WO2017139962A1 - 红景天中提取草质素的方法 - Google Patents

红景天中提取草质素的方法 Download PDF

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WO2017139962A1
WO2017139962A1 PCT/CN2016/074095 CN2016074095W WO2017139962A1 WO 2017139962 A1 WO2017139962 A1 WO 2017139962A1 CN 2016074095 W CN2016074095 W CN 2016074095W WO 2017139962 A1 WO2017139962 A1 WO 2017139962A1
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extracting
rhodiola
extract
acid
turfgrass
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PCT/CN2016/074095
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English (en)
French (fr)
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顾正兵
张�杰
陶月红
曾铖
徐丽丽
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泰州永恒生物科技有限公司
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Priority to PCT/CN2016/074095 priority Critical patent/WO2017139962A1/zh
Priority to EP16890195.7A priority patent/EP3418274A4/en
Priority to JP2018543185A priority patent/JP6654754B2/ja
Priority to CN201680002944.XA priority patent/CN107074798B/zh
Publication of WO2017139962A1 publication Critical patent/WO2017139962A1/zh
Priority to US16/104,951 priority patent/US10435384B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/41—Crassulaceae (Stonecrop family)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

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  • the invention relates to a method for separating and extracting compounds in the chemical field, in particular to a method for preparing grass quality from Rhodiola, and belongs to the field of medicinal chemistry.
  • Rhodiola The genus Rhodiola is a perennial herb or sub-shrub plant. There are more than 90 species of Rhodiola in the world, and 73 species in China. Among the many species of Rhodiola, the research of scholars at home and abroad mainly focuses on more than ten kinds of Rhodiola, Rhodiola, Rhodiola, Rhodiola, Rhodiola and Rhodiola. kind. The chemical composition of each species of Rhodiola is very different and its efficacy varies.
  • the Rhodiola contained in the 2015 edition of the Chinese Pharmacopoeia is the dry roots and rhizomes of the sedum plant Rhodiola. It is flat, sweet and bitter, and belongs to the lungs and heart.
  • Rhodiola is flavonoids, tyrosol and its glycosides, while flavonoids are mainly grass quality and its corresponding glycosides.
  • Phytoplasma can effectively scavenge DPPH free radicals and hydroxyl radicals and inhibit protein oxidation, and its antioxidant activity is stronger than the corresponding glycosides.
  • studies have shown that the pharmacological activity of the 7-position hydroxyl group of the A ring in the grass quality structure will be reduced, the more sugar, the lower the activity; the antioxidant activity of the flavonoids increases with the increase of the number of phenolic hydroxyl groups in the molecule.
  • phytohemagglutinin can induce apoptosis of human hepatoma cell line HepG2, and has the potential to develop a novel anticancer agent.
  • the grass quality is 3,4',5,7,8-pentahydroxyflavone, which is mainly found in plants of the genus Sedum. It has been found that the content of free phytoene in Rhodiola rosea is very low. We have 12 different sources. The content of free phytochemicals in Rhodiola medicinal herbs (including the Chinese herbal medicines and drug testing institute's batch number 121657-201101 and Rhodiola rosea 121412-200902) The grass quality content is only 0.0022% to 0.061%. The specific data is shown in Table 1.
  • the present invention utilizes acid hydrolysis, polyamide column chromatography, reversed-phase silica gel column chromatography to prepare phytochemicals from Rhodiola medicinal materials, and has a simple process and high yield (Table 1 No. 5, Rhodiola sinensis in Table 1)
  • the content of free turf content is only 0.06%.
  • the content of phytoene in hydrolysate is up to 4.36% and the content is increased by 72 times. It has strong specificity and is suitable for industrial production.
  • In order to explore the antioxidant activity of Rhodiola It provides a solid material basis for anti-tumor and other active ingredients as well as for the development and utilization of turf.
  • the method for extracting grass quality in Rhodiola according to the present invention comprises the following steps;
  • the grass quality extract is treated with a polyamide column chromatography, and the grass-containing fraction is collected and concentrated to dryness to obtain a crude grassy substance;
  • the Rhodiola in the step 1) is oxalic acid-7-O-rhamnoside and phytoene-7-O-(3′′- ⁇ -D-glucosyl).
  • the extraction solvent in the step 1) is selected from a methanol-water solution or a 70.0%-90.0% ethanol-water solution in a mass percentage of 30.0% to 99.9%.
  • the extraction in the step 1) described includes sequential immersion, osmosis, ultrasonication or heating.
  • the extraction in the step 2) is carried out by using petroleum ether having a boiling point of 60-90 ° C, and the ratio of the amount of petroleum ether extracted to the extract of the extract is (1-3): 1 (v). /v).
  • the acid hydrolysis in the step 2) is a direct acid hydrolysis or a two-phase acid hydrolysis method, and the acid hydrolysis uses any one of hydrochloric acid, sulfuric acid and formic acid, and the mass fraction of the acid used is 0.2%- 10.0%.
  • the organic phase in the two-phase acid hydrolysis includes any one of petroleum ether-ethyl acetate (1:1), toluene, and ethyl acetate.
  • the acid reflux temperature is from 50 ° C to 100 ° C
  • the acid hydrolysis time is from 0.5 h to 12.0 h.
  • the step 3) extracting organic solvent is petroleum ether-ethyl acetate (1:1) (v/v) or ethyl acetate, and the ratio of the organic solvent to the acid hydrolysis solution is (1). -3): 1 (v/v), the number of extractions is 3-5 times.
  • the polyamide column chromatography elution conditions in the step 4) are isocratic elution in a volume ratio (1:1) petroleum ether-ethyl acetate.
  • the reversed-phase silica gel column chromatography adopts a C18 reverse phase column
  • the C18 reverse phase column adopts a prepacked column with an inner diameter of 50 mm or a prepacked column with an inner diameter of 100 mm or a DAC preparative column.
  • the eluent of the reverse phase silica gel column chromatography in the step 5) is carried out using aqueous acetonitrile or an aqueous methanol solution, and the recrystallization is carried out using a methanol solution.
  • the invention has the advantages of complicated and complicated process, high cost, long purification cycle, and the like in the preparation of phytochemicals by using the glucosin aglycone as a raw material of the aglycone as a raw material, and the present invention uses Rhodiola as raw material. Preparation of grass quality, simple process, easy operation, economy, high yield, no by-products, environmental protection;
  • the invention adopts polyamide for chromatography as a separation material, has good adsorption performance and separation effect on grass quality, is simple in operation, economical, easy to elute and regenerate, can be repeatedly used, has short cycle, good separation effect, and price Low cost is one of the classical methods for purifying flavonoids; compared with silica gel column chromatography, the phytochemicals have large dead adsorption, serious tailing, low recovery rate and long elution period, which is effectively solved by polyamide column chromatography.
  • the invention adopts reversed-phase silica gel column chromatography, has good separation effect, is easy to operate, and is suitable for industrial production;
  • the present invention obtains high-purity phytoene, and provides a solid material basis for investigating the anti-oxidation of Rhodiola, treating cardiovascular and cerebrovascular diseases, and anti-tumor pharmacological activities.
  • Figure 1 is a chemical structure diagram of grass quality
  • Figure 2 is a chromatogram of pure grass quality
  • Fig. 6 is a chromatogram of detection before and after acid hydrolysis of Rhodiola sachalinensis in Changbai Mountain, Jilin province in Example 2.
  • the above data is consistent with the nuclear magnetic data reported in the literature, and identified as herbacetin.
  • the purity of the grass quality is as follows:
  • the sample is subjected to high performance liquid chromatography under the chromatographic conditions of the mobile phase as acetonitrile (A)-0.1% aqueous acetic acid (B), gradient elution [0 min, AB (8:92) 10 min, AB (31:69); 25 min, AB (45:55)], wavelength 275 nm, Kromasil C18 column (4.6 ⁇ 250 mm, 5 ⁇ m), flow rate 1.0 mL ⁇ min -1 , column temperature 35
  • the sample was detected at °C and the sample was a single symmetrical peak in the HPLC chromatogram.
  • the obtained fractions can be traced by thin layer chromatography.
  • the conditions for developing the thin layer chromatography are: chloroform-methanol-formic acid (12:1:1) ), after deployment, view under UV light.
  • Extract with an equal volume of petroleum ether (60-90 ° C) three times with ethanol extract extract add an equal volume of 1% sulfuric acid and 2 times the volume of ethyl acetate to the extracted aqueous layer, stir , heating and refluxing for two-phase acid hydrolysis treatment, hydrolysis for 12h, cooling, and then extracting the hydrolyzate with petroleum ether-ethyl acetate (1:1) three times, each time petroleum ether-ethyl acetate (1:1)
  • the ratio of the hydrolyzate to the hydrolyzate was 1:1 (v/v), and the extracts were combined and concentrated under reduced pressure to give an extract of 831.2 g.
  • the phytate content of the extract was 11.38%.
  • the percolate was concentrated under reduced pressure to obtain an ethanol extract extract, and the extracts were combined and concentrated under reduced pressure to obtain an ethanol extract extract (relative density: 1.136), using an equal volume of petroleum ether with an ethanol extract extract (60- 90 ° C) extraction 3 times, the volume of the aqueous layer was first added to the volume of 5% hydrochloric acid, then added 2 times the volume of ethyl acetate extract, stirred, heated reflux for acid hydrolysis for 3h, cooling, hydrolysis The liquid was extracted three times with ethyl acetate, and the ratio of the amount of ethyl acetate to the hydrolyzate was 1:1 (v/v). The extract was combined and concentrated under reduced pressure to obtain 1689.4 g of the extract. The quality content was 7.56%.
  • the extract (relative density 1.025) was extracted 3 times with 3 volumes of petroleum ether (60-90 ° C) such as methanol extract extract, and an equal volume of 1% hydrochloric acid was added to the extracted aqueous layer. , stirring, heating and refluxing for acid hydrolysis treatment, hydrolysis for 1 h, cooling, adding petroleum ether-ethyl acetate (1:1) to the hydrolyzate for 5 times, each time petroleum ether-ethyl acetate (1:1) The ratio of the hydrolyzate to the hydrolyzate was 1:1 (v/v), and the extract was combined and concentrated under reduced pressure to give 402.7 g of an extract. The phytate content of the extract was 10.69%.
  • Example 1 831.2 g of the extract in Example 1 was applied to an atmospheric pressure polyamide column having a filler of 200-400 mesh, having an aspect ratio of 1:15, eluted with petroleum ether-ethyl acetate (1:1), and a thin layer. The test was carried out, and the grass-containing fraction portion was collected, and concentrated under reduced pressure to dryness to obtain 167.4 g of crude crude material. Then, the crude grass substance was subjected to column chromatography on a reverse phase DAC preparative column (200 ⁇ 250 mm, 10 ⁇ m), eluted with a mass percentage concentration of 26% acetonitrile solution (containing 0.1% acetic acid), and detected by thin layer chromatography to merge the target stream. The solvent was recovered under reduced pressure to dryness, dissolved in methanol and recrystallized, placed at 4 ° C overnight, and the crystals were filtered and dried under reduced pressure. The quality was 54.5 g and the purity was 90.10%.
  • Example 3 402.7 g of the extract in Example 3 was applied to an atmospheric pressure polyamide column having a filler of 200-400 mesh, having an aspect ratio of 1:14, eluted with petroleum ether-ethyl acetate (1:1), and a thin layer.
  • the chromatographic test was carried out, and the grass-containing fraction was collected and concentrated under reduced pressure to dryness to obtain 90.2 g of crude grass.
  • the grass biomass mother liquor was subjected to column chromatography on a C18 preparative column (100 ⁇ 200 mm, 10 ⁇ m), eluted with a mass percentage concentration of 26% acetonitrile solution (containing 0.1% acetic acid), and detected by thin layer chromatography, and the target fractions were combined.

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Abstract

一种从红景天中提取草质素的方法，包括步骤：1）将药材用提取溶剂进行提取，再浓缩得到提取物浸膏；2）将提取物浸膏萃取，萃取后的水层再进行酸水解；3）将经酸水解后的溶液用有机溶剂萃取，合并萃取液，减压浓缩得到草质素萃取物；4）将草质素萃取物采用聚酰胺柱层析处理，收集含有草质素的流分，浓缩至干，重结晶，得到草质素纯品。

Description

红景天中提取草质素的方法 技术领域
本发明涉及化学领域化合物的分离提取方法，具体涉及从红景天中制备得到草质素的方法，属于药物化学领域。
背景技术
景天科红景天属植物为多年生草本或亚灌木植物，全世界的红景天属植物约有90多种，我国有73种。众多种类的红景天属植物中，国内外学者的研究主要集中于大花红景天、蔷薇红景天、小丛红景天、库页红景天、狭叶红景天以及圣地红景天等十余个种类。每个种类的红景天化学成分差异很大，功效也各异。2015年版《中国药典》收载的红景天为景天科植物大花红景天的干燥根和根茎，性平，味甘、苦，归肺、心经，具有益气活血，通脉平喘之效，用于气虚血瘀，胸痹心痛，中风偏瘫，倦怠气喘，其抗疲劳作用优于人参和刺五加，兴奋智力活动优于刺五加，民间常将其作为滋补强壮药服用以消除疲劳、抵御寒冷。
现代医学表明红景天主要有效成分为黄酮类、酪醇及其苷类，而黄酮主要为草质素及其相应的苷类成分。草质素能有效地清除DPPH自由基和羟自由基及抑制蛋白质氧化的作用，且其抗氧化活性强于相应的苷类成分。同时，研究表明草质素结构中A环7位羟基配糖化后，药理活性将降低，糖越多，活性就越低；黄酮类化合物的抗氧化活性随分子中酚羟基数目的增加，活性增强。此外，草质素能诱导人肝癌细胞HepG2凋亡，具有开发成一种新型抗癌制剂的潜力。
草质素为3,4′,5,7,8-五羟基黄酮，主要存在于景天科植物中，研究发现红景天药材中游离草质素的含量很低，我们对12种不同来源的红景天药材(包括中国食品药品检定研究院提供的批号为121657-201101的红景天和批号为121412-200902的高山红景天对照药材)中游离草质素含量进行分析，得出游离草质素含量仅为0.0022％～0.061％，具体数据见表1。
表1 12种不同来源红景天中游离草质素含量结果
Figure PCTCN2016074095-appb-000002
从红景天药材中直接分离游离草质素难度大，得率低，不合适工业化生产，难以满足科研及新药研发的需求，从而严重限制其深入研究。但景天科植物中以草质素为苷元的苷类成分含量较高，如草质素-7-O-鼠李糖苷、草质素-7-O-(3″-β-D-葡萄糖基)-鼠李糖苷等苷类成分。
目前关于草质素的研究报道较少，且尚未有一种适合产业化生产制备高纯度草质素的工艺路线披露。
发明内容
发明目的：本发明利用酸水解、聚酰胺柱层析、反相硅胶柱层析以红景天药材为原料制备草质素，工艺简单，得率高(表1中西藏5号红景天药材中游离草质素含量仅为0.06％，经酸水解处理后水解液中草质素含量高达4.36％，含量提高了72倍)，专属性强，适合工业化生产，为探究红景天抗氧化、抗肿瘤等活性成分以及为草质素的开发利用提供坚实的物质基础。
技术方案：本发明所述的红景天中提取草质素的方法，包括以下步骤；
1)将粉碎好的红景天药材用提取溶剂进行提取，提取液减压浓缩得到提取物浸膏；
2)将提取物浸膏萃取，萃取后的水层再进行酸水解；
3)将经酸水解后的溶液用有机溶剂萃取，合并萃取液，减压浓缩得到草质素萃取物；
4)将草质素萃取物采用聚酰胺柱层析处理，收集含有草质素的流分，浓缩至干，得到草质素粗品；
5)将草质素粗品采用反相硅胶柱层析处理，收集含有草质素的流分，浓缩至干，重结晶，得到草质素纯品。
为了进一步解决上述问题，所述的步骤1)中的红景天为含草质素-7-O-鼠李糖苷和草质素-7-O-(3″-β-D-葡萄糖基)-鼠李糖苷的总含量不低于0.08％的景天科红景天属植物中的任意一种红景天。
为了进一步解决上述问题，所述的步骤1)中的提取溶剂选取质量百分比为30.0％-99.9％的甲醇-水溶液或者70.0％-90.0％乙醇-水溶液。
为了进一步解决上述问题，所述的步骤1)中的提取包括依次通过浸泡、渗漉、超声或加热回流。
为了进一步解决上述问题，所述的步骤2)中萃取采用沸点为60-90℃石油醚进行萃取，每次萃取石油醚的用量与提取物浸膏的比例为(1-3)：1(v/v)。
为了进一步解决上述问题，所述的步骤2)中酸水解采用直接酸水解或者两相酸水解的方法，酸水解采用盐酸、硫酸、甲酸中任意一种酸，所用酸的质量分数为0.2％-10.0％。
为了进一步解决上述问题，所述的两相酸水解中的有机相包括石油醚-醋酸乙酯(1：1)、甲苯、醋酸乙酯中的任意一种。
为了进一步解决上述问题，所述的步骤2)中酸水解时加热回流温度为50℃-100℃，酸水解的时间为0.5h-12.0h。
为了进一步解决上述问题，所述的步骤3)萃取用有机溶剂为石油醚-醋酸乙酯(1:1)(v/v)或醋酸乙酯，每次有机溶剂与酸水解溶液比例为(1-3)：1(v/v)，萃取次数为3-5次。
为了进一步解决上述问题，所述的步骤4)中所述聚酰胺柱层析洗脱条件为体积比(1:1)石油醚-醋酸乙酯等度洗脱。
为了进一步解决上述问题，所述的步骤5)中反相硅胶柱层析采用C18反相柱，所述的C18反相柱选用内径50mm的预装柱或内径100mm的预装柱或DAC制备柱。
为了进一步解决上述问题，所述的步骤5)中反相硅胶柱层析处理的洗脱剂采用含水的乙腈或者含水的甲醇溶液，所述重结晶采用甲醇溶液。
有益效果：
1)应用本发明，可制备得到高纯度的草质素，草质素含量达到90.0％-99.9％；
2)应用本发明，可得到公斤级的草质素，满足产业化生产；
3)相对于以草质素为苷元的苷类单体成分为原料制备草质素时具有的工艺复杂繁琐且成本高、纯化周期长等弊端而言，本发明以红景天药材为原料制备草质素，工艺简单，操作易行，经济，得率高，无副产物，环保；
4)本发明采用层析用聚酰胺作为分离材料，对草质素有较好的吸附性能和分离效果，操作简便，经济，易洗脱再生，可反复利用，周期短，分离效果好，价格低廉，是纯化黄酮类化合物的经典方法之一；相对于采用硅胶柱层析，草质素死吸附大，拖尾严重，回收率低，洗脱周期长，采用聚酰胺柱层析有效地解决了上述问题；
5)本发明使用反相硅胶柱层析，分离效果好，操作易行，适合工业化生产；
6)本发明得到高纯度的草质素，为探究红景天的抗氧化、治疗心脑血管疾病、抗肿瘤等药理活性研究提供坚实的物质基础。
附图说明
图1是草质素的化学结构图；
图2是草质素纯品色谱图；
图3是实施例1中西藏产红景天药材酸水解前检测色谱图；
图4是实施例1中西藏产红景天药材酸水解后检测色谱图；
图5是实施例2中吉林长白山产高山红景天药材酸水解前检测色谱图；
图6是实施例2中吉林长白山产高山红景天药材酸水解前后检测色谱图。
具体实施方式
为了加深对本发明的理解，下面将结合实施例对本发明作进一步详述，这些实施例仅用于解释本发明，并不构成对本发明保护范围的限定。
草质素化学结构确定(如图1)的方法如下：使用核磁共振波谱仪，对样品进行分析，1H-NMR(400MHz,DMSO-d6):11.91(1H,s,5-OH),10.37(1H,s,7-OH),10.08(1H,s,4′-OH),9.29(1H,s,3-OH),8.62(1H,s,8-OH),8.17(2H,d,J＝8.4Hz,H-2′,6′),6.94(2H,d,J＝8.4Hz,H-3′,5′),6.27(1H,s,H-6)。以上数据与文献报道草质素的核磁数据一致,鉴定为草质素(herbacetin)。
参见图2，草质素纯度检测条件如下：样品经高效液相色谱仪在色谱条件为流动相为乙腈(A)-0.1％醋酸水溶液(B)，梯度洗脱[0min,A-B(8:92)；10min,A-B(31:69)；25min,A-B(45:55)]，波长为275nm,Kromasil C18色谱柱(4.6×250mm，5μm)，流速为1.0mL·min-1，柱温为35℃下检测，样品在HPLC色谱中为单一对称峰。
在聚酰胺柱层析及反相柱层析过程中，可采用薄层层析方法对所得流分进行跟踪，薄层层析用展开剂条件为：氯仿-甲醇-甲酸(12：1：1)，展开后，于紫外灯下检视。
具体实施例1
称取20kg西藏产红景天(经高效液相色谱仪检测草质素-7-O-鼠李糖苷、草质素 -7-O-(3″-β-D-葡萄糖基)-鼠李糖苷的含量分别为0.12％和0.11％，药材液相检测色谱图见图3，酸水解后检测色谱图见图4)，粉碎过20目筛，用10倍量质量分数为70％乙醇溶液加热回流提取2次，每次1.5h，合并提取液并减压浓缩得到乙醇提取物浸膏(相对密度为1.072)，用与乙醇提取物浸膏等体积的石油醚(60-90℃)萃取3次，向萃取后的水层加入等体积的质量分数为1％的硫酸以及2倍浸膏体积的醋酸乙酯，搅拌，加热回流进行两相酸水解处理，水解12h，冷却，然后将水解液用石油醚-醋酸乙酯(1:1)萃取3次，每次石油醚-醋酸乙酯(1:1)的用量与水解液比例为1:1(v/v),合并萃取液，减压浓缩得到831.2g萃取物。经检测，萃取物中草质素含量为11.38％。
具体实施例2
称取80kg吉林长白山产高山红景天(经高效液相色谱仪检测草质素-7-O-鼠李糖苷、草质素-7-O-(3″-β-D-葡萄糖基)-鼠李糖苷的含量分别为0.067％和0.028％，药材液相检测色谱图见图5，酸水解后检测色谱图见图6)，粉碎过20目筛，用12倍量90％乙醇溶液渗漉提取，渗漉液减压浓缩得到乙醇提取物浸膏，合并提取液并减压浓缩得到乙醇提取物浸膏(相对密度为1.136)，用与乙醇提取物浸膏等体积的石油醚(60-90℃)萃取3次，向萃取后的水层先加入体积的质量分数为5％的盐酸，再加入2倍浸膏体积的醋酸乙酯，搅拌，加热回流进行酸水解3h,冷却，将水解液用醋酸乙酯萃取3次，每次醋酸乙酯的用量与水解液比例为1:1(v/v),合并萃取液，减压浓缩得到1689.4g萃取物，经检测，萃取物中草质素含量为7.56％。
具体实施例3
称取10kg云南丽江产红景天(经高效液相色谱仪检测草质素-7-O-鼠李糖苷、草质素-7-O-(3″-β-D-葡萄糖基)-鼠李糖苷的含量分别为0.10％和0.10％)，粉碎过20目筛，用12倍量质量分数为90％甲醇溶液超声提取3次，每次1h，合并提取液并减压浓缩得到甲醇提取物浸膏(相对密度为1.025)，用与甲醇提取物浸膏等3倍体积的石油醚(60-90℃)萃取3次，向萃取后的水层加入等体积的质量分数为1％的盐酸，搅拌，加热回流进行酸水解处理，水解1h，冷却，向水解液中加入石油醚-醋酸乙酯(1:1)萃取5次，每次石油醚-醋酸乙酯(1:1)的用量与水解液比例为1:1(v/v),合并萃取液，减压浓缩得到402.7g萃取物。经检测，萃取物中草质素含量为10.69％。
具体实施例4
将实施例1中831.2g萃取物上样至填料为200-400目的常压聚酰胺柱，径高比为1:15，用石油醚-醋酸乙酯(1:1)洗脱，薄层层析检测，收集含草质素流份段，减压浓缩至干，得到草质素粗品167.4g。然后将草质素粗品经反相DAC制备柱(200×250mm,10μm)柱层析，用质量百分比浓度为26％乙腈溶液(含0.1％醋酸)洗脱，薄层层析检测，合并目标流分，减压回收溶剂至干，用甲醇溶解重结晶处理，4℃放置过夜，滤出晶体经减压干燥得草 质素54.5g，纯度为90.10％。
具体实施例5
将实施例2中563.1g萃取物上样至填料为200-400目的常压聚酰胺柱，径高比为1:17，用石油醚-醋酸乙酯(1:1)洗脱，薄层层析检测，收集含草质素流分段，减压浓缩至干，得到草质素粗品80.1g。将草质素粗品经C18制备柱(50×250mm,10μm)柱层析，用质量百分比浓度为32％甲醇溶液(含0.1％醋酸)洗脱，薄层层析检测，合并目标流分，减压回收溶剂至干，用甲醇溶解重结晶处理，4℃放置过夜，滤出晶体经减压干燥得草质素18.7g，纯度为95.25％。
具体实施例6
将实施例3中402.7g将萃取物上样至填料为200-400目的常压聚酰胺柱，径高比为1:14，用石油醚-醋酸乙酯(1:1)洗脱，薄层层析检测，收集含草质素流分段，减压浓缩至干，得到草质素粗品90.2g。然后将草质素母液经C18制备柱(100×200mm,10μm)柱层析，用质量百分比浓度为26％乙腈溶液(含0.1％醋酸)洗脱，薄层层析检测，合并目标流分，减压回收溶剂至干，用甲醇溶解重结晶处理，4℃放置过夜，滤出晶体经减压干燥得草质素23.4g，纯度为99.90％。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (12)

  1. 一种红景天中提取草质素的方法，其特征在于：包括以下步骤：
    1)将粉碎好的红景天药材用提取溶剂进行提取，提取液减压浓缩得到提取物浸膏；
    2)将提取物浸膏萃取，萃取后的水层再进行酸水解；
    3)将经酸水解后的溶液用有机溶剂萃取，合并萃取液，减压浓缩得到草质素萃取物；
    4)将草质素萃取物采用聚酰胺柱层析处理，收集含有草质素的流分，浓缩至干，得到草质素粗品；
    5)将草质素粗品采用反相硅胶柱层析处理，收集含有草质素的流分，浓缩至干，重结晶，得到草质素纯品。
  2. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤1)中的红景天为含草质素-7-O-鼠李糖苷和草质素-7-O-(3″-β-D-葡萄糖基)-鼠李糖苷的总含量不低于0.08％的景天科红景天属植物中的任意一种红景天。
  3. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤1)中的提取溶剂选取质量百分比为30.0％-99.9％的甲醇-水溶液或者70.0％-90.0％乙醇-水溶液。
  4. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤1)中的提取包括浸泡、渗漉、超声或加热回流中的任意一种方法。
  5. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤2)中采用沸程为60-90℃石油醚进行萃取，每次萃取石油醚的用量与提取物浸膏的比例为(1-3)：1(v/v)。
  6. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤2)酸水解采用直接酸水解或者两相酸水解的方法，酸水解采用盐酸、硫酸、甲酸中任意一种酸，所用酸的质量分数为0.2％-10.0％。
  7. 如权利要求6所述的一种红景天中提取草质素的方法，其特征在于：所述的两相酸水解中的有机相包括石油醚-醋酸乙酯(1：1)、甲苯、醋酸乙酯中的任意一种。
  8. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤2)中酸水解时加热回流温度为50℃-100℃，酸水解的时间为0.5h-12.0h。
  9. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤3)萃取用有机溶剂为石油醚-醋酸乙酯(1:1)(v/v)或醋酸乙酯，每次有机溶剂与酸水解溶液比例为(1-3)：1(v/v)，萃取次数为3-5次。
  10. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤4)中所述聚酰胺柱层析洗脱条件为体积比(1:1)石油醚-醋酸乙酯等度洗脱。
  11. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤5)中反相硅胶柱层析采用C18反相柱，所述的C18反相柱选用内径50mm的预装柱或内径100mm的预装柱或DAC制备柱。
  12. 如权利要求1所述的一种红景天中提取草质素的方法，其特征在于：所述的步骤5)中反相硅胶柱层析处理的洗脱剂采用含水的乙腈或者含水的甲醇溶液，所述重结晶采用甲醇溶液。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113181167A (zh) * 2021-05-28 2021-07-30 浙江大学 草质素在制备治疗心肌肥厚药物中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151173A1 (en) * 2008-06-12 2009-12-17 Korea Research Institute Of Bioscience And Biotechnology Pharmaceutical compositions for prevention and treatment of viral diseases containing rhodiola extracts, fractions, the isolated flavonoid compounds therefrom, derivatives compounds thereof or the pharmaceutically acceptable salts as an active ingredient

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151173A1 (en) * 2008-06-12 2009-12-17 Korea Research Institute Of Bioscience And Biotechnology Pharmaceutical compositions for prevention and treatment of viral diseases containing rhodiola extracts, fractions, the isolated flavonoid compounds therefrom, derivatives compounds thereof or the pharmaceutically acceptable salts as an active ingredient

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, TAO ET AL.: "Study on the Chemical Constituents of Rhodiola Crenulata", WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 27, no. 4, 30 April 2012 (2012-04-30), pages 367 - 370, XP009508426 *
See also references of EP3418274A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113181167A (zh) * 2021-05-28 2021-07-30 浙江大学 草质素在制备治疗心肌肥厚药物中的应用
CN113181167B (zh) * 2021-05-28 2022-09-16 浙江大学 草质素在制备治疗心肌肥厚药物中的应用

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