CN104478686B - The preparation method of aryl turmerone reference substance in Rhizoma Curcumae Longae volatile oil - Google Patents
The preparation method of aryl turmerone reference substance in Rhizoma Curcumae Longae volatile oil Download PDFInfo
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Abstract
The invention discloses the preparation method of aryl turmerone reference substance in a kind of Rhizoma Curcumae Longae volatile oil, the present invention is with Rhizoma Curcumae Longae volatile oil for raw material, with silica gel column chromatography and preparative high performance liquid chromatography for separation means, with a certain proportion of petroleum ether-ethyl acetate, methanol-water is eluent system, the prepared aryl turmerone sterling obtained, detect through HPLC, it is a mass-tone spectral peak in different chromatographic columns and mobile phase measurement result, change chromatographic column and mobile phase measures and anomaly peak all do not occur, reference substance purity is measured more than 99% with area normalization method, meet the requirement of assay traditional Chinese chemical contrast.
Description
Technical field
The present invention relates to a kind of separating and purifying technology, the preparation method of aryl turmerone reference substance in especially a kind of Rhizoma Curcumae Longae volatile oil.
Background technology
Rhizoma Curcumae Longae is the dry rhizome of Zingiberaceae Curcuma (Curcuma) plant Rhizoma Curcumae Longae. Rhizoma Curcumae Longae, as Chinese medicine, begins to be loaded in Tang Materia Medica, has the function of removing blood stasis circulation of qi promoting, inducing menstruation to relieve menalgia, and modern medicine study shows, Rhizoma Curcumae Longae has antiinflammatory, antioxidation, scavenging free radicals, antimicrobial and antitumor action. It is used to treatment hyperlipidemia and has anti-liver toxicity in recent years.
Aryl turmerone is as a kind of important component of Rhizoma Curcumae Longae, belong to terpene compound, research in medical science relates to the effects such as inducing apoptosis of tumour cell, resisting gram-positive bacteria and gram negative bacteria, antifungal, antifertility, venom, and clinical development treatment leukemia, malignant lymphoma, bacterial inflammation, fungus inflammation, the even aspect such as the Metabolic syndrome such as diabetes, obesity disease and birth control are all held out broad prospects.
The effective ingredient of eliminating acne turmeric liniment is Rhizoma Curcumae Longae volatile oil, and volatilization main body of oil is aryl turmerone. Measure eliminating acne turmeric liniment quality control system to being further ensured that curative effect using aryl turmerone as its functional attributes, strengthen quality controllability significant. From Rhizoma Curcumae Longae volatile oil, how to obtain highly purified aryl turmerone reference substance, be need to solve.
Summary of the invention
The technical problem to be solved is to provide in a kind of Rhizoma Curcumae Longae volatile oil the preparation method of aryl turmerone reference substance, and it can separate the aryl turmerone obtaining very high purity from Rhizoma Curcumae Longae volatile oil.
The preparation method that the present invention is achieved in that in Rhizoma Curcumae Longae volatile oil aryl turmerone reference substance, with Rhizoma Curcumae Longae volatile oil for raw material, comprises the technical steps that:
(1) pressurization normal phase silicagel column crude separation: raw material and silica gel are pressed the mass ratio of 1:10-1:15, gradient elution is carried out for eluant with the petroleum ether-ethyl acetate that volume ratio is 30:1-15:1, pressurized column chromatography, by a lamellae, launching with petroleum ether-ethyl acetate, the volume ratio of petroleum ether and ethyl acetate is 15:1-25:1, observes under ultraviolet, the mixture of collector similar temperament, separates and obtains crude separation thing;
(2) pressurization normal phase silicagel column segmentation from: crude separation thing is added silica gel, the raw material added first and segmentation from the mass ratio of silica gel be 1:10-1:15, gradient elution is carried out for eluant with the petroleum ether-ethyl acetate that volume ratio is 100:1-50:1, pressurized column chromatography, by a lamellae, launches with petroleum ether-ethyl acetate, the volume ratio of petroleum ether and ethyl acetate is 25:1-15:1, observe under ultraviolet, collect polarity mixture closely, separate and obtain thin separator;
(3) preparative high-performance liquid chromatographic separates and obtains aryl turmerone reference substance: will add acetone in thin separator, the volume ratio of thin separator and acetone is 1:5-1:10, with first alcohol and water for mobile phase, the volume of methanol and water is 60:40-90:10, flow velocity is 5-10mL/min, within 15-17 minute, going out peak, 20-22 minute terminates, and collects and obtains the aryl turmerone sterling that purity is more than 99%.
In order to verify the technique effect of the present invention further, carry out following experiment:
The separation of aryl turmerone monomeric compound;
1.1 silica gel column chromatography crude separation;
Weigh Rhizoma Curcumae Longae volatile oil 10g, with petroleum ether dissolution in porcelain evaporating dishes, with 12g silica gel mixed sample, volatilize solvent, standby; By 1:10 with 100g silica gel (300-400 order), with petroleum ether-ethyl acetate (15:1) wet method upper prop; Loading again after layer of silica gel in post no longer moves down, and carry out pressurization eluting with petroleum ether-ethyl acetate (15:1), by a lamellae, launch with petroleum ether-ethyl acetate (15:1), observe under 254nm, collect the mixture that Rf0.7 place polarity is close, obtain 7.3g.
1.2 silica gel column chromatographies segmentation from;
Weigh the mixture 7.3g that crude separation obtains, with petroleum ether dissolution in porcelain evaporating dishes, with 10g silica gel mixed sample, volatilize solvent, standby; With 110g silica gel (300-400 order), with petroleum ether-ethyl acetate (50:1) wet method upper prop; Loading again after layer of silica gel in post no longer moves down, and carry out pressurization eluting with petroleum ether-ethyl acetate (50:1), by a lamellae, launch with petroleum ether-ethyl acetate (25:1), observe under 254nm, collect Rf0.5 place polarity mixture closely, obtain 5.2g.
Detected by HPLC separating the mixture obtained. HPLC condition is pillar HederaC18(200mm), mobile phase methanol-water (90:10), column temperature 25 DEG C, detect wavelength 254nm, sample size 15 μ L, measurement result is as shown in Figure 2, it has been found that mixture is 3 components, is decided to be 1,2,3 respectively from left to right.
1.3 preparative high-efficient liquids are separated;
To silica gel column chromatography segmentation from three components obtained by using Agilent1260 type to prepare liquid phase, with Agilent company AgilentZORBAXSB-C18(21.2 × 250mm, 5 μm) chromatographic column is easily separated, and takes silica gel column chromatography and segments from the mixture 1mL obtained, being dissolved in 5mL methanol and be diluted, mobile phase chooses methanol-water respectively: 95:5,90:10,85:75,80:20,75:25, flow velocity chooses 2mL/min respectively, 5mL/min, 8mL/min, 10mL/min, 12mL/min, therefrom determines Optimum separation condition. Finally determine that Optimum separation condition mobile phase is methanol-water (80:20), flow velocity 10mL/min. Collecting 3 components respectively, solvent is spin-dried for by rotated evaporimeter, obtains purified product 1, and 2,3, through MS,1H-NMR,13C-NMR is measured.
1.4 conclusions;
By MS,1H-NMR,13C-NMR is measured, and finally determines that 1 for aryl turmerone.MS measures, and is 98% with the aryl turmerone Data Matching rate in spectrum storehouse;1H-NMR,13It is correct that C-NMR measures structure,1HNMR(CDCl3,400MHz)δ:1.17(d,J=7.2Hz,3H),1.78(d,J=1.2Hz,3H),2.03(d,J=0.8Hz,3H),2.23(s,3H),2.50-2.56(m,1H),2.61-2.66(m,1H),3.19-3.24(m,1H),5.95(t,J=1.2Hz,1H),7.03(d,J=1.2Hz,4H);13CNMR(CDCl3, 100MHz) and δ: 20.72,20.99,22.00,27.65,35.30,52.70,124.11,126.68,129.12,135.56,143.71,155.10,199.89. Result is such as shown in Fig. 3, Fig. 4 and Fig. 5.
2.1 high performance liquid chromatography-purity test;
Adopt Agilent1260 type high performance liquid chromatograph (quaternary pump, automatic sampler, column oven, DAD UV-detector, ChemStation chromatographic work station), with Han Bang company HederaC18(4.6 × 200mm, 5 μm) chromatographic column, column temperature 25 DEG C, mobile phase is methanol-water (80:20), DAD sets wave-length coverage as 190 ~ 400nm, set 204,220,254,280,310nm five detect wavelength, and observe the detection case of other wavelength simultaneously, to investigate the purity of aryl turmerone.
Take aryl turmerone appropriate, every 1mL solution containing 1mg is made with methanol, as need testing solution, take blank solvent (methanol) and each 10 μ L of need testing solution, it is injected separately into chromatograph of liquid, after the chromatographic peak that result deduction blank solvent produces, the chromatographic peak area normalization content of aryl turmerone is all higher than more than 99% under each wavelength, in Table 1.
2.2 mobile phases are investigated;
Take aryl turmerone test liquid, using acetonitrile-water (80:20), methanol-water (80:20), methanol-0.05% phosphate aqueous solution (80:20), methanol-0.1% phosphate aqueous solution (80:20), methanol-0.3% phosphate aqueous solution (80:20) respectively is mobile phase, obtain HPLC collection of illustrative plates, all not occurring anomaly peak when result shows each mobile phase, when measuring each mobile phase with area normalization method, the content of aryl turmerone is all higher than 99%. Result is in Table 2, Fig. 6-10.
The investigation of 2.3 pillars;
Take aryl turmerone test liquid, use HederaC respectively18(4.6 × 200mm, 5 μm) chromatographic column, AgilentC8(4.6 × 150mm, 5 μm) chromatographic column, WatersC18(4.6 × 250mm, 5 μm) chromatographic column, obtains HPLC collection of illustrative plates, and result shows that changing chromatographic column anomaly peak does not all occur, with area normalization method measure the content of aryl turmerone is all higher than 99%. Result is in Table 3, Figure 11-13.
Owing to have employed technique scheme, compared with prior art, the present invention is with Rhizoma Curcumae Longae volatile oil for raw material, with silica gel column chromatography and preparative high performance liquid chromatography for separation means, with a certain proportion of petroleum ether-ethyl acetate, methanol-water is eluent system, the prepared aryl turmerone sterling obtained, detect through HPLC, it is a mass-tone spectral peak in different chromatographic columns and mobile phase measurement result, change chromatographic column and mobile phase measures and anomaly peak all do not occur, reference substance purity is measured more than 99% with area normalization method, meet the requirement of assay traditional Chinese chemical contrast.
Accompanying drawing explanation;
Fig. 1 is the HPLC testing result of the product of embodiments of the invention;
The thin separating mixture HPLC that Fig. 2 is the present invention measures collection of illustrative plates;
Fig. 3 is the aryl turmerone mass spectrum EI of the present invention;
Fig. 4 is the aryl turmerone of the present invention1H-NMR;
Fig. 5 is the aryl turmerone of the present invention13C-NMR;
Fig. 6 is the HPLC collection of illustrative plates of acetonitrile-water 80:20;
Fig. 7 is the HPLC collection of illustrative plates of methanol-water 80:20;
Fig. 8 is the HPLC collection of illustrative plates of methanol-0.05% phosphate aqueous solution 80:20;
Fig. 9 is the HPLC collection of illustrative plates of methanol-0.1% phosphate aqueous solution 80:20;
Figure 10 is the HPLC collection of illustrative plates of methanol-0.3% phosphate aqueous solution 80:20;
Figure 11 is HederaC18(4.6 × 200mm, 5 μm) chromatographic column;
Figure 12 is AgilentC8(4.6 × 150mm, 5 μm) chromatographic column;
Figure 13 is WatersC18(4.6 × 250mm, 5 μm) chromatographic column.
Detailed description of the invention;
Embodiments of the invention: the preparation method of aryl turmerone reference substance in Rhizoma Curcumae Longae volatile oil, with Rhizoma Curcumae Longae volatile oil for raw material, comprise the technical steps that:
(1) pressurization normal phase silicagel column crude separation: 10g Rhizoma Curcumae Longae volatile oil is added the silica gel 100g of 300-400 order, with volume ratio be 15:1 petroleum ether-ethyl acetate for eluant, pressurized column chromatography, by a lamellae, launch with petroleum ether-ethyl acetate (15:1), observe under 254nm, collect the mixture that Rf0.7 place polarity is close, i.e. crude separation thing, for 7.3g;
(2) pressurization normal phase silicagel column segmentation from: 7.3g crude separation thing will add the silica gel 110g of 300-400 order, with volume ratio be 50:1 petroleum ether-ethyl acetate for eluant, pressurized column chromatography, by a lamellae, launch with petroleum ether-ethyl acetate (25:1), observe under 254nm, collect Rf0.5 place polarity mixture closely,, i.e. thin separator, for 5.2g;
(3) preparative high-performance liquid chromatographic separates and obtains aryl turmerone reference substance: will add acetone in thin separator, the thin separator of every 1ml adds 5ml acetone and is diluted, each sample introduction 0.4mL, with first alcohol and water for mobile phase, the volume of methanol and water is 80:20, and flow velocity is 10mL/min, within 16 minutes, goes out peak, 21 minutes terminate, and once obtain aryl turmerone sterling 30mg.
The aryl turmerone sterling that will obtain, is detected by HPLC, and with first alcohol and water for mobile phase, the volume of methanol and water is 80:20, and column temperature is 25 DEG C, and flow velocity is 0.8ml/min, and detection wavelength is 242nm, and pillar is HederacC18200mm, detection was met as it is shown in figure 1, purity is 99.95%.
Claims (1)
1. the preparation method of aryl turmerone reference substance in a Rhizoma Curcumae Longae volatile oil, it is characterised in that: with Rhizoma Curcumae Longae volatile oil for raw material, comprise the technical steps that:
(1) pressurization normal phase silicagel column crude separation: raw material and silica gel are pressed the mass ratio of 1:10-1:15, gradient elution is carried out for eluant with the petroleum ether-ethyl acetate that volume ratio is 30:1-15:1, pressurized column chromatography, by a lamellae, launching with petroleum ether-ethyl acetate, the volume ratio of petroleum ether and ethyl acetate is 25:1-15:1, observes under ultraviolet, collect the mixture that Rf0.7 place is close with aryl turmerone polarity, separate and obtain crude separation thing;
(2) pressurization normal phase silicagel column segmentation from: crude separation thing is added silica gel, the raw material added first and segmentation from the mass ratio of silica gel be 1:10-1:15, gradient elution is carried out for eluant with the petroleum ether-ethyl acetate that volume ratio is 100:1-50:1, pressurized column chromatography, by a lamellae, launch with petroleum ether-ethyl acetate, the volume ratio of petroleum ether and ethyl acetate is 15:1-25:1, observe under ultraviolet, collect Rf0.5 place and aryl turmerone polarity mixture closely, separate and obtain thin separator;
(3) preparative high-performance liquid chromatographic separates and obtains aryl turmerone reference substance: will add acetone in thin separator, the volume ratio of thin separator and acetone is 1:5-1:10, with first alcohol and water for mobile phase, the volume of methanol and water is 60:40-90:10, flow velocity is 5-10mL/min, collects and obtains the aryl turmerone sterling that purity is more than 99%.
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| CN110051550A (en) * | 2019-05-30 | 2019-07-26 | 佛山市康伲爱伦生物技术有限公司 | A kind of ar-turmerone inclusion compound and preparation method thereof with sterilization anti-inflammatory effect |
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Inventor after: Wang Huijuan Inventor after: Zhou Ying Inventor after: Guo Donggui Inventor after: Pan Bowen Inventor after: Shi Yang Inventor after: Cui Kai Inventor after: Liu Xiongli Inventor before: Zhou Ying Inventor before: Pan Bowen Inventor before: Shi Yang Inventor before: Wang Huijuan Inventor before: Cui Kai Inventor before: Liu Xiongli |
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Effective date of registration: 20201109 Address after: 533000 Donghai Industrial Park, Tiandong County, Baise City, Guangxi Zhuang Autonomous Region Patentee after: Guangxi Mojiang biopharmaceutical Co., Ltd Address before: 550025 science and Technology Department, north campus, Guizhou University, Huaxi, Guizhou, China Patentee before: Guizhou University |
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