CN103421054A - Preparation method of phyllaemblicin B - Google Patents
Preparation method of phyllaemblicin B Download PDFInfo
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- CN103421054A CN103421054A CN2013103616082A CN201310361608A CN103421054A CN 103421054 A CN103421054 A CN 103421054A CN 2013103616082 A CN2013103616082 A CN 2013103616082A CN 201310361608 A CN201310361608 A CN 201310361608A CN 103421054 A CN103421054 A CN 103421054A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- RLZZBHBWPWGOSA-MYVHNFIBSA-N [(2s,3r,4s,5s,6r)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl] (2s,3ar,4r,4's,5'r,6s,7ar)-4'-benzoyloxy-3a,4-dihydroxy-5'-methyl-3-oxospiro[5,6,7,7a-tetrahydro-4h-1-benzofuran-2,2'-oxane]-6- Chemical compound O([C@H]1C[C@@]2(OC[C@H]1C)C([C@@]1(O)[C@H](O)C[C@@H](C[C@H]1O2)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)=O)C(=O)C1=CC=CC=C1 RLZZBHBWPWGOSA-MYVHNFIBSA-N 0.000 title abstract description 7
- XQMUICMLSDHQEH-UHFFFAOYSA-N Phyllaemblicin B Natural products CC1COC2(CC1OC(=O)c3ccccc3)OC4CC(CC(O)C4(O)C2O)C(=O)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O XQMUICMLSDHQEH-UHFFFAOYSA-N 0.000 title abstract description 6
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
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- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
技术领域: Technical field:
本发明涉及化学领域化合物的分离提取方法,具体涉及植物天然生理活性物质余甘根苷B的制备方法。 The invention relates to a method for separating and extracting compounds in the chemical field, in particular to a method for preparing plant natural physiologically active substance emblicin B. the
背景技术: Background technique:
余甘根苷B(phyllaemblicin B),为天然存在的具有复杂结构的高度氧化倍半萜类配糖体。该化合物最初从叶下珠属植物的中分离得到,但存在分离纯化过程繁琐,得率低等问题。因该化合物对柯萨奇病毒B3有显著抑制活性,可用于制备治疗病毒性心肌炎药物,本申请人曾申请相关专利并获得授权(专利号为:ZL200810058110.8),但该专利未涉及其制备方法。余甘根苷B为水溶性的倍半萜类配糖体,极性大,分离纯化困难,迄今没有工业化批量生产的制备工艺。 Phyllaemblicin B is a naturally occurring highly oxidized sesquiterpene glycoside with a complex structure. The compound was initially isolated from Phyllostachys genus, but there were problems such as cumbersome separation and purification process and low yield. Because the compound has significant inhibitory activity against Coxsackie virus B3, it can be used to prepare drugs for the treatment of viral myocarditis. The applicant has applied for a related patent and obtained authorization (patent number: ZL200810058110.8), but the patent does not involve its preparation method. Emlical glycoside B is a water-soluble sesquiterpene glycoside with high polarity and difficult separation and purification. So far, there is no preparation process for industrialized mass production. the
发明内容: Invention content:
本发明的目的旨在提供一种成本低、得率高、纯度高、制备过程简单易行、生产周期短,易于批量制备和工业化生产的余甘根苷B制备方法。 The object of the present invention is to provide a method for preparing emblical glycoside B with low cost, high yield, high purity, simple preparation process, short production cycle, and easy batch preparation and industrial production. the
为了实现本发明的上述目的,本发明提供了如下的技术方案: In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:
取任何一种含余甘根苷B的植物,烘干、粉碎,用含水乙醇加热回流提取2-3次,每次2-3小时,经过滤、浓缩、干燥得乙醇提取物,然后经大孔吸附树脂柱层析分离,在薄层层析的检测指导下,用不同浓度的含水甲醇梯度洗脱,收集含有余甘根苷B的洗脱液,浓缩,经正相硅胶柱柱层析纯化,用氯仿、甲醇与水的混合液进行洗脱, 再经葡聚糖凝胶柱层析和反相硅胶柱柱层析纯化,用低级醇与水的混合液进行洗脱,得余甘根苷B粗品,进一步用乙醇水重结晶,得余甘根苷B。 Take any plant containing emblical glycoside B, dry and pulverize, heat and reflux with water-containing ethanol for 2-3 times, each time for 2-3 hours, filter, concentrate, and dry to obtain ethanol extract, and then adsorb through macropores Separation by resin column chromatography, under the detection guidance of thin layer chromatography, use gradient elution with different concentrations of aqueous methanol, collect the eluate containing emblicalin B, concentrate, purify by normal phase silica gel column chromatography, and use chloroform , a mixture of methanol and water for elution, and then purified by Sephadex column chromatography and reverse-phase silica gel column chromatography, and eluted with a mixture of lower alcohols and water to obtain the crude emblical glycoside B, further Recrystallize with ethanol water to obtain emblicin B. the
上述技术方案中用于提取余甘根苷B的植物原料为:大戟科叶下珠属(Phyllanthus)植物的根及地上部分,如,余甘子(P.emblica)、泰国余甘子(P.acidus),渐尖叶下珠(Phyllanthus acuminatus)等;以及大戟科算盘子属(Acalypha)植物的根及地上部分,如,算盘子(Glochidion puberum)、红算盘子(Gl.coccineum(Buch-Ham.)Muell.Arg)、毛果算盘子(G.eriocarpum Champ.ex Benth)、革叶算盘子(Gl.daltonii(Muell.Arg.)Kurz),等。 The plant raw materials used for extracting emblica B in the above technical scheme are: the roots and aerial parts of plants of the genus Phyllanthus in the Euphorbiaceae family, such as P.emblica, P.acidus , Phyllanthus acuminatus, etc.; and roots and aerial parts of plants of the genus Acalypha in the family Euphorbiaceae, such as Glochidion puberum, Gl. coccineum (Buch-Ham. ) Muell.Arg), G.eriocarpum Champ.ex Benth, Gl.daltonii (Muell.Arg.) Kurz, etc. the
上述技术方案中用于柱层析分离的大孔吸附树脂为聚苯乙烯型大孔吸附树脂。可以是:如:MCI gel CHP-20P,DiaionHP20,DiaionHP20SS,D-101,KB-8等。提取物与大孔吸附树脂的重量比为1∶8-10,柱径高比为1∶6-8。 The macroporous adsorption resin used for column chromatography separation in the above technical scheme is a polystyrene type macroporous adsorption resin. Can be: such as: MCI gel CHP-20P, DiaionHP20, DiaionHP20SS, D-101, KB-8, etc. The weight ratio of the extract to the macroporous adsorption resin is 1:8-10, and the diameter-to-height ratio of the column is 1:6-8. the
上述技术方案中用于柱层析分离的正相硅胶为不同厂家生产的各种正相硅胶的产品型号与规格。例如:硅胶H(30-40m),硅胶G(200m)等。 The normal phase silica gel used for column chromatography separation in the above technical scheme is the product model and specification of various normal phase silica gels produced by different manufacturers. For example: silica gel H (30-40m), silica gel G (200m), etc. the
上述技术方案中用于柱层析分离的葡聚糖凝胶为不同厂家生产的各种葡聚糖凝胶的产品型号与规格。例如:Sephadex LH-20,Toyopearl HW-40F等。 The dextran gel used for column chromatography separation in the above technical scheme is the product model and specification of various dextran gels produced by different manufacturers. For example: Sephadex LH-20, Toyopearl HW-40F, etc. the
上述技术方案中用于柱层析分离的反相硅胶为不同厂家生产的各种反相硅胶的产品型号与规格。例如:Rp18,ODS等。 The reversed-phase silica gel used for column chromatography separation in the above technical scheme is the product model and specification of various reversed-phase silica gels produced by different manufacturers. For example: Rp18, ODS, etc. the
上述技术方案中用于检测和指导柱层析分离的薄层层析法是7:3:0.5的氯仿:甲醇:水为展开剂,10%硫酸乙醇液为显色剂。 The thin-layer chromatography that is used to detect and guide the separation of column chromatography in the technical scheme is 7:3:0.5 chloroform:methanol:water as a developer, and 10% sulfuric acid ethanol as a chromogenic agent. the
上述技术方案中的重结晶步骤是将余甘根苷B粗品用少量低级醇(可以是甲醇或乙醇)充分溶解,加入适量水微微加热,室温下放置过夜,结晶。将析出的结晶用水反复洗涤,过滤,干燥,得到白色粉末,即为余甘根苷B的纯品。 The recrystallization step in the above technical solution is to fully dissolve the crude emblical glycoside B with a small amount of lower alcohol (which can be methanol or ethanol), add an appropriate amount of water and heat slightly, and place it at room temperature overnight to crystallize. The precipitated crystals were repeatedly washed with water, filtered, and dried to obtain a white powder, which was the pure product of emblicin B. the
上述技术方案中余甘根苷B干燥方法可用加热干燥、冷冻干燥、喷雾干燥或微波干燥。 The drying method of emblical glycoside B in the above technical solution can be heat drying, freeze drying, spray drying or microwave drying. the
经过上述制备工艺提取分离得到的余甘根苷B,得率高,产品的纯度大于98%,无有机溶剂的残留,可直接用作为制药的天然原料或作为标准对照品使用。 The emblical glycoside B extracted and separated through the above preparation process has a high yield, a product purity greater than 98%, and no organic solvent residue, and can be directly used as a natural raw material for pharmacy or as a standard reference substance. the
本发明制备余甘根苷B的具体步骤如下: The concrete steps that the present invention prepares emblical glycoside B are as follows:
(1)植物原料去杂、洗净、烘干、粉碎成粗粉(过20-40目筛)。 (1) Plant raw materials are removed from impurities, washed, dried, and crushed into coarse powder (passed through a 20-40 mesh sieve). the
(2)将该植物粗粉用70%含水乙醇加热回流提取3次,每次提取时间为2小时,过滤、合并滤液,浓缩、回收溶剂,干燥,得乙醇提取物。 (2) Heat and reflux the plant meal with 70% ethanol to extract 3 times, each extraction time is 2 hours, filter, combine the filtrate, concentrate, recover the solvent, and dry to obtain the ethanol extract. the
(3)用少量水将乙醇提取物溶解,过滤,缓缓倾入大孔吸附树脂(如:DiaionHP20SS,D-101,KB-8等)层析柱的上端。层析柱的柱径高比为1:8-10,乙醇提取物与大孔吸附树脂的重量比为1:6-8。以不同浓度的乙醇梯度洗脱,同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得乙醇洗脱物。 (3) Dissolve the ethanol extract with a small amount of water, filter, and slowly pour into the upper end of the macroporous adsorption resin (such as: DiaionHP20SS, D-101, KB-8, etc.) chromatography column. The diameter-to-height ratio of the chromatographic column is 1:8-10, and the weight ratio of the ethanol extract to the macroporous adsorption resin is 1:6-8. Gradient elution with different concentrations of ethanol, while using thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain ethanol eluate. the
(4)将乙醇洗脱物用少量甲醇水溶解,倾入葡聚糖凝胶层析柱的上端。柱径高比为1:10-15,乙醇洗脱物与葡聚糖凝胶的重量比为1:5。以不同浓度的乙醇梯度洗脱,用薄层层析法检测指导洗脱。薄 层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得到淡黄色粉末。 (4) Dissolve the ethanol eluate with a small amount of methanol water and pour it into the upper end of the Sephadex chromatography column. The ratio of column diameter to height is 1:10-15, and the weight ratio of ethanol eluate to Sephadex is 1:5. Gradient elution with different concentrations of ethanol was used to detect and guide the elution by thin layer chromatography. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain a light yellow powder. the
(5)将该淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain crude emblical B. the
(5)将该余甘根苷B粗品用少量甲醇水溶解,缓缓倾入反相硅胶(如:Rp-18等)层析柱上端。层析柱的柱径高比为1:10-15,余甘根苷B粗品与大孔吸附树脂的重量比为1:8。以不同浓度的甲醇梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品。 (5) Dissolve the crude emblical glycoside B in a small amount of methanol water, and slowly pour it into the upper end of a reverse-phase silica gel (such as: Rp-18, etc.) chromatography column. The diameter-to-height ratio of the chromatographic column is 1:10-15, and the weight ratio of the crude emblicin B to the macroporous adsorption resin is 1:8. Gradient elution with different concentrations of methanol. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水微微加热,放置过夜,析出结晶。将结晶用水反复洗涤,过滤,干燥,得到白色粉末,即为余甘根苷B纯品。 (6) Fully dissolve the crude emblical glucoside B with a small amount of ethanol, add an appropriate amount of water and heat slightly, and leave it overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered, and dried to obtain a white powder, which was pure emblicin B. the
(7)余甘根苷B纯品的干燥,可用电热恒温真空干燥法,也可采用冷冻干燥、喷雾干燥和微波干燥法等。 (7) The drying of pure emblical glycoside B can be done by electrothermal constant temperature vacuum drying, freeze drying, spray drying and microwave drying, etc. the
(8)余甘根苷B的高压液相色谱(HPLC)定量分析按以下的方法进行: (8) Quantitative analysis of emblical glycoside B by high pressure liquid chromatography (HPLC) is carried out according to the following method:
仪器与试剂:HPLC仪器为Alliance高效液相色谱仪,自动进样器,PDA二级管阵列可变波长检测器。乙腈为色谱纯,水为超纯水,其余溶剂均为分析纯。 Instruments and reagents: HPLC instruments are Alliance high-performance liquid chromatography, automatic sampler, PDA diode array variable wavelength detector. Acetonitrile was chromatographically pure, water was ultrapure water, and the rest of the solvents were analytically pure. the
色谱条件及检测波长的选择:用十八烷基硅胶为填充剂(如:Agilent ZORBAX C18酸性柱,4.6×150mm);乙腈:水(4:96→30:70,线形梯度)为流动相。柱温:30℃,流速:1ml/min。 Selection of chromatographic conditions and detection wavelength: Octadecyl silica gel is used as filler (such as: Agilent ZORBAX C18 acidic column, 4.6×150mm); acetonitrile: water (4:96→30:70, linear gradient) is used as mobile phase. Column temperature: 30°C, flow rate: 1ml/min. the
含量测定:由上述工艺制备的余甘根苷B的含量>98%。 Content determination: the content of emblical glycoside B prepared by the above process is >98%. the
经过上述制备工艺提取分离得到的余甘根苷B,得率高,产品的纯度大于98%,无有机溶剂的残留,可直接用作为制药的天然原料或作为标准对照品使用。 The emblical glycoside B extracted and separated through the above preparation process has a high yield, a product purity greater than 98%, and no organic solvent residue, and can be directly used as a natural raw material for pharmacy or as a standard reference substance. the
与现有技术相比,本发明的方法具有如下优益性: Compared with prior art, method of the present invention has following advantage:
1、本发明是从叶下珠属、算盘子属植物中的任何一种含余甘根苷B的植物中提取余甘根苷B。 1. The present invention extracts emblical glycoside B from any plant containing emblical glycoside B in the genus Phyllophyllum and the genus Abacus. the
2、本发明采用含水低级醇(可以是甲醇或乙醇)为溶剂从植物原料中提取余甘根苷B,得率高,成本低。 2. The present invention uses a water-containing lower alcohol (which can be methanol or ethanol) as a solvent to extract emblical glycoside B from plant raw materials, with high yield and low cost. the
3、本发明采用薄层层析法作为检测手段,指导柱层析的分离纯化,所得产品的得率高,纯度高。 3. The present invention uses thin-layer chromatography as a detection means to guide the separation and purification of column chromatography, and the resulting product has a high yield and high purity. the
4、本发明使用的柱层析填充剂为聚苯乙烯型大孔吸附树脂、葡聚糖凝胶以及反相硅胶,这些填充剂材料均可以重复使用。 4. The column chromatography filler used in the present invention is polystyrene macroporous adsorption resin, dextran gel and reversed-phase silica gel, and these filler materials can be used repeatedly. the
5、本发明采用水重结晶的方法纯化高纯度的余甘根苷B,不仅产品形状好、纯度高、无溶剂残留,而且简单易行、成本低。 5. The present invention adopts the method of water recrystallization to purify high-purity emblical glycoside B, which not only has good shape, high purity and no solvent residue, but also is simple and easy to operate and low in cost. the
具体实施方式: Detailed ways:
下面以本发明的实施例来进一步说明本发明的实质性内容,但 本发明的内容并不局限于此。 Further illustrate substantive content of the present invention below with the embodiment of the present invention, but content of the present invention is not limited thereto. the
本发明实施例中制备余甘根苷B的具体步骤可概括如下: The specific steps for preparing emblical glycoside B in the embodiments of the present invention can be summarized as follows:
(1)植物原料去杂、洗净、烘干、粉碎成粗粉(过20-40目筛)。 (1) Plant raw materials are removed from impurities, washed, dried, and crushed into coarse powder (passed through a 20-40 mesh sieve). the
(2)将该植物粗粉用70%含水乙醇加热回流提取3次,每次提取时间为2小时,过滤、合并滤液,浓缩、回收溶剂,干燥,得乙醇提取物。 (2) Heat and reflux the plant meal with 70% ethanol to extract 3 times, each extraction time is 2 hours, filter, combine the filtrate, concentrate, recover the solvent, and dry to obtain the ethanol extract. the
(3)用少量水将乙醇提取物溶解,过滤,缓缓倾入大孔吸附树脂(如:DiaionHP20SS,D-101,KB-8等)层析柱的上端。层析柱的柱径高比为1:8-10,乙醇提取物与大孔吸附树脂的重量比为1:6-8。以不同浓度的乙醇梯度洗脱,同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得乙醇洗脱物。 (3) Dissolve the ethanol extract with a small amount of water, filter, and slowly pour into the upper end of the macroporous adsorption resin (such as: DiaionHP20SS, D-101, KB-8, etc.) chromatography column. The diameter-to-height ratio of the chromatographic column is 1:8-10, and the weight ratio of the ethanol extract to the macroporous adsorption resin is 1:6-8. Gradient elution with different concentrations of ethanol, while using thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain ethanol eluate. the
(4)将乙醇洗脱物用少量甲醇水溶解,倾入葡聚糖凝胶层析柱的上端。柱径高比为1:10-15,乙醇洗脱物与葡聚糖凝胶的重量比为1:5。以不同浓度的乙醇梯度洗脱,用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得到淡黄色粉末。 (4) Dissolve the ethanol eluate with a small amount of methanol water and pour it into the upper end of the Sephadex chromatography column. The ratio of column diameter to height is 1:10-15, and the weight ratio of ethanol eluate to Sephadex is 1:5. Gradient elution with different concentrations of ethanol was used to detect and guide the elution by thin layer chromatography. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain a light yellow powder. the
(5)将该淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂, 10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain crude emblical B. the
(5)将该余甘根苷B粗品用少量甲醇水溶解,缓缓倾入反相硅胶(如:Rp-18等)层析柱上端。层析柱的柱径高比为1:10-15,余甘根苷B粗品与大孔吸附树脂的重量比为1:8。以不同浓度的甲醇梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品。 (5) Dissolve the crude emblical glycoside B in a small amount of methanol water, and slowly pour it into the upper end of a reverse-phase silica gel (such as: Rp-18, etc.) chromatography column. The diameter-to-height ratio of the chromatographic column is 1:10-15, and the weight ratio of the crude emblicin B to the macroporous adsorption resin is 1:8. Gradient elution with different concentrations of methanol. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水微微加热,放置过夜,析出结晶。将结晶用水反复洗涤,过滤,干燥,得到白色粉末,即为余甘根苷B纯品。 (6) Fully dissolve the crude emblical glucoside B with a small amount of ethanol, add an appropriate amount of water and heat slightly, and leave it overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered, and dried to obtain a white powder, which was pure emblicin B. the
(7)余甘根苷B纯品的干燥,可用电热恒温真空干燥法,也可采用冷冻干燥、喷雾干燥和微波干燥法等。 (7) The drying of pure emblical glycoside B can be done by electrothermal constant temperature vacuum drying, freeze drying, spray drying and microwave drying, etc. the
(8)余甘根苷B的高压液相色谱(HPLC)定量分析按以下的方法进行: (8) Quantitative analysis of emblical glycoside B by high pressure liquid chromatography (HPLC) is carried out according to the following method:
仪器与试剂:HPLC仪器为Alliance高效液相色谱仪,自动进样器,PDA二级管阵列可变波长检测器。乙腈为色谱纯,水为超纯水,其余溶剂均为分析纯。 Instruments and reagents: HPLC instruments are Alliance high-performance liquid chromatography, automatic sampler, PDA diode array variable wavelength detector. Acetonitrile was chromatographically pure, water was ultrapure water, and the rest of the solvents were analytically pure. the
色谱条件及检测波长的选择:用十八烷基硅胶为填充剂(如:Agilent ZORBAX C18酸性柱,4.6×150mm);乙腈:水(4:96→30:70,线形梯度)为流动相。柱温:30℃,流速:1ml/min。 Selection of chromatographic conditions and detection wavelength: Octadecyl silica gel is used as filler (such as: Agilent ZORBAX C18 acidic column, 4.6×150mm); acetonitrile: water (4:96→30:70, linear gradient) is used as mobile phase. Column temperature: 30°C, flow rate: 1ml/min. the
含量测定:由上述工艺制备的余甘根苷B的含量>98%。 Content determination: the content of emblical glycoside B prepared by the above process is >98%. the
经过上述制备工艺提取分离得到的余甘根苷B,得率高,产品的 纯度大于98%,无有机溶剂的残留,可直接用作为制药的天然原料或作为标准对照品使用。 The emblical glycoside B extracted and separated by the above-mentioned preparation process has a high yield, a product purity greater than 98%, and no organic solvent residue, and can be directly used as a natural raw material for pharmacy or as a standard reference substance. the
实施例1: Example 1:
从余甘子(P.emblica)根中提取余甘根苷B: Extract emblical glycoside B from the root of P.emblica:
(1)余甘子根原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) The raw material of Emlical root is removed from impurities, washed, dried, and crushed into a coarse powder passed through a 30-mesh sieve. the
(2)余甘子根粗粉109kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物7.8Kg。回收溶剂可用于反复提取。 (2) 109 kg of emblica root coarse powder was extracted with 70% ethanol under reflux for 3 times, each extraction time was 2 hours, filtered, combined filtrates, concentrated under reduced pressure, recovered solvent, and dried to obtain 7.8 kg of ethanol extract of emblica root. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物400g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 400 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末260g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 260 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗 脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品(120g)。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain crude emblical B (120 g). the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品68g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 68 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B54.238g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain emblical glycoside B54.238g. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例2: Example 2:
从泰国余甘子(P.acidus)根中提取余甘根苷B: Extract emblical glycoside B from the root of P.acidus in Thailand:
(1)泰国余甘子根原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) Thai emblica root raw materials are removed from impurities, washed, dried, and crushed into a coarse powder passed through a 30-mesh sieve. the
(2)泰国余甘子根粗粉6kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物700g。回收溶剂可用于反复提取。 (2) 6 kg of Thai emblica root coarse powder was extracted with 70% ethanol under reflux for 3 times, each extraction time was 2 hours, filtered, combined filtrates, concentrated under reduced pressure, recovered solvent, and dried to obtain 700 g of ethanol extract of emblica root. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测 指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物200g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, use thin-layer chromatography to detect and guide elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblical glycoside B was collected and concentrated under reduced pressure to obtain 200 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末90g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblicalin B was collected and concentrated under reduced pressure to obtain 90 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品20g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 20 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品12g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 12 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根 苷B4.356g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 4.356 g of emblical glycoside B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例3: Example 3:
从泰国余甘子(P.acidus)地上部分中提取余甘根苷B: Extraction of emblical glycoside B from the aerial part of P.acidus in Thailand:
(1)泰国余甘子茎原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) Remove impurities from Thai emblica stems, wash them, dry them, and crush them into a coarse powder that passes through a 30-mesh sieve. the
(2)泰国余甘子茎粗粉4kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物500g。回收溶剂可用于反复提取。 (2) Extract 4 kg of Thai emblica stem powder with 70% ethanol under reflux for 3 times, each extraction time is 2 hours, filter, combine the filtrates, concentrate under reduced pressure, recover the solvent, and dry to obtain 500 g of ethanol extract of emblica root. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物160g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 160 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末65g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblical glycoside B was collected and concentrated under reduced pressure to obtain 65 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析 柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品16g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 16 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品8g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. Collect the eluate containing emblical glycoside B and concentrate under reduced pressure to obtain 8 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B1.861g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 1.861 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例4: Example 4:
从渐尖叶下珠(Phyllanthus acuminatus)地上部分提取余甘根苷B: Extraction of emblicalin B from the aerial part of Phyllanthus acuminatus:
(1)渐尖叶下珠地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) The above-ground part of the acuminate leaves is removed from impurities, washed, dried, and crushed into a coarse powder passed through a 30-mesh sieve. the
(2)渐尖叶下珠地上部分粗粉10kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物800g。回收溶剂可用于反复提取。 (2) Extract 10kg of the coarse powder of the aboveground part of the acuminate leaf with 70% ethanol under reflux for 3 times, each extraction time is 2 hours, filter, combine the filtrate, concentrate under reduced pressure, recover the solvent, and dry to obtain ethanol extraction of Emlical root Material 800g. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物500g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 500 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末57g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 57 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品8.9g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 8.9 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩, 得到余甘根苷B粗品3.6g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 3.6 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B1.236g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 1.236 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例5: Embodiment 5:
从渐尖叶下珠(Phyllanthus acuminatus)根中提取余甘根苷B: Extraction of emblicin B from the root of Phyllanthus acuminatus:
(1)渐尖叶下珠地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) The above-ground part of the acuminate leaves is removed from impurities, washed, dried, and crushed into a coarse powder passed through a 30-mesh sieve. the
(2)渐尖叶下珠地上部分粗粉5kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物200g。回收溶剂可用于反复提取。 (2) Extract 5kg of the coarse powder of the aboveground part of the acuminate leaf with 70% ethanol under reflux for 3 times, each extraction time is 2 hours, filter, combine the filtrate, concentrate under reduced pressure, recover the solvent, and dry to obtain ethanol extraction of emblica root Material 200g. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物20g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblical glycoside B was collected and concentrated under reduced pressure to obtain 20 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压 浓缩,得到淡黄色粉末12g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. Collect the eluent containing emblical glycoside B, concentrate under reduced pressure, obtain light yellow powder 12g. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品3.5g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 3.5 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品2.7g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 2.7 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B0.982g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 0.982 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例6: Embodiment 6:
从算盘子(Glochidion puberum)地上部分中提取余甘根苷B: Extraction of emblical glycoside B from the aerial part of Glochidion puberum:
(1)算盘子地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) The above-ground raw materials of the abacus are removed from impurities, washed, dried, and crushed into a coarse powder passing through a 30-mesh sieve. the
(2)算盘子地上部分粗粉10kg用70%乙醇加热回流提取3次, 每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物650g。回收溶剂可用于反复提取。 (2) 10 kg of coarse powder of the aboveground part of Abacus was extracted with 70% ethanol under reflux for 3 times, each extraction time was 2 hours, filtered, combined filtrates, concentrated under reduced pressure, recovered solvent, and dried to obtain 650 g of ethanol extract of emblica root. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物260g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 260 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末120g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblicalin B was collected and concentrated under reduced pressure to obtain 120 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品6.1g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 6.1 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导 洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品1.9g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 1.9 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B0.531g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 0.531 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例7: Embodiment 7:
从红算盘子(Gl.coccineum(Buch-Ham.)Muell.Arg)地上部分中提取余甘根苷B: Extract emblical glycoside B from the aerial part of red abacus (Gl.coccineum (Buch-Ham.)Muell.Arg):
(1)红算盘子地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) Remove impurities, wash, dry, and crush the raw materials above the ground of Hongsuanzi into a coarse powder that passes through a 30-mesh sieve. the
(2)红算盘子地上部分粗粉15kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物700g。回收溶剂可用于反复提取。 (2) 15kg of coarse powder of the aboveground part of Red Abacus was extracted with 70% ethanol under reflux for 3 times, each extraction time was 2 hours, filtered, combined filtrate, concentrated under reduced pressure, recovered solvent, dried to obtain 700g ethanol extract of Emlical root . The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物320g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 320 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱 径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末160g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 160 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品4g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 4 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品2g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. Collect the eluate containing emblical glycoside B and concentrate under reduced pressure to obtain 2 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B0.826g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 0.826 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例8: Embodiment 8:
从毛果算盘子(Gl.eriocarpum Champ.ex Benth)地上部分中提 取余甘根苷B: Extraction of emblicalin B from the aerial part of Gl.eriocarpum Champ.ex Benth:
(1)毛果算盘子地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) Remove impurities, wash, dry, and crush the raw materials above the ground of Maoguo Abacus into a coarse powder passing through a 30-mesh sieve. the
(2)毛果算盘子地上部分粗粉20kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物800g。回收溶剂可用于反复提取。 (2) Extract 20kg of the coarse powder of the aboveground part of Abacus edulis with 70% ethanol under reflux for 3 times, each extraction time is 2 hours, filter, combine the filtrate, concentrate under reduced pressure, recover the solvent, and dry to obtain the ethanol extract of emblica root 800g. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物80g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 80 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末30g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblica B was collected and concentrated under reduced pressure to obtain 30 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩, 回收溶剂,得余甘根苷B粗品2g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical B was collected, combined, concentrated, and the solvent was recovered to obtain 2 g of emblical B crude product. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品1.3g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 1.3 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B0.631g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve it, and let it stand overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 0.631 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
实施例9: Embodiment 9:
从革叶算盘子(Gl.daltonii(Muell.Arg.)Kurz)地上部分中提取余甘根苷B: Extract emblical glycoside B from the aerial part of leather leaf abacus (Gl.daltonii(Muell.Arg.)Kurz):
(1)革叶算盘子地上部分原料去杂、洗净、烘干、粉碎成过30目筛的粗粉。 (1) The aboveground part of the leather leaf abacus is cleaned of impurities, washed, dried, and crushed into a coarse powder passed through a 30-mesh sieve. the
(2)革叶算盘子地上部分粗粉18kg用70%乙醇加热回流提取3次,每次提取时间2小时,过滤、合并滤液、减压浓缩、回收溶剂、干燥,得余甘子根乙醇提取物780g。回收溶剂可用于反复提取。 (2) 18kg of the coarse powder of the above-ground part of the leaf abacus was extracted with 70% ethanol under reflux for 3 times, and the extraction time was 2 hours each time, filtered, combined with the filtrate, concentrated under reduced pressure, recovered the solvent, and dried to obtain the ethanol extract of emblica root 780g. The recovered solvent can be used for repeated extractions. the
(3)用少量水将乙醇提取物溶解,缓缓倾入D101大孔吸附树脂层析柱的上端,乙醇提取物与大孔吸附树脂的重量比为1:8,柱径高比为1:10,以不同浓度的甲醇梯度洗脱。同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂, 10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到乙醇洗脱物100g。 (3) Dissolve the ethanol extract with a small amount of water, and slowly pour it into the upper end of the D101 macroporous adsorption resin chromatography column. The weight ratio of the ethanol extract to the macroporous adsorption resin is 1:8, and the column diameter-to-height ratio is 1: 10. Gradient elution with different concentrations of methanol. At the same time, thin-layer chromatography is used to detect and guide elution, and the conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developer, and 10% sulfuric acid ethanol solution as the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 100 g of ethanol eluate. the
(4)将乙醇洗脱物用少量水溶解,缓缓倾入葡聚糖凝胶Sephadex LH-20层析柱的上端,乙醇洗脱物与葡聚糖凝胶的重量比为1:5,柱径高比为1:10,以不同浓度的甲乙醇梯度洗脱,同时用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到淡黄色粉末25g。 (4) Dissolve the ethanol eluate with a small amount of water, and slowly pour it into the upper end of Sephadex LH-20 chromatographic column. The weight ratio of ethanol eluate to Sephadex is 1:5. The diameter-to-height ratio of the column is 1:10, and the gradient elution is carried out with different concentrations of methanol and methyl alcohol. At the same time, the thin-layer chromatography is used to detect and guide the elution. The conditions of the thin-layer chromatography are: chloroform-methanol-water (7:3:0.5 ) as the developer, and 10% sulfuric acid ethanol solution as the developer. The eluate containing emblicalin B was collected and concentrated under reduced pressure to obtain 25 g of light yellow powder. the
(5)将淡黄色粉末用少量甲醇溶解,缓缓倾入适量硅胶,搅拌均匀,加入预先准备好的硅胶(如:硅胶H等)层析柱上端。层析柱的柱径高比为1:20-25,预制备的样品与正相硅胶的重量比为1:30。以不同比列的氯仿-甲醇-水梯度洗脱。同时用薄层层析法检测指导洗脱。薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,合并,浓缩,回收溶剂,得余甘根苷B粗品3.5g。 (5) Dissolve the light yellow powder with a small amount of methanol, slowly pour in an appropriate amount of silica gel, stir evenly, and add the pre-prepared silica gel (such as: silica gel H, etc.) to the upper end of the chromatography column. The diameter-to-height ratio of the chromatographic column is 1:20-25, and the weight ratio of the pre-prepared sample to normal-phase silica gel is 1:30. Elute with different ratios of chloroform-methanol-water gradient. At the same time, the thin layer chromatography was used to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as developing solvent, and 10% sulfuric acid ethanol solution as developing reagent. The eluate containing emblical glycoside B was collected, combined, concentrated, and the solvent was recovered to obtain 3.5 g of crude emblical glucoside B. the
(6)将余甘根苷B粗品用少量甲醇水溶解,缓缓倾入Rp-18反相硅胶的上端,余甘根苷B与反相硅胶的重量比为1:8,柱径高比为1:10。以不同浓度的甲醇水梯度洗脱,同时,用薄层层析法检测指导洗脱,薄层层析的条件为:氯仿-甲醇-水(7:3:0.5)为展开剂,10%的硫酸乙醇液为显色剂。收集含有余甘根苷B的洗脱液,减压浓缩,得到余甘根苷B粗品1.5g。 (6) Dissolve the crude emblical B in a small amount of methanol water, and slowly pour it into the upper end of Rp-18 reversed-phase silica gel. The weight ratio of emblical B to reversed-phase silica gel is 1:8, and the ratio of column diameter to height is 1:10. . Elute with different concentrations of methanol and water gradient, and at the same time, use thin-layer chromatography to detect and guide the elution. The conditions of thin-layer chromatography are: chloroform-methanol-water (7:3:0.5) as the developing solvent, 10% Sulfuric acid ethanol solution is the color developer. The eluate containing emblical B was collected and concentrated under reduced pressure to obtain 1.5 g of crude emblical B. the
(6)将余甘根苷B粗品用少量乙醇充分溶解,加适量水溶解,放置过夜,析出结晶。反复用水洗涤结晶,过滤,干燥,得到余甘根苷B0.895g。 (6) Fully dissolve the crude emblical glycoside B with a small amount of ethanol, add an appropriate amount of water to dissolve, and leave it overnight to precipitate crystals. The crystals were repeatedly washed with water, filtered and dried to obtain 0.895 g of emblicin B. the
(7)应用HPLC定量分析方法,对分离制备得到的余甘根苷B进行纯度检测。 (7) HPLC quantitative analysis method was used to detect the purity of the separated and prepared emblical glycoside B. the
由上述实施例所制备的余甘根苷B的物理常数和光谱数据为: The physical constants and spectral data of emblical glycoside B prepared by the foregoing embodiments are:
分子式:C33H44O19 Molecular formula: C 33 H 44 O 19
分子量:744 Molecular weight: 744
结构式: Structural formula:
性状:白色无定型粉末;易溶于甲醇、乙醇、丙酮 Properties: white amorphous powder; easily soluble in methanol, ethanol, acetone
旋光:[a]25 D+10.4°(c0.58,MeOH) Optical rotation: [a] 25 D +10.4°(c0.58,MeOH)
红外光谱νmax(KBr):3205,2933,1778,1718,1604,1452,1294,1124,1012cm-1 Infrared spectrum νmax (KBr): 3205, 2933, 1778, 1718, 1604, 1452, 1294, 1124, 1012cm -1
紫外光谱λmax(logε,MeOH)272(2.99),279(2.91)nm UV Spectrum λmax(logε, MeOH)272(2.99),279(2.91)nm
质谱FABMS m/z767[M+Na]+(18),745[M+H]+(31),583(29),421(28),299(49),105(31) Mass spectrum FABMS m/z767[M+Na] + (18),745[M+H] + (31),583(29),421(28),299(49),105(31)
元素分析:anal.C50.19%,H6.25%,calcd for C33H44O19·5/2H2O,C50.13%,H5.91% Elemental analysis: anal.C50.19%, H6.25%, calcd for C 33 H 44 O 19 5/2H 2 O, C50.13%, H5.91%
氢谱:aglycon:δ8.16(2H,dd,J=7.5,1.5Hz,H-3',7'),7.66(1H, br t,J=7.5Hz,H-5'),7.57(2H,br t,J=7.5Hz,H-4',6'),5.36(1H,q,J=3.0Hz,H-10),4.29(1H,br s,H-5),4.03(1H,t,J=11.0Hz,H-12a),3.93(1H,br s,H-1),3.58(1H,br d,J=11.0Hz,H-12b),2.94(1H,tt,J=13.5,2.5Hz,H-3),2.36(1H,br d J=13.5Hz,H-4a),2.28(1H,dd,J=15.0,3.0Hz,H-9a),2.18n(1H,m H-11),2.01(1H,dd,J=15.0,3.0Hz,H-9b),2.04(1H,br d,J=13.65Hz,H-2a),1.90(1H,dt,J=13.5,4.0hz,H-4b),1.77(1H,dt,J=13.5,2.5Hz,H-2b),0.89(3H,d,J=7.0Hz,H-14);inner glucose,5.59(1H,d,J=8.0Hz,glc H-1),4.08(1H,dd,J=9.0,8.0Hz,glc H-2),3.89(1H,dd,J=12.0,2.5Hz,glc H-6a),3.75(1H,dd,J=12.0,5.0Hz,glc,H-6b),3.62(1H,t,J=9.0Hz,glc H-3),3.46(1H,dd,J=9.5,9.0Hz,glc H-4),3.39(1H,m glc H-5),terminal glucose:4.18(1H,d,J=8.0Hz,glc H-1),3.62(1H,dd,J=12.0,2.5Hz,glc H-6a),3.56(1H,dd,J=12.0,4.5Hz,glc,H-6b),3.23(1H,dd,J=9.5,9.0Hz,glc H-4),3.11(1H,dd,J=8.0,9.0Hz,glc H-2),2.76(1H,m glc H-5)。 Hydrogen spectrum: aglycon: δ8.16(2H, dd, J=7.5, 1.5Hz, H-3', 7'), 7.66(1H, br t, J=7.5Hz, H-5'), 7.57(2H ,br t,J=7.5Hz,H-4',6'),5.36(1H,q,J=3.0Hz,H-10),4.29(1H,br s,H-5),4.03(1H, t,J=11.0Hz,H-12a),3.93(1H,br s,H-1),3.58(1H,br d,J=11.0Hz,H-12b),2.94(1H,tt,J=13.5 ,2.5Hz,H-3),2.36(1H,br d J=13.5Hz,H-4a),2.28(1H,dd,J=15.0,3.0Hz,H-9a),2.18n(1H,m H -11),2.01(1H,dd,J=15.0,3.0Hz,H-9b),2.04(1H,br d,J=13.65Hz,H-2a),1.90(1H,dt,J=13.5,4.0 hz,H-4b),1.77(1H,dt,J=13.5,2.5Hz,H-2b),0.89(3H,d,J=7.0Hz,H-14);inner glucose,5.59(1H,d, J=8.0Hz, glc H-1),4.08(1H,dd,J=9.0,8.0Hz,glc H-2),3.89(1H,dd,J=12.0,2.5Hz,glc H-6a),3.75 (1H,dd,J=12.0,5.0Hz,glc,H-6b),3.62(1H,t,J=9.0Hz,glc H-3),3.46(1H,dd,J=9.5,9.0Hz,glc H-4),3.39(1H,m glc H-5),terminal glucose:4.18(1H,d,J=8.0Hz,glc H-1),3.62(1H,dd,J=12.0,2.5Hz,glc H-6a),3.56(1H,dd,J=12.0,4.5Hz,glc,H-6b),3.23(1H,dd,J=9.5,9.0Hz,glc H-4),3.11(1H,dd, J=8.0, 9.0Hz, glc H-2), 2.76 (1H, m glc H-5). the
碳谱:13C NMR(CD3OD,125MHz)δ213.7(C-7),175.8(C-13),167.8(C-1'),134.4(C-5'),132.1(C-2'),130.8(C-3',7'),129.9(C-4',6'),100.5(C-8),76.3(C-5),75.4(C-6),71.5(C-1),70.9(C-10),63.4(C-12),34.3(C-11),32.7(C-9),32.2(C-2,3),29.3(C-4),13.1(C-14);glucose,93.7(glc C-1),83.1(glc C-2),78.9(C-5),77.8(glc C-3),70.8(glc C-4),62.3(glc C-6);terminal glucose,105.9(glc C-1),77.7(glc C-3),77.6(glc C-5),75.9(glc C-2),70.7(glc C-4),62.0(glc C-6)。 Carbon spectrum: 13 C NMR (CD 3 OD, 125MHz) δ213.7(C-7), 175.8(C-13), 167.8(C-1'), 134.4(C-5'), 132.1(C-2 '),130.8(C-3',7'),129.9(C-4',6'),100.5(C-8),76.3(C-5),75.4(C-6),71.5(C- 1),70.9(C-10),63.4(C-12),34.3(C-11),32.7(C-9),32.2(C-2,3),29.3(C-4),13.1(C -14);glucose,93.7(glc C-1),83.1(glc C-2),78.9(C-5),77.8(glc C-3),70.8(glc C-4),62.3(glc C- 6); terminal glucose, 105.9 (glc C-1), 77.7 (glc C-3), 77.6 (glc C-5), 75.9 (glc C-2), 70.7 (glc C-4), 62.0 (glc C -6).
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