the nuclear magnetic resonance spectrum of Luo Han Guo Xanthophyll: 13 C-NMR (125MHZ, DMSO-d6)
具体实施方式 Detailed ways
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.
实施例1Example 1
本实施例是在以下实施条件和技术要求条件下实施的:This embodiment is implemented under the following conditions of implementation and technical requirements:
1)成熟罗汉果干果200g,粉碎成粗粉,加入1000mL水煎煮0.5h,4800转/分钟离心20min,得水溶液。1) Crush 200g of mature Luo Han Guo dried fruit into a coarse powder, add 1000mL water to decoct for 0.5h, and centrifuge at 4800 rpm for 20min to obtain an aqueous solution.
2)取水溶液,缓慢流过HZ-801大孔树脂(6.5cm×80cm,流速5mL/min),先用50%(v/v)酒精洗脱(流速10mL/min)至洗脱液无色,收集乙醇洗脱液,减压回收乙醇供下次使用;继用60%(v/v)的酒精洗脱(流速10mL/min)至洗脱液无色,收集洗脱液,减压回收乙醇得罗汉果黄素浸膏4.2g。2) Take the aqueous solution and slowly flow it through HZ-801 macroporous resin (6.5cm×80cm, flow rate 5mL/min), first elute with 50% (v/v) alcohol (flow rate 10mL/min) until the eluent is colorless , collect the ethanol eluate, recover the ethanol under reduced pressure for the next use; continue to elute with 60% (v/v) alcohol (flow rate 10mL/min) until the eluate is colorless, collect the eluate, and recover under reduced pressure Ethanol was used to obtain 4.2 g of mogrosin extract.
3)将罗汉果黄素浸膏进行SephadexLH-20柱层析(4.0cm×100cm),60%甲醇洗脱(流速3mL/min,30mL/流份)至洗脱液无色,减压回收甲醇供下次使用,TLC检测(展开剂:氯仿-甲醇-水60:35:5;显色剂:10%硫酸-乙醇;显色方法:加热显色),合并主要含罗汉果黄素流份,减压回收溶剂得罗汉果黄素粗品351mg。3) Carry out the SephadexLH-20 column chromatography (4.0cm×100cm) of Mogroside flavin extract, and elute with 60% methanol (flow rate 3mL/min, 30mL/fraction) until the eluate is colorless, recover methanol under reduced pressure for For the next use, TLC detection (developing agent: chloroform-methanol-water 60:35:5; color developing agent: 10% sulfuric acid-ethanol; color developing method: heating to develop color), combined fractions mainly containing mogroside, recovered under reduced pressure The solvent was used to obtain 351 mg of crude mogroside.
4)罗汉果黄素粗品甲醇溶解,再次加入Sephadex LH-20柱(2.5cm×60cm)中,用60%(v/v)甲醇水溶液洗脱(流速1mL/min,10mL/流份)至洗脱液无色,TLC上述条件检测,合并主要含罗汉果黄素流份,减压回收溶剂得罗汉果黄素精制品219mg,经HPLC检验其纯度为97.1%。4) Dissolve the crude Mogrosvenin in methanol, add it to Sephadex LH-20 column (2.5cm×60cm) again, and elute with 60% (v/v) methanol aqueous solution (flow rate 1mL/min, 10mL/fraction) until elution The liquid was colorless, detected by TLC under the above-mentioned conditions, combined fractions mainly containing monk fruit flavin, and the solvent was recovered under reduced pressure to obtain 219 mg of refined product of monk fruit flavin, whose purity was 97.1% by HPLC.
实施例2Example 2
本实施例是在以下实施条件和技术要求条件下实施的:This embodiment is implemented under the following conditions of implementation and technical requirements:
1)成熟罗汉果干果200g,粉碎成粗粉,加入1000mL水煎煮0.5h,4800转/分钟离心20min,取上清液;药渣再次加水800mL,再次煎煮0.5h,离心,取上清液。合并上清液得罗汉果水溶液。1) 200g of mature Luo Han Guo dried fruit, crushed into a coarse powder, add 1000mL water to decoct for 0.5h, centrifuge at 4800 rpm for 20min, take the supernatant; add 800mL of water to the dregs, decoct again for 0.5h, centrifuge, take the supernatant . The supernatants were combined to obtain the Luo Han Guo aqueous solution.
2)取水溶液,缓慢流经D101大孔树脂柱(6.5cm×80cm,流速5mL/min),先用50%(v/v)酒精洗脱(流速10mL/min)至洗脱液无色,收集乙醇洗脱液,减压回收乙醇供下次使用;继用80%(v/v)的酒精洗脱(流速10mL/min)至洗脱液无色,收集洗脱液,回收乙醇得罗汉果黄素浸膏6.7g。2) Take the aqueous solution and slowly flow it through a D101 macroporous resin column (6.5cm×80cm, flow rate 5mL/min), first elute with 50% (v/v) alcohol (flow rate 10mL/min) until the eluent is colorless, Collect the ethanol eluate, recover the ethanol under reduced pressure for the next use; continue to elute with 80% (v/v) alcohol (flow rate 10mL/min) until the eluate is colorless, collect the eluate, recover the ethanol to get Luo Han Guo Flavin extract 6.7g.
3)将含罗汉果黄素的浸膏进行Sephadex LH-20柱(4.0cm×100cm)层析,100%的甲醇洗脱(流速3mL/min,30mL/流份)至洗脱液无色至洗脱液无色,减压回收甲醇供下次使用,TLC检测主要含罗汉果黄素流份(展开剂:氯仿-甲醇-水60:35:5;显色剂:10%硫酸-乙醇;显色方法:加热显色),合并这些流份,减压回收溶剂得罗汉果黄素粗品508mg。3) Chromatography on Sephadex LH-20 column (4.0cm×100cm) for the extract containing mogrosin, eluting with 100% methanol (flow rate 3mL/min, 30mL/fraction) until the eluate is colorless to elution Deliquification is colorless, and methanol is recovered under reduced pressure for the next use. TLC detection mainly contains mogroside flavin fraction (developing agent: chloroform-methanol-water 60:35:5; color developing agent: 10% sulfuric acid-ethanol; color developing method: Heating for color development), these fractions were combined, and the solvent was recovered under reduced pressure to obtain 508 mg of crude mogroside.
4)罗汉果黄素粗品甲醇溶解,再次加入Sephadex LH-20柱(2.5cm×60cm)中,用100%的甲醇洗脱(流速1mL/min,10mL/流份)至洗脱液无色,TLC上述条件检测主要含罗汉果黄素流份,减压回收溶剂得罗汉果黄素精制品340mg,经HPLC检验其纯度为96.2%。4) Dissolve the crude Mogrosvenin in methanol, add it to a Sephadex LH-20 column (2.5cm×60cm) again, and elute with 100% methanol (flow rate 1mL/min, 10mL/fraction) until the eluent is colorless, TLC The above-mentioned conditions mainly contained the fraction of monk fruit flavin, and the solvent was recovered under reduced pressure to obtain 340 mg of the refined product of monk fruit flavin, whose purity was 96.2% by HPLC.
实施例3Example 3
本实施例是在以下实施条件和技术要求条件下实施的:This embodiment is implemented under the following conditions of implementation and technical requirements:
1)成熟罗汉果干果200g,粉碎成粗粉,加入1000mL水煎煮0.5h,4800转/分钟离心20min,得水溶液。1) Crush 200g of mature Luo Han Guo dried fruit into a coarse powder, add 1000mL water to decoct for 0.5h, and centrifuge at 4800 rpm for 20min to obtain an aqueous solution.
2)取水溶液,缓慢流经D101大孔树脂柱(6.5cm×80cm,流速5mL/min),先用50%(v/v)酒精洗脱(流速10mL/min)至洗脱液无色,收集乙醇洗脱液,减压回收乙醇供下次使用;继用70%乙醇洗脱,收集洗脱液,减压回收乙醇得醇浸膏5.1g。2) Take the aqueous solution and slowly flow it through a D101 macroporous resin column (6.5cm×80cm, flow rate 5mL/min), first elute with 50% (v/v) alcohol (flow rate 10mL/min) until the eluent is colorless, Collect the ethanol eluate, recover the ethanol under reduced pressure for the next use; continue to elute with 70% ethanol, collect the eluate, recover the ethanol under reduced pressure to obtain 5.1 g of alcoholic extract.
3)将含罗汉果黄素的浸膏进行Sephadex LH-20柱层析(4cm×100cm),80%(v/v)的甲醇水溶液洗脱(流速3mL/min,30mL/流份)至洗脱液无色,回收甲醇供下次使用,TLC检测主要含罗汉果黄素流份(展开剂:氯仿-甲醇-水60:35:5;显色剂:10%硫酸-乙醇;显色方法:加热显色),合并这些流份,减压回收溶剂得罗汉果黄素粗品422mg。3) Perform Sephadex LH-20 column chromatography (4cm×100cm) on the extract containing Mogroside The liquid is colorless, and methanol is recovered for the next use. TLC detection mainly contains mogroside flavin fraction (developing agent: chloroform-methanol-water 60:35:5; color developing agent: 10% sulfuric acid-ethanol; color developing method: heating to develop color ), these fractions were combined, and the solvent was recovered under reduced pressure to obtain 422 mg of crude mogroside.
4)罗汉果黄素粗品甲醇溶解,再次加入Sephadex LH-20柱(2.5cm×60cm)中,用80%(v/v)的甲醇水溶液洗脱(流速1mL/min,10mL/流份)至洗脱液无色,TLC上述条件检测主要含罗汉果黄素流份,合并这些流份,减压回收溶剂得罗汉果黄素273mg,经HPLC检验其纯度为96.7%。4) The crude Mogrosvenin was dissolved in methanol, added to a Sephadex LH-20 column (2.5cm×60cm) again, and eluted with 80% (v/v) methanol aqueous solution (flow rate 1mL/min, 10mL/fraction) until washing The deliquified colorless, TLC above-mentioned conditions mainly contain mogroside flavin fractions, these fractions are combined, and the solvent is recovered under reduced pressure to obtain 273 mg of mogroside flavin, the purity of which is 96.7% by HPLC.
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CN105708879A (en) * | 2016-04-26 | 2016-06-29 | 上海交通大学 | Maca extract as well as preparation method and application thereof |
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CN111233958A (en) * | 2020-03-23 | 2020-06-05 | 湖南华诚生物资源股份有限公司 | A kind of Luo Han Guo flavin metal zinc complex and preparation method thereof |
CN111848708A (en) * | 2020-07-27 | 2020-10-30 | 湖南华诚生物资源股份有限公司 | Method for separating various active components such as squalene from Luo Han Guo extraction residue |
CN111848708B (en) * | 2020-07-27 | 2021-07-20 | 湖南华诚生物资源股份有限公司 | Method for separating various active components such as squalene from Luo Han Guo extraction residue |
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